RAB7L1 INTERACTS WITH LRRK2 TO MODIFY INTRANEURONAL PROTEIN SORTING AND PARKINSON'S DISEASE RISK

Abstract

The present invention relates to methods and compositions for treating Parkinson's disease in a subject.

Description

All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.

This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.

BACKGROUND

Parkinson's disease (PD) is a degenerative disorder of the central nervous system. It results from the death of dopamine-containing cells in the substantia nigra, a region of the midbrain; the cause of cell-death is unknown. Early in the course of the disease, the most obvious symptoms are movement-related, including shaking, rigidity, slowness of movement and difficulty with walking and gait. Later, cognitive and behavioral problems may arise, with dementia commonly occurring in the advanced stages of the disease. Other symptoms include sensory, sleep and emotional problems. PD is more common in the elderly with most cases occurring after the age of 50.

Parkinson's disease is diagnosed by a physician exam, and diagnosis is based on the medical history and a neurological examination of the patient. There is no laboratory or molecular test that will clearly identify the disease. Brain scans are sometimes used to rule out disorders that could give rise to similar symptoms. Patients may be given levodopa, or other dopamine affecting agent, and resulting relief of motor impairment tends to confirm diagnosis. The finding of Lewy bodies in the midbrain on autopsy is usually considered proof that the patient suffered from Parkinson's disease. Thus there is need for biomarkers for PD disease or treatment.

SUMMARY

Recent genome-wide association studies have linked common variants in the human genome to Parkinson's disease (PD) risk. In certain aspects, the invention describes that the consequences of variants at 2 such loci, PARK6 and LRRK2, are highly interrelated, both in terms of their broad impacts on human brain transcriptomes of unaffected carriers, and in terms of their associations with PD risk. Deficiency of the PARK16 locus gene RAB7L1 in primary rodent neurons, or of a RAB7L1 orthologue in Drosophila dopamine neurons, recapitulated degeneration observed with expression of a familial PD mutant form of LRRK2, whereas RAB7L1 overexpression rescued the LRRK2 mutant phenotypes. PD-associated defects in RAB7L1 or LRRK2 led to endolysosomal and Golgi apparatus sorting defects and deficiency of the VPS35 component of the retromer complex. Expression of wild-type VPS35, but not a familial PD-associated mutant form, rescued these defects. Taken together, these studies implicate retromer and lysosomal pathway alterations in PD risk.

In certain aspects the invention provides that genetic variants at PARK16 and LRRK2 interact to modify Parkinson's disease risk.

In certain aspects the invention provides that splicing of the PARK16 locus gene RAB7L1 is modified by genetic variants.

In certain aspects the invention provides that RAB7L1 and LRRK2 coordinately regulate protein sorting through the retromer pathway.

In certain aspects the invention provides that expression of the retromer component VPS35 can suppress LRRK2 mutant pathology.

In certain aspects the invention provides human functional genomics combined with cell and animal model studies, to provide convergent evidence of a critical role for RAB7L1 at the PARK16 locus and of retromer pathway dysfunction in Parkinson's disease etiology.

In certain embodiments, the subject is suspected of or evaluated for having predisposition or risk to sporadic (non-familial) PD.

In certain aspects, the invention provides methods to determine risk or predisposition to develop PD in a subject in need thereof comprising: (a) providing a sample from a subject in need thereof, (b) determining the presence or absence of a genetic variant(s) at PARK16 and LRRK2 locus, and (c) comparing the genetic variant(s) at PARK16 and/or LRRK2 locus from the subject sample to the PARK16 and/or LRRK2 locus variant in a reference sample, wherein the reference sample is associated with a non-PD status. In certain embodiments, the methods further comprise determining whether the gene variant(s) lead to a deficiency of the PARK16 locus gene RAB7L1. In certain embodiments, the deficiency is reduced level of the full-length RAB7L.

The methods can determine the protein level of full-length RAB7L, the full-length RAB7L mRNA levels, or a combination thereof. In other embodiments, the methods can determine the level of VPS35 protein or mRNA, or a combination thereof. The methods can determine the protein level of full-length RAB7L, the full-length RAB7L mRNA levels, the full-length RAB7L mRNA levels, or a combination thereof.

In certain embodiments, the predisposition or risk that is determined is to sporadic (non-familial) PD.

In certain embodiments, the variants are associated with familial PD. In other embodiments, the variants are associated with sporadic PD.

In certain aspects, the invention provides methods to determine risk or predisposition to develop PD in a subject in need thereof comprising: (a) providing a sample from a subject in need thereof, (b) determining the presence or absence of a genetic variant(s) at the PARK16 locus gene RAB7L1, and (c) comparing the genetic variant(s) at PARK16 locus gene RAB7L1 from the subject sample to the PARK16 locus gene RAB7L1 variant(s) in a reference sample, wherein the reference sample is associated with a non-PD status. In certain embodiments, the genetic variant(s) at the PARK16 locus affect the PARK16 locus gene RAB7L1. In certain embodiments, the PARK16 locus gene variant(s) lead to a deficiency of the PARK16 locus gene RAB7L1.

In certain aspects, the invention provides methods to determine a risk or predisposition to develop PD in a subject in need thereof comprising: (a) providing a sample from a subject in need thereof, (b) determining the level of full-length RAB7L, and (c) comparing the level of full-length RAB7L from the subject sample to the full-length RAB7L1 level in a reference sample, wherein the reference sample is associated in non-PD status, and wherein a reduced level of the full-length RAB7L is indicative of an increased risk or predisposition to PD. The methods can determine the protein level of full-length RAB7L, the full-length RAB7L mRNA levels, or a combination thereof. In certain embodiments, the methods comprise determining the full-length RAB7L1 level in a reference sample. In certain embodiments, the methods comprise determining whether the levels the full-length RAB7L1 level in the subject sample are reduced compared to these levels in a reference sample.

In certain aspects, the invention provides methods to determine a risk or predisposition to develop PD in a subject in need thereof comprising: (a) providing a sample from a subject in need thereof, (b) determining the level of VPS35 protein or mRNA, and (c) comparing the level of VPS35 protein or mRNA from the subject sample to the VPS35 protein or mRNA level in a reference sample, wherein the reference sample is associated in non-PD status, and wherein a reduced level of the VPS35 protein or mRNA is indicative of an increased risk or predisposition to PD. In certain embodiments, the methods comprise determining the level VPS35 protein or mRNA in a reference sample. In certain embodiments, the methods comprise determining whether the levels the VPS35 protein or mRNA level in the subject sample are reduced compared to these levels in a reference sample.

In certain embodiments, the methods determine protein or mRNA levels, or a combination thereof.

Certain PARK16 and/or LRRK2 locus variants, including but not limited to variants as described herein, are associated with increased PD risk. In certain embodiments, the PARK16 locus gene is RAB7L1. In certain embodiments, PARK16 and LRRK2 locus variants cooperatively determine PD risk. In certain embodiments, the effect of a risk associated variant at the LRRK2 locus is dependent (or correlated) on the presence of the risk variant at the PARK16 locus, and vice versa. In certain embodiments, there is a genetic interaction between PARK16 and LRRK2 locus variants that affects PD predisposition and risk.

In certain embodiments, the methods further comprise determining whether PD-associated variants or defects in RAB7L1 or LRRK2 lead to endolysosomal, Golgi apparatus sorting defects, deficiency of the VPS35 component of the retromer complex, or any combination thereof.

In certain aspects, the invention provides methods to determine risk or predisposition to develop PD in a subject in need thereof comprising: (a) providing a sample from a subject in need thereof, (b) determining the presence or absence of an endolysosomal, Golgi apparatus sorting defects, deficiency of the VPS35 component of the retromer complex, or any combination thereof, compared a reference sample, wherein the reference sample is associated with a non-PD status.

In certain embodiments, the splicing of the PARK16 locus gene RAB7L1 is modified by genetic variants and is associated with increased risk of PD. In certain embodiments, the PD-associated variants or defects in RAB7L1 or LRRK2 lead to endolysosomal, Golgi apparatus sorting defects, deficiency of the VPS35 component of the retromer complex, or any combination thereof.

In certain embodiments of the methods, the subject is diagnosed with PD and is not administered dopamine affecting agents (i.e. not treated for PD).

In certain embodiments of the methods, the subject is diagnosed by clinical symptoms, imaging of dopamine uptake, or combination thereof.

In certain embodiments, the methods comprise isolating nucleic acids from the subject's biological sample. In the instant methods, the subject's sample is a biological sample, including but not limited to a blood sample, plasma sample, serum, CSF, tissue, cell or any combination thereof. Methods to isolate nucleic acid sequences from biological samples are known in the art. Methods for quantitative determination of amount of nucleic acids in a biological sample are known in the art.

In certain embodiments, the methods comprise quantifying the nucleic acid levels of RAB7L1, VPS35, or any combination thereof, wherein the nucleic acid levels are quantified by RT-qPCR, or any other suitable method. In other embodiments, the methods comprise quantifying the protein levels of RAB7L1. VPS35, or any combination thereof. Methods to determine protein levels in a quantitative manner are known in the art.

In certain embodiments, the sample is a cerebro-spinal fluid (CSF) sample, blood sample, plasma, serum, or any other suitable sample, or any combination thereof.

The invention provides a kit comprising PCR primers to carry out the methods of any of the steps, may also include instructions to carry out steps (a), (b) and (c) of these methods.

A kit comprising at least one nucleic acid, for example but not limited to a primer or a probe, to selectively quantify the levels of RAB7L1. VPS35, or any combination thereof, so as to determine the levels of RAB7L1. VPS3, and instructions to carry out steps (a) and (b) of the method of any of the methods.

In certain aspects, the invention provides methods for treating PD, comprising administering to a subject in need thereof a therapeutic amount of the retromer component VPS35.

An aspect of the invention provides for a method of treating Parkinson's Disease (PD) in a subject. In one embodiment, the method comprises determining the presence or absence of a genetic variant at the PARK16 and LRRK2 loci in a sample from a subject, wherein the presence of a PD-associated genetic variant at both the PARK16 and LRRK2 loci in the subject sample indicates the subject has an increased risk or predisposition to PD; and administering a treatment if the subject has an increased risk or predisposition to PD. In one embodiment, the genetic variant at the PARK16 locus comprises a genetic variant in the RAB7L1 gene. In another embodiment, the genetic variant at the RAB7L1 gene is SNP rs1572931. In a further embodiment, the PD-associated genetic variant at the PARK16 locus comprises a guanine (G) nucleotide at SNP rs1572931. In some embodiments, the PD-associated genetic variant at the PARK16 locus encodes a RAB7L1 mRNA, wherein exon 2 is excluded from the RAB7L1 mRNA sequence. In other embodiments, the the PD-associated genetic variant comprises SEQ ID NO: 5. In further embodiments, the PD-associated genetic variant at the PARK16 locus results in loss of expression of a RAB7L1 protein. In some embodiments, the genetic variant at the LRRK2 locus comprises SNP rs11176052. In other embodiments, the PD-associated genetic variant at the LRRK2 locus encodes the protein of SEQ ID NO: 27 or 28. In yet further embodiments, the PD-associated genetic variant at the LRKK2 locus results in loss of expression of a LRKK2 protein. In one embodiment, the treatment comprises administering to the subject an effective amount of a nucleic acid encoding a protein comprising 90% of SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 11, SEQ ID NO: 14, or a combination or fragment thereof. In some embodiments, the treatment comprises administering to the subject an effective amount of a protein comprising 90% of SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 11, SEQ ID NO: 14, or a combination or fragment thereof. In further embodiments, the PD disease status is determined by any suitable method, including but not limited to a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In another embodiment, the subject is not diagnosed with PD. In yet other embodiments, the method further comprises a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In some embodiments, the subject is suspected of having PD or is at risk of developing PD based on the presence of any one of parkinsonism symptoms. In further embodiments, the sample is a CSF sample, blood sample, plasma sample, serum sample or any combination thereof.

An aspect of the invention provides for a method of treating Parkinson's Disease (PD) in a subject. In one embodiment, the method comprises determining the presence or absence of a genetic variant at the PARK16 and LRRK2 loci in a sample from a subject, wherein the presence of a PD-associated genetic variant at both the PARK16 and LRRK2 loci in the subject sample indicates the subject has an increased risk or predisposition to PD; and administering a treatment if the subject has an increased risk or predisposition to PD, wherein the treatment comprises administering to the subject an effective amount of a protein comprising 90% of SEQ ID NO: 6. SEQ ID NO: 26, SEQ ID NO: 11. SEQ ID NO: 14, or a combination or fragment thereof. In one embodiment, the genetic variant at the PARK16 locus comprises a genetic variant in the RAB7L1 gene. In another embodiment, the genetic variant at the RAB7L1 gene is SNP rs1572931. In a further embodiment, the PD-associated genetic variant at the PARK16 locus comprises a guanine (G) nucleotide at SNP rs1572931. In some embodiments, the PD-associated genetic variant at the PARK16 locus encodes a RAB7L1 mRNA, wherein exon 2 is excluded from the RAB7L1 mRNA sequence. In other embodiments, the the PD-associated genetic variant comprises SEQ ID NO: 5. In further embodiments, the PD-associated genetic variant at the PARK16 locus results in loss of expression of a RAB7L1 protein. In some embodiments, the genetic variant at the LRRK2 locus comprises SNP rs11176052. In other embodiments, the PD-associated genetic variant at the LRRK2 locus encodes the protein of SEQ ID NO: 27 or 28. In yet further embodiments, the PD-associated genetic variant at the LRKK2 locus results in loss of expression of a LRKK2 protein. In further embodiments, the PD disease status is determined by any suitable method, including but not limited to a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In another embodiment, the subject is not diagnosed with PD. In yet other embodiments, the method further comprises a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In some embodiments, the subject is suspected of having PD or is at risk of developing PD based on the presence of any one of parkinsonism symptoms. In further embodiments, the sample is a CSF sample, blood sample, plasma sample, serum sample or any combination thereof.

An aspect of the invention provides for a method of treating Parkinson's Disease (PD) in a subject. In one embodiment, the method comprises determining the presence or absence of a genetic variant at the PARK16 and LRRK2 loci in a sample from a subject, wherein the presence of a PD-associated genetic variant at both the PARK16 and LRRK2 loci in the subject sample indicates the subject has an increased risk or predisposition to PD; and administering a treatment if the subject has an increased risk or predisposition to PD, wherein the treatment comprises administering to the subject an effective amount of a nucleic acid encoding a protein comprising 90% of SEQ ID NO: 6, SEQ ID NO: 26. SEQ ID NO: 11, SEQ ID NO: 14, or a combination or fragment thereof. In one embodiment, the genetic variant at the PARK16 locus comprises a genetic variant in the RAB7L1 gene. In another embodiment, the genetic variant at the RAB7L1 gene is SNP rs1572931. In a further embodiment, the PD-associated genetic variant at the PARK16 locus comprises a guanine (G) nucleotide at SNP rs1572931. In some embodiments, the PD-associated genetic variant at the PARK16 locus encodes a RAB7L1 mRNA, wherein exon 2 is excluded from the RAB7L1 mRNA sequence. In other embodiments, the the PD-associated genetic variant comprises SEQ ID NO: 5. In further embodiments, the PD-associated genetic variant at the PARK16 locus results in loss of expression of a RAB7L1 protein. In some embodiments, the genetic variant at the LRRK2 locus comprises SNP rs11176052. In other embodiments, the PD-associated genetic variant at the LRRK2 locus encodes the protein of SEQ ID NO: 27 or 28. In yet further embodiments, the PD-associated genetic variant at the LRKK2 locus results in loss of expression of a LRKK2 protein. In further embodiments, the PD disease status is determined by any suitable method, including but not limited to a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In another embodiment, the subject is not diagnosed with PD. In yet other embodiments, the method further comprises a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In some embodiments, the subject is suspected of having PD or is at risk of developing PD based on the presence of any one of parkinsonism symptoms. In further embodiments, the sample is a CSF sample, blood sample, plasma sample, serum sample or any combination thereof.

An aspect of the invention provides for a method of treating Parkinson's Disease (PD) in a subject. In one embodiment, the method comprises determining the presence or absence of a genetic variant at the LRRK2 locus in a sample from a subject, wherein the presence of a PD-associated genetic variant at the LRRK2 locus in the subject sample indicates the subject has an increased risk or predisposition to PD; and administering a treatment if the subject has an increased risk or predisposition to PD. In some embodiments, the genetic variant at the LRRK2 locus comprises SNP rs11176052. In other embodiments, the PD-associated genetic variant at the LRRK2 locus encodes the protein of SEQ ID NO: 27 or 28. In yet further embodiments, the PD-associated genetic variant at the LRKK2 locus results in loss of expression of a LRKK2 protein. In further embodiments, the treatment comprises administering to the subject an effective amount of a nucleic acid encoding a protein comprising 90% of SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 14, or a combination or fragment thereof. In some embodiments, the treatment comprises administering to the subject an effective amount of a protein comprising 90% of SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 14, or a combination or fragment thereof. In further embodiments, the PD disease status is determined by any suitable method, including but not limited to a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In another embodiment, the subject is not diagnosed with PD. In yet other embodiments, the method further comprises a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In some embodiments, the subject is suspected of having PD or is at risk of developing PD based on the presence of any one of parkinsonism symptoms. In further embodiments, the sample is a CSF sample, blood sample, plasma sample, serum sample or any combination thereof.

An aspect of the invention provides for a method of treating Parkinson's Disease (PD) in a subject. In one embodiment, the method comprises determining the presence or absence of a genetic variant at the LRRK2 locus in a sample from a subject, wherein the presence of a PD-associated genetic variant at the LRRK2 locus in the subject sample indicates the subject has an increased risk or predisposition to PD; and administering a treatment if the subject has an increased risk or predisposition to PD, wherein the treatment comprises administering to the subject an effective amount of a nucleic acid encoding a protein comprising 90%/o of SEQ ID NO: 6. SEQ ID NO: 26, SEQ ID NO: 14, or a combination or fragment thereof. In some embodiments, the genetic variant at the LRRK2 locus comprises SNP rs11176052. In other embodiments, the PD-associated genetic variant at the LRRK2 locus encodes the protein of SEQ ID NO: 27 or 28. In yet further embodiments, the PD-associated genetic variant at the LRKK2 locus results in loss of expression of a LRKK2 protein. In further embodiments, the PD disease status is determined by any suitable method, including but not limited to a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In another embodiment, the subject is not diagnosed with PD. In yet other embodiments, the method further comprises a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In some embodiments, the subject is suspected of having PD or is at risk of developing PD based on the presence of any one of parkinsonism symptoms. In further embodiments, the sample is a CSF sample, blood sample, plasma sample, serum sample or any combination thereof.

An aspect of the invention provides for a method of treating Parkinson's Disease (PD) in a subject. In one embodiment, the method comprises determining the presence or absence of a genetic variant at the LRRK2 locus in a sample from a subject, wherein the presence of a PD-associated genetic variant at the LRRK2 locus in the subject sample indicates the subject has an increased risk or predisposition to PD; and administering a treatment if the subject has an increased risk or predisposition to PD, wherein the treatment comprises administering to the subject an effective amount of a protein comprising 90% of SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 14, or a combination or fragment thereof. In some embodiments, the genetic variant at the LRRK2 locus comprises SNP rs11176052. In other embodiments, the PD-associated genetic variant at the LRRK2 locus encodes the protein of SEQ ID NO: 27 or 28. In yet further embodiments, the PD-associated genetic variant at the LRKK2 locus results in loss of expression of a LRKK2 protein. In further embodiments, the PD disease status is determined by any suitable method, including but not limited to a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In another embodiment, the subject is not diagnosed with PD. In yet other embodiments, the method further comprises a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In some embodiments, the subject is suspected of having PD or is at risk of developing PD based on the presence of any one of parkinsonism symptoms. In further embodiments, the sample is a CSF sample, blood sample, plasma sample, serum sample or any combination thereof.

An aspect of the invention provides for a method of treating Parkinson's Disease (PD) in a subject. In one embodiment, the method comprises determining the presence or absence of a genetic variant at the PARK16 locus in a sample from a subject, wherein the presence of a PD-associated genetic variant at the PARK16 locus in the subject sample indicates the subject has an increased risk or predisposition to PD; and administering a treatment if the subject has an increased risk or predisposition to PD. In one embodiment, the genetic variant at the PARK16 locus comprises a genetic variant in the RAB7L1 gene. In another embodiment, the genetic variant at the RAB7L1 gene is SNP rs1572931. In a further embodiment, the PD-associated genetic variant at the PARK16 locus comprises a guanine (G) nucleotide at SNP rs1572931. In some embodiments, the PD-associated genetic variant at the PARK16 locus encodes a RAB7L1 mRNA, wherein exon 2 is excluded from the RAB7L1 mRNA sequence. In other embodiments, the the PD-associated genetic variant comprises SEQ ID NO: 5. In further embodiments, the PD-associated genetic variant at the PARK16 locus results in loss of expression of a RAB7L1 protein. In one embodiment, the treatment comprises administering to the subject an effective amount of a nucleic acid encoding a protein comprising 90% of SEQ ID NO: 11, SEQ ID NO: 14, or a combination or fragment thereof. In another embodiment, the treatment comprises administering to the subject an effective amount of a protein comprising 90% of SEQ ID NO: 1, SEQ ID NO: 14, or a combination or fragment thereof. In further embodiments, the PD disease status is determined by any suitable method, including but not limited to a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In another embodiment, the subject is not diagnosed with PD. In yet other embodiments, the method further comprises a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In some embodiments, the subject is suspected of having PD or is at risk of developing PD based on the presence of any one of parkinsonism symptoms. In further embodiments, the sample is a CSF sample, blood sample, plasma sample, serum sample or any combination thereof.

An aspect of the invention provides for a method of treating Parkinson's Disease (PD) in a subject. In one embodiment, the method comprises determining the presence or absence of a genetic variant at the PARK16 locus in a sample from a subject, wherein the presence of a PD-associated genetic variant at the PARK16 locus in the subject sample indicates the subject has an increased risk or predisposition to PD; and administering a treatment if the subject has an increased risk or predisposition to PD, wherein the treatment comprises administering to the subject an effective amount of a nucleic acid encoding a protein comprising 90% of SEQ ID NO: 11, SEQ ID NO: 14, or a combination or fragment thereof. In one embodiment, the genetic variant at the PARK16 locus comprises a genetic variant in the RAB7L1 gene. In another embodiment, the genetic variant at the RAB7L1 gene is SNP rs1572931. In a further embodiment, the PD-associated genetic variant at the PARK16 locus comprises a guanine (G) nucleotide at SNP rs1572931. In some embodiments, the PD-associated genetic variant at the PARK16 locus encodes a RAB7L1 mRNA, wherein exon 2 is excluded from the RAB7L mRNA sequence. In other embodiments, the the PD-associated genetic variant comprises SEQ ID NO: 5. In further embodiments, the PD-associated genetic variant at the PARK16 locus results in loss of expression of a RAB7L1 protein. In further embodiments, the PD disease status is determined by any suitable method, including but not limited to a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In another embodiment, the subject is not diagnosed with PD. In yet other embodiments, the method further comprises a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In some embodiments, the subject is suspected of having PD or is at risk of developing PD based on the presence of any one of parkinsonism symptoms. In further embodiments, the sample is a CSF sample, blood sample, plasma sample, serum sample or any combination thereof.

An aspect of the invention provides for a method of treating Parkinson's Disease (PD) in a subject. In one embodiment, the method comprises determining the presence or absence of a genetic variant at the PARK16 locus in a sample from a subject, wherein the presence of a PD-associated genetic variant at the PARK16 locus in the subject sample indicates the subject has an increased risk or predisposition to PD; and administering a treatment if the subject has an increased risk or predisposition to PD, wherein the treatment comprises administering to the subject an effective amount of a protein comprising 90% of SEQ ID NO: 11, SEQ ID NO: 14, or a combination or fragment thereof. In one embodiment, the genetic variant at the PARK16 locus comprises a genetic variant in the RAB7L11 gene. In another embodiment, the genetic variant at the RAB7L1 gene is SNP rs1572931. In a further embodiment, the PD-associated genetic variant at the PARK16 locus comprises a guanine (G) nucleotide at SNP rs1572931. In some embodiments, the PD-associated genetic variant at the PARK6 locus encodes a RAB7L1 mRNA, wherein exon 2 is excluded from the RAB7L1 mRNA sequence. In other embodiments, the the PD-associated genetic variant comprises SEQ ID NO: 5. In further embodiments, the PD-associated genetic variant at the PARK16 locus results in loss of expression of a RAB7L1 protein. In further embodiments, the PD disease status is determined by any suitable method, including but not limited to a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In another embodiment, the subject is not diagnosed with PD. In yet other embodiments, the method further comprises a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In some embodiments, the subject is suspected of having PD or is at risk of developing PD based on the presence of any one of parkinsonism symptoms. In further embodiments, the sample is a CSF sample, blood sample, plasma sample, serum sample or any combination thereof.

An aspect of the invention provides for a method of treating PD in a subject. In one embodiment, the method comprises measuring the expression levels of a set of genes in a sample from a subject, wherein the set of genes comprises at least one gene selected from the genes listed in Table 2; comparing the expression levels of the set of genes in the subject sample to expression levels of the same set of genes in a reference sample or samples, wherein the reference sample or samples are from an individual who has a PD-associated SNP, and wherein similar expression levels of the set of genes in the subject sample and the set of genes in the reference sample(s) indicates the subject has an increased risk or predisposition to PD; and administering a treatment if the subject has an increased risk or predisposition to PD. In one embodiment, the treatment comprises administering to the subject an effective amount of a nucleic acid encoding a protein comprising 90% of SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 11, SEQ ID NO: 14, or a combination or fragment thereof. In some embodiments, the treatment comprises administering to the subject an effective amount of a protein comprising 90% of SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 11, SEQ ID NO: 14, or a combination or fragment thereof. In further embodiments, the PD disease status is determined by any suitable method, including but not limited to a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In another embodiment, the subject is not diagnosed with PD. In yet other embodiments, the method further comprises a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In some embodiments, the subject is suspected of having PD or is at risk of developing PD based on the presence of any one of parkinsonism symptoms. In further embodiments, the sample is a CSF sample, blood sample, plasma sample, serum sample or any combination thereof.

An aspect of the invention provides a method of treating PD in a subject. In one embodiment, the method comprises determining a level of full-length RAB7L1 in a sample from a subject; comparing the level of full-length RAB7L1 from the subject sample to a full-length RAB7L1 level in a reference sample, wherein the reference sample is associated with a non-PD status, and wherein a reduced level of the full-length RAB7L in the subject sample indicates the subject has an increased risk or predisposition to PD; and administering a treatment if the subject has an increased risk or predisposition to PD. In one embodiment, the method comprises the level of full-length RAB7L is protein level of full-length RAB7L, or mRNA levels of the full-length RAB7L, or a combination thereof. In one embodiment, the treatment comprises administering to the subject an effective amount of a nucleic acid encoding a protein comprising 90% of SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 11, SEQ ID NO: 14, or a combination or fragment thereof. In some embodiments, the treatment comprises administering to the subject an effective amount of a protein comprising 90% of SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 1, SEQ ID NO: 14, or a combination or fragment thereof. In further embodiments, the PD disease status is determined by any suitable method, including but not limited to a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In another embodiment, the subject is not diagnosed with PD. In yet other embodiments, the method further comprises a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In some embodiments, the subject is suspected of having PD or is at risk of developing PD based on the presence of any one of parkinsonism symptoms. In further embodiments, the sample is a CSF sample, blood sample, plasma sample, serum sample or any combination thereof. In another embodiment, the method further comprises a step of sequencing nucleic acids isolated from the subject's sample to determine the presence or absence of a PD-risk associated SNP, wherein the presence of a PD-risk associated SNP is further indicative that the subject is at risk of developing PD or is suffering from PD.

An aspect of the invention provides for a method of treating Parkinson's Disease (PD) in a subject. In one embodiment, the method comprises determining a level of isoform 3 of RAB7L1 in a sample from a subject; comparing the level of isoform 3 of RAB7L1 from the subject sample to an isoform 3 of RAB7L1 level in a reference sample, wherein the reference sample is associated in non-PD status, and wherein an increased level of isoform 3 of RAB7L1 in the subject sample indicates the subject has an increased risk or predisposition to PD; and administering a treatment if the subject has an increased risk or predisposition to PD. In another embodiment, the level of isoform 3 of RAB7L1 is a protein level. In a further embodiment, the method further comprises determining the level of transcript variant 4, 5, or a combination thereof of RAB7L1. In one embodiment, the treatment comprises administering to the subject an effective amount of a nucleic acid encoding a protein comprising 90% of SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 11. SEQ ID NO: 14, or a combination or fragment thereof. In some embodiments, the treatment comprises administering to the subject an effective amount of a protein comprising 90% of SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 11, SEQ ID NO: 14, or a combination or fragment thereof. In further embodiments, the PD disease status is determined by any suitable method, including but not limited to a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In another embodiment, the subject is not diagnosed with PD. In yet other embodiments, the method further comprises a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In some embodiments, the subject is suspected of having PD or is at risk of developing PD based on the presence of any one of parkinsonism symptoms. In further embodiments, the sample is a CSF sample, blood sample, plasma sample, serum sample or any combination thereof. In another embodiment, the method further comprises a step of sequencing nucleic acids isolated from the subject's sample to determine the presence or absence of a PD-risk associated SNP, wherein the presence of a PD-risk associated SNP is further indicative that the subject is at risk of developing PD or is suffering from PD.

An aspect of the invention provides a method of treating Parkinson's Disease (PD) in a subject. In one embodiment, the method comprises determining a level of transcript variant 4, 5, or a combination thereof of RAB7L1 in a sample from a subject; comparing the level of transcript variant 4, 5, or a combination thereof of RAB7L1 from the subject sample to a transcript variant 4, 5, or a combination thereof of RAB7L1 level in a reference sample, wherein the reference sample is associated in non-PD status, and wherein an increased level of transcript variant 4, 5, or a combination thereof of RAB7L1 in the subject sample indicates the subject has an increased risk or predisposition to PD; and administering a treatment if the subject has an increased risk or predisposition to PD. In another embodiment, the level of transcript variant 4, 5 or a combination thereof of RAB7L1 is a mRNA level. In a further embodiment, the method further comprises determining the level of isoform 3 of RAB7L1. In one embodiment, the treatment comprises administering to the subject an effective amount of a nucleic acid encoding a protein comprising 90% of SEQ ID NO: 6, SEQ ID NO: 26. SEQ ID NO: 11, SEQ ID NO: 14, or a combination or fragment thereof. In some embodiments, the treatment comprises administering to the subject an effective amount of a protein comprising 90% of SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 11, SEQ ID NO: 14, or a combination or fragment thereof. In further embodiments, the PD disease status is determined by any suitable method, including but not limited to a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In another embodiment, the subject is not diagnosed with PD. In yet other embodiments, the method further comprises a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In some embodiments, the subject is suspected of having PD or is at risk of developing PD based on the presence of any one of parkinsonism symptoms. In further embodiments, the sample is a CSF sample, blood sample, plasma sample, serum sample or any combination thereof. In another embodiment, the method further comprises a step of sequencing nucleic acids isolated from the subject's sample to determine the presence or absence of a PD-risk associated SNP, wherein the presence of a PD-risk associated SNP is further indicative that the subject is at risk of developing PD or is suffering from PD.

An aspect of the invention provides a method of treating Parkinson's Disease (PD) in a subject. In one embodiment, the method comprises determining a level of retromer components in a sample from a subject; comparing the level of retromer components from the subject sample to a retromer component level in a reference sample, wherein the reference sample is associated with a non-PD status, and wherein a reduced level of the retromer components in the subject sample indicates the subject has an increased risk or predisposition to PD; and administering a treatment if the subject has an increased risk or predisposition to PD. In another embodiment, the level of retromer component is protein level of retromer component, or mRNA levels of retromer component, or a combination thereof. In a further embodiment, the retromer component is VPS35, VPS29, VPS26 or a combination thereof. In some embodiments, the level of VPS35. VPS29, or VPS26 is protein level of VPS35, VPS29, or VPS26, or mRNA levels of VPS35. VPS29, or VPS26, or a combination thereof. In one embodiment, the treatment comprises administering to the subject an effective amount of a nucleic acid encoding a protein comprising 90% of SEQ ID NO: 6. SEQ ID NO: 26, SEQ ID NO: 11. SEQ ID NO: 14, or a combination or fragment thereof. In some embodiments, the treatment comprises administering to the subject an effective amount of a protein comprising 90% of SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 11, SEQ ID NO: 14, or a combination or fragment thereof. In further embodiments, the PD disease status is determined by any suitable method, including but not limited to a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In another embodiment, the subject is not diagnosed with PD. In yet other embodiments, the method further comprises a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. In some embodiments, the subject is suspected of having PD or is at risk of developing PD based on the presence of any one of parkinsonism symptoms. In further embodiments, the sample is a CSF sample, blood sample, plasma sample, serum sample or any combination thereof. In another embodiment, the method further comprises a step of sequencing nucleic acids isolated from the subject's sample to determine the presence or absence of a PD-risk associated SNP, wherein the presence of a PD-risk associated SNP is further indicative that the subject is at risk of developing PD or is suffering from PD.

An aspect of the invention provides for a composition for evaluating the existence of, or predisposition to, PD in a subject, said composition comprising polynucleotides or oligonucleotides, wherein each polynucleotide or oligonucleotide hybridizes to a gene, gene fragment, or gene transcript of at least two different markers in a subject sample, wherein the markers comprise LRRK2, RAB7L1 and VPS35.

An aspect of the invention provides for a composition for evaluating the existence of, or predisposition to, PD in a subject, said composition comprising polynucleotides or oligonucleotides, wherein each polynucleotide or oligonucleotide hybridizes to a gene, gene fragment, or gene transcript of a different marker in a subject sample, each marker being one of the genes listed in Table 2. In one embodiment, the composition comprises a microarray, a microfluidics card, a chip, or a chamber.

An aspect of the invention provides a kit for determining the levels of RAB7L1, LRRK2, VPS35, or a combination thereof, the kit comprising at least one oligonucleotide or polynucleotide to selectively quantify the levels of RAB7L1. LRRK2. VPS35, or a combination thereof. In one embodiment, the oligonucleotide or polynucleotide comprises SEQ ID NO: 15, 16, 17, or 18.

An aspect of the invention provides for a diagnostic kit for determining whether a sample from a subject exhibits a presence or absence of a PD-associated genetic variant, the kit comprising at least one oligonucleotide or polynucleotide for sequencing nucleic acids isolated from the subject's sample to determine the presence or absence of a PD-risk associated SNP, wherein the presence of a PD-risk associated SNP is further indicative that the subject is at risk of developing PD or is suffering from PD. In one embodiment, the oligonucleotide or polynucleotide comprises SEQ ID NO: 24, or 25.

An aspect of the invention provides for a diagnostic kit comprising the microarray, microfluidics card, chip, or chamber described herein.

An aspect of the invention provides for a synthetic nucleic acid comprising SEQ ID NO: 15, 16, 17, 18, 19, 24, or 25.

BRIEF DESCRIPTION OF THE FIGURES

To conform to the requirements for PCT patent applications, many of the figures presented herein are black and white representations of images originally created in color, such as many of those figures based on immunofluorescence microscopy. In the below descriptions and the examples, this colored staining is described in terms of its appearance in black and white. For example, GFP staining which appeared green in the original appears as a grey stain when presented in black and white. The original color versions of FIGS. 1-13 can be viewed in MacLeod et al., (2013) Neuron. 77(3):425-39 (including the accompanying Supplementary Information available in the on-line version of the manuscript available on the Neuron web site). For the purposes of the PCT, the contents of MacLeod et al., (2013) Neuron. 77(3):425-39, including the accompanying “Supplementary Information.” are herein incorporated by reference.

FIGS. 1A-1B. LRRK2 and PARK16 PD risk-associated variants function in a common genetic pathway. (1A) PD risk-associated variants exert functional effects in the CNS of unaffected individuals that is assessed in terms of a global transcriptome impact. Similar to the one observed in PD affected brain, it may reflect a pre-disease prodromal state. (1B) Schematic of GPI analysis. Without being bound by theory, PD risk-associated genotypes at 2 independent loci (upper panels) differentially alter the function of a nearby gene (red star in middle panel). This secondarily impacts the brain transcriptome (lower panels), with significant overlap for different PD-risk genotype shows.

FIGS. 1C-1D. LRRK2 and PARK16 PD risk-associated variants function in a common genetic pathway. (1C) Hierarchical clustering dendrogram shows that the gene expression signatures across 7 PD-associated variant GPIs (“Risk GPI”; in unaffected cerebral cortex Broadmann Area 9 [BA9]) are most similar to the signatures seen in PD brain (BA9 or substantia nigra; SN; in red (e.g., PD/S.N. and PD/Cx.) rather than in other CNS diseases such as Alzheimer's disease, Huntington's disease, Bipolar Disorder or Schizophrenia. 352 gene transcript expression patterns—corresponding to the intersection of the PD risk variants GPIs (FIG. 8A-C)—were interrogated. Clustering was performed using Pearson's distance with complete linkage (see Methods). (1D) Genetic interaction between PARK16 and LRRK2 alleles revealed by GPI analysis in 185 unaffected brain samples (GEO GSE15222 “Initial”) and in an independent cohort of 143 unaffected brain samples (GEO GSE15745. “Replication”), as established by the interaction factor between pairs of GPIs as indicated, in a linear regression model (see Methods). The p-value (“p”) associated with the interaction term as well as its orientation (“Dir.”) are presented. Results combined across both cohorts presented (“Combined”) with the resulting Z-scores and p-values for interaction.

FIGS. 1E-1F. LRRK2 and PARK16 PD risk-associated variants function in a common genetic pathway. (1E) The PARK16 genotype modifies LRRK2 associated risk in sporadic PD. A table presents the odds ratios for PD at the LRRK2 locus as a function of the PARK16 genotype in 4 independent GWAS cohorts: 1 of Ashkenazi Jews (“AJ”, n=417) and 3 of Caucasians (“NGRC”, n=4008; “NINDS”, n=537; “MAYO”, n=886). (1F) Manhattan plot of the Chr1 region reported as a modifier of age of disease onset in familial PD with LRRK2 mutation (Latourelle et al., 2011). Epistasis was evaluated for 74 SNPs in 4 independent sporadic PD GWAS datasets. X-axis represents chromosomal location, Y-axis represents −log 10 of the combined p-value for epistasis of each SNP with the PD risk SNP rsl 1176052 at the LRRK2 locus. The PARK16 locus PD-associated SNP rs823114 (arrow) exhibited the most significant association (p=4.6 E-6; red line (shown as grey in black and white image) represents the significance threshold after correction for multiple testing).

FIG. 2A shows schematics of the PARK16 locus on chromosome 1.

FIG. 2B. Overexpression of the PARK16 locus gene RAB7L1 specifically rescues a LRRK2 mutant phenotype. RAB7L1 modifies a LRRK2-associated neurite process length phenotype. Rat primary cortical neuron cultures transfected with a vector expressing G2019S mutant LRRK2 displayed reduced total neurite length relative to vector alone (cells are co-transfected with GFP for visualization by fluorescence microscopy). (i) (ii) Co-transfection of a wild-type or constitutively active (CA) RAB7L1 expression vector (1 μg/well) along with LRRK2 G2019S (0.5 μg/well) significantly rescued neurite length; other PARK16 genes—NUCKS1, SLC45A3, PM20D1, and SLC41A1—failed to rescue. CA or inactive (IN) RAB vectors were also tested as indicated (left panel; GFP-tagged at the N-terminus; 1 μg/well). (iii) Knock-down of RAB7L1 by shRNA vector transfection led to a similar decrease in neurite length as with LRRK2 G2019S expression. n=20 neurons in 4 independent cultures per group. Mean total neurite lengths are displayed; error bars represent SEM, *: p<0.05, **: p<0.01, ***: p<0.001 for ANOVA followed by Tukey's HSD post hoc analysis.

FIGS. 3A-3C. Evidence of a RAB7L1-LRRK2 complex. (3A) Immunoprecipitation (IP) analysis of RAB7L1 from lysates of HEK293T cells transfected with plasmids encoding a GFP-RAB7L1 fusion protein (or vector alone) and a 3×flag (3FL) epitope-tagged LRRK2 construct (either wild type [WT], G2019S [GS], K1906M [KM], or empty vector). IP with an anti-flag antibody was followed with immunoblot (IB) analysis with an anti-GFP or an anti-LRRK2 antibody as indicated. Arrowheads indicate the expected protein sizes. (3B) Co-immunoprecipitation of LRRK2 with RAB7L1 from lysates of HEK293T cells transfected with a plasmid encoding a 3×flag LRRK2 construct and a plasmid encoding a GFP-RAB7L1 fusion protein (either WT, CA, IN, or GFP only). (3C) Immunoprecipitation using an anti-LRRK2 antibody from whole brain lysates of non-transgenic (NT), LRRK2 wild type transgenic (WT), LRRK2 R1441C (RC) transgenic, or LRRK2 knockout (−/−) mice. IB was subsequently performed for RAB7L1, RAB11 and LRRK2.

FIG. 3D. Evidence of a RAB7L1-LRRK2 complex. (3D) Subcellular co-localization of RAB7L1 and LRRK2. Human neuroblastoma SH-SY5Y cells were transfected with GFP-tagged RAB7L1 vectors (in green (shown as light grey in black and white image)); either WT, CA, or IN forms, as well as a RAB7L1 construct lacking exon 2 and 3 and corresponding to an alternatively spliced RAB7L1 transcript, “AT”) and a 3×flag-tagged LRRK2 vector (in red, left panel (shown as grey in black and white image)). Subcellular localization was determined by immunostaining with a marker for the Golgi apparatus (Golph4; in blue (shown as dark grey in black and white image)). The CA form leads to a reduced localization to the Golgi apparatus. Co-localization is evaluated by quantifying the fraction of RAB7L1/Golph4, RAB7L1/LRRK2 and LRRK2/Golph4 staining overlap (Upper, lower and middle right panels respectively). Results represent mean±SEM (n=15 per group).

FIG. 4A. RAB7L1 rescues lethality and dopamine neuron loss in a Drosophila model of LRRK2 G2019S neurodegeneration. Modifier screen for suppressors of an early adult lethality phenotype seen with expression of LRRK2 G2019S selectively in tyrosine hydroxylase (TH)-positive dopamine neurons. Left, a panel of 16 Drosophila RAB transgenes was screened (of 31 total; see Table 3). Adult survival (days post-eclosion) curves are presented for individual strains harboring different RABs along with the LRRK2 G2019S transgene. Non-transgenic survival curve is shown for comparison. n>25 for all conditions. Right, Adult survival (days post-eclosion) of Drosophila is presented in the context of transgenic expression of LRRK2 (WT or G2019S), with or without RAB7L1 (WT, CA or IN), using a tyrosine hydroxylase promoter GAL4 driver for dopaminergic neuron expression. Non-transgenic survival is also shown for comparison. n>25 for all conditions.

FIG. 4B. RAB7L1 rescues lethality and dopamine neuron loss in a Drosophila model of LRRK2 G2019S neurodegeneration. (left) Confocal microscopy of mushroom bodies of the CNS from transgenic Drosophila as in (FIG. 4A), with dopaminergic neuron nuclei visualized using an additional marker transgene, a nuclear localization sequence (NLS)-GFP fusion, also driven by TH-Gal4. (Right) Quantitation of surviving dopaminergic neurons in the PPM1 and PPL1 clusters of Drosophila CNS mushroom bodies. Means are displayed; error bars represent SEM; ***:p<0.001 by ANOVA followed by Tukey's HSD post hoc analysis for (4A) and (4B).

FIGS. 5A-5D. PARK16 PD risk-associated variants modify RAB7L1 splicing and protein accumulation. (5A) Exonic structure of the human RAB7L1 gene. (5B) Analysis of RAB7L1 alternative splicing in human cortical brain samples. The rs1572931 allele G, linked to the PD high-risk haplotype (R), is associated with an increase in the fraction of RAB7L1 transcripts that lack the exon 2 to exon 3 junction region (termed exon 2 skipping; presented relative to the extent of exon 2 skipping seen in carriers of the rs1572931 protective allele A; quantified by qrtPCR using primers as depicted by red and black arrows in (5A) detecting respectively the amount of total and unskipped RAB7L1 mRNA; n=15 and 57 for P and R respectively; details in Table 6). (SC) (i) Schematic of predicted splice site enhancer and silencer motifs upstream of RAB7L1 exon2 and affected by rs1572931 variants G (associated with increased PD risk, “R”) and A (protective, “P”, associated with decreased PD risk). (ii) Structure of a minigene construct to assess the effect of rs1572931 variants on RAB7L1 exon2 inclusion in vitro. Green arrows indicate the position of the primers used to assess exon 2 inclusion. (5D) Impact of PARK16 variants on splicing in vitro. The rs1572931 allele G (associated with increased PD risk, R; relative to the allele A associated with decreased PD risk, P) leads to a relative decrease in RAB7L1 exon 2 inclusion in transfected human SH-SY5Y cells as assess by PCR gel quantification (pictures in FIGS. 12D-E; n=6/group).

FIG. 5E is a bar graph showing the impact of rs1572931 on RAB7L1protein level in human cortical brain samples. rs1572931 allele G is associated with a decrease in RAB7L1 protein level in non-PD post-mortem human cortical brain samples, as assessed by Western Blot from individuals homozygous for the risk allele (R, n=25) and from carriers of the protective allele (P, n=13). Mean levels are displayed; errors bars are SEM; *: p<0.05, **: p<0.01, ***: p<0.001 by two-tailed t-test (5B, 5D) or by linear regression analysis (5E).

FIGS. 6A-6C. RAB7L1 and LRRK2 modulate lysosome and Golgi apparatus sorting in a retromer-dependent manner. (6A-6C) Analysis of MPR sorting in primary rat neuron cultures transfected with vectors encoding LRRK2 G2019S mutant (GS), RAB7L1, VPS35, or VPS35 D620N: or with shRNA plasmids for VPS35, RAB7L1 or vector only, co-transfected with GFP vector for visualization and immunostained for MPR as well as either the Golgi marker Golph4 (6A, upper panel), the lysosome marker Lamp2 (6B, upper panel) or with the early endosome marker EEA1 (6B, upper panel). MPR colocalization with either the Golph4 or LAMP2 marker was reduced with G2019S LRRK2, VPS35 D620N, or knockdown of either RAB711 or VPS35 (6A, lower panel; 6B, lower panel). These manipulations also increased total LAMP2 staining (but not Golph4 staining). Scale bar represents 10 um. Quantifications of the MPR co-localization and of total organelle marker analyses are presented in the lower panels. Error bars represent SEM. n>10 cells in 3 independent wells per group. *: p<0.05, **: p<0.01, ***: p<0.001 for comparisons with “vector” group, ++: p<0.01, +++: p<0.001 for comparisons with “LRRK2 G2019S” group by ANOVA followed by Tukey's HSD post hoc analysis.

FIG. 6D is a schematic showing cell sorting phenotype associated with defects in the LRRK2-Rab7L1 pathway or knockdown of the VPS35 retromer component. MPR accumulation at Golph4-positive structures (trans-golgi network [TGN]) and at LAMP2-positive structures (lysosomes and late endosomes [LE]) is reduced, and lysosomes appear swollen.

FIG. 7A. Evidence of retromer Insufficiency in the context of LRRK2-RAB7L1 pathway defects. Transfection of rat primary cortical neuron cultures with a wild-type (WT) VPS35 expression vector rescued the reduced neurite length phenotype associated with LRRK2 G2019S (GS) mutant expression or with Rab7L1 (R7L1) knockdown. Overexpression of a familial PD mutant VPS35 D620N vector leads to reduced neurite length relative to vector alone. Knockdown of VPS35 by shRNA leads to similarly reduced neurite length relative to vector alone, which is not rescued by Rab7L1 overexpression (n=20 neurons in 4 cultures per group).

FIG. 7B. Evidence of retromer insufficiency in the context of LRRK2-RAB7L1 pathway defects. (Left) Confocal microscopy of mushroom bodies of the CNS from transgenic Drosophila with dopaminergic neuron nuclei visualized using a TH-Gal4-driven nuclear localization sequence (NLS)-GFP fusion. (Right) Quantitation of surviving dopaminergic neurons in the PPM1 and PPL1 clusters of Drosophila CNS mushroom bodies.

FIG. 7C. Evidence of retromer insufficiency in the context of LRRK2-RAB7L1 pathway defects. Relative quantification by western blot of VPS35 (left) or VPS29 (right) protein levels in lysates from mouse neuroblastoma (N2a) cells transfected with vectors encoding VPS35 WT, PS35 shRNA, VPS35 D620N, LRRK2 WT, LRRK2 G2019S (GS), RAB7L1, RAB7L1 shRNA, or vector control (N=3/group).

FIG. 7D. Evidence of retromer insufficiency in the context of LRRK2-RAB7L1 pathway defects. LRRK2 impacts the levels of retromer components in mouse brain. Relative quantification by Western blotting of VPS35 (left), VPS29 (middle) and VPS26 (right) levels in brain tissue samples from non-transgenic (“nTg”), LRRK2 wild-type (“LRRK2-WT”) and LRRK2 R1441C mutant (“LRRK2-RC”) BAC transgenic mice (N=3/group).

FIG. 7E. Evidence of retromer insufficiency in the context of LRRK2-RAB7L1 pathway defects. Immunoprecipitation (IP) analysis of RAB7L1 from lysates of SH-SY5Y cells transfected with plasmids encoding a GFP-VPS35 fusion protein with VPS35 wild-type sequence (“WT”) or the familial PD mutant D620M (“D620N) or vector alone, along with a LRRK2 construct or an empty vector. IP with an anti-GFP antibody was followed with Western immunoblot analysis with an anti-LRRK2 or anti-GFP antibody as indicated. Arrowheads indicate the expected protein sizes.

FIG. 7F. Evidence of retromer insufficiency in the context of LRRK2-RAB7L1 pathway defects. IP using an anti-LRRK2 antibody from whole brain lysates of non-transgenic (NT), LRRK2 wild type transgenic (WT), LRRK2 R1441C (RC) transgenic, or LRRK2 knockout (−/−) mice as in FIG. 3D. Immunoblot was subsequently performed for VPS35 and β-Actin.

FIG. 7G. Evidence of retromer insufficiency in the context of LRRK2−RAB7L1 pathway defects. VPS3S mRNA levels in substantia nigra tissue as determined by meta-analysis of 5 gene expression microarray datasets (Table 5) in 63 unaffected individuals and 81 PD patients samples (left panel) and in laser-microdissected substantia nigra dopaminergic neurons from 8 unaffected individuals and 10 PD patients samples (right panel, GEO GSE20141). Expression levels are normalized to mean of the unaffected group.

FIGS. 7H-7I. Evidence of retromer insufficiency in the context of LRRK2-RAB7L1 pathway defects. (H) VPS35 mRNA in cerebral cortex tissue as determined by high-throughput sequencing of the 3′UTR ends of polyadenylated mRNA transcripts on a cohort of 17 unaffected and 17 PD cerebral cortical tissue samples. Levels are expressed as reads per million (rpm). (I) VPS35 mRNA levels in Globus Pallidus Interna (Gpi) samples (n=10/group GEO GSE20146). Expression levels are normalized to mean of the unaffected group. For all graphs means are displayed, error bars represent SEM; p<0.05(*) p<0.01(**) for ANOVA followed by Tukey's HSD post hoc analysis (7A, 7B, 7C) or by two-tailed t-test (7G, 7H).

FIG. 8A is a graph depicting GPIs of SNPs at 7 PD-associated loci that show a high degree of overlap. Overlap was quantified in terms of number of gene expression profiles that were impacted in the same direction by 7 disease risk-associated haplotypes (at the SNCA, MAPT, LRRK2, PARK16, HLA-DRA, STK39 and LAMP3 loci). 352 were impacted in the same orientation (up or down, of 8560 queried) by all 7 SNPs. Such overlap is highly significant, as shown by analysis of randomly chosen sets of SNP variants. Analysis of 25000 randomly chosen sets of 7 SNPs is shown for comparison, with average of 19.5 overlapping genes and count distribution as shown. p=1E-5 using Wald statistics (see Methods).

FIG. 8B is a histogram of the resampling result for the estimation of the significance between the PD-risk GPI and the expression profile characteristic of PD in prefrontal cortex.

FIG. 8C is a schematic that shows the correlation pattern for each of the genes belonging to the PD-risk intersection GPI with a FDR<5% and that also shows a significant difference (p<0.05, two tailed t test) in their expression levels in either BA9 or SN for a PD vs unaffected comparison.

FIGS. 8D-8E shows Gene Ontology categories enriched in genes whose expression levels are positively (red (first 5 rows)) or negatively (blue (last 5 rows)) associated with the PD risk-associated allelic load for all PD loci (8D) and specifically for the LRRK2 and PARK16 loci (8E). Analysis were conducted using DAVID.

FIG. 9A is a bar graph depicting total neurite length of rat primary cortical neurons transfected with vector or LRRK2 WT or LRRK2 R1441C (0.5 ug per well) and RAB7L1 expressing vector or empty vector (1 ug per well) as indicated. Means are represented, errors bars are SEM. N=20 per group.

FIGS. 9B-9C. Rab7L1 knockdown efficiency measured by Western blot quantitation in 3 independent vector or Rab7L1 shRNAtransfected cultures (9B). Graph shows relative band intensity+/−SEM * p<0.05 by two-tailed t-test. Validation of PARK16 locus genes overexpression vectors by Western Blot (9C). Lysates from cells tranfected with the PARK16 gene indicated (+) or control vector (−) probed by immunoblot using corresponding antibodies that recognize both endogenous and exogenous PARK16 gene expression. Constructs were transfected in cell lines of matching species (human SH-SY5Y for RAB7L1 and NUCKS1; mouse N2a for SLC41A1 and SLC45A3). 30 ug protein was loaded per lane. Beta-actin loading control is shown below.

FIG. 10A is a photographic image of Rab7L1 and LRRK2 immunohistochemistry of substantia nigra section from non-transgenic, LRRK2 WT, and LRRK2 R1441C transgenic mice. Tyrosine hydroxylase (TH) staining (in green (shown as light grey in black and white image)) marks dopaminergic neurons.

FIG. 10B is a photographic image of an immunoblot analysis of N2a cells transiently expressing wildtype or mutant forms RAB7L1 as indicated. 30 μg of cell lysate was loaded in each lane. Arrrowheads indicate RAB7L1 as detected by an anti-GFP antibody; the DN form leads to a smaller product as expected.

FIG. 11 is a bar graph showing a negative geotaxis analysis of lrrk mutant Drosophila. Lrrk mutant (−/−) Drosophila defective negative geotaxis can be rescued by pan-neuronal expression of human LRRK2 WT or G2019S transgenes. Bars represent Mean times to climb 10 cm upward in a cylinder are represented. Error bars are SEM. ***: p<0.001, for comparison with the non-transgenic group by ANOVA (df=3, F=13.14) followed by Tukey HSD post hoc analysis.

FIG. 12A is a schematic showing the exons/introns structure of the RAB7L1 gene and its different known isoform products.

FIG. 12B is a schematic showing the exons/introns structure of an artificial RAB7L1 minigene and expected isoform products.

FIG. 12C is a schematic showing the RAB7L1 protein functional domains as predicted by CD-search, in parallel with the exonic structure of the CDS. The dashed red line indicates the alternative start site of the CDS in the event of exon 2 exclusion.

FIG. 12D is a photographic image of a gel showing rtPCR products to assess the splice of a RAB7L1 exon 2 reporter in SH-SY5Y cells transfected with a minigene bearing one of the two rs1572931 alleles. The numbered arrows correspond to the different isoforms expected from the minigene as depicted in FIG. 12B.

FIG. 12E is a graph showing the relative quantification of the different isoforms produced by the RAB7L1 minigene. The numbers correspond to the different isoforms expected from the minigene and shown in FIG. 12A.

FIG. 12F is a bar graph showing that alternatively spliced RAB7L1 (AT) does not functionally rescue LRRK2 G2019S neurite length assay. Total neurite length of primary rat cortical neurons transfected with expression vectors as indicated. (n=20 per group; ***p<0.001 vs vector alone by ANOVA followed by Bonferroni correction).

FIG. 13 is a photographic image of an immunoprecipitation using an anti-LRRK2 antibody from whole brain lysates of nontransgenic (NT), LRRK2 wild type transgenic (WT). LRRK2 R1441C (RC) transgenic, or LRRK2 knockout (−/−) mice. IB was subsequently performed for VPS35 and betaactin. VPS35 but not beta-actin were co-precipitated with LRRK2. Neither VPS35 nor beta-actin were immunoprecipitated by a control IgG antibody, or from LRRK2 KO mice.

DETAILED DESCRIPTION

The singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.

The term “about” is used herein to mean approximately, in the region of, roughly, or around. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20%.

Parkinson's disease (PD) is a common neurodegenerative disorder of aging, characterized by slowed movements and a distinctive tremor at rest (Lang and Lozano, 1998). Defining pathological features of the disease include neurodegeneration that is most prominent among midbrain dopamine neurons (DNs) in the Substantia Nigra (SN) and Lewy body protein aggregates that are composed in part of alpha-Synuclein (aSyn) protein. As the course of PD is thought to last decades, and as at the time of autopsy the vast majority of DNs are long lost, the molecular pursuit of initial etiological events has proven difficult.

In rare inherited familial forms of PD, specific causative mutations have been identified, and this has significantly advanced the field (Abeliovich and Beal, 2006; Hardy et al., 2006). For instance, autosomal dominantly inherited mutations in aSyn, including missense mutations and triplication of the locus, lead to a familial PD variant, implicating aSyn directly in the disease process. Another familial genetic cause of PD is the presence of autosomal dominantly inherited mutations in the Leucine rich-repeat kinase-2 (LRRK2) protein, which encodes a large multidomain protein with GTPase and kinase activities. Although the precise functions of aSyn and LRRK2 in neurons remain to be determined, both proteins have been broadly implicated in intraneuronal protein sorting. aSyn mutations have been reported to modify synaptic vesicle kinetics (Abeliovich et al., 2000) as well as trafficking to the Golgi apparatus in a variety of model systems (Cooper et al., 2006; Thayanidhi et al., 2010), whereas LRRK2 mutations are implicated in defective lysosomal protein degradation and macroautophagy, which is a cellular process that delivers cytosolic proteins and protein agregates to the lysosome (Dodson et al., 2012; Heo et al., 2010; MacLeod et al., 2006), and Golgi Apparatus integrity (Stafa et al., 2012). The recent identification of rare autosomal dominant familial PD mutations in VPS35 (Vilarino-Guell et al., 2011; Zimprich et al., 2011), which encodes a component of the retromer complex that guides protein sorting from the endosome-lysosome degradation pathway retrogradely to the Golgi Apparatus (Bonifacino and Hurley, 2008; Seaman, 2009; Seaman et al., 1998), suggests that defective protein sorting in vesicular compartments may play a role in PD.

Several genome-wide association studies (GWAS) have described common genetic variants (at single nucleotide polymorphisms; SNPs) that modify PD risk in non-familial ‘sporadic’ cases (Hamza et al., 2010; Simon-Sanchez et al., 2009). Strikingly, a subset of these common variants lie within genomic loci previously associated with familial disease, such as aSyn or LRRK2, supporting the notion that common pathogenic pathways underlie familial and sporadic forms of PD. However, mechanisms that underlie the impact of non-familial genetic loci on PD risk, or that relate the functions of such loci to familial PD genes, remain unclear.

Described herein is a series of human brain transcriptome, human genetic, and cell biological studies, that together implicate a PD-associated genetic and cellular pathway. RAB7L1—one of 5 genes within the PARK16 non-familial PD risk-associated locus—functions together with LRRK2 to impact non-familial PD risk in the human population. This genetic interaction is apparent even in unaffected individuals who carry both risk alleles, as quantified in terms of a broad transcriptomic analysis of brain gene expression. Similarly, these genes together modify neuronal survival and neurite integrity in model systems. At a cellular level, defects in this PD-associated RAB7L1-LRRK2 pathway lead to abnormal lysosomal structures and defective retromer complex function, that normally links the endolysosomal protein degradation system with the Golgi apparatus (Bonifacino and Hurley, 2008; Seaman. 2009; Seaman et al., 1998). Consistent with a role for such cellular defects in disease pathology, mutations in a retromer complex component, VPS35, have recently been associated with rare forms of autosomal dominantly inherited familial PD (Vilarino-Guell et al., 2011; Zimprich et al., 2011).

Molecules of the Invention

As used herein, a “RAB7L1 molecule” refers to a RAB7L1 protein, or a fragment thereof. A “RAB7L1 molecule” can also refer to a nucleic acid (including, for example, genomic DNA, complementary DNA (cDNA), synthetic DNA, as well as any form of corresponding RNA) which encodes a polypeptide corresponding to a RAB7L1 protein, or fragment thereof. For example, a RAB7L1 molecule can comprise the nucleic acid sequences shown in SEQ ID NOS: 1, 2, 3, 4, or 5, or comprise the amino acid sequences shown in SEQ ID NOS: 6, 7, 8, or 26. For example, a RAB7L1 molecule can be encoded by a recombinant nucleic acid encoding a RAB7L1 protein, or fragment thereof. The RAB7L1 molecules of the invention can be obtained from various sources and can be produced according to various techniques known in the art. For example, a nucleic acid that encodes a RAB7L1 molecule can be obtained by screening DNA libraries, or by amplification from a natural source. A RAB7L1 molecule can include a fragment or portion of a RAB7L1 protein. A RAB7L1 molecule can include a variant of the above described examples, such as a fragment thereof. Such a variant can comprise a naturally-occurring variant due to allelic variations between individuals (e.g., polymorphisms), mutated alleles, or alternative splicing forms (e.g. SEQ ID NOS: 2-5). In one embodiment, a RAB7L1 molecule is encoded by a nucleic acid variant of the nucleic acid having the sequence shown in SEQ ID NOS: 1, 2, 3, 4, or 5 wherein the variant has a nucleotide sequence identity to SEQ ID NOS: 1, 2, 3, 4, or 5 of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. In another embodiment, a variant of the RAB7L1 protein comprises a protein or polypeptide encoded by a RAB7L1 nucleic acid sequence, such as the sequence shown in SEQ ID NOS: 1, 2, 3, 4, or 5.

As used herein, a “LRRK2 molecule” refers to a LRRK2 protein, or a fragment thereof. A “LRRK2 molecule” can also refer to a nucleic acid (including, for example, genomic DNA, complementary DNA (cDNA), synthetic DNA, as well as any form of corresponding RNA) which encodes a polypeptide corresponding to a LRRK2 protein, or fragment thereof. For example, a LRRK2 molecule can comprise the nucleic acid sequences shown in SEQ ID NOS: 9, or 10, or comprising the amino acid sequences shown in SEQ ID NO: 11, 27, or 28. For example, a LRRK2 molecule can be encoded by a recombinant nucleic acid encoding a LRRK2 protein, or fragment thereof. The LRRK2 molecules of the invention can be obtained from various sources and can be produced according to various techniques known in the art. For example, a nucleic acid that encodes a LRRK2 molecule can be obtained by screening DNA libraries, or by amplification from a natural source. A LRRK2 molecule can include a fragment or portion of a LRRK2 protein. A LRRK2 molecule can include a variant of the above described examples, such as a fragment thereof. Such a variant can comprise a naturally-occurring variant due to allelic variations between individuals (e.g., polymorphisms), mutated alleles, or alternative splicing forms. In one embodiment, a LRRK2 molecule is encoded by a nucleic acid variant of the nucleic acid having the sequence shown in SEQ ID NOS: 9, or 10 wherein the variant has a nucleotide sequence identity to SEQ ID NOS: 9 or 10 of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. In another embodiment, a variant of the LRRK2 protein comprises a protein or polypeptide encoded by a LRRK2 nucleic acid sequence, such as the sequence shown in SEQ ID NOS: 9 or 10.

As used herein, a “VPS35 molecule” refers to a VPS35 protein, or a fragment thereof. A “VPS35 molecule” can also refer to a nucleic acid (including, for example, genomic DNA, complementary DNA (cDNA), synthetic DNA, as well as any form of corresponding RNA) which encodes a polypeptide corresponding to a VPS35 protein, or fragment thereof. For example, a VPS35 molecule can comprise the nucleic acid sequences shown in SEQ ID NOS: 12 or 13, or comprising the amino acid sequences shown in SEQ ID NO: 14. For example, a VPS35 molecule can be encoded by a recombinant nucleic acid encoding a VPS35 protein, or fragment thereof. The VPS35 molecules of the invention can be obtained from various sources and can be produced according to various techniques known in the art. For example, a nucleic acid that encodes a VPS35 molecule can be obtained by screening DNA libraries, or by amplification from a natural source. A VPS35 molecule can include a fragment or portion of a VPS35 protein. A VPS35 molecule can include a variant of the above described examples, such as a fragment thereof. Such a variant can comprise a naturally-occurring variant due to allelic variations between individuals (e.g., polymorphisms), mutated alleles, or alternative splicing forms. In one embodiment, a VPS35 molecule is encoded by a nucleic acid variant of the nucleic acid having the sequence shown in SEQ ID NOS: 12, or 13 wherein the variant has a nucleotide sequence identity to SEQ ID NOS: 12, or 13 of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. In another embodiment, a variant of the VPS35 protein comprises a protein or polypeptide encoded by a VPS35 nucleic acid sequence, such as the sequence shown in SEQ ID NOS: 12 or 13.

The nucleic acid can be any type of nucleic acid, including genomic DNA, complementary DNA (cDNA), synthetic or semi-synthetic DNA, as well as any form of corresponding RNA. For example, a nucleic acid encoding a RAB7L1, a LRRK2, or a VPS35 protein can comprise a recombinant nucleic acid encoding such a protein. The nucleic acid can be a non-naturally occurring nucleic acid created artificially (such as by assembling, cutting, ligating or amplifying sequences). It can be double-stranded or single-stranded.

The invention further provides for nucleic acids that are complementary to a RAB7L1, a LRRK2, or a VPS35 molecule. Complementary nucleic acids can hybridize to the nucleic acid sequence described above under stringent hybridization conditions. Non-limiting examples of stringent hybridization conditions include temperatures above 30° C., above 35° C., in excess of 42° C., and/or salinity of less than about 500 mM, or less than 200 mM. Hybridization conditions can be adjusted by the skilled artisan via modifying the temperature, salinity and/or the concentration of other reagents such as SDS or SSC.

According to the invention, protein variants can include amino acid sequence modifications. For example, amino acid sequence modifications fall into one or more of three classes: substitutional, insertional or deletional variants. Insertions can include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. These variants ordinarily are prepared by site-specific mutagenesis of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture. In one embodiment, a RAB7L1, a LRRK2, or a VPS35molecule can be modified with an amino acid sequence inserted as a carboxyl terminal fusion. For example, carboxyl terminal fusions may be used to increase the stability of a RAB7L1, a LRRK2, or a VPS35 molecule.

In one embodiment, a RAB7L1 molecule comprises a protein or polypeptide encoded by a nucleic acid sequence encoding a RAB7L1 protein, such as the sequences shown in SEQ ID NOS: 6, 7, 8, or 26. In another embodiment, the polypeptide can be modified, such as by glycosylations and/or acetylations and/or chemical reaction or coupling, and can contain one or several non-natural or synthetic amino acids. An example of a RAB7L1 molecule is the polypeptide having the amino acid sequence shown in SEQ ID NOS: 6, 7, 8, or 26. Such variants can include those having at least from about 46% to about 50% identity to SEQ ID NOS: 6, 7, 8, or 26 or having at least from about 50.1% to about 55% identity to SEQ ID NOS: 6, 7, 8, or 26, or having at least from about 55.1% to about 60% identity to SEQ ID NOS: 6, 7, 8, or 26, or having from at least about 60.1% to about 65% identity to SEQ ID NOS: 6, 7, 8, or 26, or having from about 65.1% to about 70% identity to SEQ ID NOS: 6, 7, 8, or 26, or having at least from about 70.1% to about 75% identity to SEQ ID NOS: 6, 7, 8, or 26, or having at least from about 75.1% to about 80% identity to SEQ ID NOS: 6, 7, 8, or 26, or having at least from about 80.1% to about 85% identity to SEQ ID NOS: 6, 7, 8, or 26, or having at least from about 85.1% to about 90% identity to SEQ ID NOS: 6, 7, 8, or 26, or having at least from about 90.1% to about 95% identity to SEQ ID NOS: 6, 7, 8, or 26, or having at least from about 95.1% to about 97% identity to SEQ ID NOS: 6, 7, 8, or 26, or having at least from about 97.1% to about 99% identity to SEQ ID NOS: 6, 7, 8, or 26. In another embodiment, a RAB7L1 molecule can be a fragment of a RAB7L1 protein.

In one embodiment, a LRRK2 molecule comprises a protein or polypeptide encoded by a nucleic acid sequence encoding a LRRK2 protein, such as the sequences shown in SEQ ID NOS: 11, 27, or 28. In another embodiment, the polypeptide can be modified, such as by glycosylations and/or acetylations and/or chemical reaction or coupling, and can contain one or several non-natural or synthetic amino acids. An example of a LRRK2 molecule is the polypeptide having the amino acid sequence shown in SEQ ID NOS: 11, 27, or 28. Such variants can include those having at least from about 46% to about 50% identity to SEQ ID NOS: 11, 27, or 28 or having at least from about 50.1% to about 55% identity to SEQ ID NOS: 11, 27, or 28, or having at least from about 55.1% to about 60% identity to SEQ ID NOS: 11, 27, or 28, or having from at least about 60.1% to about 65% identity to SEQ ID NOS: 11, 27, or 28, or having from about 65.1% to about 70% identity to SEQ ID NOS: 11, 27, or 28, or having at least from about 70.1% to about 75% identity to SEQ ID NOS: 11, 27, or 28, or having at least from about 75.1% to about 80% identity to SEQ ID NOS: 11, 27, or 28, or having at least from about 80.1% to about 85% identity to SEQ ID NOS: 11, 27, or 28, or having at least from about 85.1% to about 90% identity to SEQ ID NOS: 11, 27, or 28, or having at least from about 90.1% to about 95% identity to SEQ ID NOS: 11, 27, or 28, or having at least from about 95.1% to about 97% identity to SEQ ID NOS: 11, 27, or 28, or having at least from about 97.1% to about 99% identity to SEQ ID NOS: 11, 27, or 28. In another embodiment, a LRRK2 molecule can be a fragment of a LRRK2 protein.

In one embodiment, a VPS35 molecule comprises a protein or polypeptide encoded by a nucleic acid sequence encoding a VPS35 protein, such as the sequences shown in SEQ ID NO: 14. In another embodiment, the polypeptide can be modified, such as by glycosylations and/or acetylations and/or chemical reaction or coupling, and can contain one or several non-natural or synthetic amino acids. An example of a VPS35 molecule is the polypeptide having the amino acid sequence shown in SEQ ID NO: 14. Such variants can include those having at least from about 46% to about 50% identity to SEQ ID NO: 14 or having at least from about 50.1% to about 55% identity to SEQ ID NO: 14, or having at least from about 55.1% to about 60% identity to SEQ ID NO: 14, or having from at least about 60.1% to about 65% identity to SEQ ID NO: 14, or having from about 65.1% to about 70% identity to SEQ ID NO: 14, or having at least from about 70.1% to about 75% identity to SEQ ID NO: 14, or having at least from about 75.1% to about 80% identity to SEQ ID NO: 14, or having at least from about 80.1% to about 85% identity to SEQ ID NO: 14, or having at least from about 85.10% to about 90% identity to SEQ ID NO: 14, or having at least from about 90.1% to about 95% identity to SEQ ID NO: 14, or having at least from about 95.1% to about 97% identity to SEQ ID NO: 14, or having at least from about 97.1% to about 99% identity to SEQ ID NO: 14. In another embodiment, a VPS35 molecule can be a fragment of a VPS35 protein.

In one embodiment, a RAB7L1, a LRRK2, or a VPS35 molecule, according to the methods described herein can be administered to a subject as a recombinant protein. In another embodiment, a RAB7L1, a LRRK2, or a VPS35 molecule, can be administered to a subject as a modified recombinant protein. In a further embodiment, a RAB7L1, a LRRK2, or a VPS35 molecule, according to the methods described herein can be administered to a subject by delivery of a nucleic acid encoding a RAB7L1, a LRRK2, or a VPS35 protein, or fragment thereof. For example, nucleic acids can be delivered to a subject using a viral vector.

Polypeptides can be susceptible to denaturation or enzymatic degradation in the blood, liver or kidney. Accordingly, polypeptides can be unstable and have short biological half-lives. Polypeptides can be modified to increase their stability, for example, a fusion protein can be generated for increased stability and to cause a longer biological half-life to the polypeptides in circulation.

The term “biological half-life” is the time required for the activity of a substance taken into the body to lose one half its initial pharmacologic, physiologic, or biologic activity.

The invention provides for a nucleic acid encoding a RAB7L1 protein, or fragment thereof.

For example, the human genomic nucleotide sequence corresponding to the sense strand of the human RAB7L1 gene is depicted in SEQ ID NO: 1 (9752 bp). Sequence information related to RAB7L1 is accessible in public databases by GenBank Accession number NC_000001.10, 205737114, 205744615, complement (nucleotide).

SEQ ID NO: 1:

1    ctgaaagaaa aagataatca tcagagaagt acggggatga caaagaaaga acagcgtcat
61   agaaggcata agggaaacaa atgtcaagga gtggtcaact atgtcaaaac gaataagaac
121  agagaaaact ggatccttaa agatgagtag cttgaactaa cctcctgtac ctgggtaacg
181  aacattctgg gcaaaatata ttgtgaacca tgctgatgtt ctttcgccac aaaacataag
241  tgatagcctc tctatcaaga gacctagatt tcctagtgtc tggtcctggg ctgttttccc
301  atggccccgc tggttctctt cctctattcc tagaaaagta tagaaaatgg agctctcttt
361  gcttgccatt gagctcatgt cctgccaact tctacccaag tgacaggtag gtcagtaaag
421  ttacaggctg agaaattaaa attatttgcc aaacactagt tttatgatta tctattaaca
481  acagccacaa caaatcaaga aggagtccca gtgtatttca attagcttct agctcaagtg
541  atacatgagt agtatggcta gaataggcat tgtagaagta tgaatggaga actgtctctt
601  gttttctgtc catctttatt gttattgctg gatattaaat gctgtaattt ttggcatagg
661  tttttttcca aagactggtc tcttgggtca agttttcctg aacttgcctg gtattgggca
721  agttcacaca gaaaatatga gtcatttcac aaagtcctgg gcttgtctat ctacttgaag
781  gaataggaaa tggacttcac ttggagcatg atggatgagg cttgggcttt tactaatgaa
841  ataagtttca atcttaactc agttaaacct ttaaagacag gagaaaacta tctggggatg
901  gattcaggcc agtgagatca cgttgcacaa atctcccctt aagggatctt tactgaccta
961  gaattgtttc aaaataagat tccaaactgt cgagggagtt tgaagccttg aaacttggtg
1021 taggtttgct taaaacaaaa taacgaatat actacatttc ccaggaggct tcgcggtgag
1081 cctccgcact cggctggttc tctttaccgc gaggaaagct gggaaatgta gtgccacagg
1141 caaccctgca cgtgacgctt gcggaggaag gggagagaga ggcgcgcggg agggcgtcta
1201 gggaatcgag gtgccggctg ctccttcctc acaatttggt ttgtgctgca aggggagggt
1261 ccccatcatc tggccccagt ggtgtaagga gctgactggg attcagtcac tgacttggag
1321 ccgctcgggg gaagtcccgg tgggtgaggt tccgcggcgc ctggtccagt ttctcggcag
1381 tcaggccagg agggggtggg gaaggtgcga acagccgggg gctcggagct cgcgccctgc
1441 gccccaccca ggttgcggcc gcccggggag gaagcgcgta taggttgagt gcaaagtttc
1501 cttttttgct ttctcgtcag agcagcccag acaggcggtg ggtgggaatg cctcacttca
1561 gtttgaagag ggtccggatc caaaggggtt aaaacgagcg acccccgatc cccgaccaca
1621 cttcccgcct ccctaaaacg cacaccccgc tagccatggg cagccgcgac cacctgttca
1681 aagtgctggt ggtgggggac gccgcagtgg gcaagacgtc gctggtgcag cgatattccc
1741 aggacagctt cagcaaacac tacaagtcca cggtgggagg tgagacgtct cgggggtggg
1801 gagggggagg gggaggagga ggccccgacc gcggactcga gctggggacc caagtggggt
1861 ggggatttgc tgtcggttgt tgggtccttc ttcgggtcag gaagtactgt ctgggaacct
1921 agatggtgta attgccgggt gtttggttta cacgcctctc ttgagtttcc cgttagattt
1981 tcccaaactg agcccactgc caggagaaag gtaagagcaa aggcgtgaag atgagaaata
2041 gggaagggag gatgaggatg agcaattgct gtttattgca ggcgtactgt gaacttaagt
2101 ctccagcctt tcagctgtcc ggcaaatctc tccaatcagc agaagcagct gagatcctct
2161 gtggggcctc ctggttaggt gccggctggg gctgtagtcc tgcccctctg ttgagggagg
2221 aactgcaagg tgtttaacct ggcttcagtg ggagaacgca ggaagcggaa ctggtctgaa
2281 ggactgtagt tttgttgtgg ggctgtgggt gggatggaag acacaaaatc cccgaacgag
2341 ccccttggct acgcagtcag atgcccatga gggctgatga gctgtgtttt tgtccgttga
2401 ttgcttaaat aaaggtaacc aaggaattct tccaagaatg tgaaatatac cacttctctg
2461 acctatcttc ccaccacttt tttcctcaaa ctggagaagg tagggaggaa ggggacaccg
2521 agttcattca tttctttccc gggtgtggcc tttggactca actatttttt cagaggcaga
2581 aatgccttat tgttaattag aaggtattta gctaggaaga gctggtgatg ggggagagta
2641 acttaaaatt ttatttctct ctgttaaaag aacttgtttc aaggggggaa aactcgtacc
2701 aatggaaaaa ttgaaaatgt gtgctcctgt tcatatggaa gtttaaaaag aaattaacaa
2761 acatttacat tgcaattaaa acattctcct taagaacccc cagtagggcc cagtaccgtg
2821 gttcatactt gcaatcccag cactttagga ggctgaggcg ggaggattgc ttgagcccag
2881 gagttcgaga ccagccatgg gaaacatggc aagaccccca cctctacaaa aattaaagaa
2941 gaaagtagcc gtggtcccag ctactcggga aactgagacg agaggattgc ttgaactggg
3001 gaggtcgagg cttcagtgag ccccgttcgc accactcact gcactgtagc ctgggcaaca
3061 gagtgagacc ctgtctcaaa aacaaaggaa caaaaaattt aagtctggca aaattaaagg
3121 ttcaaagttt cagacactct atcctgaccg ctctgccctc ccactcctaa ataatataag
3181 ggtccctatt tcacggattt gggttaaaag tggcctgcta ttaatagtta atttttaaca
3241 attgctgtag ttttccttta gcaaagaata taattggcta attttttttt cagtgtttag
3301 tctaaatttc ccttagcttt gattcctgtc acagatgtga aagataatat ttccaaacct
3361 cagaaattac tctgtttctc tccttagcca aaaggctttc cccatggaat acttactcag
3421 ccttaaggag tctcagcacc tgcagacttg gtggtagatt ccagcaggac tcttcagcaa
3481 catccacaca gtgtcttctt cttgggactc ctctgggacc ttttccattt attccattct
3541 tgcccaagat aatacctgca accttatggt ggatgtagag ttccccaggg tgtgttcttt
3601 tcataataca atttgattag aaaccaacag tgaaattgaa tgtggaagat acgaaagcaa
3661 ccacacttgt ctcatttccc ccttctcccc catcacccct tccctgagaa aaccccctaa
3721 atctcccaga aactatttaa cctgagaatg agaattgttt tcatagttac tcccaccccc
3781 agtcttggag acaaacagta gcaaataaat gagcttgaca gtagagtaaa agctggaaac
3841 ccctttggag agtgagtatg aatcatccat ctcacactaa atacacgcat gtggactcat
3901 ctcagtgaat tgagggctta aagttaaaca tatgggattg gagttgtgtg tccatagggt
3961 ttcactgcct atttgatttg agtttatccc tattaatttt ttacagtgaa attttattaa
4021 agtataatgt acatatattt tcagtggatt ttgctctgaa ggttctccag tggtctgact
4081 acgagatagt gcggcttcag ctgtgggata ttgcaggtaa agtgggaggg ctagatagga
4141 acaagggaag aaattttgat ttaactagaa agctctgagg aaaaaatggg gcagcaaagc
4201 cagaaattgt ttttctgaag ctggaatgat tgagctgctc tgaggacaca tgagctgaat
4261 aggtgcagag cttttgtaga tgttttgccc tgaatagatc tggagcagtc tctatgtgga
4321 tggaggggct ggcaaggtgg tggattgtcc agactatagg tggacactcc cacagcaggg
4381 gagggtgctt tttctaggtt taaggctgac cttggtgaaa aacaagatcc ccagaaaata
4441 gatacagtaa aaatagtgaa tccttactat gtgccaagca ttcttctaag tattttaaat
4501 tcactgactc attaatcctc acatcaaccc tatcataaaa gctactgctt ttggagaatt
4561 gcttgaacct gggaggtgga ggttgcagtg agcctagatg gcaccactgc actccagcct
4621 gggcaacagg agtgcaggaa actgtttcaa aaaaaaaaaa aaaaagcgac tgcttttatc
4681 ttcactttac aaatgagaaa tctgaagcac tgagagcttg atttgcccga agtcaaatag
4741 tatgcagcag agcagggatt tggactggag caatctgggt gcggaatctg ccttggaaat
4801 aattgggctt cattatccct ttatgggata gaaaaccaag agtcagaggg ggaaagtgag
4861 tggccccatc tgacacatat gcttactgcc ctcttctaca aggaatgggg agaggccggc
4921 tcttgggtca ccttgagaac tctccccttt ctccccaggg caggagcgct tcacctctat
4981 gacacgattg tattatcggg atgcctctgc ctgtgttatt atgtttgacg ttaccaatgc
5041 cactaccttc agcaacagcc agaggtggaa acaggaccta gacagcaagc tcacactacc
5101 caatggagag ccggtgccct gcctgctctt ggccaacaag gtatgtggcc aatctcagaa
5161 aatggtccct agcctctatc tgtggttaga aaggaagtaa aatgctctct gattctgggc
5221 atccatttcc ccttcttaag ttggcaaaat catctctacc tccttcaaag gatgttgagg
5281 gtgagtgaga ctttctttct tttttttttt tttgagacgg agtctcgctc tgttgctcag
5341 gctggagtgc agtggcgcga tctcggctca ctgccagctc tgcctcccag gttcacgcca
5401 ttctcttgcc tcagcctccc gagtagctgg gactacaggt gcccaccacc acacccagct
5461 aatttttttg tatttttagt agagactggg tttcattgtg ttagccagga tggtctcgct
5521 ctcctgacct catgatctgc ccgcctcagc ctcccaaagt gctgggatta caggcgtgag
5581 ccactgcgcc cagccaaggg tgagactttc tttgggaaga atgttgatta tagatttccg
5641 atgctggtgt tcttatccat tggtggaaat atgtttaaaa tccacagtat ctgagccata
5701 ttctttctta gttgcaggaa gtttatgata ctgcctttta ttttttctct ttgctagtgt
5761 gatctgtccc cttgggcagt gagccgggac cagattgacc ggttcagtaa agagaacggt
5821 ttcacaggtt ggacagaaac atcagtcaag gagaacaaaa atattaatga ggctatgagg
5881 taagtagctc atgctgatag gcatgcagat gtatccatct tccactgtgg ccctgtggac
5941 cctcttttct tatttctgag ccaacaatga cagactgagc cattggaatg ctagccccta
6001 aaggtcaggg atactgtctt tcctgagata actggggaca aaggggcatt taaaccttct
6061 ggctttacca aatactctct gatgataagt cagactacat ctgccatttc tgcttttcct
6121 ccaaaataag cttctcaggc ttattttttg tcttttagag tcctcattga aaagatgatg
6181 agaaattcca cagaagatat catgtctttg tccacccaag gggactacat caatctacaa
6241 accaagtcct ccagctggtc ctgctgctag tagtgtttgg cttattttcc atcccagttc
6301 tgggaggtct tttaagtctc ttccctttgg ttgcccacct gacaatttta ttaagtacat
6361 ttgaattgtc tcctgactac tgtccagtaa ggaggcccat tgtcacttag aaaagacacc
6421 tggaacccat gtgcatttct gcatctcctg gattagcctt tcacatgttg ctggctcaca
6481 ttagtgccag ttagtgcctt cggtgtaaga tcttctcatc agccctcaat ttgtgatccg
6541 gaattttatg agaaggatta gaaatcagca cctgcgtttt agagatcata attctcacct
6601 acttctgagc ttatttttcc atttgatatt cattgatatc atgacttcca attgagagga
6661 aaatgagatc aaatgtcatt tcccaaattt cttgtaggcc gttgtttcag attctttctg
6721 tcttggaatg taaacatctg attctggaat gcagaaggag ggggtctggg catctgtgga
6781 tttttggcta ctagaagtgt cccagaagtc actgtatttt tgaaacttct aacgtcataa
6841 ttaagtttct cttgtcttgg catcaagaat agtcaagttt tttggccggg catggtggct
6901 catgcctgta atcccagcac ttggggaggc caaggcaggc ggatcacatg aggccaggaa
6961 ttcgagacca acctggtcag catggcaaaa ccccgtctct actaaaagta caaaaattag
7021 ccaggcgtga tggcacgtgt ctgtaatccc agctactctg gagactgagg tgggagaatc
7081 gcttgagact gggaggcaga ggttgcagtg aaccgagatc atgccaccgc acttcagcct
7141 gggtgacaga gaaggactcc gtctcaaaaa aaaaagaaaa aagaatagtc atttttaaac
7201 tacctatctc atgcaatgaa agcattttct tccacaaaga gcttaatcct catgatagga
7261 ttgcctagtg tctcccattt gcaggtttct gggttgatgt cttaatgcat aatactgcaa
7321 gtgacatcag ctggctgtga tgcttcgaaa taggtctgct cctcacagct ttgggaatct
7381 gaatggaaga agaaaagaga gaagttaaca acctccactg gggcaacttt gtgaacacgt
7441 aggcacttag tcataggaaa catattatgt gcaggtccta gcctggggga ggaaagtaga
7501 tagacagaaa atcattaggt aatttaagta ctaaattggg cagggctttt tagtatcaaa
7561 tcactactag accatttaat ttgttaaatt atctctagga tggtgattta taacctaccc
7621 aaagttatcg atattcttag taaactctga ggcctgaagt tctgtgatag accttaaata
7681 agtgtcctaa gtcagtggtt cccaaatctg gctggtcggg aatacctggg aagtttgtta
7741 aaatttttta aaaatgtttt aagatttttg ggtcctgagc cagccgtggt ggctcacacc
7801 tgtaatccca gcactttggg aggctgaggc aggtggatcg cctgaggtca ggagttcaag
7861 atcaacctgg ccaacatact gaaaccccgt ctctactaaa aataagaaaa attagctggg
7921 cgtggtggcg ggcacctgta atcccagcta cttgggagac tgaggcagga gaatcgcttg
7981 aacctgggag ttagaggttg cagtgagctg agatcacacc attgcgcttc agcctgggca
8041 acaagagtga aactccatct ccaaaaaaaa aaaacaaaaa gaaaaagatt tttgggtccc
8101 gacctcaaac ctactgaatc agaatttcta gggatgaagc ctaggaatgt gttgttgttt
8161 tcagagcttc cctggtgatt gtgataagcc tggtttggaa accattgctg gagaactttg
8221 taaagataca gagacccaga ccttttgtat ttacatttaa atacaaatac aaatcctggg
8281 tttctatata ttctgttagc ttttcaggtg attctgctac acagacgttg aaaaccactg
8341 ccctaagaaa gagatcagag gccacatatc agagagaaaa gggaccaaac cttcggtggt
8401 ttgttgtgtg tcgttttaat gccaattatt ttaacttgca cagtcttctg aaaccttgta
8461 ttaatagttc tcttttgtat taccattttc aggtagggtt ttgatcacta tgattctgaa
8521 gataatagtg aaatagtgaa tttcattgat atgaagagat aattgatttt cattcattgg
8581 tttgaacacc tgcaaaatca caaataaatg agaactaagt cttgtattca tggtggttat
8641 tggcctttaa tgtgagtttg tcaaagtgct gttttatact gatagctcaa gaggattgcg
8701 gaaatggaga ctttattttt taaatacctt tttctaaatt ttcaattcaa ggggtacgtg
8761 tgcaagtttg ttacatggat atattgtgtg atgctaaagc ttgggtttct gcttagcctg
8821 tctcccaagt agtgaacata gcacccagtc ggcacttttt caacgcttac tccctctctc
8881 cccacttttg ggagtctcca gtgtctgttg ttcccatctt tatgtgtgtg cccaatgttt
8941 acctcccact tacaagtgag aaatgtggtt aaataccttc ttttaaaatg aaggtaaata
9001 ttattctgtt tacctgttaa cttactaata ccaattgctt agatccatct gcctagtacc
9061 aattcagtac ttgatggaac atgccaaata ctacttggaa acgtgacctt tttgaaaaac
9121 tgaatgtgct tgcttttccc atttgctgac tcaggattta cctgcctttt caagcattgt
9181 tagaggctgg ccagccagcc agcccttctg gatattctct cccaactggt gaacattata
9241 ggcctgaaac taattaaaga aggaactgaa atatacctaa ttcctcttcg tgcccctatc
9301 cataagagaa aagcaattat gattaggaaa aataaaatat ttacatgaat ggaaatgcga
9361 tgctactaaa aataattagg tcttatcact cggatgctac ctctttggtg cattcaaaag
9421 aggtagcgtc caaaattgtt tggaacgtag atatcgtagc ccctccctag aggaggtcct
9481 acatttgaga caagatgcct ctccaaccac tcaggtggct tttggtcaaa aaagaaaaca
9541 gttcccccag agtgttctca gaaaccctaa gggagggctg agaacacagc tgtggggtag
9601 tgtctgccct gactttcttc tttgaacaga agaaatttgg tcacttagct gagtgaccaa
9661 ataagtggag cagaggagat tgtaggtcag ggaaggccct ttagcagtaa agaatggtct
9721 gctgctttat cactactatt gcgccagaat ta

For example, the nucleotide sequence corresponding to the mRNA of the human RAB7L1 (transcript variant 1) is depicted in SEQ ID NO:2 (3324 bp), wherein the underscored bolded “ATG” denotes the beginning of the open reading frame. Nucleotides 1130-1238, 1526-1779, 4045-4116, 4959-5140, 5758-5879, and 6159-8626 of SEQ ID NO: 1 can be spliced together to form RAB7L1 (transcript variant 1). Sequence information related to RAB7L1 (transcript variant 1) is accessible in public databases by GenBank Accession number NM 003929.2 (nucleotide).

SEQ ID NO: 2:

1    agtgccacag gcaaccctgc acgtgacgct tgcggaggaa ggggagagag aggcgcgcgg
61   gagggcgtct agggaatcga ggtgccggct gctccttcct cacaatttgg tttgtgctgc
121  aaggggaggg tccccatcat ctggccccag tggtgtaagg agctgactgg gattcagtca
181  ctgacttgga gccgctcggg ggaagtcccg cccagacagg cggtgggtgg gaatgcctca
241  cttcagtttg aagagggtcc ggatccaaag gggttaaaac gagcgacccc cgatccccga
301  ccacacttcc cgcctcccta aaacgcacac cccgctagcc atgggcagcc gcgaccacct
361  gttcaaagtg ctggtggtgg gggacgccgc agtgggcaag acgtcgctgg tgcagcgata
421  ttcccaggac agcttcagca aacactacaa gtccacggtg ggagtggatt ttgctctgaa
481  ggttctccag tggtctgact acgagatagt gcggcttcag ctgtgggata ttgcagggca
541  ggagcgcttc acctctatga cacgattgta ttatcgggat gcctctgcct gtgttattat
601  gtttgacgtt accaatgcca ctaccttcag caacagccag aggtggaaac aggacctaga
661  cagcaagctc acactaccca atggagagcc ggtgccctgc ctgctcttgg ccaacaagtg
721  tgatctgtcc ccttgggcag tgagccggga ccagattgac cggttcagta aagagaacgg
781  tttcacaggt tggacagaaa catcagtcaa ggagaacaaa aatattaatg aggctatgag
841  agtcctcatt gaaaagatga tgagaaattc cacagaagat atcatgtctt tgtccaccca
901  aggggactac atcaatctac aaaccaagtc ctccagctgg tcctgctgct agtagtgttt
961  ggcttatttt ccatcccagt tctgggaggt cttttaagtc tcttcccttt ggttgcccac
1021 ctgacaattt tattaagtac atttgaattg tctcctgact actgtccagt aaggaggccc
1081 attgtcactt agaaaagaca cctggaaccc atgtgcattt ctgcatctcc tggattagcc
1141 tttcacatgt tgctggctca cattagtgcc agttagtgcc ttcggtgtaa gatcttctca
1201 tcagccctca atttgtgatc cggaatttta tgagaaggat tagaaatcag cacctgcgtt
1261 ttagagatca taattctcac ctacttctga gcttattttt ccatttgata ttcattgata
1321 tcatgacttc caattgagag gaaaatgaga tcaaatgtca tttcccaaat ttcttgtagg
1381 ccgttgtttc agattctttc tgtcttggaa tgtaaacatc tgattctgga atgcagaagg
1441 agggggtctg ggcatctgtg gatttttggc tactagaagt gtcccagaag tcactgtatt
1501 tttgaaactt ctaacgtcat aattaagttt ctcttgtctt ggcatcaaga atagtcaagt
1561 tttttggccg ggcatggtgg ctcatgcctg taatcccagc acttggggag gccaaggcag
1621 gcgcatcaca tgaggccagg aattcgagac caacctggtc agcatggcaa aaccccgtct
1681 ctactaaaag tacaaaaatt agccaggcgt gatggcacgt gtctgtaatc ccagctactc
1741 tggagactga ggtgggagaa tcgcttgaga ctgggaggca gaggttgcag tgaaccgaga
1801 tcatgccacc gcacttcagc ctgggtgaca gagaaggact ccgtctcaaa aaaaaaagaa
1861 aaaagaatag tcatttttaa actacctatc tcatgcaatg aaagcatttt cttccacaaa
1921 gagcttaatc ctcatgatag gattgcctag tgtctcccat ttgcaggttt ctgggttgat
1981 gtcttaatgc ataatactgc aagtgacatc agctggctgt gatgcttcga aataggtctg
2041 ctcctcacag ctttgggaat ctgaatggaa gaagaaaaga gagaagttaa caacctccac
2101 tggggcaact ttgtgaacac gtaggcactt agtcatagga aacatattat gtgcaggtcc
2161 tagcctgggg gaggaaagta gatagacaga aaatcattag gtaatttaag tactaaattg
2221 ggcagggctt tttagtatca aatcactact agaccattta atttgttaaa ttatctctag
2281 gatggtgatt tataacctac ccaaagttat cgatattctt actaaactct gaggcctgaa
2341 gttctgtgat agaccttaaa taagtgtcct aagtcagtgg ttcccaaatc tggctggtcg
2401 ggaatacctg ggaagtttgt taaaattttt taaaaatgtt ttaagatttt tgggtcctga
2461 gccagccgtg gtggctcaca cctgtaatcc cagcactttg ggaggctgag gcaggtggat
2521 cgcctgaggt caggagttca agatcaacct ggccaacata ctgaaacccc gtctctacta
2581 aaaataagaa aaattagctg ggcgtggtgg cgggcacctg taatcccagc tacttgggag
2641 actgaggcag gagaatcgct tgaacctggg agttagaggt tgcagtgagc tgagatcaca
2701 ccattgcgct tcagcctggg caacaagagt gaaactccat ctccaaaaaa aaaaaacaaa
2761 aagaaaaaga tttttgggtc ccgacctcaa acctactgaa tcagaatttc tagggatgaa
2821 gcctaggaat gtgttgttgt tttcagagct tccctggtga ttgtgataag cctggtttgg
2881 aaaccattgc tggagaactt tgtaaagata cagagaccca gaccttttgt atttacattt
2941 aaatacaaat acaaatcctg ggtttctata tattctgtta gcttttcagg tgattctgct
3001 acacagacgt tgaaaaccac tgccctaaga aagagatcag aggccacata tcagagagaa
3061 aagggaccaa accttcggtg gtttgttgtg tgtcgtttta atgccaatta ttttaacttg
3121 cacagtcttc tgaaaccttg tattaatagt tctcttttgt attaccattt tcaggtaggg
3181 ttttgatcac tatgattctg aagataatag tgaaatagtg aatttcattg atatgaagag
3241 ataattgatt ttcattcatt ggtttgaaca cctgcaaaat cacaaataaa tgagaactaa
3301 gtcttgtaaa aaaaaaaaaa aaaa

For example, the nucleotide sequence corresponding to the mRNA of the human RAB7L1 (transcript variant 2) is depicted in SEQ ID NO: 3 (3223 bp), wherein the underscored bolded “ATG” denotes the beginning of the open reading frame. Nucleotides 1130-1238, 1526-1779, 4045-4116, 4959-5140, 5758-5879, 6159-8626 of SEQ ID NO: 1 can be spliced together to form RAB7L1 (transcript variant 2). Sequence information related to RAB7L1 (transcript variant 2) is accessible in public databases by GenBank Accession number NM_001135662.1 (nucleotide).

SEQ ID NO: 3:

1    agtgccacag gcaaccctgc acgtgacgct tgcggaggaa ggggagagag aggcgcgcgg
61   gagggcgtct agggaatcga ggtgccggct gctccttcct cacaatttgc ccagacaggc
121  ggtgggtggg aatgcctcac ttcagtttga agagggtccg gatccaaagg ggttaaaacg
181  agcgaccccc gatccccgac cacacttccc gcctccctaa aacgcacacc ccgctagcca
241  tg              ggcagccg cgaccacctg ttcaaagtgc tggtggtggg ggacgccgca gtgggcaaga
301  cgtcgctggt gcagcgatat tcccaggaca gcttcagcaa acactacaag tccacggtgg
361  gagtggattt tgctctgaag gttctccagt ggtctgacta cgagatagtg cggcttcagc
421  tgtgggatat tgcagggcag gagcgcttca cctctatgac acgattgtat tatcgggatg
481  cctctgcctg tgttattatg tttgacgtta ccastgccac taccttcagc aacagccaga
541  ggtggaaaca ggacctagac agcaagctca cactacccaa tggagagccg gtgccctgcc
601  tgctcttggc caacaagtgt gatctgtccc cttgggcagt gagccgggac cagattgacc
661  ggttcagtaa agagaacggt ttcacaggtt ggacagaaac atcagtcaag gagaacaaaa
721  atattaatga ggctatgaga gtcctcattg aaaagatgat gagaaattcc acagaagata
781  tcatgtcttt gtccacccaa ggggactaca tcaatctaca aaccaagtcc tccagctggt
841  cctgctgcta gtagtgtttg gcttattttc catcccagtt ctgggaggtc ttttaagtct
901  cttccctttg gttgcccacc tgacaatttt attaagtaca tttgaattgt ctcctgacta
961  ctgtccagta aggaggccca ttgtcactta gaaaagacac ctggaaccca tgtgcatttc
1021 tgcatctcct ggattagcct ttcacatgtt gctggctcac attagtgccagttagtgcct
1081 tcggtgtaag atcttctcat cagccctcaa tttgtgatcc ggaattttat gagaaggatt
1141 agaaatcagc acctgcgttt tagagatcat aattctcacc tacttctgag cttatttttc
1201 catttgatat tcattgatat catgacttcc aattgagagg aaaatgagat caaatgtcat
1261 ttcccaaatt tcttgtaggc cgttgtttca gattctttct gtcttggaat gtaaacatct
1321 gattctggaa tgcagaagga gggggtctgg gcatctgtgg atttttggct actagaagtg
1381 tcccagaagt cactgtattt ttgaaacttc taacgtcata attaagtttc tcttgtcttg
1441 gcatcaagaa tagtcaagtt ttttggccgg gcatggtggc tcatgcctgt aatcccagca
1501 cttggggagg ccaaggcagg cggatcacat gaggccagga attcgagacc aacctggtca
1561 gcatggcaaa accccgtctc tactaaaagt acaaaaatta gccaggcgtg atggcacgtg
1621 tctgtaatcc cagctactct ggagactgag gtgggagaat cgcttgagac tgggaggcag
1681 aggttgcagt gaaccgagat catgccaccg cacttcagcc tgggtgacag agaaggactc
1741 cgtctcaaaa aaaaaagaaa aaagaatagt catttttaaa ctacctatct catgcaatga
1801 aagcattttc ttccacaaag agcttaatcc tcatgatagg attgcctagt gtctcccatt
1861 tgcaggtttc tgggttgatg tcttaatgca taatactgca agtgacatca gctggctgtg
1921 atgcttcgaa ataggtctgc tcctcacagc tttgggaatc tgaatggaag aagaaaagag
1981 agaagttaac aacctccact ggggcaactt tgtgaacacg taggcactta gtcataggaa
2041 acatattatg tgcaggtcct agcctggggg aggaaagtag atagacagaa aatcattagg
2101 taatttaagt actaaattgg gcagggcttt ttagtatcaa atcactacta gaccatttaa
2161 tttgttaaat tatctctagg atggtgattt ataacctacc caaagttatc gatattctta
2221 ctaaactctg aggcctgaag ttctgtgata gaccttaaat aagtgtccta agtcagtggt
2281 tcccaaatct ggctggtcgg gaatacctgg gaagtttgtt aaaatttttt aaaaatgttt
2341 taagattttt gggtcctgag ccagccgtgg tggctcacac ctgtaatccc agcactttgg
2401 gaggctgagg caggtggatc gcctgaggtc aggagttcaa gatcaacctg gccaacatac
2461 tgaaaccccg tctctactaa aaataagaaa aattagctgg gcgtggtggc gggcacctgt
2521 aatcccagct acttgggaga ctgaggcagg agaatcgctt gaacctggga gttagaggtt
2581 gcagtgagct gagatcacac cattgcgctt cagcctgggc aacaagagtg aaactccatc
2641 tccaaaaaaa aaaaacaaaa agaaaaagat ttttgcgtcc cgacctcaaa cctactgaat
2701 cagaatttct agggatgaag cctaggaatg tgttgttgtt ttcagagctt ccctggtgat
2761 tgtgataagc ctggtttgga aaccattgct ggagaacttt gtaaagatac agagacccag
2821 accttttgta tttacattta aatacaaata caaatcctgg gtttctatat attctgttag
2881 cttttcaggt gattctgcta cacagacgtt gaaaaccact gccctaagaa agagatcaga
2941 ggccacatat cagagagaaa agggaccaaa ccttcggtgg tttgttgtgt gtcgttttaa
3001 tgccaattat tttaacttgc acagtcttct gaaaccttgt attaatagtt ctcttttgta
3061 ttaccatttt caggtagggt tttgatcact atgattctga agataatagt gaaatagtga
3121 atttcattga tatgaagaga taattgattt tcattcattg gtttgaacac ctgcaaaatc
3181 acaaataaat gagaactaag tcttgtaaaa aaaaaaaaaa aaaa

For example, the nucleotide sequence corresponding to the mRNA of the human RAB7L1 (transcript variant 3) is depicted in SEQ ID NO: 4 (3438 bp), wherein the underscored bolded “ATG” denotes the beginning of the open reading frame. Nucleotides 1130-1779, 4959-5140, 5758-5879, 6159-8626 of SEQ ID NO: 1 can be spliced together to form RAB7L1 (transcript variant 3). Sequence information related to RAB7L1 (transcript variant 3) is accessible in public databases by GenBank Accession number NM_001135663.1 (nucleotide).

SEQ ID NO: 4:

1    agtgccacag gcaaccctgc acgtgacgct tgcggaggaa ggggagagag aggcgcgcgg
61   gagggcgtct agggaatcga ggtgccggct gctccttcct cacaatttgg tttgtgctgc
121  aaggggaggg tccccatcat ctggccccag tggtgtaagg agctgactgg gattcagtca
181  ctgacttgga gccgctcggg ggaagtcccg gtgggtgagg ttccgcggcg cctggtccag
241  tttctcggca gtcaggccag gagggggtgg ggaaggtgcg aacagccggg ggctcggagc
301  tcgcgccctg cgccccaccc aggttgcggc cgcccgggga ggaagcgcgt ataggttgag
361  tgcaaagttt ccttttttgc tttctcgtca gagcagccca gacaggcggt gggtgggaat
421  gcctcacttc agtttgaaga gggtccggat ccaaaggggt taaaacgagc gacccccgat
481  ccccgaccac acttcccgcc tccctaaaac gcacaccccg ctagccatgg gcagccgcga
541  ccacctgttc aaagtgctgg tggtggggga cgccgcagtg ggcaagacgt cgctggtgca
601  gcgatattcc caggacagct tcagcaaaca ctacaagtcc acggtgggag ggcaggagcg
661  cttcacctct atgacacgat tgtattatcg ggatgcctct gcctgtgtta ttatgtttga
721  cgttaccaat gccactacct tcagcaacag ccagaggtgg aaacaggacc tagacagcaa
781  gctcacacta cccaatggag agccggtgcc ctgcctgctc ttggccaaca agtgtgatct
841  gtccccttgg gcagtgagcc gggaccagat tgaccggttc agtaaagaga acggtttcac
901  aggttggaca gaaacatcag tcaaggagaa caaaaatatt aatgaggcta tgagagtcct
961  cattgaaaag atgatgagaa attccacaga agatatcatg tctttgtcca cccaagggga
1021 ctacatcaat ctacaaacca agtcctccag ctggtcctgc tgctagtagt gtttggctta
1081 ttttccatcc cagttctggg aggtctttta agtctcttcc ctttggttgc ccacctgaca
1141 attttattaa gtacatttga attgtctcct gactactgtc cagtaaggag gcccattgtc
1201 acttagaaaa gacacctgga acccatgtgc atttctgcat ctcctggatt agcctttcac
1261 atgttgctgg ctcacattag tgccagttag tgccttcggt gtaagatctt ctcatcagcc
1321 ctcaatttgt gatccggaat tttatgagaa ggattagaaa tcagcacctg cgttttagag
1381 atcataattc tcacctactt ctgagcttat ttttccattt gatattcatt gatatcatga
1441 cttccaattg agaggaaaat gagatcaaat gtcatttccc aaatttcttg taggccgttg
1501 tttcagattc tttctgtctt ggaatgtaaa catctgattc tggaatgcag aaggaggggg
1561 tctgggcatc tgtggatttt tggctactag aagtgtccca gaagtcactg tatttttgaa
1621 acttctaacg tcataattaa gtttctcttg tcttggcatc aagaatagtc aagttttttg
1681 gccgggcatg gtggctcatg cctgtaatcc cagcacttgg ggaggccaag gcaggcggat
1741 cacatgaggc caggaattcg agaccaacct ggtcagcatg gcaaaacccc gtctctacta
1801 aaagtacaaa aattagccag gcgtgatggc acgtgtctgt aatcccagct actctggaga
1861 ctgaggtggg agaatcgctt gagactggga ggcagaggtt gcagtgaacc gagatcatgc
1921 caccgcactt cagcctgggt gacagagaag gactccgtct caaaaaaaaa agaaaaaaga
1981 atagtcattt ttaaactacc tatctcatgc aatgaaagca ttttcttcca caaagagctt
2041 aatcctcatg ataggattgc ctagtgtctc ccatttgcag gtttctgggt tgatgtctta
2101 atgcataata ctgcaagtga catcagctgg ctgtgatgct tcgaaatagg tctgctcctc
2161 acagctttgg gaatctgaat ggaagaagaa aagagagaag ttaacaacct ccactggggc
2221 aactttgtga acacgtaggc acttagtcat aggaaacata ttatgtgcag gtcctagcct
2281 gggggaggaa agtagataga cagaaaatca ttaggtaatt taagtactaa attgggcagg
2341 gctttttagt atcaaatcac tactagacca tttaatttgt taaattatct ctaggatggt
2401 gatttataac ctacccaaag ttatcgatat tcttactaaa ctctgaggcc tgaagttctg
2461 tgatagacct taaataagtg tcctaagtca gtggttccca aatctggctg gtcgggaata
2521 cctgggaagt ttgttaaaat tttttaaaaa tgttttaaga tttttgggtc ctgagccagc
2581 cgtggtggct cacacctgta atcccagcac tttgggaggc tgaggcaggt ggatcgcctg
2641 aggtcaggag ttcaagatca acctggccaa catactgaaa ccccgtctct actaaaaata
2701 agaaaaatta gctgggcgtg gtggcgggca cctgtaatcc cagctacttg ggagactgag
2761 gcaggagaat cgcttgaacc tgggagttag aggttgcagt gagctgagat cacaccattg
2821 cgcttcagcc tgggcaacaa gagtgaaact ccatctccaa aaaaaaaaaa caaaaagaaa
2881 aagatttttg ggtcccgacc tcaaacctac tgaatcagaa tttctaggga tgaagcctag
2941 gaatgtgttg ttgttttcag agcttccctg gtgattgtga taagcctggt ttggaaacca
3001 ttgctggaga actttgtaaa gatacagaga cccagacctt ttgtatttac atttaaatac
3061 aaatacaaat cctgggtttc tatatattct gttagctttt caggtgattc tgctacacag
3121 acgttgaaaa ccactgccct aagaaagaga tcagaggcca catatcagag agaaaaggga
3181 ccaaaccttc ggtggtttgt tgtgtgtcgt tttaatgcca attattttaa cttgcacagt
3241 cttctgaaac cttgtattaa tagttctctt ttgtattacc attttcaggt agggttttga
3301 tcactatgat tctgaagata atagtgaaat agtgaatttc attgatatga agagataatt
3361 gattttcatt cattggtttg aacacctgca aaatcacaaa taaatgagaa ctaagtcttg
3421 taaaaaaaaa aaaaaaaa

For example, the nucleotide sequence corresponding to the mRNA of the human RAB7L1 (transcript variant 4) is depicted in SEQ ID NO: 5 (3070 bp), wherein the underscored bolded “ATG” denotes the beginning of the open reading frame. Nucleotides 1130-1339, 4045-4116, 4959-5140, 5758-5879, 6159-8626 of SEQ ID NO: 1 can be spliced together to form RAB7L1 (transcript variant 4). Sequence information related to RAB7L1 (transcript variant 4) is accessible in public databases by GenBank Accession number NM_001135664.1 (nucleotide).

SEQ ID NO: 5:

1    agtgccacag gcaaccctgc acgtgacgct tgcggaggaa ggggagagag aggcgcgcgg
61   gagggcgtct agggaatcga ggtgccggct gctccttcct cacaatttgg tttgtgctgc
121  aaggggaggc tccccatcat ctggccccag tggtgtaagg agctgactgg gattcagtca
181  ctgacttgga gccgctcggg ggaagtcccg tggattttgc tctgaaggtt ctccagtggt
241  ctgactacga gatagtgcgg cttcagctgt gggatattgc agggcaggag cgcttcacct
301  ctatgacacg attgtattat cgggatgcct ctgcctgtgt tattatgttt gacgttacca
361  atgccactac cttcagcaac agccagaggt ggaaacagga cctagacagc aagctcacac
421  tacccaatgg agagccggtg ccctgcctgc tcttggccaa caagtgtgat ctgtcccctt
481  gggcagtgag ccgggaccag attgaccggt tcagtaaaga gaacggtttc acaggttgga
541  cagaaacatc agtcaaggag aacaaaaata ttaatgaggc tatgagagtc ctcattgaaa
601  agatgatgag aaattccaca gaagatatca tgtctttgtc cacccaaggg gactacatca
661  atctacaaac caagtcctcc agctggtcct gctgctagta gtgtttggct tattttccat
721  cccagttctg ggaggtcttt taagtctctt ccctttggtt gcccacctga caattttatt
781  aagtacattt gaattgtctc ctgactactg tccagtaagg aggcccattg tcacttagaa
841  aagacacctg gaacccatgt gcatttctgc atctcctgga ttagcctttc acatgttgct
901  ggctcacatt agtgccagtt agtgccttcg gtgtaagatc ttctcatcag ccctcaattt
961  gtgatccgga actttatgag aaggattaga aatcagcacc tgcgttttag agatcataat
1021 tctcacctac ttctgagctt atttttccat ttgatattca ttgatatcat gacttccaat
1081 tgagaggaaa atgagatcaa atgtcatttc ccaaatttct tgtaggccgt tgtttcagat
1141 tctttctgtc ttggaatgta aacatctgat tctggaatgc agaaggaggg ggtctgggca
1201 tctgtggatt tttggctact agaagtgtcc cagaagtcac tgtatttttg aaacttctaa
1261 cgtcataatt aagtttctct tgtcttggca tcaagaatag tcaagttttt tggccgggca
1321 tggtggctca tgcctgtaat cccagcactt ggggaggcca aggcaggcgg atcacatgag
1381 gccaggaatt cgagaccaac ctggtcagca tggcaaaacc ccgtctctac taaaagtaca
1441 aaaattagcc aggcgtgatg gcacgtgtct gtaatcccag ctactctgga gactgaggtg
1501 ggagaatcgc ttgagactgg gaggcagagg ttgcagtgaa ccgagatcat gccaccgcac
1561 ttcagcctgg gtgacagaga aggactccgt ctcaaaaaaa aaagaaaaaa gaatagtcat
1621 ttttaaacta cctatctcat gcaatgaaag cattttcttc cacaaagagc ttaatcctca
1681 tgataggatt gcctagtgtc tcccatttgc aggtttctgg gttgatgtct taatgcataa
1741 tactgcaagt gacatcagct ggctgtgatg cttcgaaata ggtctgctcc tcacagcttt
1801 gggaatctga atggaagaag aaaagagaga agttaacaac ctccactggg gcaactttgt
1861 gaacacgtag gcacttagtc ataggaaaca tattatgtgc aggtcctagc ctgggggagg
1921 aaagtagata gacagaaaat cattaggtaa tttaagtact aaattgggca gggcttttta
1981 gtatcaaatc actactagac catttaattt gttaaattat ctctaggatg gtgatttata
2041 acctacccaa agttatcgat attcttacta aactctgagg cctgaagttc tgtgatagac
2101 cttaaataag tgtcctaagt cagtggttcc caaatctggc tggtcgggaa tacctgggaa
2161 gtttgttaaa attttttaaa aatgttttaa gatttttggg tcctgagcca gccgtggtgg
2221 ctcacacctg taatcccagc actttgggag gctgaggcag gtggatcgcc tgaggtcagg
2281 agttcaagat caacctggcc aacatactga aaccccgtct ctactaaaaa taagaaaaat
2341 tagctgggcg tggtggcggg cacctgtaat cccagctact tgggagactg aggcaggaga
2401 atcgcttgaa cctgggagtt agaggttgca gtgagctgag atcacaccat tgcgcttcag
2461 cctgggcaac aagagtgaaa ctccatctcc aaaaaaaaaa aacaaaaaga aaaagatttt
2521 tgggtcccga cctcaaacct actgaatcag aatttctagg gatgaagcct aggaatgtgt
2581 tgttgttttc agagcttccc tggtgattgt gataagcctg gtttggaaac cattgctgga
2641 gaactttgta aagatacaga gacccagacc ttttgtattt acatttaaat acaaatacaa
2701 atcctgggtt tctatatatt ctgttagctt ttcaggtgat tctgctacac agacgttgaa
2761 aaccactgcc ctaagaaaga gatcagaggc cacatatcag agagaaaagg gaccaaacct
2821 tcggtggttt gttgtgtgtc gttttaatgc caattatttt aacttgcaca gtcttctgaa
2881 accttgtatt aatagttctc ttttgtatta ccattttcag gtagggtttt gatcactatg
2941 attctgaaga taatagtgaa atagtgaatt tcattgatat gaagagataa ttgattttca
3001 ttcattggtt tgaacacctg caaaatcaca aataaatgag aactaagtct tgtaaaaaaa
3061 aaaaaaaaaa

For example, other mRNA transcript variants of human RAB7L1 can exist. For example nucleotides 1130-1238, 4045-4116, 4959-5140, 5758-5879, 6159-8626 of SEQ ID NO: 1 can be spliced together to form RAB7L1 (transcript variant 5). Sequence information related to RAB7L1 transcript variants is accessible in public databases such as GenBank.

For example, the polypeptide sequence corresponding to human RAB7L1 (isoform 1) is encoded by the nucleic acid sequence of SEQ ID NOS: 2 or 3 and is depicted in SEQ ID NO: 6 (203aa). Sequence information related to RAB7L1 (isoform 1) is accessible in public databases by GenBank Accession numbers NP_003920.1 and NP_001129134.1 (protein).

SEQ ID NO: 6:

1   MGSRDHLFKV LVVGDAAVGK TSLVQRYSQD SFSKHYKSTV GVDFALKVLQ WSDYEIVRLQ
61  LWDIAGQERF TSMTRLYYRD ASACVIMFDV TNATTFSNSQ RWKQDLDSKL TLPNGEPVPC
121 LLLANKCDLS PWAVSRDQID RFSKENGFTG WTETSVKENK NINEAMRVLI EKMMRNSTED
181 IMSLSTQGDY INLQTKSSSW SCC

For example, the polypeptide sequence corresponding to human RAB7L1 (isoform 2) is encoded by the nucleic acid sequence of SEQ ID NO: 4 and is depicted in SEQ ID NO: 7 (179aa). Sequence information related to RAB7L1 (isoform 2) is accessible in public databases by GenBank Accession numbers NP_001123135.1 (protein).

SEQ ID NO: 7:

1   MGSRDHLFKV LVVGDAAVGK TSLVQRYSQD SFSKHYKSTV GGQERFTSMT PLYYRDASAC
61  VIMFDVTNAT TFSNSQRWKQ DLDSKLTLPN GEPVPCLLLA NKCDLSPWAV SRDQIDRFSK
121 ENGFTGWTET SVKENKNINE AMRVLIEKMM RNSTEDIMSL STQGDYINLQ TKSSSWSCC

For example, the polypeptide sequence corresponding to human RAB7L1 (isoform 3) is encoded by the nucleic acid sequence of SEQ ID NO: 5 and is depicted in SEQ ID NO: 8 (131 aa). Sequence information related to RAB7L1 (isoform 3) is accessible in public databases by GenBank Accession numbers NP_001129136.1 (protein).

SEQ ID NO: 8:

1   MTRLYYRDAS ACVIMFDVTN ATTFSNSQRW KQDLDSKLTL PNGEPVPCLL LANKCDLSPW
61  AVSRDQIDRF SKENGFTGWT ETSVKENKNI NEAMRVLIEK MMRNSTEDIM SLSTQGDYIN
121 LQTKSSSWSC C

The invention provides for a nucleic acid encoding a LRRK2 protein, or fragment thereof.

For example, the human genomic nucleotide sequence corresponding to the sense strand of the human LRRK2 gene is depicted in SEQ ID NO: 9 (144275 bp). Sequence information related to LRRK2 is accessible in public databases by GenBank Accession number NG_011709.1 (nucleotide).

SEQ ID NO: 9:

1      gcgctggctg cgggcggtga gctgagctcg cccccgggga gctgtggccg gcgcccctgc
61     cggttccctg agcagcggac gttcatgctg ggagggcggc gggttggaag caggtgccac
121    catggctagt ggcagctgtc aggggtgcga agaggacgag gaaactctga agaagttgat
181    agtcaggctg aacaatgtcc aggaaggaaa acagatagaa acgctggtcc aaatcctgga
241    ggatctgctg gtgttcacgt actccgagcg cggtaatcac ttgaaaataa actgtgcttt
301    tatttttgca aactttctcc ccctccttac atttgcaaat tttgtcctcc tccccttgac
361    cctgctcaaa cccggactct taaggagccg caaactccca tatcctttcc ttagggcaga
421    aagcagctga gaatttcagg aaggtcttca cctttttgac ttttctcccc gtttcagact
481    aaaaaggaga gggggtgctg tggattgtga ctttgcttct tttccccacc cacttgtttt
541    ccagcctcca agttatttca aggcaaaaat atccatgtgc ctctgttgat cgtcttggac
601    tcctatatga gagtcgcgag tgtgcagcag gtaaaggcat tgttttcact tcaactcatt
661    ctcccttctg tttggaagga gacgttttac tggcaatgtt aatatagccg agagttcttg
721    gttattccca aaatttggct tgaggaacct ctgactgtga ttttaagatg ggaatattgt
781    taaatcatta cgcaatgtaa acgggatgaa gagccccagt atgtgttccc tgagtgtctt
841    taagaagtaa ctttataaaa ccaacagtat ggatggtggt agaaggagga taaaaatggg
901    ttcggtttta gtctcgttat tggcaagatg aattcattag tgtttagact tgactattcc
961    aagtatcttc ccaatacaga gcatgtccta gatgagaaga ttatgaatag tttggaaaag
1021   gggaataatt aatagtgata aaatgcaact ttgtcactag caaactcttg tagagttcag
1081   cactttttaa aattcaaaga tttctagcct ttagttgtag tataccttgt agtatctaaa
1141   gaaagtgatg tcttatgaga ccctcatagt ttgcaactgt tgtcatataa aatgcatgta
1201   gaagtgaaac ttttacaatc tgtaccatag gaaacccaga aatttgctat gtatcttgga
1261   ttttttttta aagggggcct taaaaatggt aattaagaat gatttacagt caaaacaaaa
1321   ttataggcca aggtgataac ttccttcgga gcacttagag atttggggaa ctgaaatcag
1381   ttttgtcatc tgcatgttaa ctcatgcaga gaaagagaat tggactttga actccttgga
1441   ggtgcagtca gaaagccaat gtttcttaat ggttgagagg cttgacagac atgaggcatc
1501   tcaatcttta aagtggtgtg ggtctatctt tatcttgatg tttatctctg tatctagctg
1561   tatctagtct gggtgaacca tctagcttct ttgatatgag gacatttaca tctggaagaa
1621   atattttaat ttgttttcaa ctgtgaaata ttttccatct gactattata gattttcacg
1681   ctgctatcaa accaaaccaa gaaaagatgg aggcataata aagatgctgt tcttttaaga
1741   ctcaaagtcg gaattttgcc tgtggaatat gagtcacttt ttgggcactg gcctattgtg
1801   cttcctgctc tgcacccacg tcatcccttc ttacttgtct ctgctttggt gttcagaagt
1861   gcctgattct ggccaccttc attccctaga ctctgtactt gatagagtca ctcctgcttg
1921   atactgctca ggacagtcag atcctgggta ggcgttttgg tctgcagggt ctagataagg
1981   cagtgctata cttgacaacc caggggagcc tggaacatac ttcctaattc ttaattttag
2041   aaattgccca agcctgagca tacttgtccg gagtagttat gagtgtcact tagtatttct
2101   gcctagagag taccagaggc aaagtatgct ggaaaataag gaagagtttt tttaaaagta
2161   attaattact tttttggata tatcatagtt gtatatattt tggggataca tatgctattt
2221   gatacatgta tacaatgtgt aatgttcaaa tcagggtaac tggaatatcc gtcacctcga
2281   acatttttct ttgtgttggc aacgttgcaa tttctttctt ctagctattt taaaatatgc
2341   aatgaattat taaccataat ttccctgcta tactattaaa tattaactta attgcttgta
2401   tctaattata tttttgtaca cattaaccac cttctcttta tccctcccca tcctttcatt
2461   tccagtctct ggtaaccacc attctactct cttcctccat gagatccacc ttttccgctc
2521   ctacatatga gtgagattat gcaatatttt atttctgtac ctggcttatt taatttaacc
2581   taatgacctc cagtcccacc catgctgttg caaatgacag gatttcattt tttatgactg
2641   aataatattc cattgtgtat gtataccaca ttttctttta tttttagtta agtaattaat
2701   ttagagacag ggtctcactc tgttgcccag actggagagc agtggtgtga tcaaagctca
2761   ctgcaggcct gcaatcctgg gctgaagtag tcctcctgcc tcagcctccc aggtagctag
2821   gactataagc atgtgccacc atgctcagct aatttttttt tcttttttta ctttttgtag
2881   agatggagtc ttgctatgtt gcccaggtta gtttcaaact cctgacctca agtaatcctc
2941   ctgcctcggc ctccatattt tctttgttaa tctgttgata gacatataag gtgattctgt
3001   attttaacta ttgtgaacag ggctgcaata aacatgggag ttcagatata tctttgatat
3061   actgatgttc tttttttgga tatataccca gcgatgagat tactggatca tatgaaaatt
3121   ctctttttag ttttttaaga tacctccata ctgtgtttca tcatggctgt gctactttat
3181   attcccatca gcagtgtacc accattcccc tttttctgca tccttaccag catttgttat
3241   tttttgtctt tttgataata gccattctgc ctgtggtgag ataatatctc attatggttt
3301   tgatttgcct ctccctaatg attagtgaag tttaggattt ttttttcatg tacctgttag
3361   ccatttgtct gtcttctttt gagaaatgtc tatttggatc ttttgtccat ttaaaaataa
3421   gacttttttt tttttttttt tttttgctaa ttgagttttt ttagttcctt atgtattctg
3481   gttgttaatc ttttgttgaa tggatatttt gcaaatattt tctccctttc tttatgttgt
3541   ctcttcactt tgttaattat tttctttgtt gtgcagaagc tttttagctt gatataatcc
3601   catttgccta tttttgttgt aattgcctgt gcttttgagg tcctacccca aaaatcattg
3661   cacagaccaa tgtcctgtag catttcccca gtgttttctt ctagtagttg catattttca
3721   ggtctgagat gtaagtcttt aatccatttt gaaatgattt gtgtatatgg tgaaagctgt
3781   ggatctagtt tccttctttt gcacagccaa tatttgattc tcactgaaat ctcactgccc
3841   tcctggaaat attaccggat gttttactgt catagcgaag tgagagtaag ctgctcactg
3901   aggatcaaag agcttgtgac agacctaaga ctcaagtctt ctcacacctt caaaatctct
3961   ttccatcata caatctacta gctgctgaat tcgcaagctt tttgtgcaag ctagtaaaaa
4021   gcaaaatggt ttgatacaaa tactgtggcc atgctaggta caatgacatc aatttaaatt
4081   atcattggtc ttaacaaggg gatgtagaaa ggggtctcct actgacattt taatactcac
4141   ttaaaagtag tatttttcct tcagatttct ttatattatt agtataatta ctgtaagtat
4201   cctttactgc tttatatgtt gaattagctg gaagtgccaa aagaaaaact cttaatgata
4261   aatttaaggt attaaggtaa atttctcctt catttaattt aatagtaatt cttaattact
4321   atttaaaata aagattaagg tttgtttcta gatgccattt aacatgatat tccagactgc
4381   cagttttatt ttcaaagttt gtttcatatt ttattaatgt ttcttcataa atgacagtct
4441   ttagaaaatt gacggttaag ctaggtgctt tatatttttc ttttcctgcc tatcttttca
4501   ctgtgctcct aaattttaca tctctttatt ctcaagggtt caacctttga agaaggggag
4561   caaaataaat gaaagtggct aaaatttttt ctttaacccc tagactcttt cctgttgtgc
4621   attaattaca tgcttgagtt tttagaataa ttataataaa gtaaaactac caatttaatt
4681   gtattgtaac tgtgcaagat gggaaccttc tctcttagag agataagctt ttaattgaat
4741   agattaatgg atcaattgtt acctctgctt tgctgccaga gattctattt aatcacagaa
4801   gttccatgta gtgctggaga gctcagttgc ctgaatcttt ttgcaaagcg tttactgata
4861   ctgttgcttc accaaccaaa acaaacaggt tttttccttg agtcagcttt gtaggtacag
4921   agatgagttt ggcatcctat gtgacttttt tttttttttt tttttttttt tttaccacca
4981   gaagctgttc agaatgttat tttcttaaat agttcggaaa aaagtcttga tgtattctat
5041   gaaagcacaa aaatagtcag tttctatgac agctggattg tcaacgtctg ttcagcttac
5101   gtggaggagg atgtcctact tgagtagtat aggtagaaat agctatcaga aattgccgcc
5161   tttgaaagca atttgaaatt atgtaaaagg aagtaatgac aaaataaagc aatttatgtt
5221   taatctggaa aagatccaaa agtaatattg taaagagatc ttgagtaatc atttttatct
5281   tcctaaaata gccgttgttt actcccgtaa gcgagtaaga aacttgtgcc attattcctt
5341   attgggtgca tatagatttc tcaccttgtc attcaactcc ttgcaatatt caactttact
5401   tatgcatcca gccttatccc aaaatagcct cttccctgta gcagcttcct tatcatgtag
5461   cagcctactc tcctcaccgc tcatccgttc ttatacaatc tggcttaagc tctacctctt
5521   catattatat tcttctctga gtaatttcaa ctcacactga gtcttacttt cagtatttct
5581   attatattgg tatttattac acatcacact tagatacttt tccattagtc tctagagggt
5641   acatatatgc actttctctt tttttttttt tttttgacca tgtcaagtat agttctatag
5701   tataatagaa tgaattggag atcctttaca tttagagagg gaggagtcta cagtaggaag
5761   aataggttaa ttttcatctc cggtttgaaa tcaggacttt caattttttt ttcagaggta
5821   aagagcacct tagtcaagtt ggcatcttgt aaacagactg agtgaagata tacttaaaat
5881   gcatctataa tttcatattt tatttcgaaa tgtgaaagag cctactaggg gtgtctgtga
5941   tctctagacc ttatcaattc attctagaga aatctggagg gagccattga ggagttctac
6001   ctcctgtcta ttttatagag ctctttctct ttttctccta tcagatgtag attcaattgc
6061   taaaaatgcc acgtttcttg cctctattat tctagcttca ttacttgggg agcagccatt
6121   ctgataactt acattttgct actaaaatct ccaacttcac ctaatccttc attataagcc
6181   acttcatttt ttcctataat taaaatttta aatatgtgga ggaaattctg tcaggtagat
6241   atgacttaaa acctactaag ggccaggtgc agaggctcac gcctgtaatc ccagcacttt
6301   gagaggccaa gtcaggagga ttgcttgagc ccaggagctt gagaccagcc tgggcaacag
6361   agcgagaccc ctgtcttcac aaaaaaaaaa aaaaaaaaat tagctgggtg tggtggcaca
6421   tacctgtagt cccagctact tgggaggctg aagtgggtgg atcacctaag gacaggagtt
6481   ctaggctgca gtgatctatg attgcaccat tgcactccag cctgggtgac agagtgagac
6541   cctatctcaa aaacaaaaac aaaaccaaaa aaaaaaaaaa aacaagaaaa aaaaagtact
6601   aaggatctgg tatagccatt cttgcactta ataatcttgg tacaacctct aaaactattt
6661   tttatagttt attttatttt gccttattta gacaattggc atgtctatgt tcttcataat
6721   ttagaaatta tatgtattta tatatataaa ttatatatat tatatattat gtataaatta
6781   aattatatat atatataata tatatatatt tgaaatggat tcttgctctg tcatccaggc
6841   tagagtgcag tggcacaatc ttggctcact gcaccctctg cctcctgggt tcaagcgatt
6901   ctccttcctc agcctcccga gtagctgaga ttacaggcgc ccatcaccat gcctggctaa
6961   tttttgtatt tttagtagag actgggtttc accatcttgg ccacattggt ctcaaactgc
7021   cggctgaaat gttgtatttt ttgtatgtct ttctggtatg atttttggag aaaggtgtat
7081   cctaagaata cggcttgctt ttgtttctgg gtaagcattt tagggtatca ttttgttgta
7141   taaccattgt ttacaagtga gataagcatc tattccacta agattgaaga gattcatgtt
7201   tgactgagta tgctctatta acattcttta aaacatgtga atatatgtct ttcttgtttt
7261   caggtgggtt ggtcacttct gtgcaaatta atagaagtct gtccaggtac aatgcaaagc
7321   ttaatgggac cccaggatgt tggaaatgat tgggaagtcc ttggtgttca ccagtaagta
7381   tgatagatat gtaaaacaaa tggccttgag tatttatttg tacacatgac aaccttccct
7441   tgatacactg tgtttgcaat ccaaggctac tcctgtggaa ttctttaaaa tacagatatt
7501   tttccttgag tcaatgattt acatttatag agagctttaa actcagaagt ttgatttaga
7561   aagcaaacat ttaaggtaac atgtcagaag ttattatttt aataatataa tcatataatt
7621   ataaaactgg ttaagttgta gatttttgat gagtactttt gaattcaaac catgaagaga
7681   ttttggcttt taataataga atcgatacaa accactagtt cttaaaaaaa tgggaactga
7741   gaaaagttag ttctgtaagt agtaatttga aagttgatgt tctactgtct ttaaatagta
7801   catttatata tatattccta tatatacagt aagtttaaac tatggctttc agaaagagtt
7861   aagaaagagg aaattaactt tcagcacatc tgtagccaaa tcttgatagt aattttacca
7921   gctatgtttt tgcagtttgc agcataatgg cttcttagat gagactactt ccttagccat
7981   cattaattaa gaaaatattt tctcaagaag aatgtgtttc caggaaaata cattttggat
8041   agctttgttt cttgacagtt aaaaaatatc ttctaagcta ctgaggaggc tgaggaagga
8101   gaatcacttg aacctgggag gcggaggttg cagtgagcgg aggttgcagt gagccgagat
8161   tgcaccactg cactccagcc tgggtgacag agcgacactc tgtctcaaaa acaaacaaac
8221   aaacaaacct tttgtcatta actttaaatc ttttttatac ctaatatgac ttttctttat
8281   cacagaaaag gaaattgtga atattttttg gcttccaatg gtatatggtt tatgaaaatt
8341   taatttatga aaaattttca ggtgtttgta ttgctgatca gtgtcaagta gtgctataaa
8401   tttagacaaa ttagagctat gtgtttgtcc ataagtgaac atgtctgtgc ttatacattt
8461   tcccctcttt gacaaatgtg ttgctcttct tgttttcagt acataaaggg tgtgttttgg
8521   aaagagcata tttacaatta attggagttc tcgtcttcaa tctaatctct gtaattctat
8581   gtatcagttc taaagtatac agcatttgat gaggaattac tcaaaatata ccagtaatta
8641   ggaattgtaa ctttaaatgt cccttggttt gggtgataat ttccaggaag tccaaagatg
8701   agccagtcta taacctcagg gagtgtttgg gaaactcatc tagtcatatt cctgtacaaa
8761   ccaactgttc aaattaaatt acataaaagt ttatgtagga aatttcattc actcactcac
8821   ttactcattc actcactttg ttcatccagt cattcatctt ctattcattg aatgtttttg
8881   aagcctgtcc tctgggtcag aaaccatgca gttgtgaaga agatagacac actgctgtct
8941   ccagtggagt gtattagatc actcccagca aaaattgatt gtaaaacaga tttctctttt
9001   ttcaaggcct tttccctcca aagacttacc agtactgaag aaaaatttct tccgtggtaa
9061   taaagtcagg aattgtggga atggtatagg gagaggtagg ggcagggtga ttaggaggaa
9121   ggctggcaga gaatcgaaga ctggcttcat tcaggtcctc caattgccaa atggagatta
9181   tgcaacgttt cttgaataca tacaaaactc tagatgtggc cagctcagtc ttcttccaat
9241   aatgtaaagc caaacaatgc tttgcaggaa tagactagag attatatttt gggattaata
9301   acatagggat taaaatctta tcttgaacta actaaacatt attgatatgc taaattcact
9361   tttttttttt tttttttttt ttttttttag acagagtctt tctctgttgc caggctggcg
9421   tgcagtggcg tgatctcggc tcactgtaac ctctgtgtcc cgggttcaag tgattctcct
9481   gcctcagcct ctcgagtagc tgggactgca ggagtacgcc accacgccca gctaattttt
9541   gtatttttag tagagacagg gtttcatcat gttggccagg atggtcttga tctattgacc
9601   tcatgatccc cccgcctcgg cgtcccaaag tgctgagatt acaggcgtga gcaacggcca
9661   ccggcccact actttttaat atatcattaa tttctctttt aaaaacagta gcaatcaata
9721   atttaaatat tcaaatgaat tcttaattta tatacacaaa ctaacatctt tattatatct
9781   ctatatttta atatatcaac atgtctaaga taatttataa atttacatca tatataaaaa
9841   tgggtttgct ctcgatgtat ataaggcttc atgatatttt gaatatggag ttgggtgaaa
9901   atagtgaatc tgaatatttg aatttgaata tttattggaa aataagtagt gcttttaact
9961   ttttaaatga gacacataat agtcccctgt tgattttttt ttattttttt aactttatta
10021  aagtatagtt gacaattaaa aattgtttat atttaaggta tacaattgat gatttgacat
10081  gtgtacattg tgaattattc accacaatca agctaattaa cattccctgt tagtttttat
10141  aagcctggtt caggtttgta gaaagaaaca aacacacatg gccaggcacg gtggctcaca
10201  cctataatcc cagactttgg gaggccaagg caggaggatc acttgaaccc agaagtttag
10261  accagcctag gcaaaatagc aaaccctgtc tctccaaaaa agaatgaaaa aattagcctg
10321  gtgtggtggc atgtacctgt atccctagca actcaggagg ctgaggcagg aggattgctc
10381  acttgagccc aggagtttga ggtttcagtg agctatgatt gcaccattgc attccagcct
10441  gggtgacaca gcaagaccct gtctctaaaa gcaggcaaca aaaacacatg agcttcacta
10501  cagggaatta aatacaatga gagtaataaa aaataggtga gcaaaaaaaatgcaaaataag
10561  caaacttttg agtatgatat ttcattctta tcttgatttc tgtttttaac tccagattga
10621  ttcttaaaat gctaacagtt cataatgcca gtgtaaactt gtcagtgatt ggactgaaga
10681  ccttagatct cctcctaact tcaggtaata tgtgtatatg ttttttgtgt tgattcaaat
10741  taaaaaaaaa gttgatacca ttaagtaaat gtgtgtgtgt gtgttttttt tttttttttt
10801  ttgaagatca ggattagggt agcttgattt aaatgtccta aaattgcatc tgtttttaga
10861  cctagtgatg ggacagccat aatataatct aaatatcagt tatttccaaa attctttctg
10921  tttccatctc ttctccttat ctcttttctc tatactttgc ctctcaaaaa tctcattcaa
10981  tacattggtt ttaaacatta ccttatatat tatccccaaa tctctgttgg tagtccgatg
11041  tttgctccca aaatctggac ctacatctca tattccccca ggttagtggt cattcctgcc
11101  cttgccatta ttacttcttt ctccctatat atctttaata aattttctat aatatatgtt
11161  ctggagtatg ccataatcgt tacattttga aaacacatag tattacttct tgagtatttg
11221  ctagatgcca ggcttcacaa gtaaaatgct tcacgtgctt ttaaacacct gaatctgaaa
11281  acaccccttg agatagggat tttatctcag ttttctaagt gacaaacact gaagtgcaga
11341  gaagttgctt tggccactaa gaggtagaaa cagggtctga ttctccatgt caggttcctt
11401  ccctgagaaa actttggcct ggtagataat ggacctgaaa acaaaaaatc ttgaaatgat
11461  gcaacagttg tgggcattgc tgtgctggac actggctatt atataaggtt ccgagaagaa
11521  aggccgctca cagggagcta atcttgaagg gctgggagga gtttccttcc atgtagggga
11581  gggcttttta ggttgagaga agtatgggtg cagaggcctg ggaggatagc atgagagagg
11641  ctggacgtgt gattgggaag gtttgaattg tcctcatcag tcctgctaag agatgtaaag
11701  accatgctgg agaaagagga agatgagagt atgagggaaa aacaagaagg tactcaacat
11761  ttcactacag ctttttatga ccatgttgta tggcatgcac taagagtctt taaccatgat
11821  ttaatttaac ctcacccttg ggaggtattt tttttttgac ggagattcat tctccatttg
11881  gcgatggaca ggaagatgag ggtttattaa tatgaaaaat ctaccaacac tggaatatat
11941  tgaagttagc ctcatacagt actactactc ctattccagt attattattt ttattgacag
12001  aatagatgct gtttgtgtta agttttggat tatgatagga aatgtttggt atagtaaaag
12061  gcaagagtgt gacatgcagt tagtccaagt acgaagagat accaaaaaaa aaatgtttag
12121  tgaggagcag agtttagcat atttggagtg aagacaatgt gcggaaggaa aggagctgat
12181  gagatacatg tactatagtc ggttgtgtga aaggtcttgt ttttcatact aaggatcatg
12241  agaagatctt agtagattcc agcaagggat ttgcaagacc acatttgtgt tttagaaata
12301  taatgcaggc aataaaccag ttggatagaa attggggact gtagagcaat taagcaactg
12361  tttttgcatt ctagatgaga atgcaaaaca caataggaat gaaggtgcct tgttcaaaag
12421  gagttttgtt caaaaggaat cttcaagatg tgtaggagat attcttaaag gacttggtaa
12481  tgaattgatt tgttgcttgg atagagaatg agaagagaaa ggagggtgga agaaggaaga
12541  tgacttagga gtttctcttg ggtagctagt ggattatggt atcattgatg aagacaggga
12601  acaggagtag gccaggtttg gggtaactgt ggaatattca gatgttgtct aagaggcata
12661  agaatgtatt tcagatgttt ggggcaagtt gtctaggcta gaagtactga ttgagactca
12721  tgaaattata gtaaagtgaa ctgggagttt atctcattta taaagatcta gagcttgata
12781  agtctaacat ctagggcagt taagtagttt atcaacaaac aaacaaacaa acaaacaaga
12841  aaaccatggg tctacaaacc attcacagtc ttcatgtaaa aattaattca tgtaaaaatt
12901  aacacattaa atgttaaagc agctctttac tcagagcata ttattctctt taaaataggt
12961  aaaatcacct tgctgatatt ggatgaagaa agtgatattt tcatgttaat ttttgatgcc
13021  atgcactcat ttccagccaa tgatgaagtc cagaaacttg gatgcaaagc tttacatgtg
13081  ctgtttgaga gaggtatttt aaaatgtcaa attccttaaa gtatatataa gaaaaaaagg
13141  cttatactgg gaaaagtaga acacagttat aataagaaga aggtttctaa aatcctacta
13201  tttattaaga agtgggagtt gtctgtcaag ggtgaggaat ggggttaatt cagaagtatt
13261  gcttgttttg gtggggtgaa tttcattcgt gggttataaa tcatgcccct ggagtagact
13321  ttcttcaatt gcttaacaag gcataaggtt tactttgaaa actggatgtg tgggtgctat
13381  gaaagaaaaa ataaaactgt gaagccaagc ataggttaca ctgggattat gatgttgagt
13441  catcaccaga aatcatagaa attgcataaa gagcctgaag gtttacaaag tgtccttcag
13501  gaaaaagact aatatgcatt tcatagcctg gccctgagat tgataactga gattattatg
13561  taattttaga gttggttgga gtccttgttt agtctttcca ttgaccttag gaggaagtgg
13621  gtcacagcag tgaagtgagc atcctgcctg aggacacaga gcttgtgaca gtacagttca
13681  attagcaatt attttaagag ccccttttgt atcattatga gagccaactg tgctaggggt
13741  ttagataaga atgatttatg tgggccctgt gtcagttatc agtttaccag tctaatttct
13801  tgcagttccc agaatgggat agatcacctg ataactgttg aattccctgt ctcctcccag
13861  aaggatttta aacagcttat agataattat aatacacaag agtaaacaaa atggatgaga
13921  aaataggtga agggacaata atataaagct agattaagtt tactgtgttt ctaaggtcct
13981  gcatatttac aaaggggtgg gccagaaatt tgtctgtttg cttcctatct gacaaagaaa
14041  agaggtaaat atcagtggtt acaaagttcc ttaagataaa agtaaaccta ttattcagga
14101  gaagcaattg gtctcatggg agatctgaga aacatcttcc catgggtttt cctggatgag
14161  acaataaagg acatacattt tgcaaggaat acaaagtgta ttgcagcgag agtgactctg
14221  tcaaaagtca gaatagcatg ggcctggtac ccagctcttt gataatcata caccgtgaag
14281  tagaagatag tttacagcga gtacggaatt ccttcaggct gtcatgtata aatgttctat
14341  cttgcaacta agctttcgat gacaattagg ataaagtttg aggttctatt gtcttgcagg
14401  gtctgtaatc ttctgtgtgg aaggttaggg gcacattctt cttcctggaa ggagggctag
14461  catcacttta tcaccatcgt tgtttagtcc atctaagaca ctggaggtag accatagaat
14521  gttacaaaga agaatgttgc tcaatagaaa aaccatcagt gctgagaggg ttatgactat
14581  aaatgtagag tagaaaaatt tctgattttt ccaggagtat caggttctcc aggactcagg
14641  ggtgactata aagttaattt tcaaaatttg aaagtgtact gtggaaacta gaccataaag
14701  tgagaaagtt ccatgatatt cttcacttgt taggaaaact taactgattt cacattatat
14761  tatagggaca ctctggcata aaattaaaaa aaatgaatgt tgatcactta gagtgctgtg
14821  ttttctaaca tatttctggc gccattctca agctagataa actatazttt tatacatgtt
14881  tttcaggttg ttgcccaata acaatgactc caaatggaac ttactggctt gatcaaatga
14941  ctttaattgt gaaaattaat gatttatatt tttgctgtct gatggaaaac cactaagaca
15001  gagtatttca aagtctgatt acttgccatt tgctcaagtt gacaactctt gaactgaaac
15061  atttagccga gctgcccttc agcagcctac cattaatgcc tcccttttaa atattgcaat
15121  atgtccagtt ccagttggcc atctttatta gtcactgtca gttttctcta gaatttccca
15181  aatgaaattg taaataattt tgtttttctg agaactgctt gctgactagc acttttacat
15241  ttcaaaacat ggagtaccta acataggccg aaacaaaatt atttgaatct ccgtagcttg
15301  ttttctcatt ataacattct taggaagggc tgcttcacag aaatatattt tttatttaag
15361  gagattacac ttgatgtatc tcacacaact ataatgaata ttgtaatttt tgaataatta
15421  aactttcata tcatctttaa gcttattcag tattttgtct ttcattttta agtctcagag
15481  gagcaactga ctgaatttgt tgagaacaaa gattatatga tattgttaag tgcgttaaca
15541  aattttaaag atgaagagga aattgtgctt catgtgctgc attgtttaca ttccctagcg
15601  attccttgta agtagcattt aaatgttatt tattttttgt atctgaaaaa ttacaatata
15661  tctcattctg agtatatttt aacaatattt ttattattta gaaacttgtg gatgctcaac
15721  ccattcattc atttattcat ttaattaatt tacattcact gacattatac tgaagttggc
15781  tgtgggcttg gtgctggaga aacaatcgtg gaaaatacag atgtgttcct taccttttca
15841  gagcttgtag tacaatgggg gacacagata agtacagagg tgattacagt ggcagaagtg
15901  atggcagatg gcagaagtac ctagagttag gagatcaaat aggaagtgag gcagtgtctc
15961  ttagcaaaga tttaataagt ggagcttcct gtgcatgaag gtgtgacctg aagtgagaat
16021  gcaggcaaag tggcccaggc agtgggcatg gttaatgtaa agatgctgga gcaagagaga
16081  gcagactgcc ttcaagaaga caaaagtagc tcagtaaagg tgtggggtta tgagtgtgcg
16141  tgcatgcatg cgtgtgtgcc gctgtgcatg cacatcccca aatatcctat ccgtttgtgt
16201  ttcattgaca gaggcaaggg agagcttgat aagaggcagt aaatgaggcc agagacatgg
16261  agtggagagc atgaagggcc taaaaagcca catgaaggag tttgaatttt attgtgactc
16321  ttgattagca ttttaatgag gctttgaaat ttagccacat ttttcaccaa aaatattaat
16381  cagaagaaat taatttgatg tgtatgctac caatgattgc tattaggcta aaataatggt
16441  tcatattctg ttttgttttg tattaatggt tcatattctg ttttgttttg taagtgacca
16501  ttaacacttt gtattttatg tattacttgt gtgggtttct acaggatata catatgcatt
16561  tatctagtga tattttcatc ctcacacatg tgaagttttg aggattagag ttaaacaatg
16621  tacctggtat gtaataagtg ttctaaaatc actgacagga ttattagaca atatgtattt
16681  tatatgtgtg ttgtatacta tatgtaattg catttatggt ttcagatatg gaaatcactg
16741  tgtcaatctg aaggtgtgag ccttcggtgt aggcagagta aaacccaatg cccttgtgaa
16801  agaatgcttt tttttggtga tgtttataaa atcacaatgt tttcttatcc acaggaaatt
16861  aaacactgga aagtgggtgg ggctgaacaa taatagagaa aggccatggt tttacatttc
16921  tctgagacat cactgccaac aaactgaata tgtttttcat tatacttttt ccttggctat
16981  atttattcat ttatttattt attttgggct ggaggttttt ggaatccatt gttttccacc
17041  cacattggac ataactccag taaaaatgtg ttgattcata atgcaaaagt caagaaagta
17101  gcagctaaaa attaagaaat caaaagtttt taaaacactg attctaactg aaaaacattt
17161  gcttttcagt ctttaagtct attgttctga gtcaaagcag ttcatttcct tacgttgtta
17221  attttttttc tatgtttaag cattgtaata tactttttgt gaaaacagtt gattagtttt
17281  ggttgtgcca aaacaaatac taaaatgttt tgcaaacagc ctttttttaa acaaaaaaag
17341  aacagttaac atttgatgca gagatataca tgttttctcc atgtaggttc acacctcact
17401  tcctttattg attaattgct ttttctggta gagtctttct ttcctttctg ttttacctgt
17461  gtttgtccct aagacttata ttttaatatt atgcctcctc tctttcgttc tcccatcttt
17521  tcttccacct attttggagc cttcaggaag cttgattttg ctgccttgta cattggttgc
17581  ccttctggaa tggaggaaac aggtcatagc tgattttaac tgttccatct ggtgacatat
17641  tcttgatttt ctttcttttg gttggggaaa aaaaaacaat gcaaaagtca ttctccaatg
17701  gggttgagcc tcgttaagaa atagaccctc cacaatggtt gaactagttt acagtcccac
17761  caacagtgta aaagtgttcc tatttctcca catcaaaaaa aaaaaaaggt aagcaatata
17821  acatgagcca tatctaatag gacttcagaa attatctatc ctatagttcc aggatgacga
17881  tgatgatggt tgtgataatg atgaagattg tgatgatagt tatatgggag ataaaacttt
17941  aagcacttta catattaaat tctataatat tcaccacatc tattaaaata tgttacatta
18001  ttgtccctat tttaccaaca agaaaactga ccaacaagat taaacaacgt gactaagttc
18061  acacaacctg taacagcaga atctatgtca atcacaacac aattagcagc tatttctgtg
18121  gcaattttca ataaagatgt gtctggaaaa aaaaaaaaga aatagaccct ccaatttatt
18181  tatctgaaaa cttatgacca atacattaca tttccagact ttcattttca gtacttttcc
18241  tttcattttc agtactaaaa gtactctgaa tttttccttt ttttgatctt aaggctttaa
18301  gccaagaaac aggaataaag taaattttcc ttaatgccaa agattagtcc tacaccccat
18361  tatgttatta atgaacagca tagtattttt tacagctact taaagaacat gatgtttaaa
18421  tttggaaatg cagtcattat gctgccatct atttacagtc tatataagac gtctttgtat
18481  gcatatttga aaggagaaca tggttacctt attgataatt atgatctctt taaattcagg
18541  caataatgtg gaagtcctca tgagtggcaa tgtcaggtgt tataatattg tggtggaagc
18601  tatgaaagca ttccctatga gtgaaagaat tcaagaagtg agttgctgtt tgctccatag
18661  gcttacatta ggtgagtttc ttagttaata tgtcatcaca cactgtatga tatacatata
18721  catataaaac atatatatgt tgcataataa tggataagta gcatattgac atactttgaa
18781  tgaaaatatt gtaaaatccc agaaaaaata aattaaaaca aaaagaaaat actgtaaatt
18841  accaaactgt tctgctgtgc ttagatggac ttttaaaagg agtgtcaaaa atagatgtgt
18901  agaatgtaaa agaagtatta tcttaatctt atttttatag atgtagttct atagatgagt
18961  ttttttattg ttgaggctat atttaaaata taattatgta agaattgata catacaaaaa
19021  tatgcataac atatacaata taaagcataa tgcaaataac tcactatcca acttaaatgt
19081  tgtatattcc cagtggggga agctaccctg ggcttcttcc tgcccttgct ttctctatag
19141  aggttaacac tatccagaat tttgtgttta caaatctttt gtttataaat atgatttact
19201  acattttcat gtttctctaa gcaaaattgt taatgtttgc ctgcttttgc ttcatcaaaa
19261  tgtaatcata ctgtatgttg tcttctgcaa ttttcaaata tcagtgctat gattataaga
19321  atcagaaata tttttgcatg tggttgcatg tagtattcta ttatttggaa ataccacaat
19381  ttatttttcc atttttctat ccatggacat ttggattctt tcttatttta tgctactact
19441  atctgtgtta aaacagataa ctgaaaaaga acagttaaca tttggtgcag agatatacat
19501  attttctcca tatataaata agggttaata ttttaaaaaa tatttatttg ccattggtat
19561  gtccttcttt gtgaaatatg caatctgtca tttatcaata ttgttcacag gatagtctgt
19621  ctttttaaaa ccgattcata gattctggat aataatgttt tggtggtagg ttatacgtat
19681  tgaaaatacc ttcccttgaa catcacactc tggggactgt tgtggggtgg ggggaggggg
19741  gagggatagc attgggagat atacctaatg ctagatgacg agttagtggg tgcagcgcac
19801  caacatggca catgtataca tatgtaacta acctgaacat tgtacacatg taccctaaaa
19861  cttaaagtat aataataatt ttaaaaaaaa ggagatgaag aggtagctgc aggttgactg
19921  agcaagggtc attgtctatt tgaagtttca aaggtatctc tgaaaataaa cacagttttt
19981  tgcaagagtg aaaaaaaaaa aaaaaagaaa ataccttccc ttgacttgta gcatgtcttt
20041  tcaccgtctt tatggtggct tttgtgatat agttaaattt aataatcagt tcctttgtga
20101  tttactcttt tttatgtatc ttgtttaaga aatcttaatc tgtcctttcc ctaagataat
20161  aaatatattg tatattttat tccaaatctt actcgtttgt taaactgttg cagtttgttt
20221  ttgtaaagaa cccaattccc cctctttttt tcagtgtgga gagctagtta ccatcacagc
20281  acaatttatt agaggttcac ctgtttccca gtcatgtggt atataaatat gtaaacatat
20341  atgcttatgt ttctgtcctt ggcgttctgt tgcattaatc tgatttgtcc gaatcagatt
20401  taaatctgat gttaaatata acactattta aattaacagt tctgtaagtc ctgatatctg
20461  aaaagctaat aggtcaagtc accatacatt cttttttagg cacagcttgc cttttttctt
20521  gggcctttac tattccatgc aaattacagg attaacttgt ccagttcctt ggaaaacact
20581  gttgagattt tgactggaat tgcacaaaat atgtggatca ttttaagaag aactgacatc
20641  tttacattat aatttcttct atacatgtct tgcacatctt agtttttgct acgtatctta
20701  ttttttatta ttattgtaaa tggtatttct ttttaaatca tatttttgaa ctgtgttttc
20761  ataaagaaat gcaaatgatt ttttgtatat tgaactaacc tacctttcta aactcttcgt
20821  taattctgac aatttgtttc taaatgcact taagttctct acctagcaat tatataattt
20881  gtaaataatg acagtctgag ttcttccttt ctgcttctta tattctttat ttcatttcct
20941  tgtcttctta catgagtcag aattattaat atagtgttaa atagagccat tgatactaga
21001  catccttgtc ttgctctttt tcctgatttt aaacagaata tttttaatat tctcccatcc
21061  aaaataatgc atcttacaag ggtttaaact tttaaaaaaa attttgttaa gaaattttat
21121  tttatttctt ctttgctttt gttttttcaa tgtgggcttt taaatgtttg cttttatttt
21181  tacaatgtgg gcttaaaaat attaaaatat ttaattttat caaatatact taaaatgtag
21241  taagtctttt ttttcctttt cattatgtta atatgaagaa atacatctac aggttttcta
21301  atactaagct actgtgcact tcctgataaa tttaacttgg tcatttatta gatttttaaa
21361  aacacttcta aaaaatcttc tctgtttatt ttcacatata ccaggtgagt tttggcagcc
21421  tcttgttttt tcttttttac cctttctttt ttgttaaaaa catcctttta tttatttaat
21481  gattaataca tggctatttt gtgttttgta tctgataact atatataaag ttctggggag
21541  tctaaaccaa ctgcttctag gctgactgcc tgtcagttgc ggagccttgt tttcttgtat
21601  ggttgtgagt ttctctttta ttgagcatcc tgaggactta aattaaagat gctctttcag
21661  aggtgtttgc caggagtcag agcacaggac tgacctggga gcagtttagg atatgatcca
21721  ggcttaatat gggagactct ggttgagacc ttaccttgca gagagtctga aactggtttg
21781  ttgaatgcag cgccaggatt catgctttcc cacaagacta ctctggcgtt caactcacag
21841  ctcttgtttc agcttctttt tgaactccct ctgcccctca acacacacct tggaaatttc
21901  cttgatattt ttgtgaggac aacaatgcat ttaaaagtga gagtggttgc tgaataataa
21961  ggaatgatcg ttaactgttg gacattgatt ctgatgacat tttctcaaaa ggataaccag
22021  gaatattttg tacaaaatta ggattattat aataggcatt tagtttttca ttgttacaaa
22081  ttttgaggaa aatcattaat catttgaaaa aactaattga catgtctcca ttgtagcaac
22141  ttgtattttc acttctagta tcatgattat atcccctgtg taatttggaa attgatttta
22201  gcattaggaa atttcctagt ttcagttaaa atgaattttt tgtaagctga attctatttt
22261  acatgcacaa ctttagtttg ttatttcttt ccttgacaag catttattga atgttgttat
22321  atgactaaaa ctgtaagatg ataatgtttt aatattttca catttctttc atagcttcca
22381  aagtgtatat atatactcac atacttcata tatatgaatc tacttatata ctgtatataa
22441  aaatatgtat ataaatatat acacattgta tataaatgtg tatatatatt tacacatgta
22501  tataaatata catacaaatg tatataaata tacatattta tacacagatg tatataaata
22561  tacatattta tacacagatg tatataaata tacatattta tacacagatg tatataaata
22621  tacatattta tacacagatg tatataaata tacaaatata aatatataca tttatatata
22681  aatatacata tttatacaca gatgtatata aatatacaaa tataaatata tacatttata
22741  tataaatata catatttata cacagatgta tataaatata caaatataaa tatatacatt
22801  tatatataaa tatatatatt ttttgatacg tacaactatc ttgggagatg ggatattgtt
22861  attatttcca catttacaga tgaggtcctg tggttcatca atctttgtgt tttattcaag
22921  attatgtaag cagtaaggga tgaaatcagg ctgagaaccc aaatttcttc atccctaaac
22981  aaagtatttc ttctaaacat ggtatccatt tactagttta tctctatcag gtgacccttt
23041  attcattatt tttcatgaga aggttgtagt tgtacaaagt ggctgatatc tgataatgtt
23101  ttaatctaat tcaaagtcga tttcttaaat ccaggtgtca gagtagcaca ctactgtaca
23161  attctctgtt tcatgtttta caatttacca cagtcaagtt acaacttgca catgttacat
23221  taaaatgtga attcacctta atttcttgaa atgagccaga agaagaagtg gttttgtttt
23281  gtgatcaggg aaatgctact tgactgccaa attgtctaga acagcacatt aaagttgctt
23341  gattttatac tgttaaaatt aaataaaaca tggcaactgt catgtcatat gtaacttttg
23401  tattattctg taactttttt tgaaaataaa aagtgatcaa attgatcttc aggtaaagaa
23461  tcttttttct cttgattatc tcttagtgga tgatgatttg ttccatttaa tgggtagaga
23521  atttattgtt gctgttattg tttcaaaagt agctgaatga aactcttaac tttttcttca
23581  tagttaaaat taaaacctca agtaaataaa tatttaacgt tttgccaaac tatgtaatct
23641  aattatgggc ttaatgcatt taaaggcttt gtataatttg ctacgtattt tcacacaagt
23701  tacctgaaca taagtccagt cttcctgctg ttttctgagt cacacagtga ttcagtgacc
23761  gaacagctgt tggcagtggt gtgggtataa tagggaaaag agactgatgg ggacaaccca
23821  agtttagaca agctggtaaa agtagaagaa aatcttcttg aaaacactag tgatcactaa
23881  gggctgtgga gaatttttgc ttggtgggtg aatgtggaag aggcaacagc atgggaaggt
23941  gttggtaaag gagctccata cttgcttaaa ctgccttttg attgtgaggc cgttgatgaa
24001  tatttagttt gggctttagg ttttttatga tacaggatat tttccatttc tggctttgta
24061  tctcagagat cacttagtta cacttataga tgaataggag tttcaattcc ttgttttaga
24121  aagaagcttg gtaactgtta gtgagttaca aataagccaa atagaagaag gtacatattt
24181  ctgcagtatc aggtaaagtt tttcctcata aggatttaga ctcttggata tcatattaat
24241  tctcagaaga gtgggtataa aaaggtatgg gacttcttcc tggggtgggt tggaggtggt
24301  gaaatacctt tttttttttt tttctgagat catcatagac aagatcaaat aatggtaaac
24361  atgccaatga attttctaag cactattcct ttaagtgaaa gaagagtgtt tcagtaaaat
24421  gatttaatat tgggtcttcc aaaagatgga tttaagagtt tcaactttaa aagacagaaa
24481  aattaagtta ttttacacaa tgaatattgt cgtgccgtgt gtcacagaca tgacatgaga
24541  gggaatcaga gaacatacag ttaatacaac gcaaactagt atcattactt ttgctcaatc
24601  acttccattg tctaagtaag ataattaaag gacagcataa aataaaattt caaaacttta
24661  ctcaatcata ttaagctatt ttaattaaag taaatgtttt aatgccattg aatattcatc
24721  accattcaaa attattgatg taaatagtgt tatatgttaa aggtaattta acttccatgg
24781  atgagaattc agctaatgtt tcacttaact tttaggtaat tttttcaata tcctggtatt
24841  aaacgaagtc catgagtttg tggtgaaagc tgtgcagcag tacccagaga atgcagcatt
24901  gcagatctca gcgctcagct gtttggccct cctcagtaag taacttcact aaaaagggga
24961  ttcttacaga ggcatttgac atcaaatatg aacattgtaa caagagaatc atatgtacag
25021  atggaagcat tcaatgcctt ttctgtcctg tgtagctcat tttccagtag aggatacttt
25081  caaggaaact aacagttgtg acaaatatac acatctcaat gtagagtttt gctttacatc
25141  attcttgatt tagctttgtc attaagcagc taatctgttt taaaaaaatt tttatttgtg
25201  cctgggcatg gtggctcacg cctgtaatct cagaactttg ggaggccgag gcgggtggat
25261  cacaaggtca ggagttcgag accagcctgg ccaacatggt gaaaccccat ctctactaaa
25321  aatataaaaa ttagtctggc atggtggcgg gcacctataa tcccagctgc tcgggaggcc
25381  gaggcaggag tatcgcttgg aactggaggg taggagttga agtgagctga gattgtgcca
25441  ctgcactcca gcctgggcaa caagaatgaa actccatctc aaaaaaaata atttatttgt
25501  gttttaagtt ctggtgtaca cgtgcaggat gtgcaggttt ttcacatagg tagacgtgtg
25561  ccatggtggt ttgctatacc tattaaccca tcacctaggt attaagccca gcatgcatta
25621  gctatttttc ctaatgctct cccttctcca gtcccatccc ctaacaggcc ccagtgtgta
25681  ttgtccctct ccctgtgttc atgtgttctc attgttcagc tcccatttat aagtgagaac
25741  acgcagtgtg tggttttctc ttcatgtgtt agttatctga ggataatggt ttccagctcc
25801  atccgtgtcc ctgcaaagga catgagataa ttccttttta tggctgcata gtattccatg
25861  gtatatatgt accacatttt ctttatctag tctatcattg atgggcattt gggttgattc
25921  catgtctttg ctcttgtgaa tagtgctgtg atgaactaat cttttcaaat aatcctcctc
25981  tcgtctatta ggtttttttt ttttttggta ccttcttcct cattttatta tttatctgga
26041  taggatggta gcattatgag acgtataata tattaaaaat tatctttata attgaccaag
26101  gcttctccta aagcacacct cattctgttg gtaatatttc aaaatatgga cttagagttg
26161  gtcaaactgt taagtagata atatatataa tgtttttata tatttttcaa tttttttcaa
26221  gctgagacta ttttcttaaa tcaagattta gaggaaaaga atgagaatca agagaatgat
26281  gatgaggggg aagaagataa attgttttgg ctggaagcct gttacaaagc attaacgtgg
26341  catagaaaga acaagcacgt gcaggtagga ctctcataaa tattagagtt attcaaaatt
26401  atgttttcca gtcatttata ttttgacaga tttctttttt ctcccctaat ccaggaggcc
26461  gcatgctggg cactaaataa tctccttatg taccaaaaca gtttacatga gaagattgga
26521  gatgaagatg gccagttagt agttttgatt ttatatgata gaaaatttca gttatatttt
26581  aaatcaatac ctataaaata ccttaaccgt aacttttatt gttagaaata tttttgatat
26641  aggcatttag ttttagatgt tgctgcaaaa tagtagtagg tatgtagtat tttgatctca
26701  tcaccttcag gagttagaaa aggtagaatg agagttatta ttgagagatt tggaatcaag
26761  ggtcatttgg taattcatga atcatggaga aagaatcttt ctattttctg gctgatcgtt
26821  ttaaaatgcc atattaattc atcttgggtg atagaaattg cagagccatc tgtgatcttt
26881  tcttctatgg tgactagcca gcaggttgtc aatatcagaa attagatttg gttagagagc
26941  ttcctatgat gagttcccaa ctgatgtgac agagttgacc tgtcttcttt cgagagggtt
27001  atttgaagct gtcatctctg gataactctt tcaataggag tgccattcaa acatcataag
27061  accggcactc tctcccaaag atacaagctg tagcaaggag ttttgtgcat atcaggtttg
27121  tttcatatcc gtgagccttt gtgttttatg gcaactgatt gattatactt gtgctatttg
27181  caatgggtat cctctgggtt ttaaatagtg aatgacttat tttggaaaca aatagtagta
27241  ttctatgtct gaaatcttga ccgtctattt gtttaattat ctattgctga taagaaggga
27301  attaataaca cagatgctac ttaattaaat attttcattt tgacaagaaa cagtaattct
27361  tttgaaaact atgctaaatt ggcatcttaa taatctcatg ttgagcaagg cttttggaga
27421  ttagggtaag ggagattcat gtcgcagttt ataaatttca gttcatagga tacctatttt
27481  tcaatatcca taagataact ttaaaataaa tatattatta aaacaaagaa aatatattta
27541  cgttatacat ctttaaaacc taccttgctt ctttacaatt ttcagttttt cctctcttgt
27601  ttccattctc ttctcctcac tttacctttt tcctcagcat ctctttacat atctgctgag
27661  cttttttatt tctctctgct gcatatgctt ttgaaaaagc ataaaaatac taatttgtta
27721  ggatttaatt aattcgagtc ttaaaaaatg aactataatt cactcttgta agtggaggtg
27781  gcatgaaata ttgtttatat gctcttaatt gttgttagag atatttgata atggcaagtg
27841  agaatttgag atagttattt aaaagattac tactaacatt ttgtttgaat ttttgaaagt
27901  ttcccagctc atagggaagt gatgctctcc atgctgatgc attcttcatc aaaggaagtt
27961  ttccaggcat ctgcgaatgc attgtcaact ctcttagaac aaaatggtaa gcagtgggcc
28021  atgttttcaa ataaagggaa acacattttt gtggtatttt taattataga agctatatac
28081  tgtgaaaaat ttacataatt tataaagcta tatattgtga aagatatctc tatgtgtaga
28141  gatgtattga catatggatt atgaatatat aggtaaaagg atgaagaata aaataaacat
28201  ttgttgtata tttttccgga cttctatgtg taaactcaca catgacatat acaaaatttt
28261  atgtattttg tagaaatggg atcatattat actcttttat aaccaaattt tatttattct
28321  cttttttatg ttgatacatg tgtattctca cattatcatt ttatttttaa ttttttaaat
28381  taatttttgt ttttagatat ggtctcactc tgtcacctag gctggagtgc agtaccatga
28441  tcatggctca ctacaacctc aaacttttgg actcaggtga tgctcccacc tcagcttcct
28501  gagtatctgg gactacaggc atgcactgcc atacctggct aattttttgc agagatggtg
28561  ttttgccata ttgcccaggc tggtctcgaa ttcctgggct caagcaatcc atctgccttg
28621  gcctcccaag tgctgggatt acaggcatga accactgtgc ccggcccaaa ttataatttt
28681  taatagcttt gtagtttttc agtgcttgcc tgtattctgt tttatgaacc aatttcttac
28741  tgatgaagtt ttagtttgtg tccagtggga tgtttctgga acccatagta aacaccagtg
28801  atcactttcc tcctctgttt tcctttatac atggttccta ttattttctt gggaaaattt
28861  catagaaatg caactgggta gagagctatt caccatttta aaatctggta aaattgtcta
28921  ttataaattt ccacactata cttttttaaa aaatcgttct ttaatgtaat tcttataaat
28981  cttatagctt tgtataatta tgaagagaaa aatggcttgt atccctttag aaagacatga
29041  gttttaagat tatggttcag gctgctcaaa tttcttcccc cataaacagg aatactgcca
29101  gaaatcctta ggtgaaaact catcataaag gcattgggac ttggcagctt ttgcaggaca
29161  tttttagagg gcaaadaata gagaaaaaca ctgaaagtca gagacagaga ccagtgtaag
29221  catcagcttt taatagagaa actgggtagg gtggaaaaaa aataaagcaa ccacctcatg
29281  catgtttctt tatattatta ttgaagtcaa ataaagagga aaatcatttc ttcttcctct
29341  tcctcctttt tcatctcacc tctccaatgg cactttaata aaacgcttgg agtggccagg
29401  gcactgacag acagacaggg gctgctctca aggataatga gtcaaagggg aaggagaggg
29461  aatcgctgtt ctcgaatctc tcttattcta ctgtgcagtt aaagaggtct ggacagggat
29521  ttcactcctg aaaatgagga ctggactttg tggcttctgt tggggcacct ttagagtgga
29581  ggtagacttt tactatgtac agacaacatt gtgttggtga catcattcat aaccacctgg
29641  aaatctcctt tgatatgcaa atcaaacaac cataactttg tgaaatttcg actgcttcct
29701  attgtggtgt ctgaggactg gttacattca gagtccaccc tgatgtcttt gttcagtttt
29761  ctgctctttc tagttcctta cttctttgtt cctgatcacc tgtcaagtaa aatgtcctca
29821  gatccttttg tattgtcttt ggagttctgc cttaataaag catgaagaac ttgagtagct
29881  cgttcatcaa ctttcttggg caatttctca ttgaaagaca cttgggtgtc tttgggtgtg
29941  cagagctgag catggcttta tgtttttaga aaaaatggct acattggcag gcagaagaac
30001  tgcgtccttg gaatcatgga ggtcccaagg ttgcatacat tttgtgtgac atttttccta
30061  ttcaattaat taactaatat ttattgagct cccaagtgtg tagtgtctgt aggcacttgg
30121  gatgcattca ttaagtaaaa atcccaggct catggagttt aaactgtagt agggaagata
30181  ataagattaa ctaaaatatg taatatttga ggcagttaag aataaaaaat gaagcaagga
30241  aggagaatat gatatgttag acatcagagg agatgaagtt gtgaataggc agccaggaaa
30301  gaaggtgact attgagtaag acctgcaggg cgtcgcatat tgttttttgc ctctgaaagc
30361  agttaatttc ctgttaaaat ggagtggatg agatcaagag tattacgtag atagctggta
30421  aatgcgatag tgtgtaaaat gttctataaa gtctaacgtg actttatgat gaaatttctt
30481  cttctaggtt tattgcttgc aattttcaaa ccacacattg ggttactgtc taggatagtg
30541  attcttaaag tgtggttcct ggaccagcag catttgctgg gcgaacttga taaacagtgt
30601  aaattcaagg accccataca gaccttctca atcacaaacc ctggagttga gacccagcaa
30661  tccatgtttt aacaagctct ccaggtgatt ctgatgcaca ctaaagtttg agaaccacta
30721  acccagtgtc atttttgtct tttaaagtgt cttcttggct agaagctagc cactttggga
30781  aaggttatta caacttctga tgtgatcaag caaagtaacc aactctttat tgtatcttaa
30841  tatgtgatat tctgaatgtg tttaaaaggt atgagttttt caggctctgg cagtatttta
30901  gaatgtgtat gtgatttcta tttatttcca tgttttgtcc tatcttctta agatagacta
30961  cttattttaa aagcagtact taagttaaaa ctttttatgt ttctttttct gccactttca
31021  aagtgttgaa tcacagtgtg taatgttgga actgatattt ttatagcggc ttcaagacaa
31081  ttgatattta tgtggaaact tgaagacagt aggtttatgt ttagtgaagg aagtttatta
31141  caaagaggaa aattggccag ttgtggtggc tcacgcctgt aatcccagca ctttgggagg
31201  ccaaggcagg aggattgctt gagctcagga gttcaagacc aggctgggca acatagtgag
31261  atcccctctc tacaaaatat taacaaaaat tagccaggca tggtggcaca cttgtagtcc
31321  ctggtacttg gaggctgaga caggaggatc acttgaggcc aggaggttaa gactgcagtg
31381  agctatgatc atgctactgc actccagcct tggcaacaga gagagatgct gtcacaaaaa
31441  gaaacaccaa caaaaaaaga ggaaaattat tccttaatca ttattgctgg aatatagtta
31501  ctttccacaa atagtgaagt gccagttgta aagcatatct atatgtttcc tagactttgg
31561  cattactttg tgaaaataac tgtaattact tatgttctat gtaaatgctt tccattcatt
31621  tgtatatgat ggcatatata gaaattataa tgtttgtaaa gtccactggg ataaatggac
31681  aaagcagctg aaggctgaaa gcaaccaagc cttttacagc cccttcattc cccacactcc
31741  caaaaagctg agtgaatggt cgatacctcc acatgcttat aactcattcc cagcccacca
31801  gtgtctagca tatctggtta gtcttagctt tatataagtg cagttatttg tgaacttgtt
31861  ttaagtattg gaatacaatt taactttcat tcttattttg gagaccatta tttaaacaga
31921  tttctttttt cctgcaaaaa cactcttttc acaatggaca gagacacggt gattacatta
31981  aaaccatcta ctctatgaat aaaaatgtta aaaccaaaat cccaacaaag ggttaataaa
32041  ggcaaaaaaa attggaaatg acatgtgttt taaagaaata aacatgaatt atctttaagc
32101  tgtcaatgaa ctataaatta tgtgtgctct tgtatatgct ttcctgtaaa tttggactat
32161  attaatattc taaagcttat ggtaaaatta tgaaaatatg ctttcatatc tataagtaac
32221  attttaaaaa atctcagtta atttcagaaa aatactgtta tcaaaaggaa tacacctgaa
32281  tgttttggag ttaatgcaga agcatataca ttctcctgaa gtggctgaaa gtggctgtaa
32341  aatgctaaat catctttttg aaggaaggta atatagattc attaacttgt acagaatata
32401  tcatattggg ccaggtagaa tatcaatatt tcaagcatat ttctaacaat gaaaagaaaa
32461  agaaaaacat aagacacttg aaaactgaag cattttgcaa tgtaatctcg tgtcactagt
32521  accatagact tactttatct gaacactgaa aggaatggca agattgtgga aacatgttga
32581  aggtttgctt ttgaacctga tgcttgatgt tgactatatt ttgaaaagtg gtaattgtat
32641  agcacatagc atacagcagt ttttctaatt attgtgtgtg tgaaagttat aaaagataaa
32701  atcagtttat ggctaaattt tgctctttca caacgaatat attattcctt catctgaatg
32761  aactttgtct tctcttcctg ctctcaatcc ttagttaggg aaaattttaa ctacatctag
32821  tccaagtgca gagatgctaa gattatatag ctggcggtta gtggcacaac agagaccata
32881  tcctttgatc tatgtgtgga tggtggtggg gcagggtggg atgaggtggg ggtgaggggt
32941  ggtaggtgtg ggcagaactc tcatgtgtaa aaaaaataat tggcacagaa gttgcagtga
33001  aaactaattt tgttcctggt tttgccacta atttgagcca actgtttcat ctctaaaact
33061  tcacgttcct cattgataaa gaggaatgat aataacaact ttgaaagttg taagcttaga
33121  atgtaaggat taaataaatt aatttttaca aagggattag gataatgcct gctgcatttt
33181  aagcactcaa caaattgtgt ctattgttgt tatactgtta ctaagtgtga ataaatgaag
33241  tgcatatagc atgaaatgta gcgtaactgc agacttgtaa gaagtagggt tacactgttt
33301  ttaacatcag tctaactaat ctatgtttat atatctttct aagctgtatt tctcttattt
33361  aagtgttgtt tttgaacact gagatgaaaa gtttatctta aatgttgatt ttaatggggg
33421  cggaagtgtg caaaccttta caaatgaggc aaaaacacag cggaataaac tccagtctag
33481  gattctgtag attctgggca aggcatttaa tgtttctcgc tgcatgctct tacgtaaaat
33541  gtgtacagtt gcagccctgg agattccgca ttggctctga cagtgtgtct gcccctacag
33601  aactcatgtg agtcgagaga ctgataagta aacagattat tataatacag tctcagaatg
33661  cagtggcagt agtgtgtaaa agacgcaatg gtaagagtag agtggactca gctggggtta
33721  cccaaggagg ggaggctcca atggagggat gtgtttaaac tgggacttta agttggaaaa
33781  gaaggaatgt atctcagtgt ccacagaacc atgcaaagtg agaacatggt tctgatatgc
33841  acaagtttca gttaacaagc aaagcaagga ttgactgtat taaagttcat agtacctact
33901  gcattctagt caagtgacat ttgctcatat gtaaaagaaa gaatagctta aatacctgag
33961  agaaaccaag actgtaaaac aaattaaaaa taattaaaaa taccttataa gaagtccagt
34021  gatgttaatc tggaagagga aggttcgtgt gatgtaaaga ggccttgtct tggggtcaga
34081  caagtttggg taccagcctt gtctctgtca ctttctagtg ataagacctg aatatttaac
34141  ctctatctgt ctaagttctt catgtagaaa atggggataa taacacctac ctgctgggat
34201  tgttgttatt gacccatcgt aggtcagaag gatgttgtta gttttatgaa gtgaaataat
34261  tcccggatta ctatgaattc tatcttatga gttcaaagtt tagacaatta aaattatgta
34321  tgctcatact actgatttca aatgcatttt catatagtct ttcctgataa aatatattgg
34381  ttctgccctc ctgtacttat ttcaatttgg tgtttatacc attgaatcag atcagtcttt
34441  caataagcat gccaatttta tatccccagc aacacttccc tggatataat ggcagcagtg
34501  gtccccaaaa tactaacagt tatgaaacgt catgagacat cattaccagt gcagctggag
34561  gcgcttcgag ctattttaca ttttatagtg cctggtaagt tacatagttg attgtgggaa
34621  gagataacaa tttaaatgga tttttgattt ttcatgaaat agcaatattc taggcaaata
34681  ttaaaagact agtttctgtc gactaaatgt aaatctttct gttaaaccaa aaagaggtta
34741  aatatgatgc agaagagtca cttagattaa tttttataag aaagcaatat gaattcagta
34801  atttatttat acaaagtaac tacaatgtaa aatgtggagc ttttattttt aaggagggtg
34861  ttcatctctg ataattcttt tctatttttg ttgccatgac ctgagttcaa gctttttttc
34921  tcttgtttga attgtactat tagctaattt tcatacctgt tctcttcctt catttctatc
34981  cttttcttac tctatgattg aattaatctt tctccaatgt ggcttgtact catttacctc
35041  aatggcttcc accctccacc cacctttaat tgttactgac atctgttatc accttatttg
35101  ttctccaaag cccctttaaa acacctatgt ctgtattcat ccaacacatt tattaagtgc
35161  ttcttatgtt ctaagcactg tgatgctgtt aaactttaaa agatgaattg gaaaaaaagc
35221  acaactgtac tattatagga gcttagaggt agatggaagt gtaaatagat aattaacagg
35281  cagcgtgaga gaggcgatga tagatgcata tataaaaggc tttgagaata tcgatggagg
35341  cacaaataat ttcttcaaag gagtaggaca gagattgtaa tatttgaggt ggaagggtag
35401  atagagctgg cctggtagaa aggccaagga agacatttca agcagaagaa agtgcacatg
35461  gtcgggggtg agtgaggcat ggaggataag gagaggagtg cttggggatg agtgtggaag
35521  gaaaggctga ggcccaaaga aaagggcctt gaatgcagtg ctaaaaattt tttgcctttt
35581  aaaaatggaa gccaacccaa gtttattaaa attgtttgca gattgtagtg tcacaccgaa
35641  atttgcaact aaagacaata gcattgtggt gcagaggatg atagacaggg agagagacta
35701  gatacaagga gattgggttg gaggtccatg gtagtcaagt gagacccagt gaagggaccc
35761  agcaatggaa acacagatga gagggcagat tggacagatg gttgggagat ctgtttgatg
35821  tgacatcgtg atccctaagt atgagggatg atactttgtt ctgtgaagtt cccaatcttt
35881  tgaagtcatt tttgctttgt tcttttaaaa tgaaatcttc ctaaatctat cattttctct
35941  agttatatag gtattttgtt tctctgtata agagtattac tcaatataaa agttttttca
36001  aggacagagc tttcttcagt catttttttc tgggcccccc agtcctctgg tggctattca
36061  atcagtattt taaaaattga atcatggtgt cctattcata gtttcagctt agttttgaga
36121  cgtaatgtaa cagatgtaat tttacttgaa aatattattg catgttttat tgaattttat
36181  ttttagattg taattaaaaa caataaaaat gccttttgat tatccttaaa gttgacagcc
36241  ttattctttt gagggaggtt ttgggtttta aagataacca aaggacactc aaaaaccgtt
36301  tctgttagtt aataaaataa tactctttta gtccaaaagc aagttttgaa tacagtattt
36361  cttttctttt attgttctta aactgatcct gaaggaaaat tgttagttaa caatcaatca
36421  gatgtattat gggtgccctt gaaaataatc acttgaggac tgtacttaat gtaaaaaaat
36481  acattttata agcatatcag tatgtaagta cattcttcta gtaggtaaag gcttaatcat
36541  ttactgtatg ctaacatatt atactaccac agtacaccat actgtactgt acccaacgta
36601  atgtaccatg ccgtgcagta ccgtatcatt ctaggtatca acaagtaact atggtagcaa
36661  tactccagta gttttgaggt tgaatatgat tctggtttga acatgttaag ttgaggttct
36721  tatgagatac ctaggtgtac atgtattcat ttattagcta gattatattc aggtttttta
36781  gtggcaaaga tgtacattat ggcaaatgtg tataatttag ggtccaatta aggatagcat
36841  attgcatcta gtgcatatat cttaagtttc tttcaatcta ttactgtact ttttctcttc
36901  tactttataa taacttgtta tatagacatt gagctggttg ctttaagaaa ataataatga
36961  agacccagtt actttaataa aatatcattt gacttgtttc ttgagttact gctagtttta
37021  gaggaaataa tgtgaaattc tttaagaagg gatataggca aattgggaag tatatagagg
37081  agagtgatta gggagataaa agcacatcaa aaagagcaat aagaggaact gagattgttt
37141  tatttgaaga agagatgact caaagggata tgaaactcta ttcaaatcta cttacttata
37201  accagtaaca gtaataatac caaacaatat ggaggactta aaaagtatta ggcattgttt
37261  gaagtatttt aagtatatta tctcttctga ttctaacagc actccatgag gcagatatta
37321  ttattattat tattttggta ttgtatggac tacaaaactg aggcatagaa ttgtagagat
37381  ttttaaggtc aggcctgata taacagcacc agattttcag cttatgcagg ctgactccag
37441  agtaggcatt ttaaaataaa tatttgtgca acaatcttgt cctgaacaac tgttgtattt
37501  aaagcactat gtctagaatc cttaaggtat cgggagatga tgagattatg gatactgctc
37561  tcaaaaaaat ttacagtcca cacgacctaa aggactgtta tgtagaaagg aaattatatc
37621  catttgaata gtaggacctc aagtgataga acaaagactg gagaagaaat ttacaggaat
37681  gttcatttgg tcttaattta aggaagatgt taaataagac ccattacatg tagcatgatg
37741  tttttactac tcaggccagt tttagtggtc acatcttctt aaggtgtaat aggcagcctc
37801  agaagttact gtggtcttgc tcactggaaa tgatacacag acagtttaag ggacttgccc
37861  cagaccacac ggcaagaggt gaatgtcaga acctctttga gtacatttta aaataaggac
37921  tgaaagttgg aggagggtgg ttatcaaggc tgccttcctt accatagtat tcccagcatt
37981  aacaaaatcc ttggcatgta attggaattc agatgcttct taaataaatg aaaagcctgt
38041  tgtagccagc ttacagtttg cattaatgca gattattaaa gtggaagatc ataaatgatt
38101  ttttattaat atttatgtct ataatcttag gtttggaaaa cattattcat tcataataat
38161  tttaattata tgttacatta ccacattttt gacttgtagt gtttttagca tagttcagct
38221  acagtgtagc ttaataaaga atatgatttt ttaaaatagc aatgctatta tatagccttt
38281  acagaacttc taaaaaatga catgttctct accaccttaa tactgaaact caaatcttat
38341  tttttgctac gattattcca gctactcttt tttgtctata tttcatttct gcctttttat
38401  gttgtggtcg aagtaactct gagcttttct catgttgtcc attgttgcat aaaaatcttc
38461  cagcatctta aagcacagcc tactcacaca aaaaagtgat tgtttgctac agaaaatttc
38521  ttcaccatcg taattttttg ctacttcaaa ttcagtaagc attcttacac attatattta
38581  ttttatattc agtgatgaac tatttttata gattccttaa aatttctggt tatttatttg
38641  ataaggaaac atgtactaga aaaaagtaca acacatatat tgtgagatta attatgacaa
38701  tttctagaaa gtaacagtct gttcaactca aatgtttata agaaaattct ttctttattt
38761  atttatctgt gcatttaggc atgccagaag aatccaggga ggatacagaa tttcatcata
38821  agctaaatat ggttaaaaaa cagtgtttca agaatgatat tcacaaactg gtcctagcag
38881  ctttgaacag ggtatgttga atataagttt tctgtattta tactattaac taaaatatta
38941  aatttggaga actaggggcg ctttttcagt ctaagttttc tgttctccgt ttgctatgat
39001  aggaggaagt catgtggtta gagacataag atgacagtgg ggatgtggga agtgaaaaga
39061  tatgtactaa gctaagtcca gctaagtgta ttatcaatta tagatgtagg caagattctt
39121  ttgattgcca gtaacataaa tccactctag tttgctcaac cagaaagaga accaaagagc
39181  catatatgca gctagacctt gtgagtcatg ctgggtacta tggctgctgt tttctctttc
39241  tgtcctctgg ctacttgtct ttcttttctg gtctcatagt atatggttta gcccatgaag
39301  acataccagt gttaacagta aagtcttcgg ctgggcacag tggcccacac ctgtaatccc
39361  agcactttgg gaggctgagg tgggtggatc acgaggtcag gagttcgaga gcaacctggc
39421  caacatggtg aaaccctgtc tctactaaaa atacaaaaat taactgggca tggtggcacg
39481  tgcctgtaat cccagctact caggaggctc aggcaggaga atcgcttgaa cccaggaggc
39541  agaggttgca gtgagctgag atcacaccac tgcactccag cttgggcgac aagagtgaga
39601  cttcgtctca aaacaaaaac aaaaacagac aaacaaaaac agtaaagtct tctttgattc
39661  cctacgctca ttttcattgt tctccggaga aataacctct gaaatgattt ggtatacatt
39721  gtttccattt tttagcattt acatatccat gttcctacat tataattaaa gtatccataa
39781  atcatactga gtatgaaaaa gagaagaagg gaattacatt taaattgtgt aatgcaaaaa
39841  gtattggtgg aattaagaag ttttggaaat tttgcataag atgaattggt tctttattaa
39901  agatgttaag aataaagaca taattagtgt gaacattttt ataaaaggag gagcctattt
39961  aaaataatta atggaaatga ttccatgtga tttgatatac tttgatgaat gtcataaatt
40021  aattaaagtg gcttccagag agatctccct taaaaattca ttttaaattg aactttatac
40081  tgtcactcac tgcctataat atgtttgagt catttatact caaactttaa tacaatcctt
40141  gagtatggca agaatttatg ttgtaatggg ttaaatttat cttgagaaat atttgttgaa
40201  aataagtata tggaaggaag gggttaggca tttagaagat aaataaatat gctttgtact
40261  cttctctcct gaatctcata agccggttgt tgatggctgt tgtgaaacct tggttctttt
40321  ctttaaacaa gagacacaca gcagaggaga tgcagcatcg agtaatttat tgcaaaagaa
40381  aaagaatatt ttgcaagtta agtgaggaat agacacttat accctgacag aattcagggt
40441  gggcttacta gtaaggatga gacagcgtaa attggcacta ggaagactcc ctttgtggga
40501  gttgtacatg atttttcata agtgggtggg aagaagtgtt actagtaagc atattctagg
40561  ttgtcctctg agtgaacatg tgcagtagct gtacatgctt gttcatatat cgcatgtctc
40621  ataagtatct gaaatctcca cccaggggtg tgtgttttac tattataatg agcaaagggt
40681  cagtctgagg acaaggaaaa tcaaaatgtg catgctcccc acgctacctg acttcaaact
40741  atactacaaa gctacagtaa ccaaaacagc atggtactgg taccaaaaaa gagatatagg
40801  ccaatggaac agaacagagc cctcagaaat aatgccgcat atctacaacc atctgatctt
40861  tgacaaacct gacaaaaaca agaaatgggg aaacgattcc ctatttaata aatggtgctg
40921  ggaaaactgg ctagccatat gtagaaagct gaaactggat cccttcctta caccttatac
40981  aaaaataaat tcaagatggt ttaaagactt aaatgttaga cctaaaacca taaaaaccct
41041  agaagaaaac ctaagcaata ctattcagga cataggcata ggcaaggcct tcatgtctaa
41101  aacaccaaaa gcagtggcaa caaaagccaa aattgacaaa tgggatctaa ttaaactaaa
41161  gagcttctgc acagcaaaag aaactaccat cagagtgaac aggcaaccta cagaatggga
41221  gaaaattttt gcaatctact catctgacaa agggctaata tctagaatct acaatgaact
41281  ccaacaaatt tacaagaaag aaaaaacaac cccatcaaaa agtgggcaaa ggatatgagc
41341  agacacttct caaaagaaga catttatgca gccaacaggc acatgaaaaa atcctcatca
41401  tcattggcca tcagagaaat gcaaatcaaa accacaatga tataccatct cacaccagtt
41461  agaatggcga tcattaaaaa gtcaggaaac aacagatgct ggagaggatg tggagaaata
41521  ggaacacttt tacactgttg gtgggactgt aaactagttc aaccattgtg gaagacagtg
41581  tggcgattcc tcagggatct agaactagaa ataccatttg acccagccat cctgttagtg
41641  ggtatatacc caaaggatta taaatcatgc tgctataaag acacttgcac acctatgttt
41701  attgtggcac tattcacaat agcaaagact tggaaccaac ccaaatgtcc aacaatgata
41761  gactggatta agaaaatgtg gcacatatac accatggaat actaagcagc cataaaaaat
41821  gatgagttca tgtcctttgt agggacatgg atggtactca gcaaagtatg ccaaggacaa
41881  aaaaccaaac accatatgtt ctcactcata agtgggaatt gaacaatgag aacacatgga
41941  cacaggaagg ggaacatcac actctggggc ctgttgtggg gtggggggag gggggatagc
42001  atttggagat atacctaatg ttaaatgaca agttactggg tgtagcacac caacatggca
42061  catgtataca tatgtaacta acctgcacgt tgtgcacatc taccctaaaa cttaaagtat
42121  aattaaaaaa aaatgtgcat gctccataca ggggcaattc cctactggag atagctttgc
42181  ttaaatgagc tggactacaa tgcaaatgct gaaacttact atattgacag taagattgcc
42241  acagttgccg cgtcctgagg acatggttac ttccttttaa tacctatcct gtctcattgt
42301  gagaggatta acaactgtgc ataaaaccag ttgttctaca tgagcactta ggagggatac
42361  cagcattgtg aacatagttt aagtacgtag aggagggaac agttaagttt attcatggtg
42421  agtgttggtg aaaagtggaa gaggtaccaa aacagccgta tagataactg gttccagtta
42481  gccaacattc tctaaagtta ttagagaagc ctaagtgagg tgtaacctca gcagtcggga
42541  gccaagagag caagtaagtg ctgtgatgtg gagaaaatca ctttgttcca actgagaaga
42601  aatggttgag cactgctttt cccccatgcc agtactgacg cacagccttt cacttagcac
42661  tgattatcga taggggtggg gagttaaggt atggggaaac acaagtaaca atattttatt
42721  tcaaaaacct ctccactgta attcccctaa tccttcatca tggttgagga aaatggctct
42781  aaaaaatgag agcaattact gtagctccaa aattctgtga ttgcatgtct tactctgaat
42841  agcaattaca aagcatcaga ggatttaggt ccaaatattg cagacacaag aaaatgaatt
42901  acattttaat acatctaaac ttggagagca gagttccaaa taaggtagaa cttgagattc
42961  aactctgatt tataaagcag agactaagaa gagtatttat aaagcgaatc catgtttgga
43021  tacataaaag tgcaataaaa ttcaagctga agttaaaatc tctgtctaga acagcgatgt
43081  tccatttatg cctgatcctt ttagcttttc cacagatgaa gactttgtca cctgttccag
43141  agagatatat ttgttcatta ttgtttccag agagcaaaat ggaaaataaa ctctgcacat
43201  tttggccgca tctgtgtttt atatgcggtg acactcctgt tctcttcagt gaggaaatcc
43261  agtaaagtaa aaccagtctt ctgatgaaat gggcacaaat caaagaactt gtgagcttca
43321  caaaaacctt gaagcaaaat ataccaagct taaatattga atgtattgat ttcagtagtc
43381  aaaaacagag ctcatctgca aaagcaacaa caacaatgac aacaacaaat tacatataag
43441  taaaatttaa aaaaggttta caggatgaat atacagaaaa ctatgaagct taggggtaga
43501  gaggagtagt tgaatatatg aaaagataca tcttttttga tgaaggacta aatttttaaa
43561  aatgaaaatt gatctatgtt tatgaaccat tagtaaaaat aacaatagta ttttctggaa
43621  ctaagtaagc tagtgtaaaa tttgtatgaa aaaattaata catatgagta atcaggagaa
43681  ttttaaaaaa gagtgttgat tcatatggcc taactccaaa agataatcaa atgtattata
43741  caattttagt aattataatg gtgtgatact ggcactaggg gagagagatc agtgccacag
43801  aagggtggct caggaaatag actcaaataa aaatttgtaa tgttatgata ttcctcgact
43861  gcgggaggaa aatagattac tcagttactt gtgttggaac aactaactct tcagccattt
43921  gggaaaagca aagaaagctg aatatttatc ttactccttt tgccaaaaca aattacagat
43981  ggatgagatt taaagtctat aatgaaattg gtatatgtac atattaaaag tttcaactat
44041  aattgttatt aacgtaaatg acaacggaac accttgttgg agggaaattt ggtaatatct
44101  atcaaaatta aaatgccatg ttttctgatc agcaatttca ttctaagaat ttttaatgta
44161  gatatacttg ctcatgtaca taaagattat tagacatgaa tgttcactgt ggcatgattc
44221  gtaattaaaa atgtggcaac aaactaaatg cttacatggt aatcattcat gctgtttgcc
44281  agatattttt atttctccac cttgtggtga ttctggcata gtatttagta gttaggttga
44341  agcatgtgac taattttggt cagggagttg taaatggagg gaatagctgg tgagacatct
44401  tgcggaatac tgtctcaggt accatcgtac ctgcagtgtt tgagatagta gttgctccat
44461  cagtagaggt ccctgaggga ctacaacaag cagagtcccc tcctgacctg caacacatat
44521  gtagcatgag caataatgga acttattatt tttgcaccct ataaaatttt gagaattgtt
44581  actgcagctt aatttaatct ttcctgacaa tacaatacct atcaatagag actaatcaga
44641  taaattatac catttccaaa ttgtatcatc ctgcataaat attaaaaaac aatgaggtga
44701  gattcctaat gtgctgctat ggaaaaatct tcaattttct atgtgtacga atgtatttcc
44761  caggtattcg ttttttcctt cctgtatgtt gacacattat gcacactttt ggatgaaatt
44821  aatatatttc caccgcttta tgtcctctcc ctcactttta tcacccaatt gtagcaaagt
44881  atatgtttgc ttttatagct ttagctatat aaaattttct aatagctaag tttgtggttc
44941  ctggaattaa gatatctgaa tttaaatcta acactactac ctacagacta tgcaccctgg
45001  acaagtactt aatgtcttgg ttttgttatg tataaaatgg agataataac tgatttttca
45061  cttagagttg ttgaatattt tataagataa ttcatgaaaa agtgtcagta taatgcttgg
45121  cacatagtat gcgctcaata aatgttaatg ttattactat tagatttaaa agtatctttt
45181  gacccctggc tatagaagat gaggaaatca gagtatttgc acttctaata tctcctgttt
45241  tcccacctac ttttgttcaa taaattaact ccacattggc agggtagata atatttatat
45301  tcagctttct aattatgctt tctaagtttg tgtttatctt actcctaccg ttatttggaa
45361  gcagtcttca tctcaagtcc ttttgttact gttttttcac tcatctttta gttgtctgaa
45421  tttctttaga aaagtttaat tccctgaata tttaatgttt atattttgtt gttgtttttg
45481  cctttatatt tggactccta aaaaaagttt tgttggttgt aaaatattgt ttttttttcc
45541  ttgaagtgtt tgtaggcatt tcactgtgtt ctagtactga atgttttagt ggagaagtct
45601  gaggccagct tcatttgttt cttggatatt tttgcccagt gctgaaagga ttttttctct
45661  tgaaatccat ccactttatt agggtatatc tcaatgttag tcctgggata tggtatcctc
45721  attctagcta tacattcaac ttttttcctg gaaagttttc ttgagttgca tctttatata
45781  catatacata cacgaataca tatacatggt aaagatatta aggataaaga taggtgcttt
45841  ataaatacca atttgatata attttggcat gaaaaaaagc ctgtggtgca ttctgagttt
45901  ggaagtagaa ctggatatac ttacttatta ctattgtttt taatacaaaa atggagacag
45961  ggtctcacta tgttgcccag gctggtctca aactccttga ctcaagcaat cctgcctcga
46021  cctcccaaag tgctgagatt acaaatgtga gccaccgtga ccagcctaaa tatctaaatt
46081  tttctctttc attattttag ctctcttctt tggggctatc agtcatatga tgttggatct
46141  cttttgcctt aatcttctaa tttttcaaag actaatccag agaccactat ctatcaccat
46201  gccattactc atagacaagg gacatcattc cacacattta gcttatttgt caaaatcaga
46261  gatgggattt ataaaagaaa aagaataaag ggagaaaaca aaacacaaat tatctgggaa
46321  tgcgagtctg ttttcctttc tttggctaat ctacttacct agtagctagg aaagaggcca
46381  gcatgagtat ttttccattg agctggctag ctcagtcatt ggagaaacca tttcaatatt
46441  tatggattct ctgaaaaaaa cttggagagt aaaaaagcat aggtggtagg atgctgccat
46501  ccttctgttg gcatctccag agtttcactt gaaaaacacc tcctaagtag acactgataa
46561  attgatttta atccctattt gagcaccaat gcaatacatt actatttcaa gatggagcat
46621  tagataacta aaggaaattc tattgtgggc ttgtaaatat catgatagtc atacctatgt
46681  aatacatata tgtatatata aatattaatt tttaaacatt tacttagcta ttaaaaatca
46741  aatgctcata tttaaaatta ttggtttatt aatgcaccat cacatgttat catgtgatac
46801  tctgaattct ctttttgcat agacaaatca gagattacct gctgcaagtc attagtcatc
46861  attaacctgt actgaaatgg ttagagccta ggggagttcc aggtaaaagg accagtaatg
46921  agtagaagct tgttagatgt agagatattg aggacagaga taggtgcttt ataaatatca
46981  atttgatata attttagcat aaaaataagc ctatgatgct ctttgagctt gggagtagaa
47041  gtggatatgt aattttcagg gcgtagtata aaatggaaat gcacactcct tgtttgaaaa
47101  ttattaacga ttttacaagg gtgacaacag agcattgaag caagtgctct gtgagcatga
47161  agccctgtgt gaaatacaca cctgtgaagc tggccttgcc tgccaaacag gaatgctggt
47221  actgttaatc aaacagaaag ttcagaatga caatctgact tttttttgta gaagatacta
47281  aacttttggc cttgaatatc tgaatttaag atactggtat caagccaaaa gaaatttggg
47341  cttagaactt gaaagattta aacttgtgct ttgacttgtg tgctcacagc tctctgtgtc
47401  acaatttttt tcatctgtac ttcgggacaa caatagtgtg tcgacatcac aaaggttttg
47461  ggaggattaa gtaggtgaat acatatgaag ttcttaaaag aatgtctggc actgagtgag
47521  tgctacctcg gttttggcat cgttattgtg gtcattgcta ttgttattat gacttgaagt
47581  catattagtg tatgaaatcc catccatgaa tagaagacag aaagaacttt gggcagggtt
47641  tggaggtaaa agaagacatt gtaaaggaga tgggtaaagg aatgatttta aagactgaga
47701  ataattaggg gagtacgatg tcatggaaat taagggatga taagcaccaa ttaagttgtt
47761  attggacttt tttctttgaa tggttcaaat tcagaataat aaggaaagaa gtctgattat
47821  acgaaattaa agggtagggt gactgtggag gtagtgggag ttgacttttc tgctaataag
47881  tttagaaata aaggaaaaat ggtagcttga ggaaagagag gagcgattaa gggaaagtct
47941  ctcgttaact cttgtttttc atctctgagg acagcgctta gcccaaggcc tgacctgtga
48001  tcattactct ctcaaggttt attccatgga cagagctatc tcatttcatg cttataacaa
48061  ccctacatta ttagaattgt tttagagatg aggaagctga gacacacacc aaaccagcct
48121  tccaatttca ctttgcacaa ctttgaattt ctttatattt cttgaataaa agttccactt
48181  tttaacttac cacttcttag cagtcattgt ctaactgagt aattgttact tcattcattt
48241  aatggttctc agattcgcat aatttgaacc taaatttaat tggcctccaa gctgatgtgc
48301  ttacagaaac agtgacagga aacaaaaatg tcaagggaga ctatgtatta ttaagatgat
48361  aaatgaaatg atgtccaagc tgagcaatta aagtgtgaag tagaaggaca cagggtgaga
48421  aactgatgct tctcctcagc ctctataaaa aagatactga ataaagataa ttgagaggca
48481  ttaggggact agactgagaa aggattggaa atctgttcac tgagagtaca gaaatgagga
48541  agcttggaag gcagaagatt ttggtcaaag acgtctggct tgaagctatt tcagctcttt
48601  ggattatctg tggtggaggc catcacgtct ttggagtgga ggtaccatga aactagtgtc
48661  tgcaaaacat catctaaatg aaagcaaaat tcctgagaag gatggcacta taatttttaa
48721  aagaaaagct atgagttaag cattcatatc aaggtagatg tttggagtgt attgcttgtg
48781  tgtgaaaagg cagagatgac cagaataaga gttagaggta tgctgcgttt tcttcttggt
48841  tgatgagtag gatggcctgg acaaagaagt gacctctagt aaaatacctt catagtgtca
48901  aatcatctgg aggaaattca gattaaagag gttggatgat gtcgtaatta agatcctggg
48961  cttttaaggt ggacacattt atattcaagt cccaggccca atgcatatta gctctgttac
49021  ttgagctttt atttctgcat ctttaaagtt tggcaaacct attacatgaa gctgttgagg
49081  ggataaatga aataacgcat gcaaagcact tgcagtaaag actaattatc aatattttat
49141  ttgttaagag gcagcattgc gttttactgg tcaagtatgt agactctggg gtgaaacata
49201  tttggtttgg tttcatctct gcaatttata gtttgtgtag actttgagaa tagttctcaa
49261  tcattctaac cctcagtgaa ttcatcttct aatgggagtg atatcagtat ggatttcatg
49321  agattatgaa aagaaaatgc ctacaaagta tttattacaa tgcctggcac agaacaagct
49381  ctccttaatt gtaaaaatgc taactcttat tcttcataat aaataaaagt aattaatgtt
49441  atagaaaaca aaatcaagga tactgattta tatttggatt acttgattta tattttgtca
49501  gtctataact ggtcttaact aaggtaagta ttaagatctc atttttaaca gcgagtattc
49561  ttttgatttt agttcattgg aaatcctggg attcagaaat gtggattaaa agtaatttct
49621  tctattgtac attttcctga tgcattagag atgttatccc tggaaggtgc tatggattca
49681  gtgcttcaca cactgcagat gtatccagat gaccaaggtc agtacaattt gaattcagga
49741  tttagaatag atttttgtag ggcattagct ggtgactgga tgtctttaaa tatttttctt
49801  cagttttgag atttaaaaca attctttttt tttattttcc tagaaattca gtgtctgggt
49861  ttaagtctta taggatactt gattacaaag aagaatgtgt tcataggaac tggacatctg
49921  ctggcaaaaa ttctggtttc cagcttatac cgatttaagg atgttgctga aatacagact
49981  aaagtatgtg cattatcttg gaaagaattt gggaacttgt gcgaatttca cttttggagc
50041  agtttgtgta attcccactt tgcatgaatg gggtattcta gttaatggaa aaccatttat
50101  ccttttgtag tattttaatt atacaagcaa agaaaattgg attgaatctc taaagatcca
50161  gtgtttcatt atgaaatctc taaagtcagc atggttattc accatttatc ttgcccataa
50221  aagttcagag aatgtgctaa gaaatcccag ctagctgagt ttattcgctt agattttaga
50281  taaatagaat ttataaatat tccaaagttt gtcactctct gggttttatt gcaggttgct
50341  tacctttagt aattttgctt gttgattttt ttccttgcag tgaaaaaatg tttttaacat
50401  ttttcatcaa gcaaaattta aaacatgata tataataact gtctttgtaa ggaattcaag
50461  atactggcct agagttagtt cacgggagat taagaataaa tttgttttgt tttgtttttt
50521  aattgtagca aaacaaatag tttttcttca agagtttctg ccttggttgt ggagtttgca
50581  actttcataa actacaaagg aatttttttt ttttttttgg agacagggtc tcactctgtc
50641  acccaggctg gagtgtagtg gcagatttca gctcactaca acagctgctt cccgagctca
50701  agtgattctc ttgcctcagc ctcctgagta gctcagacta caggcatgca ctcccatgcc
50761  tggctaattt ttgtattttt tgtagagatg aggtttcacc atatttccca ggctggtctc
50821  aaactccctg gtctcaagca atctgtcctg ctcagcctcc caaagtgctg ggattacagg
50881  tgtgagccaa ggtgcccagc tgactcagga aatatttttt gtaactggca gcattgacca
50941  ggaataaaaa tacctggtct ctaatctttg cacagacatt atcagtaaat gagagaatat
51001  gtgtaaagtt ttttaaaaaa ttataaagtt atgaacatac aaaattctta gattaataac
51061  aacaatgtgt tttataactg cttttcataa tgtgcctcag gctaggctga ttaaaccaag
51121  ataggattga ttaaaagtaa tcttagggaa agggaaggat tttgtgccgg tatggaactc
51181  tcagttactc tggattaatt catctaggca taaattttag aatctctata gtagagttta
51241  tgaactaaat ctggcctgcc aacatatttt atttgtccag ttcagggttt tgctttgttt
51301  tttgagacag agtctcactc tgttgcccag gctgtagtgc agtggcgtag tctcagctca
51361  catcaccctc cgcctcctgg gttcaagcaa ttctcctgcc tcagcctccc tagtagctag
51421  gactacaagt atgcaccacc atgctcagct aatgtttgta tttttagctg agatggggtt
51481  tcgccgtgtt ggccaggctg gtctcaaact cctgacctca agtgattcac tcacctcgga
51541  ctcccaaagt gttgggatta caggcatgag ccactgcacc cggccttcag ttcagtgttt
51601  aaaagttttt aattcgaatg acgtactttc tgcacatttg catggcctgc tctgctgtag
51661  cattcacttg ttttcagaga cctctgctct agaggcaggt ggatcacctg tccctcagac
51721  atacataaat taaggctact ttgcttatca aatattagta ttcctagata ctcagcatca
51781  taagagttcg aagtaataat tttaatattt agatgacgta agttaattta aaattttttt
51841  gagatggggt ctcactctat ggcccaggct agagtgcagt ggcacaatct cggttcactg
51901  caacctctgc ctcctgggcc catcctcctg ggtgggctca agtgatccac ctcagcctcc
51961  tgagtagctg ggactacagg tgcatgcacg ggtaatttta aaatattttt tataggcaca
52021  agattttgcc atattgtgca ggctggtctt gaattcctgg gctcaagcaa tcccacagtg
52081  ctgagattac aggtgtgagc catggtgtct agccaatttt attaatatgt aatattagag
52141  gtaataaaat attaaaaagt taagatgatc cttggtggct ttacccaacc taaataatac
52201  taaagtcaaa agcccaatct ttcattaaaa catcacatga gtgaagagga cagactctgg
52261  ggatgtgctt aaggtggttc taaaaaagta acggtcttct ttataaataa cttattatta
52321  gaatgtaatc ctcagagtgc cctcagcgct tctcaactac actcaacata aatgaaatct
52381  aggagtccac actagccttt ctgagataaa catttcggaa gacagcgcaa aaagctgggg
52441  gatatgctag gctctctaga gaacctactg ttcaatatta taatacaaat ttttactcta
52501  ttgacctgtt tggatgtgta gttctgctga tccaaccgct ttaatcctgt ttaatatctg
52561  ggtttcatcc tataactatg gctttagaca agcatctttg aaaaccaaat ttgagggtat
52621  tagttctttt tcctgctttg ctactgaatg gtttgttaac tagcatttta ttctctgtgc
52681  ctgctatatt tcttagtcat gagagagaga gggagtattt atttacagga taaatacttt
52741  aaagcaccaa cccaatatat ctatagttaa atgaacatcc taggtattgt ttcatataca
52801  aactctctct gctttatact gtttattcat tttgcctgta attgcttatt ttattatttt
52861  ttttcttata cttttaggga tttcagacaa tcttagcaat cctcaaattg tcagcatctt
52921  tttctaagct gctggtgcat cattcatttg acttagtaat attccatcaa atgtcttcca
52981  atatcatgga acaaaaggat caacaggtac agtgtttttc acttgcatcc taaatgttat
53041  gtatttatct gactctaatt ctcatttcca ctctttttag tttctaaacc tctgttgcaa
53101  gtgttttgca aaagtagcta tggatgatta cttaaaaaat gtgatgctag agagagcgtg
53161  tgatcagaat aacagcatca tggttgaatg cttgcttcta ttgggagcag atgccaatca
53221  agcaaaggag ggatcttctt taatttgtca ggtaaatatt caaggcctca cttttgtctt
53281  tgctcagtat tcttatagaa tgtaagagcc ctgccattgt gtatctctta cttatatcat
53341  attattcttc actacagaaa tttaccagtt tattgcaatt gtttgtgtct tgtagtagat
53401  ttatagaatt ccagaagtaa tagggtcctt taggtgttat ccagtctaat ctttcatttc
53461  atctgtttac ttatcttgtt aagttgataa ataacttttc aaatgtgtcc cttagtaggc
53521  atctctacaa cttagtctcc agatacactc cacataacac atagttctaa tgttttgata
53581  atttttaaac catttttttc catggtttta gtttctttgc ctagaaagtt ctcccctgag
53641  ggctaccaca catggctatg caggctgtgg atggcacact tttgtcggtg ccattcacag
53701  tgacatgagt tgctgttggc caaagttgtg taacactggt ctttctttcc ttctctcttc
53761  cctcctgaac catgtaaaca tatatctatc tgattgttct gctctccctt caaaatataa
53821  ttcaaattat ctttctttaa agccctcccc atacctccaa acctccaaac aaaattaaga
53881  tttacttctt ttgtcagtct atgaaaatat atacatatct cttgtatact tggtgagttg
53941  tgtgaaaata acagtgtaca gtgttcatct ttgtatcatt cagaatatcg agctcattgc
54001  tttacatatg gtgtgtattc aataaatact aggttcattg cttatatttc agatttgtat
54061  tatttgtata agtgttagag tttatactag cattcaggta gcactatgtc tattttctag
54121  aaatttaata tttctaacaa agcaattatg tagtgattta atacacatta ttaaataatc
54181  aataaagtac tatgtttgcc aatagtttac tttttaaacc ttactgtatt taatatccct
54241  actgtattta atatcccact tgcctatgga ttgaaatcaa tttgttgact gttaagatta
54301  agttaatact aattagtaat caacataaaa agaaaaagaa tttgtaaccc attttcatgc
54361  attacgttta tgaattaaaa tcacataaac aatctaatta tttaaattta gtcaaatttc
54421  ttttaagcaa gcaacaatta aaatagttgc tccgctttac taaagataat taaatttttc
54481  catcaataat ttaatacatt tttactgtgc atcttttgca tgcagattat tgcattaatt
54541  ttaattgaaa ataccgaaga actaaaaaga aacttcccct tctaagtcca cattaaggaa
54601  acaacatacc taaaagcacc tgatacaact gtactacatt ccccacagga aatcatttct
54661  actattcttt caatttatcc aaatctttct acccaacagg atttttactt tattcctctt
54721  tccatattct tttggacttc atatgcttag ttttatcttt tctttttaaa acgaaatctt
54781  aaatccaagg attatgtatt aggtttaaag aatttatccc agttgtcaga ggttatttat
54841  atctagcaaa caataactgc tgattaaatc ttgtggatga gtttgtcgta tgtaccttat
54901  ttgtgccaga gcaaaataag gtaatcagga ctatttattc atttaccaag aggttacata
54961  ttgaaggact atctagagca agggtggagt tgtgttagac tttctgcaca gaatttgata
55021  atggaatgta catgattggt agagaagaat atggaagttt aatactgggt atgcaaatgc
55081  atggataaaa acctcaaggt aaaactcatc aaatcacagt ggaaaaagta tagtgaagtc
55141  tgaataaaaa taataagagg ctgggcatgg tggctcacat ctgtaatccc aacactttgg
55201  gatgttgagg tgggaggatc acttgagcca ggagttcgag accaacttga gaaacatagt
55261  gagactccat ttctacaaaa caaaccaaca agcaaaaaac catgtatgat ggcacacaca
55321  tgtagtccta gcttcatgca gggtggctca tgcctgtaat cccagggctt tgggaagtca
55381  aggcgggagg atcatttgag cccaggagtt caagaccagc ctgggcaaca tagttagacc
55441  cccatgtcta caaaaagtca aaaaattagc tgggtatggt ggtacctgct tatagtccca
55501  gctacttggg agtctgaggt gggaggatga cttgagcctg ggaggttgag gctgcagtta
55561  gctgagattg caccattgca ctccagccta ggcaacagag ccagaccctg ttaaaataaa
55621  ataaaataaa ataaaataaa ataaaataaa ataatataat aaggctgagg tgggaggatc
55681  acttgagcct aggaggtcaa ggctgcagga gctaagattg tgccactgta cagcagcctt
55741  ggtgacagag ggagactctg tctcaaaacc aaccggtcgg gtgcggtggc tcacgcctgt
55801  aatcccagca ctttgggagg ccgaggtggg tggatcatga ggtcaggaga tcgagaccat
55861  cctggctaac ccggtgaaac cttgtttcta ctaaaaaaat acaaaaaatt agccaggcgt
55921  ggtggcaggt gcctgtagtc ccagctactt gggaggctga ggcaggagaa tggcgtgaac
55981  ctggaaggcg gagcttgcag tgagcctaga tcgcgccact gcactgcagc ctgggcgaca
56041  gagtgagact ccgtctcaaa aaaaaaaaaa acaaaaaacg aaccaaccaa ccaaccaaca
56101  aaacaaacaa acaaaaaacc aacaaaacca aacacttcta tcatgctcat taccacctgg
56161  gcactgctcc aaatacttta cacaatttaa tccttacgac aacctacgaa aaggtccagt
56221  aggttctaat gttattccca ttgtgcaagt gagaagctga ggcactgagg gtttaaataa
56281  cttgcctaag aacaagctcc tggtaacagt gtgaaatctg cctccacagt gcctgcttta
56341  atttcttggc tacacagcag attcatggta gtggtggtag tggtgttcat tttctctaaa
56401  ataacagttt gaataatttg gttttgataa tgcactgcat ttattataaa ttagatgatc
56461  agagaaagat tgcagggata agaaattatg cttttgataa tctttagtta tattcttaat
56521  tttcttcatt attatttaaa tgtaaaaata aatatctgtg agcagtagta ttttcctgtc
56581  atgaagctga aattactttc ataaatatgt gtgaatattc taaagagaat gactctgtag
56641  gatttaaaga aattaattct tatttttgct ggcatttatt tattttatca gattcacttt
56701  ctcatatatg tctctcttca tggcaccata tgcctaaagt cagcttggat agtttggatc
56761  ctccaaggaa aattccttcc acaaacatgt gcagcacaca gtgctagata attaatagag
56821  aatataaaat gggtttcctg tttcaagatg gtttgtaggt ctgtatgtgt agggcattga
56881  caagagagta aaacataaat caccttagta caaagtaagg agtgaatggc atatcttaga
56941  gaaaaaaaag ttactgggct ataagagaag gcatttgtga gtttttccct ccctccccgc
57001  ttcccttccc ttcccttccc ttcccttccc ttcccttccc ttcccttccc ttcccttccc
57061  ttcccttccc ttcccttctc ttccctcccc tccccttctc ttccctcccc tcccctcccc
57121  tcccctcccc tcccttctcc tttctcttcc ccttcccctt cctcttcccc ttccccttct
57181  ccttccttcc ttccttcctt cctcttccac ctgccttcct tttaattttg ctatgagccc
57241  ttaaagagga ttttagtaat ttgctactta aattaaatat atttgctaga tgttgtgcta
57301  ggcttcagga atacaagttg gattgcagta atgtaaagcc ctttgcattc tagcaagaaa
57361  acagatgggt atgtatgttt gctcagtgct acattaaatg aaatggatgg gagccgggag
57421  gagaaatggt gtgtttggcc tgagaggtta gtagcaagga cttctctgca agaaagtttg
57481  aagccaattc ttcaagaatg aacacctttt tgctgggtga aaagtagagg aaggcatttg
57541  gggtaataga aatagcataa aaggtaatga ggtttgaaaa attacatgct gtgtttggaa
57601  gaatgtcctg gagcagcagc gttttagaag gtttttaaag acgatggtga cttgatcaga
57661  gctctgtagt gctttgagga tgggttgaag gtgggcgtac ttggagactg gtgggcattt
57721  aattggtgcc ttccaaccac ataaatgaat gtcccctcaa atcccttgga aacactttaa
57781  ttctagaaaa ttcaaaaatt gtccccaaca tctttttcct ctgagttggt accctggatc
57841  tttgggtctt cttttctttc cttttttgat gttttatttt gggtaatgaa agtcacacag
57901  gttttgaagc cagcagattt ggcttcaaat ccaagtctca gttgcttgct agctgtaagg
57961  gacaaattat atatcttttc taaatactca tctataaaat gggagtaata attgctatgg
58021  cataggattt ttttaaaaaa aagattagaa atcatgtgtg tacagaattt agcacagtaa
58081  ctgatggata ttatttctat tacctgttat cttggtcttc tagttgatag ctccttgcta
58141  gcgtctagct cctttccata gctcttcctg agtagggcca gcatgcagtg ccacagcttg
58201  ctaaggcttc tcctggattg ctgagttgtt ctagtttttg tggcacctca catgctaacc
58261  caccctgaac acatgctctg aaaacataac atttagagga aggttgaaga ctgagagaca
58321  aggtatatct ttgaggaaat tcagatgctt gtcttgagga gctcaggaaa gctagacacg
58381  agtaatgact gtcgtttgtg tgtggcatta ataaatttta caatagctat gtccccattt
58441  agttattcta tgtcacaaat aaaggcagga cagtagtatt tactgtgtta aggtactggt
58501  ttcccaggta tcttacagtg agaagacaga agctcagaaa gtgtaagcaa tgtgcatatt
58561  tggtggagtc tggatgtaaa cagagatctt gatgccaagc ctgtggagct ttgtctccat
58621  ataatgttgt ctctttcata ataactgact gtcatgtggc agattattca tgctattctg
58681  acattgatgg cattaatatc atcttatttt cccaatctat tcaaggatca gttttgcctt
58741  attttatttt gtttcattcc aaattggaga tgtagagaaa aatcacatga agtttgattt
58801  gccagtctcc taaaaggaag aaaaatgtag atttttaata tacttaattt tttgtcttta
58861  ataggtatgt gagaaagaga gcagtcccaa attggtggaa ctcttactga atagtggatc
58921  tcgtgaacaa gatgtacgaa aagcgttgac gataagcatt gggaaaggtg acagccagat
58981  catcagcttg ctcttaagga ggctggccct ggatgtggcc aacaatagca tttgccttgg
59041  aggattttgt ataggaaaag ttgaaccttc ttggcttggt cctttatttc cagataagac
59101  ttctaattta aggaaacaaa caagtaagta acaaggagaa tattttttac aattcttatt
59161  tttaatagta tttttttaag tcactagtct tttagtggtt attcatgcca gtttgaggga
59221  ccttaagcca aagatattgc aaaggtttgg attttttttt tttttggcta tgaaatactt
59281  caaaatgaca tttaagttct ttatgagata gcaaatagtt atttataaaa atagagcaaa
59341  atagtggaag ctttttgaag gggtactttt taatatatat tttttattat taaagtaaga
59401  tatccctgtt tttaaaggaa atataaaatt ataaaaaaga aaataaaaat aacttatttt
59461  atctcttata agtaattaat atggatattt ttcctaactt tttatatgct tacatgtacc
59521  tatgcattca aatgtatgta aaagcataca cacatattta tttggcattt ttaacttaga
59581  atatacttta tatttcaatt gataatgcat tttctttata ctttcaagct catgtgtatt
59641  ttgtacatat tatgtgtatt gatggtaagt taccatcttc tgacactatt tttatctttt
59701  gagctctctc atttgttcac actaaatgtg tttttagcgt gaaagctccc agctttccct
59761  gtgttaactt agtcccatgc ccatctcctt ccccatggtc atcaaactcc atgaatcaac
59821  accttaagga ccatcttgca agtaacatgt ttgcttctct catttttatg atgcactcac
59881  tagcaaaaca ccagttttgg tcagtctacc agtctacttt ttccctcagt ttcaccaaga
59941  aaactgagtg ctgctagaga aaagtaccca tccatgcaat ttggtgcctt tatacatcaa
60001  ggtttccaac cgctcagtag gctccaaaag ttccaatcag gctgaatttt cctcggtttc
60061  tcaaacactt cgtgtaccct tacttccagt ctttttccag tgttactctc tctctaccta
60121  gctctaaatt ctctcttcac ctggctgtct cttcattctt cctgtctcag tgctatcacc
60181  agtctggaag gttctcttac atgaccctat agcactttat ttctcacata tactaccatt
60241  caccacatta tataatttaa tttttcattt ttataatcta ctttttggta aattgttagt
60301  accatgaagt caatgtcaat tttgttcatg gttgtaacct taccattgat actagtgttt
60361  tgcacatagt agattatcat ttagaattaa gtattcaata ttggcaaaaa ataaaaattg
60421  tgtaatacat tatgttgata agcatgtgtg gaaacatgct tcatatattg atatgaattt
60481  aaattgtcct cttttgagga caatttggca atatctacta atatttttaa tatatgtaca
60541  tacttttttg acctcacaat gtactgttag gaatctatga tacagacatt ctcaatgtgc
60601  acaaaaatta tgtacaaaaa tgcacattaa aacattgttt ataatagcaa aagagtagga
60661  aaaaaaccta agtattcccc aaaaggaact attcaaataa ataatggtac atacatgttg
60721  tggaatgctt tgcaatcatt gaggaaaaaa aaacgtggag caaatttaat gtcctgataa
60781  agattacatt actccgtggc aaaaaaaagg gcacagacag tgtttttact atgctaatgt
60841  tgatgaaaat gcaactggaa tatgatagtt ataaaagttt gaatatgaaa taaaaccctc
60901  cagaaatggg ttccctggtt gtctctgggt ctttggaaat tactgagaca tggttagatc
60961  ccatgtttca ttacttaaac tagtcttatg ccaaaaacct gcttacttta atcttcaata
61021  tccgatggag aggaattgtg ggcccattgg agagggacag agggagattt atcattcact
61081  atattctctt tgttctgtct ggagttttta ccattgacac atcttaccca gttaaaaaaa
61141  catagaattg tcatttgatt aattggaggg tataaccatg atttcactgg cagctggtct
61201  gagtaaagaa cactttgggt catagctttc aaacattttt caggtagtat ttgcctaagt
61261  gacatatttg tgtgtgagct catcctaccg ggcttcggga taatttccca tatcataaca
61321  tattactctg gaaaaaggaa ccatttgggt atatgggtat agtgtaagcc atagtatcag
61381  ttgccttctt ggggtttatc atatgggtcc accacatatt tacagtagga atagatgtag
61441  atacatgagc atacttcact ctgctactat aattattgct attcctactg ttgtcaaagt
61501  cttttagctg attatctaca cttcacaggg gtaatatcaa atgatcccgc accatgctct
61561  gagccccagg gtttattttc ctttttacag taggaggcct aaccagcatt gtattagcca
61621  actcatcact ggatattgta ctacatgata cagcagtatc atgtagtaat atttgaagtc
61681  attacagtag taatatttga agaaatcttc ctggctgtat gtagaacaat gaggactcag
61741  ccaacttatt cttcatagta gagctaatac ataatgtaat gaagttgtga gaagaatgtt
61801  aactttgaaa ttccatcagg tttcccaata gtcataatga atcactcagc aaactttata
61861  aaaataacaa gatcctttat ttagcagttt atgtgttcta tgcattgtgc taaacatttt
61921  atatgcatca tttcaattac tctttttcat caccatatac tgtatttatt atcatcttca
61981  ttttccaggt gaggacactg atacccaggg agctcatata actcacaaat ggcatttcta
62041  tgacttgaac acaggcctgt ctggcttcaa agcctaggcc ttttcctgaa taaagttagt
62101  tccatagaga ttcagttttg ctgtctacat gaaagcattg tgtacatggt tatgtttttt
62161  taaaaaatat atgatctgcc acctgttaat tattcaggat cactagtgta aggtgacttt
62221  gaaaggaaaa atagaaatat tctccagaag catagcaata cgtaagaact ttggtcctat
62281  gtatgtttat ttttgcataa ttgttgattt ctaagttgct ggtgtatctc ttattttcag
62341  atatagcatc tacactagca agaatggtga tcagatatca gatgaaaagt gctgtggaag
62401  aagcaacagc ctcaggcagc gatggaaatt tttctgaaga tgtgctgtct aaatttgatg
62461  aatggacctt tattcctgac tcttctatgg acagtgtgtt tgctcaaagt gatgacctgg
62521  atagtgaagg tatttattat aaaaaaaaac cctttatgct ttatatttac acactgacat
62581  tgaacaatag gacccaagac aaaaacctga cctaaatcat ctggaaaaac ttgagtagaa
62641  atgtgtttat tatcgcaaac agttaagttt actaattttg gttaaagtga tgggtcaagg
62701  aagtgtgtct ctgtgcttct aaatgttata ctaattggtt aatggttaat attccaggaa
62761  acaaactctg actagactgg aacgagattc cacgctctgt cattgactag atcctttcgt
62821  ggcttgtgta agccccttaa ccttgttaaa ggtagtaatg tcgactttgc agggttatac
62881  ataataatta gaaaaaatgt atgtaaaatg tctgcaacaa tgcttggcaa acaggaagcg
62941  cttaataaaa aaggttttta tctttactat agcttaaaac aatattaata ttttaatagc
63001  tcacttgaga taacttttta aaaaattaat atggtgaaat atataatgac aatgattagg
63061  gctgatgtat ttagcattag cagtttggta aaaatggagt gaggggcttt cttattaata
63121  tagtatgatt gaaaacactg ggtgatagaa taaggatatt tgagagggca aaaaatgaga
63181  gttgttccaa aatattgtgt ctcaagtcaa accattttta aaaatcaagt gtagtgattt
63241  atatacatat ataatttata taaaataaaa tgcattcact ttaagtatat attttcatgc
63301  gctttgaaaa ttaacatatt catgtggtca ttgtcgctat taagttatgc aatattttca
63361  ttatccaaaa aagtttcttc atgcctcttc actgaaaaca tccccttccc ctggcccccc
63421  tgacccttag caatcatttg cttcctgaca atgtagatta atgttttctc tagttttata
63481  tacataggat catacagtga gtactctttt gtgtctgttt tccaaaatga ttgtactatg
63541  ttctaccccc accagcagta catgagcatt ctggttgctc tacatccttg tcagtacttg
63601  gtattttcaa gttactttta gctgttctag tggaggttta attcatttag atgtaatttt
63661  catttccctg atgactaatt atgtagagga tgttttcatg ttcttattgg ccattcttat
63721  ttttgtgtga agtgttgaag tattttgctt ttaattgggt tgtcttatta tataagagtt
63781  ctttgtatat tctagataga agttgtgaca ggtatatgta ttgcatattt ttttcccagt
63841  catagcttgt cttttcattt tactaattct atttttaaca aaacagaggt tttaaatctt
63901  ggtgaaatgc agttttccag tttttttctt ttatggtttg tgctttttgt atcccactta
63961  agaaaccttt tcttagccta aatttgtgaa tattttctcc catattttct cttagaagtg
64021  ttaaaatctc agctttggca tttaggtcta aaacatttta agttgatttt tgtgtgtggt
64081  gtgtcaatga agagttgaca tttatttctt tctgtatgga tatccagttg ttccaacatt
64141  acttgttgaa aatattatat aattcctcat tgaattaatg gaagctttgt tctctttaat
64201  tgactattta ggtatggttc tattttagca ttatttattc tgtgccactg atttatacct
64261  tattcttatg ccaataccac actgtcttga ttactctagc ttaatagcag ttcttgaaat
64321  cagatagtgt aagtcctctg gtgttctttt aaaaaaaatt gttcttatta ttctaggttc
64381  tttgcatttc catataaatt tttaatcaac taactttatg ctgggatttt tattgtaatt
64441  aagtccatat ttagatttta tatagaattt atttataaat taaatcatat tacctatgat
64501  tttaatgtaa tctataatat ataattaata cataaataat aatttatata gattatatct
64561  ataaattaat ttgagggaac taatatatta ataatgagtc ttttgacata ttaatgtgat
64621  atatagttca attagtcttt taaaatttct aacagtgtgt tgtattggat gttttctgat
64681  actattatag atggtattgt atttgaattc taatttccag tagttcactg ttgatatata
64741  gaaacataat tcatttattt gtacatgaat tttgtatcct gtaaacttac taagctcact
64801  tacgtgttcc agtaccttat tatagattct acaggatttt ctttgttcac aattatacca
64861  tttggtaata aagacagatt tgctttttct tttctaatat ttatgtcttc tttttttttc
64921  ttgtcctatt gcactggcta ggacctccag tactatgttg aatagaattg gtcagactgg
64981  gcatcattcc ctggtttcca aacttagagg aaaaacatac agtctctaat cactaattat
65041  gacattatct gtagtttttc atagatgccc ttcatcaaat tgaagaagct tcttcctagt
65101  catttttgga gagccttttt ttatcattaa taggtgttga aaaatgctct tcagcatctg
65161  ctgaggtatt catatttttc acctttattt tgttaatatg gtcaaataca ctgactgatt
65221  ttctaatgtt aaaccaactt gcattcctgg agtaaatctc acttggttat ggtacattat
65281  cctttttata tagtattaaa ttttgttttc taaattttgt taagaatttt gcatctgtga
65341  gagatattag tctgtagttt tatacttcct tgtaattatg ggagtaatgc tggcctcttg
65401  aaatgaattg ggaagtgttt cccattccac aattttctgg aagaatttgt gtaaaggtat
65461  tttcttctta aatgtctgat agacttcacc agcaaaggcc atctaggttt taaatttttg
65521  tgaggaggag atttagaatt atgaatttaa taactttgat agatgtatga ttattttaat
65581  tttcttttac ttcttaagtc agttttagta atgtgtaact ttcaaggaat atgcccattt
65641  catataagtt gccaaattta tttgtttaat gttacttata gcaattattt aattatcctg
65701  ttagtgattt aattatcctg ttaatctctt caaagaatca gatttttgtt atattgattt
65761  tctctattgt ttgtgttact ttctcacgga cttttgttct tatctttatt gttccctttt
65821  ccttacttat ttttatttta attgactctt tttatagatt cttaatttgg aagcttagaa
65881  cactggtttt tagaaccttc ttattttgta acagaaacat ttatttaagg ctgtatatgt
65941  ccttataaat atcactttca ctgcatccca atattttgat gtgtcctctt tctattcatt
66001  taaaaaaatt taatttccca aatgtccaac aatgatagac tggattaaga aaatgtggca
66061  catgtacacc atggaatact atgcagccat aaaaaatgat gagttcatgt cctttgtagg
66121  gacatggatg aagctggaaa ccatcgttct cagcaaacta ttgcaaggac aaaaaaccaa
66181  acaccgcatg ttctcactca taggtgggaa ttgaacaatg agaacacttg gacacaggaa
66241  ggggaacatc acacaccagg gcctgttgtg gggtgggggg aagggggagg gatagcatta
66301  ggagatacac ctaatgtaaa tgaggagtta atgggtgcag cacaccagca tggcacatgt
66361  atacatatgt aacaaacctg catgttgtgc atgtgtaccc tagaacttaa agtataataa
66421  aatatatata tatatatata aatttaattt cctttgtgac ttctttgatt cacgagttat
66481  ttagaaggct gtcatttatt ttccaaatat ttggagattt tctggataac tttctggtta
66541  ttggtttcag tttgactttt ttgtgctcag ataacctact ctgatgattt ctatcctttt
66601  atatttattg aaacatgctt tatggattat catatggtct gtcttggtga tgtgccatgt
66661  gcaactgaaa agaaagtata ttcagctgtt attgggtcaa atcaataggc caaattgttt
66721  ggtaggattt ctcaagtttt ctgtgacttc actgactttt ctttctattt attgtatttg
66781  atagagaaat attgaagttg taattgagga tttgttggtt gccttttcag ttttatctgt
66841  ttatgctttg tatattttga agctctgttg tttgttgtgc aaacattaag gactatataa
66901  caaaggacta tatataaaaa taatttttta aataaagtaa ccactttata ttaatctaag
66961  tttgcagtta gtatcatttt cttcctctgg aagaattccc ttttacattt ctagcgcaga
67021  tcttccagaa acccatatgc agattactgg agcacttttt ccataactgc cccagaactc
67081  ccagctatct cagtctccat aaatgttgat ctctatctcc tcatctcagg aaagccatga
67141  gactgtttaa atttcttctt atgtatatgg cccttaattt gtctctaagc agaaggccag
67201  agtgatcata gggctcacat aatttgtttc ccttctttcg ggtattacat agtcctatgc
67261  tgtctgttgc ccagtgtctg tatccagtca tttaatatat ttatccagtt ttcttgttgt
67321  ttatgtgagt aggttaagtt cacatttgtt actctatcat gcctgaaaga actctcttca
67381  agtatttttc aagaaaacat tagaaatttt aacataaaat aattataaaa tgcactatgc
67441  tgcttgtaca aataatgtgc taatagacat ttcacacaca tataaagtgg gataattatg
67501  ataatttaat aatctttata attatgtaat tgttactaaa ttataaatta tattttatta
67561  aaattcttaa gcattaaaat aatttaaact atagaattaa ttaaacttta gcaatgaacc
67621  aggcaggaac tgtttatatc aggatttata ctgaatatta taagggtggt atttggatat
67681  acagatttta taatttggtt ttgacataaa atgaattctt tctttttcta agataaagag
67741  taggggaatt taaaatcata attaattttt cacaaatgca ttcacattat ttatctttaa
67801  tatgattttt ttattgatct ttgttttctt gtaaatttat tattttgaaa gtactgcctt
67861  ttccttattt tataaaacaa ttattgccag ccaaatttat tgtgtttatt ttaataccat
67921  tccataaaag aaaccatgaa acatgaaatt caaatagaaa tttattaaaa attgctgact
67981  gttaaataat ttgtgtgatt acaacattta aagcaagatt tgaaaaattt ataagaaaaa
68041  ttcagggagt taatccactc tctttcctat gctgctagac cctattccag cggtctccat
68101  aaaaaaaatt cagagaaaca gaaagcagta acagtgatca gattgcatac aaactttctg
68161  cacacacata tatttgttat aacttatgta accgttgatt gatttggctt tttcctgctg
68221  gactatgagc tcctctgagg aatagattta ttttttttct gcatatcagt atcccgtgct
68281  tacccagtgt ctagtctgta acagccattc tataaatatt tatgggtgaa gaaaaatgtt
68341  gttgaatttt taaagtgaaa aaccaacatg gcttatcatc tctattttaa agattttaca
68401  aagggaatgg actgtgaaat ttccattaat aaaaataggt ctaatcttcc atgattgaac
68461  tatgatagaa ggatcttatg attgagtaag ctttttgtat tcaccttcat gttattttat
68521  cattttcaaa ataggaagtg aaggctcatt tcttgtgaaa aagaaatcta attcaattag
68581  tgtaggagaa ttttaccgag atgccgtatt acagcgttgc tcaccaaatt tgcaaagaca
68641  ttccaattcc ttggtaagtt aaattgtgca attgtgatta tgttgtgttt tgctgctgac
68701  attctcttga taactaaaat ttatgccaaa gctaggaaca attggtaggg atttccctga
68761  tgtatgaaaa ctataatttt gagattttta tatatgtaat agatatgaaa acatattaga
68821  tgtaaattat gctcaattca catttgtagt cttttgagta tgcagggtat gaattttttg
68881  gggcacatat atatatatat atatacttac agtacacttc aagatggttt tcttctttct
68941  tttcagaact ccatgtctga aaagagccca ggctagacct ctacctaatg gtgtgggttg
69001  gtcccatgaa cactttagct agaaatctga tagtgatttc taagaaacca gacagaagtc
69061  tgaaggacac tgaacaagat ggagtagcat aatataattc attgttcatc tatctatcta
69121  tcatctatct atctatctat ctatctatct atctatctat ctatctatca tctatctatc
69181  aatcatctat ctattttggt gtattgaaag tcatttaatt ttttagatac ctttattatt
69241  atttcaacct cttgtctgtt ttggaactat ggaaggacta tggcatattt gcatgaggag
69301  tctgataatt ctagttgagg aaattgggag ccaccttatt ctcaggttca ctttgaaaga
69361  cctgttctaa cctattctcc aattttgatt atagctgagt actaaaaata tgagggttgt
69421  tttgtgttaa ttctagatct taagatgggt gaaatgaatg actgtagttg aatcggttaa
69481  attagctgtc agtctttata tgctctttcg aatttatata taaatttagt tataaaaagt
69541  agtttggtta atgagaaatt atatggatat agctttttca ctcaaccttt ctgtttttca
69601  gtttccttat atttaaaaca aaggagaaag agtagatgct ttctaaggtc atttgagcac
69661  tgaactggag ttttctttta tcctcataat tgggttctta gtttttactt gcctattttt
69721  tcccataatt ataaatacca ttaaccctat taaaatttca tggttccttc cttataaaaa
69781  tgtcctcttc tccaataaat gacagcaatt ttattataaa ttatttttta ataggggccc
69841  atttttgatc atgaagattt actgaagcga aaaagaaaaa tattatcttc agatgattca
69901  ctcagtaagt atttggatgt aatcataagt aaatagatat tttgggcaga atgcagtgtt
69961  tggttgaatt tcctccaatt attcaaatat tcttggtgcc agtttcatct tacataatct
70021  tcatatatat ttacctaatg attccttcca taagctatag aaaaatgaaa catacattta
70081  aaaatttacc tttcttgaat attatagaac acattagtct ttttttttac aagttttctg
70141  aaatgtaaat aacacctcca ccaaggccac tcttcatcct ctcctccagt tttctttctt
70201  tctttttttt ttcacaccac ttacaccacc tgattaaatg ctttttattt actgttcatc
70261  tctgccactg gaatgtaaac tccatagttt ggttttctac tgaatcttca gtgccttgaa
70321  gaaagcctga ttgctaggag gtgctcacta aacttttact acatgaattt acattatttg
70381  cttatatttc cattgtttgt ttgtttttga caaaagggtc atcaaaactt caatcccata
70441  tgaggcattc agacagcatt tcttctctgg cttctgagag agaatatatt acatcactag
70501  acctttcagc aaatgaacta agagatattg atgccctaag ccagaaatgc tgtataagtg
70561  ttcatttgga gcatcttgaa aagctggagc ttcaccagaa tgcactcacg agctttccac
70621  aacagctatg tgaagtaaat ttaatttatc cttgtaactt tcaagacatt tgaagagctt
70681  ttgtatttat atctaatttg cataattaag tcgtttaaaa gaacattcta cttttgtgtc
70741  actgggtgat aagtcccccg tgcctctggt ttttgcacac atatcttagt ctgtgtgatg
70801  ttcaggagca tctttgaggg caggcaatgg aaacagatct gattagaaag gaattccagg
70861  ttctgtacgg agtacatgtt aaagtctgtc aagtgtatat tgattatact ttaatcattt
70921  aattcaagta agacaacttc aacaatttaa attagattag gtaaactaga attagacctg
70981  gtttggtagt actggctctg actcagctac caactgtgtg acaatatgaa catgtcactc
71041  cgcctgctct aatccttcat tttctcatct gtgaaataga gattcaacta aatgattact
71101  gaaagttttt ttttcagttt aaaataatgt gtaacttaaa gatttttttc tttttggtca
71161  aagttcctgt cttgtaagaa ttaaagtata acatagtttg ttgataggat agctctctga
71221  aaattgactt tgctcaccat ttgtatgtac tacagatcaa aatagttttg aaagccaaag
71281  aagatatcat aaaagttaaa attattttaa tgcaaatgtt taaattgtta aattctcaag
71341  gctgggcatg gtgactcatg cctataatcc cagcactttg ggaggctgag gcgggtggat
71401  ctttttgagt tcaggagttc aagaccagct taagcatcag caaaatcctg tctctaccaa
71461  aaatatgaaa aattagccag gtgtggtggg gtgcacctgt ggtcccagct acttgggatg
71521  ctgaggtggg aggattgcct gatcctggga ggcagaggtt gcagtgagcc aagatcgcac
71581  cactgtactc tagcctgggc aacagagtga gaccctgtct caaaaaaaaa aaagtcttgt
71641  tgcaaatgca tttccccctt tttaagccta aaaaattaat cataattttg agatgtttta
71701  aaggcaacat tacataaatt ttaagtatat ttaagggatg ttttttctct aaagttttta
71761  tatctggaga cagagagaag aaagaaaggt gacccactcc tccagccatg ccctaatgtc
71821  taaaatgtgt ctttctctct tctccacttc tttgccttgc taaatacttc aagccaccca
71881  gggctcaatt taactgtcac ttcatcactc tctgcttctc ttcctttttc cttctaccct
71941  ccagccaacc tacccaccta gcctccatgt ccataacacc tgatgctttc cacctaattc
72001  tcattcggtc atccaatttg atttaaaata cctttgcccc cactagactg tgaactcttt
72061  gaggacagga cttgtttcag gtttgtttct gtctattccc agtgcctagt gagatctgac
72121  atatggtaga agtttagtac ttactgaatt gatttgtgga ggaataaatg tctgaaactt
72181  ggtaatcctt caattaatat ttgttaaatg agcaagcaaa ataattttgg gatttagtct
72241  agttaaaaca aagagaattg gaagagactg tgacaaggtg agacatgccg gcattcaatg
72301  actggacaag ctcagaacct tctcttaggg aaatttcaaa atgacaccat tagatggcac
72361  tttgtttgtt tgtttgttat tgtcaaaggg tctcatctat gttcctttta taggaacatt
72421  tcctgattaa accttgggaa taattttaaa atctttactt cagaataagt taatgagggt
72481  ctgaaacaaa agcaggaatt ttgaaacaac ttctggggct aagagtggtt aataagcctc
72541  tataatgata tcaaactcta gagtttctcg tgtggataaa tatattgata aataaagaag
72601  accatagaga agtgattgat tttggtattt tagctctttg agagtattac gtacctaagt
72661  tttaaaaaat tgacataatg tgtaagtagg ggtttgctat tatcattata aaattagaaa
72721  ttgcttaaaa atagaaagta gaaatttgaa acaaaaagtt tcgtaaaaaa caggaggttc
72781  taaaatgaaa cacattataa gtaactattt ttatagttaa atcttaaata tatcaaaata
72841  tgtaaaattt ctgacagcat ttaaaacata ttcccaggat tatattgtac tttttgttaa
72901  atcattaatt caaatatttg ttgaggcgta ttctgcttcc attttgctct ttctggaaat
72961  aatttacaaa aaagctgaag gaagctttca actctatttt tgtgaacctg ctttttacaa
73021  tctacctgtt gtaattttcc tggttttacc catgctacaa gcagagacga ttgggccaat
73081  tagtatactg aaattctgtt gtggattgtg ttttcaactt tttgaaaatt cttgatggtt
73141  ctagttacca gaggtgtgta aggcagaaat attagctaga cttaagttcc tcagatggtt
73201  cactttagaa ttttaaacta ttgtcttttc agactctgaa gagtttgaca catttggact
73261  tgcacagtaa taaatttaca tcatttcctt cttatttgtt gaaaatgagt tgtattgcta
73321  atcttgatgt ctctcgaaat gacattggac cctcagtggt tttagatcct acagtgaaat
73381  gtccaactct gaaacagttt aacctgtcat ataaccagct gtcttttgta cctgagaacc
73441  tcactgatgt ggtagagaaa ctggagcagc tcattttaga agggtaagaa agagctcatt
73501  aaaaataaaa gggttgccta aatatgctga tgttaacaaa atatgctgac atttttatag
73561  caatgagttt taacaacatg gtgaaactcc atctctacta aaaatacaaa aattagccca
73621  gcgtggtggt gcgcacctta taatcccagc tactcagagg ctgaggcatg agaatcgctt
73681  gaacccagga ggcggaggtc gcagtgagcc gagatcgtgc cattgcactc cagcctgggt
73741  gacagaggcg agactctgtc tcaaagaaat atatatatat atatatataa tatatgtatt
73801  ataatatata atacatatat tatatatatt tattatatat aatacctata tattatatat
73861  atactatata taatacttat tatatatata ctatatataa tacttattat atatataata
73921  aaagaccgag gcaatgaata ttacaacttt tatcaactga cttacatttt tacaactaat
73981  ttttaaatta atgagtcctc tttgatgctg ttctttgaaa gcaaattgtt tttgatattt
74041  tttctttaaa gcatatgaat ttatgcaatt taatcattat cttgtctctt gtgactagaa
74101  ataaaatatc agggatatgc tcccccttga gactgaagga actgaagatt ttaaacctta
74161  gtaagaacca catttcatcc ctatcagaga actttcttga ggcttgtcct aaagtggaga
74221  gtttcagtgc cagaatgaat tttcttggta agtgttctgt gtgggtctcc tccttaccag
74281  gccctctaag ttgtacaaga tgagtcatat atggaccctt tagttgtgga tttaaaagtg
74341  gcatttcagt ttaaatatta tgctggattt aaaaaataaa attagcaggt tggcaataaa
74401  acaaaatgct ataaaactat gaaaagacat gaaagaaaca taaatgcata ttggtaagtg
74461  aaagaagcca atctgaaaag gctacatact attcgttccc aactataaga cattctggaa
74521  gagacaaaat tatgaggaca ttaaaaagat caatggttgt caggggttag gggagggaga
74581  gatgaataga taaaggacag aggattttta gggcagtgaa actgttttgt atactataga
74641  gggggatatg tgccatggta cacttgtcaa aacccataga atgtgcagta taaagaatga
74701  accctgatgt aaactatgga ctttgagtga taatggtgtg tcaaatattg gctcattgat
74761  tggaacaact gtgccaaact aatgcaaatt gttaataata ggggaaaatg tgtgtggggt
74821  ggtggtggtg gtgagggaag aagggattta ttgaaagtct ctggactgtg tgctcaattt
74881  tcctgtgtat ctaaagctgc cctaaaaata gtctataatt taaaaaaatt atcacatttt
74941  tattgtcaga ggttaaaatg atagttactt ggcctactgt gtagtaccct gtggttccct
75001  ttagtcttaa actaaacatg cacatggctg cctgagctgg gtaaggcatc ctgatactga
75061  gatattgttt ttcatactga agtttcttca gcaacttttt gtatgataaa tatgattact
75121  ctttgctgtt gttagaaata aaattaatca tttaatggtt ttcaaattag tgaagttaat
75181  gtatattcat tcagctcttg tgctttgcaa gacattataa taatgcaaat tattatcatc
75241  acttttatta aaagttgtag aatcccctgc cttctcttag catatgaaat aatagaggaa
75301  attatgttca tttgtatcct aaatgaacat tttaatttta aggaacaaaa tacttttatg
75361  acaataaaca ggaattcccc atattttatc ttccttcata gagaatactc acatcttgcc
75421  acccatgtgt ttattctata cctgactcag taaaataatt tttaattcta tttaatagca
75481  gaatatggct taactactta ttaatagtat tttaatttgt cagagctttc agaaactaat
75541  aatgatcaaa gccatcttaa tttggatagt attttcctcc ttttccgcgc cctcccttcc
75601  tcctaccctc ctgaatattg gccactttcc aaccatgatc ttgaaactgg ggatagagtt
75661  gttgactcac tcactagtaa gcaaagcaaa ctcggtctct gtccttaagg aacttgtgat
75721  taagttcaga gcacagacaa acattaagta atgatggcat aaataaatgt aattttatat
75781  tgtggtgcct tctgtggaaa agtttcagga tgctctgtgg tacctcaggg gaatctgatt
75841  cagactgtgg agtcaggaaa gcatttctgt gcctgtgaca tttaagttgg gtctggaaag
75901  aggagcatcc aaaaggacct tgaagcttgt gtatggtcag tgtgtcatct cagctgaatc
75961  tcacaatgat cctaatggtc atacctagga caggtatgac ctctgcccaa ttgaggaaat
76021  tgaagccgtg agattttttg tgtgatgctt acaggtgcat acaattaatt atcttgttgt
76081  gaaagtttga atggacccca gttcaaaatc tgtttgcttt gcactacaca atcttaatag
76141  tttgagaagt ggtcttacat ttgagaagat gccccatgga ggtgacatct gagcaggacc
76201  actaaccatt aaaaaataat cccatgtaaa tggtgaaacc ctgtctctac taaaaataca
76261  aaaattagct gggtgtggtg gcgtgtgact ataatcccag ctactcggga ggctgaggca
76321  gaagaatcgc ttgaaccagg gagtcagagg ttgcagtgag tggagatcag gccacagcac
76381  tccagcctgg cgacagagca agactccgtc aatcaatcaa tcaaacaatc aataaatccc
76441  atgtaaaata aagttttagt tctgtggcct tatgagtgtt ttccatacag catatgaaac
76501  tcaagactct gaagtcttaa gtggagaatc atttcgattc atttattttg cgaataggtg
76561  aggtataata gctatctttc tgcttctcag gaagacagct tctaggagtg tcctggaaca
76621  ttttgaccct tgaagattgt ctagataaag aataacccat atttttaacc ttgaaaatgc
76681  taataactaa ttcctcaatc cgttttccta gaaacggtag atttgtagat ctctcaagtg
76741  tcttagtgtt catctgatct agctacactc ttttacagat aagaaaagag actgtgtgat
76801  tttctgtttc ccaaattgtg tttcacagaa ttttatttct tggaggtacc tcatactgca
76861  tttccttctt aacaattcat aaagtaggcc aggcacggtg gctcatgcct gtaatcccag
76921  cactttggga ggccggggcg ggcagatcac aaggtcagga gatggagacc atcctggcta
76981  acatggtgaa accccatctc tactaaaaat acagaaaatt agctggacat gatggcacct
77041  gcctgtaatc ccagctactc aggaggctga ggcaggagaa tctcttgaac ctgggaggcg
77101  gagattgcat tgagctgaga tcacgctact gcactccagc ctgggcaaca gagcgagact
77161  ctgtctcaaa taacaataat aataataaaa taataaaaaa taaaaaaatc ataaaatata
77221  ccagagtatt gagaactcag atattttact ttattttact gaatgtttcc caatctctta
77281  agattatatg atcgaagaat acttttatta aataacaccc attattattc catagaacac
77341  tctttatgag ctagtaccat aaagaatttt tgtccttata atatactgta ttttagtaaa
77401  gtgttcatag ttgttttgct atagttattt aagagtaatt ctagttaatc tatatggtat
77461  ataataacat cacacataat ttgaaatgaa tattctagga atttttactt tgtacaaatg
77521  ttgggtagac aaatttatat ttaaatagct gtttagcttc caggttaaaa ggttctggta
77581  agtatagcat attattttat tgtttagtgg aagtattaag gctattacct tatttttaaa
77641  agtggatttt taaaaattgt cagtgtgaga atggaaatca aattaagtga cacactattg
77701  gtagctgttc ttatttttga atttaatgga aatctggtta aaatgattaa aatgtttatc
77761  tcattttttt tcttttagct gctatgcctt tcttgcctcc ttctatgaca atcctaaaat
77821  tatctcagaa caaattttcc tgtattccag aagcaatttt aaatcttcca cagtaagttt
77881  attgttattt taattttaaa agcacattag ctggaacaga acctttagaa acatgatttc
77941  gatttagtca tatagaggta attgatttct aaacctactc aacttgatgt ttttgtatgt
78001  atgaatgatt ttcactagat aaaagaccca actcattact taaaatggaa acttttatat
78061  ttatttagtg gatcattgtg taaaaacaac ttaagattgt ttaattaatt gctgtattag
78121  tataatgaaa tgagattata ctggctatca ctttaacttt taaaatttta attgtttgtg
78181  gaacttgata tgttgccaaa atacccttaa ctttcacatt atgcttaagt tatgtttgag
78241  tgaaattttg ggggaagatt aggcaagttt atgtagttcc aggtttttga gatattttgg
78301  ttaattcatg aaaccagaag cttcctgtta actttaaatt caggcattaa ttggatcttg
78361  agtgtgttgt aatcttaaat gctattctaa ttatatcata catgataaac caaattcata
78421  aaaatatatg tgtaaattta tcttttcctt tgttttcttg ctgtcagcta ttccttcaaa
78481  cactatggct ttttagaatt gacactaaaa tgctgcttgc atgatgctgc aatgagctct
78541  tctgtgagct tatatttagg caaataataa ttagaattta gccatagaga gtgttacaca
78601  aacctataat agctaaatta cgtctagctt tagaatgtgt ttaactgttc taactactct
78661  acagcggttc atctctttaa tcttcctaat aatgccctac gatagccact gttattatct
78721  ccacttcata agagatgaag taacttgccc agagtcatag ttcttaaaca ctggtggttc
78781  aacttcaggc tctcaaatca catgcatact catatgtcaa taatgctagt tttgacgtta
78841  cactttattc tcaccctggg gaaaattatt tgtgatgtta tttcatgtat tttggaaata
78901  ctcatttggt ttatgtcttt tctgtgtatt gttttagctt gcggtcttta gatatgagca
78961  gcaatgatat tcagtaccta ccaggtcccg cacactggaa atctttgaac ttaagggaac
79021  tcttatttag ccataatcag atcagcatct tggacttgag tgaaaaagca tatttatggt
79081  ctagagtaga gaaactgcat ctttctcaca ataaactgaa agaggtaaga cgattattgc
79141  cacttaaaaa atatacttta tgatttgcat cattacaaat tatcatttta agtgatattt
79201  agcttctaaa taccaatttc atgaaactag aagcttcctg ttaactataa attcctgtca
79261  actataaatc cagatttcca ttaaatttaa aaataagaac agctactaat gatgtgtcac
79321  ttaatttaat ttccattctc acaccgacaa tttaaaaaaa tctactttta aaaataaggt
79381  agtagcctta atacttccac taaacaataa aacaatatgc tatacttatc agaacctttt
79441  aatctgaaag ctaaacagct agatataaat ttgtctaccc aacatttgta caaagtaaaa
79501  attattagaa tattcatttc aaattatgtg tgatgttatc atataacata ttatagatta
79561  actcaaatta ttcttaactg ttacttaact atgacaaaac aacttagaac gctttgctaa
79621  tacacaagat agtataagga taaaaattct ttatagtact agctcataaa gagtgttcta
79681  tggaatagta gtggatgtca tttaataact ataaattcaa aataagcatt gtaaatatca
79741  ataccattca attttttttt gttttttaaa caagttgtaa gcctacccta tggtaaatgg
79801  atatggtaac acagcataat ttcctcaaaa aattactttt gtgatatact tttaaaggat
79861  tatatgaata tatacataat tatagatgaa tgtgatgctg tgtgtcattg tatcaccaaa
79921  tctctgtcca atctgttaac agactcttaa ataaaccatt tttctcaagt tgttactggc
79981  ctgtatactg tattacttgt ttttcagctt tccttggtac attattaaat ttctgcattc
80041  cgccaccatg ctatcaccct aatggatcaa ccttttttgt tttgctccat tctctctgtt
80101  gtaaatctga aattgataat ttgtttgtct cagaaaatat tatttttcaa gttctagcgt
80161  attgcctaca aaaaccaaaa gaattaagtg tctgacactg tgaggttcag caaaactgtg
80221  catatatttt gctacctgat tttttgccag caaatgagtg ttttctatta taaatatagt
80281  atatattgct taaaaatttg gaacagaaaa gaaattactc caattaggat gccacctaaa
80341  gtataagcat gtagctgtac tttgagaaca ctaaattgca tgcaggtttg tagtgactag
80401  gtcttcttgc ctttactgaa ggagcagaat gaagtcacag ataatggata accaaatcca
80461  ttttgtggta agaacttcct tactttcaat gtcttgaaga gatgaagtat gttaccaaag
80521  gagattgggt tttttaatat tacagatgag tgacatagat tgtttgggag taagttttta
80581  tatgtaagtt tttatgtttt taaacacata ctgacaactt atgacaaacc tttggaaagt
80641  tttaaaactc tgttgaaagg ttgtgcaagc tgctgatgga atctgtgagc ctttcttgtt
80701  tcttatcacg ttttttggca gagcacattt cttccttccc accaacaggt tttgcccttt
80761  tttttcccat taagattcct cctgagattg gctgtcttga aaatctgaca tctctggatg
80821  tcagttacaa cttggaacta agatcctttc ccaatgaaat ggggaaatta agcaaaatat
80881  gggatcttcc tttggatgaa ctgcatctta actttgattt taaacatata ggatgtaaag
80941  ccaaagacat cataaggtta gataattttt ttctatttgg ttttactaaa tttatttcag
81001  attttctact ctctgtgact ttgatggaca tatattgtta ctatttaggg aaaaataaat
81061  agtaatattt ggcattaata tgctgtgtgt catttgcctt tcatttaatg aatgtgtttc
81121  tgtggtgcca ctgtagagat ttctcattct tcttagccag actaatgttg agagcggctt
81181  ctcctccttc tgtttctttt cagtggagta gactctaaaa gaaaataagt attgctattt
81241  ggtctctggt taccaattac acaatctaaa gaaatacagc acagtataat aacttctcac
81301  actgtatttc atatagcaac tagttaacat atgcctctta catcttaaag cattatagct
81361  actgacatca tgtgaaatta ctaacttcta ttttgcccat taggatgagt aatctactca
81421  ccttgatcag ttttgaaagc accaaaactt ctcaagtatc actgtttctg gtctttacac
81481  tttaagcact ttaaatatct ttggtaatgg attttatcct cctttttgtt ccctttcagc
81541  acatcggtct tattactttc tcataaaatc ctttgctccc ttttccacag ttactgtatt
81601  aacgttgcag acctcagctc tgtcatcacc tctcaacttg actgtaatat ccaccaaggc
81661  agagaccatg gctgtgttca ctcactattc aaatcttggc acccaacaca gtgcctggca
81721  tacaattaat agttgtttaa ttaccagtga tttatactta ctcattctct tctgcctaaa
81781  atctcttaaa tttatattta acttcatctg tttttatgag gaaggatttt gttttctgaa
81841  ctcctgagct tgatttcatt ttaaaggagt ttgttatctt ttgtgctaat tgtggctacc
81901  cttcatccta cccaattatt tttctctctt gaaactggaa aagatggtca tataaaaatt
81961  ggttcagttc ttactaaaca tttagtagaa ctagctttca gtgtattata ctgtattatc
82021  taactaaata tttttaatat ttaatattta atttaatata taactaaata tttttaaaca
82081  tgtttaacat tttcagaaaa gacagaaaga cctagagcag attagaaatt gtaggcatca
82141  tttgcttttt gaagaaagac attttttcaa atagtggtgc attcttaaga aataaatcaa
82201  gaaaggtaat gttgcttttt ggtcatatca tcaggaatgt tggtcagatt cttattagtt
82261  acaggaatga attgatcact actctgatgt aaaattcact tatgatttag tctttttctc
82321  taatttgaaa ctgtggcaac attttaacat atttcaaaat atatctttct ctatccatta
82381  tatttttgat aacactttga ctctactatt agtttaaagg tggtttttta gctacctaaa
82441  cacttctatt tcattcaggt tttacattaa gatcattagg aatgaaagct aacatctgct
82501  gatagtataa tagtttatat ttatttatga tgttatgtga tctcactatc catatatact
82561  attatatgca tatgtgatat acatgaatat atagctatac atcatatata ccatatatga
82621  atatatacac acacatatat aatgtaacta atatgaccct attatcaagc tttaacagta
82681  tacatatatc tctaccttgt ttctatgtca tatggacttt gtgaaatttt gaactttata
82741  atttataggg tttttctttt cttttctttt cttttttttt ttttttttga gactgagttt
82801  cactcttgtc acccaggctg gagtgcagtg gcctgatctt ggctcactgc aacctctgcc
82861  tcttggcttc aggcgattct cctgcctcaa cctcccaagt agctggaatt acaggcacct
82921  gccactgttc ccggctactt tttggatttt taatagagac ggggtttcac tatattggcc
82981  aggctggtct caaactcctg acctcatgat ccgcccacct cggcctccca aaatgcaggg
83041  attacaggtg cgagccaccg cacctggcgt ataatttgta gggtttttca tactatttaa
83101  agacattaga atatgtatac atgtatgtat atgtgtgtat atatagaggt atatatatat
83161  tgcatatcgt attctaatta gtattgcaaa catattttgg ccttttgatt atttctggtg
83221  atagtgtaac atgttttctt tggtgatttt accaaacatt atcaactacc ctaaaatctc
83281  tagcaaaata tatgcattaa cagtactctg aaagacatgt acattattag ttatatgaga
83341  tatgcactct tctggatact atattttaga atagtgtgac atgtaaaaga actcacctaa
83401  atctcaagta tacttttaag cagtttatta ttttattttt atctttcaaa tactaggttt
83461  cttcaacagc gattaaaaaa ggctgtgcct tataaccgaa tgaaacttat gattgtggga
83521  aatactggga gtggtaaaac caccttattg cagcaattaa tgaaaaccaa gaaatcagat
83581  cttggaatgc aaagtgccac agttggcata gatgtgaaag actggcctat ccaaataaga
83641  gacaaaagaa agagagatct cgtcctaaat gtgtgggatt ttgcaggtat ttctttctat
83701  agaattttaa aattcacttt taccatttgt ttggaacagg gattcaaaaa ctgagctttc
83761  tgttctaata tccagaaacc tggtagactg tatggaatta ttccaaagcc cttcatttct
83821  cctaatttta cccttgcctc cagaatggag aagaacatgg agggatatgt taggaacaat
83881  ttggtgctag gtactttgat cggttgctga caaatatgct aaaagtggtc aatcctagta
83941  aaaacccaga atagttctct aaacatggtc tgttgttttt ctcttattag tatgctaaat
84001  aataaatagt attattctcc cagatttttt tttaaaaaag gattcttgcc tgtcgtttga
84061  aagattaaaa aaatttgtct ctaatcttta tttaggtcgt gaggaattct atagtactca
84121  tccccatttt atgacgcagc gagcattgta ccttgctgtc tatgacctca gcaagggaca
84181  ggctgaagtt gatgccatga agccttggct cttcaatata aaggtgattt gttctgatca
84241  tttgaaaata gaaaataatt catgtgtctg tgtgcgtgtg tgtgtgtgtg tgtaagttaa
84301  tttattttgg gcaaacaatt gcttcagtct ctttaaatac tttcttaaaa gaagcactaa
84361  aattttgaat tgggaaactt tccgagtaat gaagtcataa catgaaaatt gtatgttcca
84421  tgttggtgaa tgttattggt aacctgaaac tcttttatgc tgtaaaactt gaaaatatat
84481  atgttcaact gttttttaat tatattattt cttaaatgaa atctaaattt ttctaattta
84541  aaataagcta tattaaagaa aagcaatcta tatatatata tctcatcaac tttgtactca
84601  ggggccattt agtgtgaaat tcttcagatt gtatccttta agtggtccca gattattatg
84661  ctgttacatc tggaatctcc cttttgttgc ttttctatct tttcctttgt tgtcttgttg
84721  tcagctattc cttcaaacac tatggctttt tagaatggag actaaactgc tgcttgcatg
84781  atgctgcaat gaactcttct gtgcataaag tccttaaaaa gcttgtgtca ggacatttaa
84841  ccatgtaatt ggctgcatac atgcttgttt tgtaatttgg gtatttttta atgtttcttt
84901  tattaacttt tttacagcta gccaacgtga gcaaatagta cagtggcagt catatttgct
84961  tgagtggctt ttattctttc attgtagact ccaaattggt tgactttaaa acgaatttag
85021  aagattaaat tcacagataa ggaagagaaa atataaacta tatgacgtta atttgatata
85081  atttgtgggt ttatgaaatg cttattttat ttaggagtga ataactcatc ttaaggcatg
85141  aagatgggaa aggaaaacta taccactacc gttatatatg ccacctaaaa gggtgaagaa
85201  ttgggttaag aaaggccaaa aatgactttt taaaatgtcg taaggttaca tttttttctt
85261  aggtttaagg aaaaaaggac agttgttctt ttcttcttct gaagtctgct agtttctctt
85321  ttccattcaa gtgaatgtca cggaaagcaa atatcaacag gaatgtgagc aggcccagtt
85381  tgaaagcaaa cacaagaggg ttttgtgtct ttctctccag gctcgcgctt cttcttcccc
85441  tgtgattctc gttggcacac atttggatgt ttctgatgag aagcaacgca aagcctgcat
85501  gagtaaaatc accaaggaac tcctgaataa gcgagggttc cctgccatac gagattacca
85561  ctttgtgaat gccaccgagg aatctgatgc tttggcaaaa cttcggaaaa ccatcataaa
85621  cgagagcctt aatttcaagg taacatggta ggctggtaga gaaatgtaat ttattgattc
85681  tcaactgcct agaaatgtca gaaattttga gaagtgagca actcacttaa aattgtgggt
85741  tttctttcct tgttgctgtt agcattatta aagtcctttc cattttaaaa ttatttatgc
85801  cagacttcat ttctaattca tagaaatggg aacaaaaaat aattagagga acctgagaga
85861  aactaagaga ccgtttctgg gatactgaga aaatgtttct gagagagaat ctgagaaaat
85921  gtttttgatg ccttttctga ttcaacttct tatagtggtg attcaatcac aagggtaaag
85981  gtgaatactg aggtcttggg atcatctttc ttctattatt ctttaactgt tatttttcca
86041  tttcctcttt tcttttggaa ttcctgtttt atggacatct tgatcttttg tgccactcat
86101  tcatgaattt tgtcactgtg attcccattc caattttttt ccctccgtat tgtgaggcag
86161  ctgttttatt tagtcatgaa gaccactaac ttggttttca gcagtgtctc actaattact
86221  tagttcatac aaaatgggct ttttatttta ggaattatgt tttaaatgtt taaagttatc
86281  ttctcgtaag ccaaattttt ataaaatgta aataaatcag ttatcagaga gaacactttt
86341  ttttttaaat acttggcaga aaaaagaaat cttcactggg tactacaggg agtgtggtgt
86401  aaactgtact gaaaaatacc cttgatagtt ccatatgaca aacataatga tgaatttcac
86461  ttagtctgtc ttggcttagc tcaatagcac taatgatcaa gatactggct gataaataga
86521  gtcctatttg gcctgggcag tcccagcata attatgtaat agtgtcccac tatattctca
86581  aaagcattcc aatttggatg ataaattata tagtcacctt ggttataact ccatgctggc
86641  cagttagctt agttctgttc catttatata gattatgtgt gcttcactcc aaaacctaat
86701  gagccatttg taaaagtgat ggcttttgcg gtgcccaggg agagaatttg tatgtttgta
86761  tccttcaaca cacatttatt acagttatta aaaggtttta ttgatgatag atggtaatgt
86821  catgtaaaaa tgacatatta tttatttgta gactttccta ttctcttgtt ggacatgtaa
86881  ttagaaacta atatgactta aagaaaaaca aatacacaaa atttattcat ccaattaatc
86941  tcttaatcca ggtgtttttt ttttctgaga ctatacccat acttcaataa ctttgttgtt
87001  actgagaata ttttgagttt ccctttttgt cattgttgtc agagaatgta tcatatcttt
87061  aaaaagactt gttggaggat gagtttgttt tgaaaaggcc tgaatttagt tgatgcaaag
87121  tcacagataa gatggttcat taagctgtat taatactgct tttgtctaat agatatcatt
87181  accaataagt cagactagtt tttcttttgg cacttataaa tcacctttga agacaacttt
87241  ttacaaggaa ataaaacaaa tgctttgaga aataccagta ttattgaaag aaaagtatat
87301  attgctaatg gatgcagcat tctggcataa tggtttgaaa actcatttga ttgctttgta
87361  gaagaatgac tctttcagat gacccagggc ctgtgagcct gccagaactt gaaaattctt
87421  tcttccctga ggtgcttcaa cctgaattca aagagcagct tttaatctat tagagatcat
87481  tttttgtcct ctcatttatt tttcatattt gcctttgatc ttagctcttc tctaatcttt
87541  ttctgtctca accttattaa caggtgtctg tgcagacact tttaagtttt gttttttggc
87601  tcagcctgtc agttaactga taatcatgct gaaaggagaa gcaggacaaa acagagttca
87661  atgctgacaa tactcctttt aatcttgtcc agcccattag cagagcaggc atctctgtgg
87721  gccttgagac gtagtcccgt aaaactcatc ccgtttctac ttgatttgct ttctttgaga
87781  actcttgttt atttttatat ggaggtttcc tgccttggat taaaacataa acctcaatct
87841  gaagttcaat ttcatcttaa tttatgaacg actaagagag ggaacatgaa aagtggaggt
87901  tagtgaaatt atctctaatt ctctgggtta agagatacat gaaaacagtc tcttgagtaa
87961  ccatttgcag gtaaatatgg aagtaatggt tatggttgtc tctttaagtt tttagtcaca
88021  agtagaaaaa gaccaagtta atttttttct gtgtgtgctg aatttctatt tgtagtaagt
88081  gtaagaattt aagcagaaat tctgattcgt attttcagat aaaaagaata tgtaatttcc
88141  ataggtccag aaatagggag agtttgccat ctggtggttc ttaacggcac tctggatatt
88201  attaagagtt gcatttctat ttaaaattat attttaaaaa acgtttggaa gatactttta
88261  ttgtagaaac tatcctctta gggccattct ttaaaaaaat cttattttat atatttctca
88321  ttttgttgat agtgattaga ttctaagagc aacagaacaa tgatcatcct ctcctatcag
88381  aatcactgat gtttagatga tttctcattt tcccaagttc aaggttccat gaaaaacata
88441  gcttgagtgg gattttatgt ctctgcgttt cactgttgat atatatgtcc tcccaatata
88501  acattttaca aataaccaag cacaaaattt aatattttac cttgaatatt taaaatataa
88561  taatatccaa aagctcttgt aatttgtact gatatcttat actagcgtgt ctgtttcaca
88621  ttaagtttaa tgtcttagga tataaaaaat cttttttatg gttagtgatt tatcttgttt
88681  ttttttccat ggaatttctg gatagcgaga taaatatttc catactattt tatttgatat
88741  ttccaaattt gcctctgaat caacaatttt cctattttaa tttcattgta cttgttcctt
88801  acaacctaaa tagcttttta ttatattttg attttattta aaaatgtact tctgaataat
88861  atatctgttt ctgtaaaaac tgttagcact gaatttgcca accatttgac aaatacacaa
88921  ataaaataga tttttacggc ttgtcatttg taatttcata gatccgagat cagcttgttg
88981  ttggacagct gattccagac tgctatgtag aacttgaaaa aatcatttta tcggagcgta
89041  aaaatgtgcc aattgaattt cccgtaattg accggaaacg attattacaa ctagtgagag
89101  aaaatcagct gcagttagat gaaaatgagc ttcctcacgc agttcacttt ctaaatgaat
89161  caggtttgtg tttttcgttc cttattttca aagctcagct gtagtaactt ataaaagtgt
89221  ttctgaatct tttatagaat ttacattcaa agttgagaga atatccatac ggttctttaa
89281  taggccactg atttttttct ttttggaaga tcatcatgtg tgttcatgac aaatcatgta
89341  tcatgtcata agaaaacaaa tttagaaatc acctaggagt aaagcagtgg aaagagtccc
89401  tgagtgggag ttaaaatatt tgggttctag aacttgtctt tactattcag gagctgtgga
89461  accctgaata gtcaaatgac attcataatg tcaaatgagt ttagtgcatg tgaaagttat
89521  ttttatattg caaaggggaa ttattgttgg catggtctaa ctgggacgct tggagagtca
89581  atggctccct gagatgatgc agcttctgag tggaagatct agctctcttg catcaaatat
89641  tgatctcaaa gatgaaaatt ctcaaagcaa cttcagtgct aattgtgtac ttgatcatat
89701  taccttgcta gaaatgtgtg agttgtttga tagtactaga gtaagtgact gggaagctgc
89761  ttttgatccc tagattctgt tgtataaaaa atagcttccc gtggtttatg atctgttcct
87821  tttccccatc gttcttaagg tatgctgaga tatgctgtgt ttcttatctg tatttgaaaa
89881  taaaacatgt ctttgtagtg tgtattcagc aagcgaaaca gaaaattatg aatttctact
89941  tatgtgtgaa atatgctctg taatgcatgt cagtgtctca aatatgctta aatatgatca
90001  ttttatgtag tttaaaaata ctccattata atattggaac tttagaccat aggatgcaca
90061  gcttctagtc ccagctctgt cactagctat gctgaaattt cttcacctgc aaaatgagga
90121  agttggacta gattttttct aaagcccctt gatatttgtt ctagattcca tgtttcactg
90181  tttgatgact ttttactaca ggagtccttc ttcattttca agacccagca ctgcagttaa
90241  gtgacttgta ctttgtggaa cccaagtggc tttgtaaaat catggcacag gttggtgtct
90301  tttatttttg tggcacgggg gttatggtca aagcatagaa cagatggcgc ccagagcatt
90361  gagcatttta gaatttgggt ttagttaagg cagaaacttt tgtgaatttg gaaaactgtg
90421  gaacatttca catagaagac tacttgaaga gcttcatgga agaaggaaag atgtcttgag
90481  ttcacttcca tgacttggtt ttcaagccac atacagatgt ttgtatcact ctgccccatg
90541  ctgctttact agatcctgat gatgtcattg gtttggttac tgaattagtc aattgaatga
90601  tggctttgtg gaaatccttg gggtaaacac atataagaaa attaggttgc tgagcctgtg
90661  aaacctctat ctagataaca tggaggtgag ttttgactta agtgaaatga tctgagcttt
90721  aaatgcttac gattttgaaa actttggatg gccttggtta tagctatttt tttcttatat
90781  ttcacatgga aaatgatttt tttctccaaa tgataatcca ttaccaatga gtttaattag
90841  ttataataat ccatctctgt agctttgaca taaaagacca tttgagcaaa acatactacc
90901  tcagggcttt tcaaccccag catgatgaca ttttgggcca gataattctt tgttgcacat
90961  tgtaggatgt tagcagcatt tttggccttt atattcgaga cgtaagtagt atcctctagc
91021  tgtgacaagc aaaaatgtat ccagacattg ctaaatattg cttggagaat gtgaaaaatt
91081  accctagttg agaaacatta agctactgat ttgttgatga gtaaaattta tagttttgca
91141  tgtggctgcc cgagttccta aaattattat atatttttat gttagaaata tctcttccaa
91201  ttaaaccata aaggtaatta aattcactca ggcagccttg aataattgtt cctaaattcc
91261  atctaaggaa aaaaaggaag ctattgtgaa gagagaactc agttgaggct aaatcctgta
91321  ccatggaact caagagcata ttgaaacatt gcaatcagca attatttgca gtgtgtcagt
91381  tattactatt ttggtaggta tttttaaatt agattttcag ccttctgcac atatgtcatg
91441  gataatgtga ttttactcaa ttattaaatg ataatggaga cagtagtgtg acccagagca
91501  cttacttgag catcagcttg acctacgttt cagtctcttt aattacttat tagctctgtg
91561  aaatttctta atgcattaag cctttgttta cttacttttt aaataaggaa aataacaatt
91621  atcttctata ttgcctccct ggttcagtgt aagtgagggg taaatgttag ctaattttat
91681  attggatcta tttggcaatt taaagaatgt taatcaggaa attttaaaaa attcagaact
91741  ataaagaggt acttacgtag ttttggaaag tgtgtcatgt atggggacaa ataaaaaaga
91801  tgtgtaggta gctgcatcct gtacagcaaa ggaagtttta aatatatcca gcaattttgt
91861  tgtcctagct ggcgcacaat agttatcagg aggtaactca actccacata gtcaaggaaa
91921  agctaaagtt gctctctaaa gtggtgtgtt tccatgtcac tatggaacac ttgaagttgc
91981  acacatgtga acattaggat gggtatatct tatacagtag aataaggaag aggtttgcat
92041  cagaactccc cttttaaaaa aatgcagatt ttcactatga ctgcaataaa attcctgaag
92101  attctgtgga gtaattaagt tgaaactcca tgaaagttct tctcattagc atagttataa
92161  atatgataat ttaagtaaaa attaagttaa tttgagccac tcaaagttac ttttaaagac
92221  agatttaaaa tgtcaataaa atgataattt aaatttccga ttaacctaaa aaagaagtgc
92281  catcattttt atttatgcca ataaattgaa atataatgtc attttatcac taaggtttaa
92341  aggaaatgaa atctctaaat aatcaagtga aaccaagagc aacttgtctg acagctatta
92401  gcaaaaataa ataggagtat tcaccttcat gaatcaaggc aagggccgga ataatttcat
92461  ggtgcagaag ctctaatgag cccacccact ctatgcgccc cgagctgtta ggtcactaaa
92521  cttattaaaa aaaggtacca ttaaggcagg gagaagttta caagactcat ttaactgtat
92581  gataaaagag atatgaaaga gacctattca attaatcagg tggaacatta aaaagcttac
92641  atggcaattt aaccttgata aaaatacatg ggagaaatac aaaggaattt ggaaaattct
92701  ctttccttga ataaggcatc agttagctat tcaggttatg aggttgaagg aatgttagga
92761  gctcttttaa aggtgataaa gtcaagataa tgttgcagat tttattctta tgtaacaaac
92821  cccctcgaaa cttggaggct taaaatgtga acaatttatc atttctcgtt cttctgtggc
92881  ttgactgggc tcagctgcgt ggttctgctc cacatggtat tggcaagggt tattcacttg
92941  gcttcattca ttaaactgag ctggaaagtg caagaaaggt acatgcatgt ttttggagta
93001  ttggtgcttc tccatgtggc ctatcatatg gctaagttgg gcttcctcgt ggcacggtga
93061  tcacagaata attagacatc tttcatggtg gctggttacc aagagaaatg aagcagattt
93121  tttctgtcct cttaaaggct aggccaagga ctggcaaaaa tattaattct gctacattct
93181  agtaaccaga gcaaccacaa acctagctca gattaaaggg gaaggaaaag agactctata
93241  tgaatagcac ctatgtatag ggatggaaat gatgtgtcca tctttggaaa cttccactat
93301  aaatagtggt agcacgctat agatccacta ggaaaatcaa gcacaaactc tttaaaaaat
93361  aagtgtatct tagtaaaata gattagaata actagataat aatggctaac atacatgagg
93421  ttaatatgtg cttttcaaag attagctcat gtaattctca cagcaacctt tccaaatggt
93481  actttattag cccctatgat acagatgaag aaattgattg acagagaggt tgaataattt
93541  atccaacggt acacattcag gaagaggtag agttagaatt tcaaaccaag tagtttgact
93601  ccagggccta tgagtttata cattcatagg gctgatattc aaatgagaga agagaagtaa
93661  taaataaaca tataatatgt tgagtggtac agagtgctac aaagaaaata tgaagtgcag
93721  ttggagatga attgtcaaaa aaggtcttag cacttaaaaa acactaaaac agcaaacaat
93781  tctctttacc acctaaactg taagagcgat ctggaattgc tataaagtac acaacatggg
93841  agaagtctta aacaacagtt tttattattt ataggcccat tgcacactgt cattaaatac
93901  caatatgttc aatcaaccat gcattcattg attcaataaa tactgtacat acaaaataga
93961  aatacagaaa tgggtaagac aagtccttgg gcctaaggac tttataacct ggtatttcac
94021  tcaactacat gatagcataa ataatgtttg cttctgttta agtattcctt aaacattata
94081  gatctcccaa agaaaattaa atacaaacct ctttttaaag tgaatttgac aaagcaaaat
94141  aaattggaat atatagataa atatgctaaa atttgtcata tgtactttgc gtactttaca
94201  tgtgttattt cattctcagg gcaatctaag acagtcactt ttattatctc attttataga
94261  gaagaaagct gtgcagtaaa gaaatcaaat acctttccca aggttacaga gctagtagta
94321  gagcctggat ttgaatctgg gttctgactg atttttaact gccatgacaa ggatcaaagc
94381  tcaaagtgtg atctctgtgt tagaaacatc ggggttgctc tttaaaaaag ccgattctca
94441  ggcctcaacc cagacctact gacccagaca ctgcaagtag aatccatcaa aatgcagtag
94501  ttactttgag aatcatgaaa ctctgctaca cagtctgtct tcctattcat ggaagtcctc
94561  tcctagtata taaatgtgaa gtaatatttc tatttcaaac ctgtattgat aactgtctgg
94621  aagataattt tcctgggaat atattattga tgagactgca aaacagatgt gaggtattgg
94681  attagtcttt ccattgtagc tagggaaata ctgatgttca ttgtttcagt gaagttcaat
94741  gatttcctat ccgaattaac tcccttaatt taacaatttt tttttttttt ttgagagtga
94801  atgcccctct gggcttctag gccacatggt tgctagagaa attaggtact gtgttgcact
94861  tgaaaacact aaaatctttc tgactacttt cactgagcaa agagacataa aatgctttaa
94921  atttgcaaca tttcagaaaa taaattttag tgattattta tgactcgaat ctttcagatt
94981  ttgacagtga aagtggaagg ttgtccaaaa caccctaagg gcattatttc gcgtagagat
95041  gtggaaaaat ttctttcaaa aaaaaggaaa tttccaaaga actacatgtc acagtatttt
95101  aagctcctag aaaaattcca gattgctttg ccaataggag aagaatattt gctggttcca
95161  agcaggtaaa gaaaacctta aaaaattaat tgctacatgg aaattcacta tctattcttt
95221  taattgtcaa actaactgta gtctataata gatgtattaa ataaataaat atattttgct
95281  tctagtgtaa acctcctact gacatgtatc atttattttg gaataaaaca ttgcatctga
95341  cactttaaca atatagtaaa tcacttactt tatgtgtata gttactagtt ggcttatcac
95401  tgttgaaatt atttaagaaa ggtaaatagt ggagattaat gtgtgtgtgt gtctgtgttt
95461  gtgtatgtgt gtgttcttaa acaacactga gagagtttat taagcaagtt ctgagaagat
95521  agtgagtttt caacagaatt ttaaaagcat ttatggcatc acaatggatg cctatgtttt
95581  agcctatact atggaaattt ttcctactgc tctaagcaac tgggaaattt ataaagtaat
95641  atgatgttga aatgtgcaaa ttacattgat tgatggatgc agccaatttt aaaaataaat
95701  atacactttt tttctaggac atgtattttt caggatttat ataagattac atttgtctat
95761  gcataactaa ttgtaataat ttatgtatta gtgcacaggg attaccgaaa atatttcatg
95821  catctacatc tgagcatgca tttgaattgg ttattgacca ctgaattttt ggtgtaggaa
95881  aaatatgtag tgaaacaatg ttacaaaaag attacaattg tttggaatga ttaccttcat
95941  tgactttaag cagtaaaatc atttgctcaa caaggttggg tgttttgtga ggctgtataa
96001  ccatagtgtc cttttgcctt tagtttgtct gaccacaggc ctgtgataga gcttccccat
96061  tgtgagaact ctgaaattat catccgacta tatgaaatgc cttattttcc aatgggattt
96121  tggtcaagat taatcaatcg attacttgag atttcacctt acatgctttc agggagaggt
96181  aagtatctaa tgaagactta ttagattttt agagactatt aatttagact tattaatttt
96241  tagagaaatt agggagatgg catatgaaaa gtaatatgcc attttctcag agtttacttg
96301  tttggaaggc agctgaagaa ttagaaaata agctcataaa accttggagt aggcaatcta
96361  aagacacaca agcacatata acctcatcta atttgtcagg aagaaaattc cttaggtgct
96421  cactcagatc ttgactgtga ttacattgta gggactgtaa ttatctcttt tctgttgcac
96481  agccactaag acatttacaa aaaaagagca aatccggtgt ttataatgct aactctttct
96541  tctaaaataa atagagacat tttggtactc caaagggaaa atatcatttt ggggattaaa
96601  attagcttta cacaggtgtt actggtttcc aaaataaacc ttaccttgat tggaattaat
96661  caacatatag gtagttacat tgcattaaaa agttcagaaa gttttgcgtt tagcatgatc
96721  aaaaacttct ttttaaaaat tatgaggatt tatttatgat tttctttctt catctgtcga
96781  gcatattaaa ctgcttaaca gcatcaacct gaaatggatc ttaatgtgca ggggatttaa
96841  ctctttttat tgtaaagttg tggataaaat atttaataga tatggatgag gactcatatc
96901  agtaacaacc caatacttta tttcaaaatg aatagatctg tattacaatc acttgtgttg
96961  tgtgcagtag attttttccc tttaacttag gaagcagtta ataattaatg gctccatttt
97021  ttacaacgag cacttcgccc aaacagaatg tattggcgac aaggcattta cttaaattgg
97081  tctcctgaag cttattgtct ggtaggatct gaagtcttag acaatcatcc agagagtttc
97141  ttaaaaatta cagttccttc ttgtagaaaa ggtaaggaaa tcaatttgaa tgttttcaat
97201  tgcaacacta aagaaattta aacttaaaaa aaaaaaaaac tttaccttaa agctttgcga
97261  cagtatgagg tttagacaag gtgttgagct ctgttttgaa tcatgtaggc tgtattcttt
97321  tgggccaagt tgtggaccac attgattctc tcatggaaga atggtttcct gggttgctgg
97381  agattgatat ttgtggtgaa ggagaaactc tgttgaagaa atgggcatta tatagtttta
97441  atgatggtga agaacatcaa aaaatcttac ttgatgactt gatgaagaaa gcagaggaag
97501  gtatgttttg atacaactta caaatgcttt taagtgatcc ttcaatactt atgaagtgac
97561  ttttaataaa tgtaaatatt cttatccata agggatgagt tgaaaaatag tatattcaat
97621  tatagggaca gttcagaaaa ctgaattata tttattacca ataaaatctt gtattctaga
97681  ttcagaaaat gttgatttga gggtttgaat gctggcttat tgagcaacat aacctcatct
97741  gtgaaaccgg aataccaacc acatctatct catagaactg ttataaagat tcaaatagac
97801  aatacatgga cctaatttac caacatgtct gccatataat aacagctgca gcttcatgaa
97861  tgtggcaaaa gcagagagta gataactttc tagtcagatg tctggtagtc tgcagcagtt
97921  cagaattcta caagtgaacg taggaataag tttttaaaat tccaagtaga tagatactaa
97981  gtgaatcttt aaaatgttct caaatttcct agagaaatat aggattggtt agaaagggag
98041  ggattagaaa ttatagaaaa tattccatta ttttttcaca tcaaaaccac aaatttatgt
98101  atctccttaa atgttgtttt tatttaaaaa atgttttatt acttctcagg agatctctta
98161  gtaaatccag atcaaccaag gctcaccatt ccaatatctc agattgcccc tgacttgatt
98221  ttggctgacc tgcctagaaa tattatgttg aataatgatg agttggaatt tgaacaagct
98281  ccagagtttc tcctaggtaa ttctttttgt taatttgaga ataaaaatta ggatgtaatt
98341  ttctccttat aatttagaaa atagatttca taattatatt gtcatagatt ttactgtctt
98401  cqtatatttg ttataatttt tgtatttgga atgatatatt ttaaaggaat ataatattac
98461  agatctggaa tttgttttgc acataatcat gtagactagg atcaagatga ggatgagatt
98521  atcatggaag cagaaatatt tatgaaatat atctttgtat ttgccttaat tgccagggat
98581  atgggaggca aataagacag ttttcaggtg agttaagtga agcagccata ttttataaaa
98641  tgacagaata ggtaaaggaa gcacacctca gtgtagccat agcaggggtt ttatgactca
98701  gtgtgacaat gctgaattct catagaaata ttcattaaaa gccttgaaat taaagtcaaa
98761  agtgttacat ggtgacatac tcaaatactt tttttttttt ttttgatatg ctgaacaatt
98821  tacatttctt ggttccgtga attcaatcag tgattttcag tagagtatga tggaaatcat
98881  tgaattcatg tagcatgttt aggtgctcat tgagaaaagg tgaagtcatg gtaaccatgt
98941  ttcaatattc tcatttgtat cttgacttcc tgcacatgga tttttgggcc taaaagatgt
99001  ttttaaaaca tgctcataca cttcagaaga tgaaaagtgt atgcattata actactttgg
99061  gaaagaaaca gtcaacatat gttactgtat gtcattctgt atattacatg tgtggtttct
99121  catgtctctc agaataaaag ctaatgtctt tacaagacct gcgatgctgt gatctgtctg
99181  gctcctcggt tatcattttt aaaaaaagat atactttgta caaatttttt taattgacaa
99241  gtaaaaattg tatatattta tggtgtacaa catgatgttt tgatatatgt atatgttgtg
99301  gaatggagaa gtttagctat ttaacatata cattatctca aatatttatg tggtgagaac
99361  tattaaaatc tactctcata gcaatttaca agtatacagt atgttattat taactgtagg
99421  ctgacatact caagttttaa acattcctga gagtcattgg gacaactatg aaatgcatta
99481  gattgattta atataaagca tttgaagaca attttgacct tactttgttt agtttttgtt
99541  gttgttgtgt gtatacattt aattttaatc aaattacccc agaaataatg cctaagatct
99601  gtcagtcagg acataatatt attagcaaaa agttgtccaa aatttgagac atgatattta
99661  aagctaaata aactccttta tacccctctt attggcattg attgggaagt ttaggttgaa
99721  tttaaatgct ttggaactca ggaagttaat gtattagtaa tagtgggtta acataaaatg
99781  ctgaattgtc cttgctgaat cctacatctt aaccccagac ttcaaggtat acaggaaagt
99841  accagacatg gtgcatcctt cctctgaaga aatcccaaac tgtcagacac agatccctaa
99901  aatatttctt tttcctgcat taaaatgtgt ttcagatgaa tggacacgtt ttgagtagtg
99961  tatgtggaaa cgtcatttac aaagtctgtt tagttggcca ggtgtagtag ctcactcctg
100021 taatcccagc actttgggag gccgaggtgg gtgtatcacg aggtcaggag ttgaagacca
100081 gcctgaccaa gatggtgaaa cctcatctct actaaaaata caaaaaaatt aactgggtgt
100141 ggtggtgggc atctgtaatc tcagctactc gggaggctga ggcagagaat tgcttgaacc
100201 tgggaggcgg aggttgcagt gagccgaggt tgtgccactg cactccagcc taggcgacag
100261 agcgtctcaa aacaaaacaa aacaaaaaac aaaaaagcaa agtctgttta gctacccata
100321 taggaaaatg tttgtgatta ctctcccttc tctagaccca tgtcccataa atccataaat
100381 cccatgttca tttacagaaa gcagtctaga taggagtttc tcagtctttg agctgttgcc
100441 attttggctt ggataactaa ctctttctta tcgagggtca tcctgtgcac tgcagaatgt
100501 ttggcagcat ctctgtctat ccactagatg tcagtagtat ctccccttcc ctcagatgtg
100561 acaatcaaaa atgtctccgg atgttgccaa agataagggg tggggttgaa taccagtgat
100621 ttaaacaaat taggtgtatc cttctaaaaa cattttacag gtagcgactc cagcatcttt
100681 atattagagt aatctggaga aggttatgcc tctctcaatt ttccctcttt ccatttttat
100741 ttgtagggca gcaatgcatt caggcttttg gtaactcttt ttcccaagat agcagtaact
100801 attatgcagt gagtaatacg acccacctta atagatatga atagacttgt tttgtgaata
100861 tattttaaaa tataaatgta tgggattctg ttcatgcgtc tgagaagcca cagggtacat
100921 ttcctctttg tggagctatt tatttttctg gagagccaag acaggtattt ccacttcagt
100981 ggtgtgattt gaggggttag gaaaatttcc ttgccttcaa ttttctttcc aacctagatg
101041 tcacaaatac ataatagtag tccttaactt tatttttgtt ttcagtcacc tgaaagacat
101101 gacaatccat actccatatt aatgcagcgg cgattctcaa atagagaagg gctttaaaaa
101161 attagaaatc tctgccgggc gcagtggctc atgcctgtaa tctcaacact ttgggaggcc
101221 gagatgggcg gatcatgagg tcaggagatc gagaccatcc tggctaacac ggtgaaaccc
101281 catctctact aaaaatacaa aaaattagcc aggcgtggtg gtgtgggcgg ctgtagtccc
101341 agctactcgg gaggctgagt caggaaaatg gcatgaacct gggaggcgga gcttgcagtg
101401 agccgagatc gcgccactgc actccagccc gggcgacaga gcgagactct gtctcaaaaa
101461 aaaaaagaaa aaaaaaaaga aaaaaaaaac aactagaagt ccctactcca acttgaaatt
101521 tggatgtatc tccctagagt atgtttcttc tctatgctgc attgcaattt ttctttgttg
101581 ttgatagttg tccagattga ggggaggcag aacaagatgc atctatatgt ttccatctct
101641 ccgaccgatt ctctcccttc cccctctact tgctttcttt ctcttttccc tcttctgttt
101701 acccgattct atttctgatt ccagtatgta acagttccct ctgaagctct ctcaatacca
101761 acaatcctaa ctaatggttt ttaaaagtca aatattaagt actggaggga tagaatgaga
101821 gaataccaag actgataaga tgcaaataat acttttaaca tatttacaat ctaatagaaa
101881 tacaagacat gctcaaataa gttaattatt ttaatatact ctctctgagc ataaaatata
101941 attatatatg ctcattatag acatataaaa aataaatagg tagaggcttt ccatagatgt
102001 gtaatttcac cacttgaaaa ttactatatt tccttataga ctgttttgtg tgtattcact
102061 tatatccatc aagtgactac atttcaaggc actatatgag aaccataaat attgtacaaa
102121 caggatttgc taaatgtcgg tggagagtaa cagtccacgg ggctgatcat ggtcagtttg
102181 tgaggcaggc ctccaaactc cttggggatt gagatgatgg agtagcagag ctcttcaagg
102241 gtatggaggc ctgaaggtac aaagcatgct caggaaattt tggctattgc ggtttgtcta
102301 gagcacttgt tctcaacctt acctgctcat tactaattct actaagtaca gaattaaaag
102361 aagaaaaaaa tctaatgacc atttcctcct gggactaatt agatcaaaat ctttgaaccc
102421 agacattagc gttttaaaaa gctcctcaga tgtactattc agccaggact ggggcaggga
102481 aagctactga actccagcct tgagaatgag aagtagaaca agaggagaac tttaaaagga
102541 tttaggggcc actatatgac tatggagctg aatttagatt tgatttagta ggcaacgcgg
102601 aataatttgt ttctgaacag gagagtgaca caatcaaagt ggaatgatag gaaaattaat
102661 tttgcaagag agagagaatg agttggaagt aaggaactca gaaggcctcc tgggactcag
102721 cagaaagctc tgaggccacc aaatgggtgt ggtggtagtg gaaatggaga agaagggaat
102781 gtaaatgagg ctacacagtg gactgccact gttagccgtg gggttagacc acagcaagag
102841 ttaaaataat tcttcaattt taactccaga agggcctcaa aaagactttt tgtcttgtta
102901 tcatcagcta tatggaaggt agaataaaaa ctagttagga gaaaaggtaa taaatgtggc
102961 ttttgatagg ctgtgattga gttggaaggg cataccagtg aaatcaccaa cacaaagttg
103021 gaagtgtagg aaagcactta ggaggtggct ataagtgaaa atgtgaaaat tctctacatt
103081 aaagggatag atgaagtcac agaagtggat gacataattg agcagggtat gtgtagaggg
103141 aagacgggaa ggttaaggac aaaatcttta catatatctt tcttggagta gaaggaagag
103201 gaaatgttaa aggagatttg attcaatgaa acaagtaggt caggtttcta ttcaaattta
103261 caacagatat aattacaaca gatataattt atttagtttt tttcgcttgg acagcttaat
103321 ttaagtgctt tgtattttct tttcaaaagg tgatggcagt tttggatcag tttaccgagc
103381 agcctatgaa ggagaagaag tggctgtgaa gatttttaat aaacatacat cactcaggct
103441 gttaagacaa gtaagaaatt caataatata attatattaa attgcacatt attaatctac
103501 tggaactctt attttgcata cagttgtgaa aatgcaaaat aatgaccaca tttctactta
103561 agtttaatta tgcaatccta gtttgtcttt tcgttgtgga gtagaaagtt ttgtgttatt
103621 tctcctgttg agaaacaaaa cactgtatct gagaatcctt ataatcgtga tacatagtgt
103681 gttgtaaaac tttttgtaag actcacttac actcctcttt ttactttaga accttgctgt
103741 tcaaaatgtg ctccatggac aagcagccag gcattaccta ggagattgtt agaaatgtag
103801 aaacttggga cttttcagtg ccatattatt gttcctgata ctccacagta gtcagactcc
103861 tagctgcctc cacctgcttc cagaccttga agcctagcaa gctcctgact tcgccttctg
103921 ttttcttcag agtatttatc ttttactttt ctggtctagg gagagaatga tttttatttt
103981 tattgaacat gacttctgtg tgttcagggt gaaagaagaa gtttaatgca tgatctcaca
104041 ttgctaattt gattgaaggt tagaaatctt aaactaaaac tctcactgat aagcttgcac
104101 ctctcttttc tggatttatc cactttaata agaactgcta ttgattactt gctacaaaga
104161 tggagaaagt tagcatgctt atcctatttc ctactccctg tccctgtcca cttcctaaaa
104221 cttaaaattg gttgcattaa ttttcctgat atagtaacaa ttataacttg gaatgatttt
104281 caaaactttt gtttttttag tataccaact ctagacagca tggactgact ccttgctatg
104341 tgagatgagg aaaattaacg ctattctttc tccttttccc atcaccttct caagttcttt
104401 aatttattct attattttta tgtagtgaaa gtttataaca tttatattct ggtctgtact
104461 cataattaaa ttgttcacat tttgtctata gtttggttct gagaacaaaa ccaataaatg
104521 ccatttatat atttttttat ttgtacagaa ccaaaatatt tctacttcta gataaagaaa
104581 tgcaaccttc tgtcactaac ttcttttact aatagaatag taacattcca aatatcaaag
104641 tcaaatggat tctctattgt tatgtattta tcatcaattt ataaaaataa aggcatattt
104701 taatttggtc acatttttac cctgatttaa aaaaaaattt gtttttagag atggagtctc
104761 attaggttga ccaggctggt ctggaactcc tggcctcaag tgatcctctc accttaacct
104821 tctgagtacc agtggtgatt tattttatgt agctttttga ggttttctga ttatatacat
104881 atatttttaa aaaacgtact tcaggaaaag atatatattt tcatcatgac ttcaagtgtt
104941 tctaagttct taatcataca gtttgtataa cagaatctac tttcttcttg aagacattcc
105001 tcattcagca catgacttac tgctctaaac aggagagatg gatttctagg ctgcttgtgc
105061 agtgattaat ctatgagtta gtttcctcgc cctctttgat tactctcaat atttcttgga
105121 ttccatccat tctcttggtt ggattgtcct tagtttttgt tgaagaatat cttcgagtaa
105181 tttttttaag aaaaggtgtt tgtgaggtaa atgttttcag tccttacatg ttaaaaatat
105241 cttagttttg ccctcccatg tggtggatat gtcatcacac tttatttttt aggaatctag
105301 gcttgaaaca attttcttca aaatttgaag aaaattccat tgatttttag tgcccactgt
105361 tgctaatgaa aagtctgcag tcagtcagat gtttgctcct atctaggata cctttaattt
105421 cattttgaaa actgaaaatt tgaccttttg aatttcattt gttttcagtg ttctgaacct
105481 ttacaagtat gtgtttgtgt gtaggttgtt tttcattcca tctaattcat tattttgtga
105541 aaaattgtct tctgtgtatt ctcttctatt atttattatt tcctccctaa catttattaa
105601 tcatttttat tgacaactac tatgtaccag gttaggtgat gggacatatg atatatatat
105661 agtagtaagc taaacccagt caaggctctg cttctctgga gcctatatct agttacttat
105721 gattcattat tgcttatcat tgctccaaga gtatatgtta gatgacaagc cttttgggtc
105781 tatcatccat gtttgagttc cctcttcaag ttttatctat aatttgtgtt acttacttga
105841 ctgtctctta caggtttcta atatttttta gaattgcatc atctattatt tagctttctg
105901 gtgaattttg ttttgataat catattttcc atttccagca attctttcca tcccctctgg
105961 ttgttccttt gtagccatgt ttttggataa aatgtccata ggtgtttctg ttcatgtcaa
106021 ttagaatttt tttttgtatt acttgcatta ttgctttttt ctctgaggtt atttgctctg
106081 tgggttcatc ttgatctttc tcttttatct tgtcagtttt ccaaattgag tagttttggg
106141 tgacttcgta tgaagtaagt actctattga ttgttaaaga aggactgtat tgattattaa
106201 aggtaactag aatgggcatt cttcacattc atgtaggttt gcttgttcaa gttaccactt
106261 tctgaacaag aaggttagac catagacttt taagggctgc atactgcaaa gggatactct
106321 gttctttagg ttacatgggc agggatcact gctgagacca tacctgccaa aggaaggcag
106381 gctttgctct ctagatgctg gacttgaaat tgtttcccct ctgcttagtg ctgcattatt
106441 ttttttgctt cttaatctgc tgcagagtat ctagatcagg gtgtccaatc ttttggcttc
106501 cctgggccgt attggaagaa gaattgcctt gggccatata taaaatacac taacactaat
106561 gatagctgat gagcttaaaa aaattacaaa aaaaaaatct cataatgttt taagaaagtt
106621 tatgaatttg ggttgggcca cattcaaagc catcctgggc cacatgcgac ctgtgggcca
106681 caagttggac aagcttgttc tagatacttc agactctgtt ctacatctct tcatagatca
106741 ataacttgca gcaatgagtt tatcagataa attatgttca cttttcatcc ataaaaaaag
106801 tcatgggagg tactcaccat aggattggtt taatccagtc actctggcca attttcttta
106861 aaattccttt aaccttggta tattggttta attccttcag aatggctttt cccattgtga
106921 taaactggtt ccaggcttca cgagtactta ttccaagtac agaaggaaag agggtacctc
106981 ttttgagtac ttgtatttca aaggaatcaa atggcctaca taataaccca ttaaataaat
107041 agatgtctct cagccccagt gagtcatctg tgcttttctc aacaagcact atgttcaggg
107101 caatgctttg tgctaattgg ctttaggcct ttgttaccaa actgatcact gtggccctga
107161 gggtgctgtt acttagaata ttcccacctt ttctgagagg ctgatgttgg ggtcacttcc
107221 ccctgaacta aagtccaggg gctgcatggg tgagggttga gtaactcagt actctaagga
107281 ggaaaggaag gggaatatac actgttagtt aacagtggtt attcctgtat tctctcttgt
107341 ttggattcta ctggggattt ctttcttttt ttgagtcttt attgacatta ggaatgagag
107401 atagaaacag ggtgagagag gaagtaaaat taaatgtgaa ttcttccatc ttataccaga
107461 actcaactgt atttttggaa tctatatatc cttacttttc ccttgattta ttacaaaaaa
107521 ttcttagtgg ctacactaag taaattttgt aacctttaaa aaatacatag ttataatatt
107581 ttaagtactc tgagtaatag aagatttcat gtgagtacaa aatatcctgg gggcatttta
107641 attattagta acaacagtca cactgtagtt ctagaaccaa attgaacatt ttatatatag
107701 aagcttatct gtgaatcaaa tctgacataa tctcttttaa tgtgtaggta atttcttata
107761 taaattgatt ttttggcctt gctgtcagca tgcatttcaa attttacacc atgtgtttgg
107821 ggagctatgg tggaaattag tacattaaaa atgtctacag agccaattag tgtataagca
107881 gggaggcaaa ctaagggaat ggtggaagaa attaaaaaaa aaggggtgtg cataatgtgc
107941 ttcttctctt aaaaaaaaaa aaagaaaaag acaacaacaa aaagaaagaa aaatgacatt
108001 tgacggtcat tctaccagta gtcatggggc agatctatat tatactgtac tattacgctg
108061 ttttttcttt gcaattagtg agttgctttt ccaggataga aaatttggat tagacctctg
108121 tctgtgccta tgaaaacaag cagagctaac ttaactcctt ctcatcagtt ctaaccaact
108181 gacatgggca ttaaaaaaaa gattttatct actaagcaaa tatgatcaca gtctgaagct
108241 ttgttcttgg aaaatcccct ctcagggtgt tcagcctttc ttcttcagct tgcagaattc
108301 tccatgtttc agtttcctga taaatcagtg ggcgccgcta ctccacatct ttgaagctgg
108361 ttgttaagaa gcagtgcttc tgcagcaatc acagtttaaa gcatgaatca atttaacatc
108421 acacaagcta tacatttcaa cagagttaca gtttcagagt aaagtgcaat atacagtata
108481 aagcgaatct ggaattcaag cccaacaatg tcataaaaga ggctgtgaag tctcacatga
108541 tgtgggccac agagagggtt gttgccattg gatcttagct caaatactac cacatcttcg
108601 gagtggcctg ccatgggcca cccttactaa ggcaatccct ctgtccaagc caaaatgata
108661 tctcggactt tttttgaatt gcagagagat tgttgcagcc agggattgcc ttagtaaggg
108721 tggcccatgg cgggcctctc tgaggatgtg gtaatatttg agataagatc tgaatggcaa
108781 gacttcagcc atgtgaaggg tggagggatt tctagggaaa ggggtcagca caggcaaaga
108841 ccctatgatg ggaagaagct gggcacagct tgggattgaa tgaatgccaa tgtggctgaa
108901 gggtggtgat tgaagaggag ggggagacga gaaggtctgg aaggcctggg gcatgatcag
108961 atcaggtgct gaggccgtgg aagtagatgg gattttcatc taagggaaat gggaagtcat
109021 tagagagttt tcaggagggg atgatatgta tttttttaaa attgagcatt atcctcggta
109081 aacttttgta gtcgttaaac cagagattat aagcaggttt tacctcatat gccagttgca
109141 gctgattagt agtggctata gagaatcctg ggctgagaag gatactgtgg ctaaccagaa
109201 tttagtagat gagtttgacg tggcctgtta gtatgactac actgtgtgca ctgtttctgc
109261 attaaatgtc tgataaaaac agagccaaag gaaaaataga acttaaaaat ttaattctga
109321 cagtacagtt gacccttgaa aaacataaag gttggggtgc tgaccccttg tgcagtcaca
109381 aatttgcata taacttttga cctccccaaa aaactgaacc actgatagcc cactgttgac
109441 tggaagcttt actgataaca taaacacttg attaacacat gttttatatg ttatctgtat
109501 tatatactcc attcttacca taaagcaagc tagagaaaag agtattttat ttaaaaaatc
109561 ataatgaaga gaaatatatt taatcttcgt taagtagaag tggatcatta taaaggtctt
109621 caccctcatc atcttcacat tgagtgggct gaggaggggg aggtagagga aaggttggtc
109681 ttgctgtctc aagagtggca gaggtggaag aaaatttatg tatatgtgga ctcatgcact
109741 tcaatcccat gttgtccata ggtcaactgt agtttcaaaa ccagcttttt attactgaaa
109801 atacgggaaa aaaaactcag agaagaaatg gaaagtttgc tatgatccag tcatacagag
109861 aaatccatgt tcagcctgtt gatgcacttt aaagaaggag atacgtgggt aaaacctgat
109921 gttgaattac tcttacatga ttttggactt ttgcaggagc ttgtggtgct ttgccacctc
109981 caccacccca gtttgatatc tttgctggca gctgggattc gtccccggat gttggtgatg
110041 gagttagcct ccaagggttc cttggatcgc ctgcttcagc aggacaaagc cagcctcact
110101 agaaccctac agcacaggat tgcactccac gtagctgatg gtttgaggta agtaggtcat
110161 gttgttttct attcagtgca tgacaagtgt gatccagact tgctctcagg ttctgagaac
110221 acttcccagt aacactgtgc cccagtaaca atttataaac aatttggatg aaaactacca
110281 tttccctgat caaattttgt aatttcagaa aataagagta tggaaaccat gcagaacctc
110341 atagcaagta gtaatagact ttgaacccac aagttctgct ctagaaccca tcatcttaac
110401 cctgtactga tctgccttct ataaaaatgt ataagttagg cttcacagta tcaaagtaag
110461 tgtcaattac atgattccaa tgaggaaaga tgagtccata cttctcaagg ggactagagt
110521 gattcatgtt ggattcttcg gcatgaccat ctcacatgtc tcagaggcac acctaaccct
110581 gcatccagag caagctttgg agagggagca cactggagtg gaaaggctgt ggtctttgaa
110641 gacaaaaggc ctgggattca tcactattcc acacatttag taactgtgat tttatatctc
110701 tgattcccat tttttaaata gtctgtgaac catgactaat atttaatgca taaaattatg
110761 atgacttctg taataattgg agacattcca gatgaaactc ttgatgtccc ctctgccatt
110821 gctccccaac cccagtcacc ctgttacacc tgagagtcac cttacattcc tttcttcctc
110881 tctcatttca cagctaatcc ttcagcaaat cttttcagct ctgccaccaa aatatatctt
110941 aatgcttcta acaatttctc tcactaacgt ctaaatctga gccagtatca tctctcattg
111001 cctactggtc ccctgcttct acctctgtct catgatagtc ccattcctca cccagcctct
111061 ggagtgattt ttctaacatg aaagttggat caggacttgt tcctgttatt acccctcccc
111121 tgccttattt cttgggtaca gtgctcagcc actcccatcc ctgaggttcc ttgcagatac
111181 cagaggcttt atatctgctg ttgatttcac tcaggaatgt ctgactccca gatgtgctct
111241 ctacttatta taaaggatta tctgaatctt tctgaatcct ttcatttagg actctcagca
111301 gagaggatgt ccgcaacgac cctttgtctc tccagcccct ataggactat tgctgcctag
111361 gattctttat gttttcattt tttaaaaact tatttattgt ctgtcttgcc atcagaatct
111421 aagtaccatg aaagaaggga cttttcgtct tgtttgccat tgtatctcta gctcctaaaa
111481 tagtaagcct tcagaattac tgtgttgaca gtaggggaag ggggagaaag gaggaaagaa
111541 ggaaaacagt gcctggggca tagaagccaa gcagtgtatg caactttcct tctcttcttt
111601 ctcttctgaa atgctatgaa tatgcctttt aggtagtatc cagaaatgtt ccttcctgaa
111661 agggtccaga aactactgaa aactgtacag attatgaaat gaaacagggt gcagggattt
111721 ggatttgagt tgatgtttct gcttttgaac accaggggga atcttgggtt acattaatct
111781 aggtaaagtg cagaatagtc tcctgtattt cagtgccctc tttccttcat ttaactaact
111841 ctaggttcta gtttttccct aattcttcca caaatcccca aagtgtttat ttataaagtg
111901 aagaattgct attttttaac actgttcgaa acaccttatc tctaaaatga cttattctag
111961 ttctctgaaa ccttacttta aataacaaat ccagcagttt ctgatgaagt aaatgaaatg
112021 tcagcatatt ttaaaataat ttgcctaatt tgttcttagc ataatgccag aaaagctttc
112081 tggattttgt atcacaaaag gctagtagat ttcagtagct atcaatcttc taccagcact
112141 aagtatattt taaaaactca gcattaaggt ttatttttcc aagtatgttt cagcacagga
112201 aataaaatca tgctcctttg gagtccctta aatgctggag ctgtttagag tgacatacaa
112261 gaactttctt cacgttacat gctctctctt cctccatctt gcttttaact gttagcttac
112321 ttctccaatt caatccactt cgtttgaact ctttatcata attctataaa acttatgaaa
112381 atacagtcaa ctgcattttc tgtatgtttc tgtgtttcaa tatcttcaaa atggaatgta
112441 ctgccttggt acatcaccca ctatgaatct gttatttctg ttatatccca cagttgccag
112501 gccaggatac ttgtcccatc caggccaaac accttccccc gaaagcaagt atgcatttgt
112561 ccaccaggtc cttgactcta ttttacatta tctttttagt caattcattt atttttatgc
112621 cactcctgct gtcttggttc agtatgtcca gggaattatc agaatttctt ttctaaaata
112681 aaaatctgtt tatgcttgca attccttgac agttctcaat tatctgcaaa gtgcatccaa
112741 acttcttggc atagcatcaa agatctttct gtatgcctct tgcttccctt tgcggcccct
112801 gccaccccac tgcccacact gcattctagc cgtgatgaca ggcttgaatt ttcagttatg
112861 ctcatgtctg tccatcattg tatttgttat tcctctcttt cgaccaagtt gtctgcctag
112921 agagctcatt ttccttaaga atttcttcac aaaccatctc tactatgaag ctcaagtgtg
112981 tcatgaagtg ttagcttctc caacttgtgt ttcttgcaga cactctgtgc aagacattga
113041 cttaggtgct aaagagggaa agctagatat tatattgttc ttgaggttga aagcttacag
113101 tctagtagga gagtcaactt tgctgtcttt acctcagtgt ttttctccct ctgtgcttcc
113161 ctagcacgtg gtacttacat atttctggaa tcttgattaa acacctgttt gaggactgtc
113221 tgagcacaat ccttctggat tgtgacaccc tcaagggagc agagatacaa agatggcttt
113281 gtatactaaa tgactggccc tcatagatac ctagtacata tttgtcaaat aaatgaatgc
113341 attctatttt tggaataatt ctattcagaa tcagataaag tttactttaa gctatgaaga
113401 aagaagtctc ttagcaactc ttacaataat cacaatcaaa gaatgactgt ttaacttaat
113461 ataaaccagt ttgttttaat aaaatatttg acaatagtca tggttacaca atgcataaat
113521 tatggctaaa ttattatcag gaaggaaaaa tctttactta ttatttcaaa agctattttg
113581 ctagtctatt aaaagctatt agaactgcac ttcttaagat taaattctat aattgaacat
113641 tttaactaac caagatatta tctctttgcc actgacatta tttcaaatta agcttaacta
113701 tttcttttta gcctttggaa agtattctga aagagtctgt gttctataaa tatacttaaa
113761 gaggcatgtc ttataaagga tttggatact attcaatgat gtatgacttg gctttagctt
113821 ttttattctt aatctctcag cttttctctt cagcagggga agagtaccta atggcctttc
113881 agtaatccct tggtaaattt ttctttcaag cccattactt actgtgaagg tcaacttcat
113941 tagtgtattt atcttatttt tttcagccca aaataggtat attgaaatga atgggcctaa
114001 tgtcaaatgt cccgactaca tcctggaaga gagagaatct tcagctgtat tagttgatgc
114061 agttaaataa tatgtactct ccaggccctc atacaattga aagttcaggg tatcgttgct
114121 gctctgcttc taatccttcc agaagtgatt ggtgctaggt gatggagtaa ctattaattg
114181 atataatgtg agccaaaacc aacagtcacg aataagcaaa ggatttaaat ttaactccat
114241 taagtcttgt gagaaattat tttcaacata ggttataaca tacctgtgac atcacatgaa
114301 atgctgtagt caatttgaca tcatggggca gagaagacag agttggaaat cagaatttta
114361 tagacatcta atgtgataat aacattagta gctgagatgc ggtaagctct ttgaccatgt
114421 ttccagaatg gataagacct ggttgagatg aaaactttac actgtttttt tatattaact
114481 atcttttact ctttgcctga aatgtccaac tctagttgct cgtgattgcg tgggtcagtc
114541 tccagaaggt tggactttaa tattacccgt catcttttcc aagacaaaat tgtattcatt
114601 ctaactctta gccccaaatt ttctttttta accttaatat ctaacatgat taggtttatg
114661 gtaaattata tactcaaaca gaagaagaga ctaatagcaa gcaaaagtct tatattttca
114721 tttgttttca tccaaaaagt agaaaatatt ttccaaacat tgggaaacat tttagtcaga
114781 aaaataaata tcaatgataa atagaataga gaaaaatttt aaagctgagc taaacctcta
114841 tgtggtttta ggaaaatcaa aactattaaa taaatggcaa gtacaacaaa atcccatcaa
114901 ttcttattta acatacttac attttgaaat agttaaaata ttcatatgat cattgagaga
114961 attcagaatt gcctttaagt aattgttcac atatacaaaa gaaaagtctc caaaaattgg
115021 gtctttgcct gagatagatt tgtcttaaaa ttgaaatcat tcacttatca gatttgaccc
115081 ttttttaaag cataactttg ctgtgtaata ttagacttat atgttttgat ttccttctac
115141 aatatctctt aactttaagg gacaaagtga gcacagaatt tttgatgctt gacatagtgg
115201 acatttatat ttaaggaaat taggacaaaa attattataa tgtaatcaca tttgaataag
115261 atttcctgtg cattttctgg cagatacctc cactcagcca tgattatata ccgagacctg
115321 aaaccccaca atgtgctgct tttcacactg tatcccaatg ctgccatcat tgcaaagatt
115381 gctgactacg gcattgctca gtactgctgt agaatgggga taaaaacatc agagggcaca
115441 ccaggtaggt gatcaggtct gtctcataat tctatcttca ggatggataa ccactgacct
115501 cagatgtgag ttcagaagag tcaaaaggaa aacagagtct atcacattgt gaacagaggt
115561 ttattttgtg aaaaaatgca agcatcacat tgtgattttt atcattgtat tttgtaggaa
115621 aaaaacaatt gatgtaattt ttcagggcaa aaactgaata aaaagaagag aatgtttgat
115681 atcaagttat atgttttaaa gttagatttg tagattcttt agatactcta gaggtcataa
115741 aaagtaacag caaaaacttt agtgtaggta ttgttggcac ttgtgaggca aatcaaattc
115801 aggtccacaa attctttttc ataattctga aacccaaaga actctgaaaa tcccaagatt
115861 ttttaaaaaa tgactaattt ggtgtcaaaa cctaagcaag ctgacttgtt gcttattaca
115921 atctttattt ctcatgctca gtgtgaatat gcatacattt tgctgcagaa atatatacat
115981 gtttgagtac agggggctgg ccgtgaccct actgagggtt tctgtacaca tcactgtcta
116041 ccctgtggaa tcttacctcc ctttcttagt tcccaatcct gaaaagcagt tatggggcca
116101 gtgctctgta cagacatgtt gtctcagaca tcagtttgag caggaagtaa atcatttagg
116161 ggttggcatt tgtttggagt gtggggaaca ctctatcttt agggaaactt tatatagtta
116221 gttatttgta agtaaaatta caggtggcta tacatcatct tgctgattgc aactcaatta
116281 aatcaccgtg cctggcacag aagaaaatat gctacaggat atctcactag ggaaaaggtt
116341 ctagttcgtt tcctgcgcac tcaacttttg tacttagata agcaaatggc cccagattcc
116401 aatgcctggt tttatttttg ctccaaatac atatatactc ttttgttttg gatagttaca
116461 ttttagaagt agactgtgta ttctcataaa cacttcaaag tgtatgttct ggctgagagt
116521 gtctctgtgt tgttcaataa taataagact aattatcatt ttttgagtac ctgctgtgcg
116581 tcaggcccag tgccacgtat attagagaca agatctctta tcctcatgcc agggctggaa
116641 gttagctatt agtttctcat ttgccaaatg agaaaactga ggctcaggga gattatgtaa
116701 cttgcagaat atcactcagt aattggccaa gataagaatt cagtctaaat gagaaccaga
116761 tccagagata tttggcttta aattctatag tctctcctaa accatatgca actctaacat
116821 gaagaagctt atttaatctt cactattaaa aaagtcaaaa caaaacaaca gagccatgaa
116881 tagcaaatat tgtcaatgag aggtttggaa aaacagtctt aaaggatgaa attccataga
116941 cctgatatat ttccacctgg aaaaagtggg catgggacag tgattttctc ttgaaagatc
117001 tgctcatttt tgtcatggga catgaaggtg gactggacca ctcagtttct tctttctgca
117061 tctcccaacc cagtctttct gttcatgggg tgaaaatctg ttgttgaagc cttgtctgct
117121 taattggaca gtggatctct cgggtccctg tgggctgtgc gcttgtactt gagctctgct
117181 tcttcactct gtggtctagg ccagctagca gccagctgag ttcaccttgg ttcagactca
117241 tggcctttca ttttcagtat ctgacttcct ggttttgctg aaaacctgtc taaaatgtaa
117301 tatccatctg attcttcata ccaagccaca caattcttcc tgatcccttt taatctccaa
117361 tattgaatgg tggtaacata aatatggaga cagatcatgt cagaaaccca gggcctaatc
117421 ttttcttttc tgcctactct tctcacaggc tgcttagtac tttgtaagct tttttttttt
117481 ttctggctgt aacctagatt ttctctttat cattactcta tttattattg ttagagcact
117541 tctgattatc tcagccctaa actctgcctc caattttaaa taacaataac tcccactcct
117601 gctaatactg ctactactac taccatcacc aaactttttc ttccccaaag cagttctgtt
117661 tgggaaggaa acagttccct ctcatacaat ttcagttatc ttcttgtctt ttccgtttaa
117721 tgaatcttcc tgttaatgtt acatctttta acatggaaac ttctagagaa acaaaagacg
117781 atggatttgt taaacctttt gggtgtattt ttatactaac tcttactgca gcgtgtgcat
117841 tatgagtgta ggtccattac ggctgtatta ggagcagaac cttccagagc atgagcgatg
117901 tgctgggctt gtgcttagct ctatccatga gttaagtatc tcaatcctta ggaccctctg
117961 acatatgtgc tattattatt tctagtctat agatacagag actaaagttt agagaatata
118021 aaaaaacatt tacaaggtcc tatgggacaa aaactgtagg acaaaatgca aacccaagca
118081 gcctgagagc agagctcctg gtccagcact gtgatagctg gggacgcaga gacagaaaca
118141 atgcaattat tgacagggac catggtgctg tgtctgtcca cattttgaag ataattatgg
118201 tttggatatt ttcaccttta aataacttgg agagtttcaa cattaactca gtcagatgga
118261 tacatttata tcatatcctg ctgggagtga cagttaattc tgggtcctat ggcaattgca
118321 cttttgactg agatgaatgc tgactgatgg ctttaacatt taactaatgc gatagtattt
118381 aacacaccca tataaatact atagtcttcg ggtaaataaa atgttaccgg ctggacatac
118441 atgaatatct gatggagatt atggaacata ctctactcat acttctctga aagtaaaaaa
118501 taaaagatat gtttcagtac acaatgtgat atgtactcag acttaattca taaatttctc
118561 ttatccttca tccgtggatc ttttctttat ttacttattg cgtttgttaa aatgcaggct
118621 tctctgaaaa attattttta aaaatagttt ttagacaatg aatcatattt tctcaagtat
118681 tttaacattg taatcattat gataattatc caaggggaaa ttatacttat tttttattca
118741 tttattcatt catttggcaa caatacattg aacatttact aagcatcaaa ctggctctac
118801 cacttaatag tggcatgatg ttcatcaaga aattgttagt gcaatcaaga acactagaaa
118861 ttcattggat gaatttaaag aagcttttag aagggtatta tattataatt gaggcacttt
118921 atgaatatat aaataatatt atgttttcat gctagagatc atgccaatga agatatttac
118981 tttgaaaagg agaagattag aagtttaaaa gcatttccat attgaagtaa atattcattt
119041 ccatatcttc acagttatct ttctctgagt tctctgactc attgtgaaaa aaaatcccaa
119101 ccttcttcac agctctacca tcttcggatt gttgcctaga ggggtaaaaa ctattgtaaa
119161 aggatgtgtg cactggatga gaatttagaa ttagacgaaa tgacccctag agtcttttcc
119221 tgctttaaga gcctgtgatt ccaaattcta acagtacatt tatcaagaaa aaatatgctg
119281 aacatttaaa tagtttttga atagtaccta gatataatag atacctaata aatgtgctca
119341 atgaataata aataactggt taagatttaa ataagcctcc aaaatctctt ccacattcta
119401 agaagggaag cataaaggtt gttaatgaac tagtgactgt gtgggtagct cattattttt
119461 aagtactctt gactttgctg ttcattatct gtgtggcctt aggaaaatac acacatttct
119521 gaaaggatta tgtcgtttgt aaaatagaaa gtccttatct gtctaccaca gatgattctt
119581 atgcaaatca aatgaaatgt tcaataaggt gtctgtaaaa tagtagagag agatgaatta
119641 ggagctattg tgatttgttt acattatgtc acaggtgcac tttattaggg atatgtttta
119701 tcttaattac acaattcttt aacttagatt ttgagaatta tattatggtt atataggaaa
119761 atgcccttat tctaaggaaa tgtataatat atttaggtct gaaacattgt atctgtaaca
119821 atatagtatg taaattatgc taattcacat gataattata tgtaattata ttaatatatt
119881 actatgtata caatatattt acatgcatat atgtggggaa atgttatcag ttagtgtagt
119941 aggggttatc atactcaaat tcgatgtctc catccttcca actcttcatg cttttccagc
120001 atggtgagga ctgctgagct ccatcttttg ctggtagtct ctctgtcaaa tagaactgtt
120061 tccaaattca gtcatttgct ccttgaaggc tatgaattca tacttcgtta tatttttctg
120121 gctgcatatt taaattactt taacaatcat ataagttcat tgtaaaaatt ttggaaataa
120181 aaaggaagat aaaatgcaca gataatttta gcaaatgaaa taataattat attgggatgt
120241 atttcttcct agattttaat tatgtacatt cccatcaact ttttattttg aaaatgttta
120301 agcctaaaga acagttgaaa gagtagtaca ggctgggtgc agtggctcct gcctgtaatc
120361 ccaacacttt gggaggccga ggtgggtgga tcacttgagg tcaggagttc aagaccagcc
120421 tgaccaacat ggtgaaaccc tgtcactact aaaaatacaa aaatcagctg ggcacggtgg
120481 tgggcacctg taatcccagc tactcgggag gctgaggcag gagaatcgct tgaacccagg
120541 aggcagaggt tgcagtaagc caagatcaca cccctgcact ccagcctggg caagagagtg
120601 agactccatc tcaaaaaaaa aaaaaaaaaa aaaaagaaag aaagagtagt acaatataca
120661 ttcatactca tatacccgac gcataaattc attgattatt tactttttgc cctacttgtt
120721 ctttcttgct ctctttgcgt atgaatgaat cattgaaatt aagttgtaga catcatgcca
120781 tatcacctct gaacagtgtg tatatttctt tagaataagg atgtttactt acataatcat
120841 aataccatta tcacagctaa gaaaattaat tcagttgatt ttttccacat atttgataac
120901 tttctgtcta tccacgatta tgtcttacat attcttttaa tttatgtcat agcatatcat
120961 cttagaaagt gatccctaag ttactgcatg gtatacattg tttaaccatt tccctttgtg
121021 attggatgtc tttaggttga ttatattttt attattatca caaatgttga aatcactctt
121081 tttttctgaa gaatttaaaa gtaatttatc tgtcttatgg aataaaatat ttatttcccc
121141 ttaaaagaat ttcaggcatg aacccaagag agaaggcttt tttttttgtt ttagttgttg
121201 tttttatttt tattttttat tttttgggta gaaggagcag agagacaagt tcaggaaata
121261 atgagagtgt tagaattttg ttcaggttaa agtgagttgg agtgaagttt agaaatctcc
121321 tttctactca tctctcctgt ttttaaaaca ctgtcctgga aatagttaat attaggaacg
121381 agaaaaatgg tataggtttt cctagtacac tttatttctt aattatgaaa ttctacttaa
121441 taacttacca ttgaatgttt atccttatta tcattcaagg taattttatt gaagattgaa
121501 gatatttata ataaagattg aaggatttta ttgtcctgtg tggtcaacct tggggggtga
121561 gatgttatga gacaggacaa ttaattgact tgatcaaggt accttgttat aaaaataaca
121621 cagcctggtt tagaacatct cttcctgact ctcttatttg gcatatagcc taagtgtatg
121681 cctccttgga tgtatgagcc ctgatgttgg tcatatttat tattttatct gcttactttc
121741 agggtttcgt gcacctgaag ttgccagagg aaatgtcatt tataaccaac aggctgatgt
121801 ttattcattt ggtttactac tctatgacat tttgacaact ggaggtagaa tagtagaggg
121861 tttgaagttt ccaaatgagt ttgatgaatt agaaatacaa ggaaaattac ctggtaagtt
121921 ctgttttctc tacaatgaag attttttttc ttaatatcag cagcttcatt tttatttaat
121981 tgtagttgta tgcttaattc cttaaacaga tgatcatttt ttttgtttag tgcataaata
122041 ttcttaaatc ttgtgatata ttaataaaaa tcacctgaaa aaggtagcag ttttaggctt
122101 tttaaaaaat ccgcaattaa tattggtgta gttaatatta tatttagaaa catagagaag
122161 gaaattgctg ttagaactcc acatttggtg atttttaatt ttcataaaga attactgtgt
122221 actcattatc ctggaatgtt ttcgttttct tggagtgaaa taatttacat gcaggaatgg
122281 aagactgaat gatctataat aataattttt cataagaatc ggtaaatgtg tatttaatgt
112341 tatcaaagct catttggaat ggttgtctca tgctttcaag aaattagagg actttgtaat
122401 tcattcctta accattactt tagttctcac cacaaaataa cattttaagt ttatttagct
122461 ctttctcata ttttctgctt tccctttcat ttaaaaaata cttttgagtg tacacaatgt
122521 gccatgtaca ggaaatagag ctttatcttt tttgggtata acttcaagat catggcaaaa
122581 gaaaacttat tattaattgg ataaacctta gatataatct aggttatttc ccttatttta
122641 ctagttttct agtgaaaata ttcaggtctc tgctgggtac agtggcttac gcctgtaatc
122701 ccagcacttt gggaggccca ggcaggcaga tcacttgagg ccaggagctg gagaccagcc
122761 tggccaacat ggtgaaaccc tgtctctact aaaaatacaa aaagtagctg ggcatggtgg
122821 catgtgcgtg tagtcccagc taccaaggag gctgaggcac gagaatagct tgaacctggg
122881 aggttgcagt gagccgagat tgcgccactg cactccagcc taagcaacaa agtgagactc
122941 catcttaaaa aaaaaaattc agttctgtgt tctgcatcaa ccagaataag ctacgcctct
123001 tataaaaaac aaatgtgcac aaaccatctg tgaggacata aggattaaat gcttgcttac
123061 tttgagtatt aaaataaaaa gtagaagctt tattatatga gtaaaagtgt ttccaaagtc
123121 tatttgaaat gcaggtacag aatgaaaatc tgttatttta ttaaatcgtt atttgggtct
123181 ctttttattc cataaaaaaa aaatcttttc cacatctctt agtggagatc aagttaacaa
123241 aattagcttt aattttgtga caagtaaatt tacataaata taggattatg gagataatat
123301 ttttctttgc aatgtctgga ccttttataa acattgagag gaaatataac cattcttact
123361 tatttagtat gctagcatga tgttttttaa tgttttagat ccagttaaag aatatggttg
123421 tgccccatgg cctatggttg agaaattaat taaacagtgt ttgaaagaaa atcctcaaga
123481 aaggcctact tctgcccagg tattcttaaa gttttgttaa tattttgtac agaacatcat
123541 ttgcatatat gcatatatat ataatcttca ataatatata cttaaacaca taaacacaca
123601 gagacagaat taaaaatagt tataaggcaa acctcctata attttcacca tcccaggcac
123661 aaaaaaagga cattgccaaa acctcacatg ctcccatatg cctgtctcct cttctcttct
123721 ttgaaatgcc tgcccattat tttgcccatt tttctgttga ctcttttgcc tacaaatcaa
123781 atcqagaaaaa aattttata tataacatac taatcctttc ttggttttat atgttgcaat
123841 ttctttttcg agctggtggc ttgcttttta aatttcttgt agggtgcccg ttaatgaatg
123901 gaatttctta attttaatat atataaattt aacaattctt ctttttcctt cttttcctgg
123961 ttattcctat ttggtcctgt tgtagagccc atctttcttt tagagctaca aaaatattta
124021 cctgcaattt cttcaaacat tttaaaagtt tgcttttgac aagattttaa tccattggaa
124081 ttgacatttt tatctgtatc ccattccttt ttttaatgtg gaagactaat tttttcaggt
124141 tgatttacta aatggcttct ccccttttgt cccatagatc tgatgtgtcc attttgtaat
124201 ttattaaaga taatgtgcat atccacgtgt acactgtctt ccactgatca actcatttat
124261 ttttcctcca gtattaccct gtcttaattc ctgtagcttt ataatgatct cctctcccta
124321 tttatttttc tcatccagga atattttaac cagtcttagg cttagctttt ctatataaaa
124381 tttagaatca tgctgtcaaa tcttatgaaa aaccacattg tcgtttggat tggtcttgga
124441 ttgaatatgt tgacaatctg gagatgatat ccttatgata ttaagtcttt gtattctttt
124501 tcttatttat ttatttattt agaggcaggg tctcactctg tcacccaggc tggagtgctg
124561 gagtgcagtg gcaggatcac agctcaatgc agctttgacc tcctgggctc aagtgatcct
124621 gtcgcctcag tctccatcag ccaattgtgt gccaccagac ctggttaact tttcctttaa
124681 tttttttgta gagaaagggt ctcactctgt tgcccaggct ggtcttgaac tcctggcctc
124741 aagcaatcct cccgcctagg cctcccaaag tgcatgagca actgcacctg gccaagtctt
124801 cctattctgg aatggcatga attctctctg tttacttagg tcttctcaag cgtctttcaa
124861 taaagttttg taatgctaca tacaggtctt gcatatcttt tgctggattt tcttgctgca
124921 aattgcttta aatgcagttc atggcttgag atttgttttt taactgccca gttaacgtaa
124981 atctggtcta tgaattgtat tgacaacaag cttgtgagta gcaatttttc agcaaccttt
125041 tgctccctct ccccgctccc tgcacagtcc cagaacaact ttccttatat accaatgaag
125101 gtggaaatgt ggacaggttg atttctcctt cttgtttatg ctgaatatgc agtcctttgg
125161 ggtcccagct ttataggtac agtcccttta ctaagactgt ctatcttggg aaggccctag
125221 actccaactt ctctcccatg ggccccacaa agcatccaag agtatacatt tatattatct
125281 cactttgtct tggcagacaa atgtcttcag ggcaagtctg gctttagggg tccactgacc
125341 tctctggttt ctgtctctca ctgtgatttc ggcctgataa ttccttatgg tgtcagatct
125401 tcaatgtttt taatacattg tttttaaaaa aatcatttag cattttaaat tgtttttagt
125461 tggaagattt gcccaagtaa ccttgtccac catattatct gaaagagaac tcttgtcatg
125521 ttttacactg atacacattt taataaatgt gggttatctt tatgttgtga gctcttgatt
125581 tggtattata attaattgga aaagttttaa ctttaagtat tctgatcaaa atagtcaagt
125641 acaactataa tgactttatc aaatattaca taatttttct tctacttggt ttacttgttt
125701 tttaatttag gttaccatca atgttagtca catgaacttt tatatttatg tccacagtaa
125761 aaatttttca tagcttgttg tttttctttc ttgttttttc attttcaatt atacttcacc
125821 tatacttaac agaatactta acaaatatgt atatatatga caatattaaa aagcttagac
125881 atacttattt tatgtttaaa atataacata tactaggcaa gacaggaaaa ctcatcactt
125941 ttatgaaatg aggcacagac agagtaatgg gctttcttgg tgtctcctga gtggcgggca
126001 ggtggccatg tcacagctct aatcccagtt tcctgacttc tggttctgtt ttcttacggt
126061 gccttcacac tgtctctcca gatcaaaaac agaatctaga gatgacttct aaattttgtt
126121 accaaagact gaaattcctg ttcctttcaa ctactagaag ctcaactaaa ttgttggccc
126181 aagggttttt ctctcactgg cggtggctct gcaatataga attgcatgca gagtacctcc
126241 tgactccgct aaaatcctgt ttaattgacc cttgagattt tcttttcaag ttaaaaaaaa
126301 tactacacat accaaagagt atcaagcaca gtttaaaaat acatatttgt cttttcatgt
126361 aattttattt gtagtttaga ttactaatct tggtgatcta gttgggttcc agtttaacag
126421 ttttaggttt tgcttgacag agctataaca cttcagtcta tatttgattt ttcaagggaa
126481 atgagttaac tcgataagta ctgttttgtt atctttaaac tttctcaggt ctttgacatt
126541 ttgaattcag ctgaattagt ctgtctgacg agacgcattt tattacctaa aaacgtaatt
126601 gttgaatgca tggttgctac acatcacaac agcaggaatg caagcatttg gctgggctgt
126661 gggcacaccg acagaggaca gctctcattt cttgacttaa atactgaagg atacacttct
126721 gaggtaaatc caaatgctct ttaaatcttt cataatttaa agcatatacc atttggaaag
126781 ttacttagga ataaattaaa taagagccaa tgtaggatta ttattcaatt agccttctgt
126841 tagaacaaga gtattcaaga gcaaatgtgt tttgctttag aatcacagca tatgtcttag
126901 ctcagggtcc ctagaaacag agcctgagat gggttatctt gcacaagtga tttaaggaaa
126961 gagaatctta gagagaagta gaggaagcca gaaagggcag aggaaaaagc tcagcagaaa
127021 tgtggtttct gaagaggtcc agcctcagtc tctgcccaca aggagctctg gaatatgaat
127081 aggagcacag aattttccca ctgccaggca agggagccag tctttcattc tcacatattg
127141 ttcagtcatt ggctgcaatc tgctgggagg tgggggtagt gtaactttca aggcatttct
127201 gggcaagctg cctcctgtca tctgagggta ttctgtgata aatagcacat ctctgaacta
127261 tagtagccaa cactcagggt agctggggat ggcgtacctg atggataaag gagatctgga
127321 catggctcct aaaagtggat caatacattg tgttggcaaa tataagataa gtttctagac
127381 ttcaaagaca acctagtatt ttgactgctg cctgaagata aatattgtgc ctcaacatta
127441 gttctgaggt taaacaatct ttttttttta attgatcatt cttgggtgtt tctcgcagag
127501 ggggatttgg cagggtcata ggacaataat ggagggaagg tcagcagata aacaagtgaa
127561 caaaggtctc tggttttcct aggcagagga ccctgcggcc ttccgtagtg tttgtgtccc
127621 tgggtacttg agattaggga gtagtgatga ctcttaacga gcatgctgcc ttcaagcatc
127681 tgtttaacaa agcacatctt gcaccgccct taatccattt aaccctgagt ggacacagca
127741 catgtttcag agagcaccgg gttgggggca aggtcataga tcaacagcat cccaaggcag
127801 aagaattttt cttagtacag aacaaaatgg agtctcctat gtctacttct ttctacacag
127861 atacagcaac aatctgattt ctgtatcttt tccccacatt tccccctttt ctactcgaca
127921 aaaccgccaa cgtcatcatg gcctgttctc aatgagctgc tgggtacacc tcccagacgg
127981 ggtggcagcc gggcagaggg gctcctcact tcccagaagg ggcggccggg cagaggcgcc
128041 ccccacctcc cagacggggc ggcggccggg cgggggctgc cccccacctc ccggatgggg
128101 tggctgccca gcggagacgc tcctcacttc ccagacgggg cggctgctgg gcggaggggc
128161 tcctcacttc tccgacgggg cggctgctgg gcggaggggc tcctcacttc tcagacgggg
128221 cagctgccag gcaaaggggc tcctcacttc tcagacgggg cggctgccgg gcagagggac
128281 tcctcacttc tcagacaggg cggccaggca gagatgctcc tcacctccca gacagggttg
128341 cggccgggca taggctctcc tcacatccca gacggggcgg cagggcagag gcgctcccca
128401 catctcagac aatgggcggc cgggcagagc cgctcatcat ttcctagacg ggatggcggc
128461 cgggaagagg cgctcctcat ttcccagact gggcagccgg gcagaggggg ctcctcacat
128521 cccagacgat gggcggccag gcagagacgc tcctcacttc ccagacgggg tggcggccgg
128581 gcagaggctg caatctcggc actttgggag gccaaggcag gcggctggga ggtggaggtt
128641 gtaggtagcc aagatcacgc cactgcactc cagcctgggc aactttgagc actgagtgaa
128701 cgagactccg tctgcaatcc cggcacctcg ggaggccgag gctggcagat cactcgcggt
128761 taggagctgg agaccagccc ggccaacaca gcgaaacccc gtctccacca aaaaaatatg
128821 aaaaccagtc aggcgtggcg gcgcacgcct gcaatggcag gcactctgca ggctgaggca
128881 ggagaatcag gcagggaggt tgcagtgagc cgagatggca gcagtacagc ccagcttcgg
128941 ctcggcatca gagggagacg gtggaaagag agggagaggg agaccatggg gagagggaga
129001 cggagaggga gagggagagg aataatcttc ttatatggtt tgaaggaatg agaattcaca
129061 ctgaaaaata atttttaatt ttagtttcag atgtcatctt gataggcaaa acttgtctgc
129121 caattaactc atttattgct gaaaattaaa taaaattggc attgttttta aaagtaatgc
129181 aagaaagcaa aaagagttat gttgataaca gaatccttta ttctgtacaa gttctagttg
129241 cttaagctta aatcaaatcc tgctaagtat attttctttt cttaacagga agttgctgat
129301 agtagaatat tgtgcttagc cttggtgcat cttcctgttg aaaaggaaag ctggattgtg
129361 tctgggacac agtctggtac tctcctggtc atcaataccg aagatgggaa aaagagacat
129421 accctagaaa agatgactga ttctgtcact tgtttgtatt gcaattcctt ttccaagcaa
129481 aggtatggta gtgaatttga tcaatgggga aattacagat cttttaaacg actgaattgt
129541 gtgcataatt gttattgcat cagcaaagat tgttcatttt tagcctattt tcattggttt
129601 gcatatatta aagggaattg tggaaggtca cagagatatt tgttgttttt ctgaatacag
129661 atctagctga gacatttata aaataagtca accatttatt caggcctacc agccctgctc
129721 ctggtattac ctcaactgtg gctctatctc tttacttctc ctcagatcaa tgaatctttg
129781 tagggcctct tcaaggataa attctcattc attcattctt tgaaaaaaaa aaaatatata
129841 tatatatata tatgaaaccc attgtgtgcc aggcttaaac ataccagtta tctaactacc
129901 aaattaagaa aaaaattaaa taaatgaatt aataaattct taataggtga aaatgactta
129961 gctcttatca attgcagggt tcttgtccca aagaaatata tctacatagc aaaatttcag
130021 gtgtgagttg taggttggtg actgtaatat ttggggcagg atgatttcca ggaggcatta
130081 agattatacc ctatatattt ctctggttta agttagtatt ggaaaaaaag tactagaaaa
130141 atgtgaagcc tgttttttgt acctgaaata tcaactccac tggcagtttc ggagttgaaa
130201 ttatttgaat atggtcaaag aaaaatttca atggatggaa ttgggcaagg acgactttat
130261 tcaagcctat cacagcaggg gagagagatc agactgaact aaactccact gaaacaaaag
130321 gtgggagagt tttaagcgca ggggtgagct aatggaaacg tactggagca ccttgttgga
130381 aggaagtggg agcagttgtc aatgtgatta ggccatctgt gtttgctaat tgtcccttat
130441 tgaaggtagg ctcctactct cccacagaca ctggggaatt ccttccttcc ttccatccct
130501 ccctccctcc ctccctccct tccttccttc cttcttcttt tttttttttt tgaaggagtt
130561 ttgctcttgt tgcccaagct ggagtgcaat ggcatgatct tggctcactg caacttccac
130621 ctcctaagtt caagcgattc tctagcctca gcctcctgag tagctgggat tacaggcgtg
130681 caccaccaaa cctggctaat tttttacatt tttagtagaa acgggatttc accatgttag
130741 ccagactgat ctcaatctcc tgacctcagg tgatctgccc accgcagcct cccaaagtgc
130801 tgggattaca agtgtgagcc accacgccag gcctctgtct tgataattac atttcaaagg
130861 aatggctccc aggtccttgg aaaagacatt cttggggtat aaaactggga agagtctggg
130921 aaaaggggca gagaaagaat ttataattcc aagtcttcta aagtaaatac tctaagaaaa
130981 gggaggttag gaatttatag ttgagaagtc tatctaaagt ttaataaagt ggaggagaac
131041 attaaggcca ttttagtcaa catacatgtt ctttttgtaa caatttcaac atttttcctt
131101 ttagcaaaca aaaaaatttt tttttggttg gaaccgctga tggcaagtta gcaatttttg
131161 aagataagac tgttaaggta aatgttgaat gcattctaca tctaaattta ttttaagtct
131221 tttgttttat atatatctca cacccctctt atgggattat aaactccctg agagcaagaa
131281 tcataaatta tgctgtattt gtattgcttc ataaaatctt gaacacagta gatcctctga
131341 aaatacttgc tgattgactg tatattttat atgaatgaac taagaataaa atgataaatg
131401 acatctgatt gataatattg ggaatggaaa taattcaatt tgtacataac tgaggcagat
131461 aattccttat aaatatattg tggaaaaaaa acaaaaatat acttaagttt taaatatggc
131521 ttgccattaa ctttttctta agcattgaag aaatcattta attttctttt cttcagattc
131581 ctatttagtc attaaagcat tcatttctct atccatctat tcatctttgg ttccatctat
131641 tcactcaact tcctacccgt tcattctcct attgccaaaa agcttattat ctgatgagag
131701 acagggaagt agagtataac ccttaggtta tttcttttgt aatttttaca tgggaaaaag
131761 aatagattga atgtaacaat aatatttcga atatgaccta aattttttta tgtataatat
131821 ttgtacatat ttatggggta catgtgatat gttgttacat gcatagaatg tgtaatgatc
131881 aagtcagggt atttaggata tccatcacca tgagcattta tttctctgtg ctgagaacat
131941 ttcaagtctc ctagttattt tgaaatgttt ttaactgtag tcactttatt gtactattga
132001 acattagaac ttattcctcc tatctaactg tatgtttgta cccgttaacc agcctccctt
132061 catcctcccc ttctcccaca cacccatatc ctcccaagcc tctggtaact atcattctac
132121 tctctacctc catgagatca acttttttag ctcccacata tgagtgagta catgtgatat
132181 ttgtctttct gtgcttggct tatttcactt aacataatga cctccagttc catccatgtt
132241 gctgtatatg acatgatttc attccttttt atggtcaaat agtattccgt tatgtaaata
132301 cacacatttt ctttatgcat tcattcattc atgggtgctt aggttgattc cacttttttt
132361 tttagctatt gtgaatagtg ctgcgataaa catggggata taggaatccc tttgatatac
132421 tgattccctt tcctttagat tagtatcagt agtaacattg ttggattgta tggtagttct
132481 atttttaatt tttttgagaa atcaccattt tgttttccgt agtggctata gcaatttaca
132541 tacacaccaa tagcatatgg gcattcgttt ttttccgcat ccttgccagc atgttatttt
132601 ttgtcttttt tataatagcc attctaattg ggtgaagaag atttcattgt ggttttcatt
132661 tgcattttta ctgatgatta gttaatgttg agcatttttt ttcatatatc cattggccat
132721 tactatgtct tcttttgcaa atgtctattt agatcctttg ccaacttttt gttttgtttt
132781 aagacagggt cttgctttct tacccaggct ggctcacagt ggcatgatca tagcttgttg
132841 cagccttgac cttctgcact caagtgatcc tccaacatca gcttcacgag tagctgggac
132901 tacaggcgtg tgctaccata cctggctgtt tattttttgt agagatgcgg ctccactatg
132961 ttgtccagac tgatctcaaa ctcctgggct caagcaatcc tcctgcctca tcttctcaaa
133021 gtgctgggat tacaggcatg agccaccata cccagccctt tgcctacttt taaatggagt
133081 tctttttttt ttttcctgtt gaattgcttt ttcgagtttc ttgtgtattc tggatgaata
133141 gtttgcaaat atttcctcac atttaatgga tcctctctat attgttgata gtttcctttg
133201 ctgtgcagaa gctttttagt ttattatagt cccatttgtc taattttgtt tttgttgcct
133261 gtgcttttgg gatcttaacc ataaactctt tgtctagacc aatgttctga aatgtttccc
133321 ctgtttcctt ttaatagttt catagcttct ggtcttacat ttaagtcttt aatccatctt
133381 gagttgaatt ttgtaaatgg tgagagagtg gggcctactt tcatccttct gcatactgat
133441 atccagcttt tccagcacaa tttattgaag gtggtattct ttctcccatg tatgcttttg
133501 gtgcctttgt tgaaaattag ttggctttaa atatgtgggt ttatttctgg gtcctctaca
133561 ttggtctacc tacctgtgtt tttgccaata ctgtgctgtt ttggttactc tagccttgta
133621 atatattttc aagtcaggta gtgtgatgcc tccagctttg ttctttttgc tcaggattgt
133681 tttggctatt ttggctcttt ttcggttcca ctcaagtttt agaaattttt ttttctattt
133741 ctgtgagaaa tatcattgga gctttcatgg gaatttcatt gaatctgtag attgctttgg
133801 gtagtatggt cattttaaca atattaattc ttccagtctg tgagcatgaa tatctttcca
133861 tttgtttgtg tcctcttcaa tttctttcat gtttttcagg tttccttata gagatcgttc
133921 atcatctttg ttaaatttac tcctaggtat tttattaatt ttttgtagct acttcaaatg
133981 ggattgcttt cttgattttt cagctacttt gttgttgttg ttgtatagat atgctactga
134041 tttctgtatg ttgattttga atcctttact atactcattt atcagaacta agagtttttt
134101 ttaatggaat ctttaggttt ttgagtttta attttaatat ctctaatcat ttaagatgga
134161 aagtagtttt ttgaaagcac agattttatg gagttttgtt ctgtggatac tcaatttgct
134221 gagtgtgttt tctttttttt ttggcaaagc ttaaaggagc tgctcctttg aagatactaa
134281 atataggaaa tgtcagtact ccattgatgt gtttgagtga atccacaaat tcaacggaaa
134341 gaaatgtaat gtggggagga tgtggcacaa agattttctc cttttctaat gatttcacca
134401 ttcagaaact cattgagaca agaacaagcc aactgtaagt tattttttat ctgtacaagt
134461 aatttatcat tatacttttg ttttttcctt ataatcatta ataatactgt tgataattca
134521 taaggaagat cttttaaaat gcataattta ttttctatca taaaattaaa ctttcattat
134581 aaaaaatttt gaaaattcca gaaagcagaa gggtattttt aagaagtcac tcaacctaat
134641 cacttttagg gataaaatat gtaaactcat tgtaatctta gtagtattta tcaatctaaa
134701 ttttttaaca atttttatta tctgtgtttc aaattagaca tgaaattgga agacaatcaa
134761 ctttgtattt caccaaattc acggactata catatgcaat ttgggtaact tccattaagt
134821 attgattgta ggaaagatag acagcaagta ttcttgcttg tccaaagttg tttctagatt
134881 tgataattat acagatgtct actcacagcc aagttagtga taccagtttc aaacaagaat
134941 aaaataaaat attaatataa acctctttca agtttgcttt ttttcagtgg tattttaatc
135001 aaatctttgc agttggttct tatttatcac attcctcagt gataagcagt ataggattgt
135061 ggataagagc ataaatcata gttcagatat tgctttgcca tctattggtt ttgtgaactt
135121 gggcaataac ctttcacatc ttcagtcatc tcttacctga ggattgtaat attctctatc
135181 tcaaagagat atttggagga gtaaataaga atgttaatat atggatctta attaacataa
135241 tgaccagcac ctggtaagct gtcaataaac attagctatt attattatta ttagctttga
135301 gtcacaaatc cctaccttag ggaatatcct ggtttcccat tatccatcaa atttcccaag
135361 attggcactt gggagtaatc tttgactcct ttgttttttg ctccctttat ccacttgacc
135421 ctcctaattg tcatttgaat ctttgtactt ctcactgttc tcagtgctgg taccatgggc
135481 cagactgcca tccattatct gtggccacat taacaagagc atccagttct caccctctga
135541 ttatactctc ttcaacatat tttcagcatt gcagacagag ggacctttct aaaatgtaca
135601 tcaatctgat gcaattttcc tgcttaaaac ccttcagtgg tatcccattg tcctttatat
135661 gaagtccaaa ttccttaaga cagcctactg ggcccttcat aattcagtgc ttgcttacct
135721 gtctagattc atattttgca agctttttgc catctgtgct ttacccaaac tgaaattgac
135781 tcagatctct aagacaacct gttattacct ttcaccccca acctttgaat gtggtcttct
135841 ccttacctgg atgactatta tgccccctgc ctatttcagt ccaagcacca cttgctctga
135901 gtggcaggtt aggtgacttt gtgcttccat tgcatctaat gttttccttc acagtagcta
135961 taattgtatt tgttaaagta gtttctcaat accactaaat ctactggctt tcaacattgg
136021 ctgtgcattt agaaaccact aggtagctga aaataatatg atacctgggc cctacctcag
136081 accaattaaa tcagaccagt taagcctggg atggggatca gatttttttt ttcaggttct
136141 caagtgattc taaagtatat ttgaggttaa gatatactgc ggagtgcagt gtattataag
136201 ttcccatgaa tgaggatttt tatttctgtc tttacatatt tatttactag tatgtggtta
136261 acatttggat caactcattc tcattctgta atacccacat tttaaaaaat gaatgtaaaa
136321 atgtctttta ttatttttat ttttcaaatt taattttaga ttccaggaat acatgtgcag
136381 gtttgttaca aaagtatagt gcgtgatgct gaggtttgga atacaactga actcacaacc
136441 cagaaagtgg gcatagtgct tgataggtag tttttcatct ttgctcccct tcctgtaatt
136501 tccactttta acccaattat acaagcctga aaacctttaa aaagaaaggg cctccagttt
136561 atttttttat ttcatagcac attttatggg attatggata tcagttaact ctttaaagtt
136621 ccatataaga tttggagcat agatgcttta ctagagagca tccataaaga tcaagctctc
136681 aaagatgctc atctcccaaa agagattggg acctagatgc ataaacactt aaatataaat
136741 atttgctctc ttctgaacaa gtatctcctg tggccttggt ctctacccca caaaacagac
136801 atcacatcac taatcagggg ttgcctcatc atcagtaccc tcatcatcat cagtacacac
136861 cgactgagag gcatgttggg taatgaaaga tgactgcctt tgaagccgga agactccata
136921 ggacttttgg agttctaggt ctgcaacatg tttttagtcc taggtcggca agcatgatgt
136981 tagtcatagg accttgagca aattatttag catttgtaca tgtttctttc tactattaaa
137041 aacattgaga tttatcatat tttatgtttt tttattaagg atcaagcaag ataacacaca
137101 aaagtatttt ataaaatacg gaaattccat gcaaaacttt gtcctaattg gaactatttt
137161 ctattaaata cagcaaatat ccaagaagga attacctaaa gcgtagtggc tctctgacaa
137221 cacatcatat ttttacctcc ttttccagaa tagaaagaaa agggggagga aaaaaaatct
137281 tcctactcta ggatatacta atgattgtta aatctttatg gtattttcat gttatctatc
137341 tgatttaaat gcaattttga ctatttttac atattcctcg ttgttcattc atactgtagt
137401 gccttcttct attcccccac ttaacagact gcttgattat atcagaggtg cttatctgtg
137461 caactgttta ctggacgagg ggtatgtaga aaatacattg tcctcatcct tatgaaatta
137521 cactcataat ctagtgtgag ggatgcacta ataaagacaa ttttatattt aataaggtct
137581 ataatatgac agtgacacca gtggggaaaa gggactggtt ggtctatttt gataggtcag
137641 ggaagaaatc cagaggagcc gacatttaag ttcatcctca aaggccaagt aggagtttgc
137701 catgctgatg tggcccaagg tagccaatct gtttgtgaaa tatgtacaat gccagatgtc
137761 ttaggttgaa agggaaatat tttaaggtgt tcgtaatttt tctttatgtt taaaagggga
137821 aaatggcaaa tattttactt tctgtttatg tttggatgat gtggattttt gttttctata
137881 atttgactgg cttaactgca aagatatccc ttgctttaaa atttgaagac actgcaacta
137941 aattttattt cagcatttta tattttataa ctctaggtat aaaaggctaa cacttaattt
138001 tctgagcatt cqtgaaacaa agttttgcaa gaacattcaa aagttacaga tataatattt
138061 ccttcagaaa tttagatata gtacaaaatt ctacaaagag ccacatagaa ttgaaactaa
138121 aagtaagacc aaagtaaaca ttggacataa tctttatttt attatcacaa gaaattaata
138181 taaagtaacc aaaagtaagt aaagtaccaa agcatgttat atattcaatt cagaatggtt
138241 agggaagaat atgaaataat tgcaatagtc tagcttgttt agttttcaaa atagtgtttt
138301 tacattaaga actaatataa ggttgtatta cacgtagaaa ttttaagaag aaaacaaata
138361 gtgatgactt tctatttttt tttctctgta ggttttctta tgcagctttc agtgattcca
138421 acatcataac agtggtggta gacactgctc tctatattgc taagcaaaat agccctgttg
138481 tggaagtgtg ggataagaaa actgaaaaac tctgtggact aatagactgc gtgcactttt
138541 taaggtaaat tctgtggttt ttaattttat tcccaaaaga attatctttg cacttctagt
138601 gtcacagagg aaggattttt cttcctttct gcctctgaat agagaatttt tttaaaatgc
138661 agaaaaaaat ttgtaatgct tctcagcacc atcttttcag atcaagaaaa ttttgtcttc
138721 agaacataaa agaataggca cataatgtgc atagttttct catggtatta caaagaatgt
138781 tctcgaatga aaatactaca ttattgaaaa tgagcatatt ggagtctctg ctagctttga
138841 catagttctg tcacagtgtc aaatatacta tttataatta aattatgggc cccaggatta
138901 tctgctctaa agaaaaagag tcacaaaata atagacaaat atggggggaa atgcaatgga
138961 ctgaccgagg cgctaaggag tggggatcaa gaccccagaa tgagagcata gtgcttagtc
139021 tgatgcagcc tgtgagtgac aaatccatag caagcacatt ctttctgtgc tggtgctgag
139081 aaacaggacc attttcaagc ttatttgcta gccactttat atttttattt tgttttgatt
139141 ttaccatata gatctatgat actcttgaga acattttaga ttacacacta tatctgtaaa
139201 aggatacttc aaagtttcct gtcttagatt catctgacag tttttctatg gattgtgaga
139261 agggctcaca gtttatgttc agaagggcca acaggtctcc ttgataaagg gttccttact
139321 tcctgaagta ccaaaacgat taggattctt ttatttctgg acacttcatt tttgtcatga
139381 attagactat tcactggttc tgggaaaaaa ttcagtggtt tgtatcgata tcttttacat
139441 gtgaatgact ataattttat gttcctttgt aacattgaga cttcatgtaa aacttttgac
139501 tctaactttt ttttttcttt atcctgggca catgtatgct atttttactg aattagatag
139561 ctttggtatt tataaaaatt gtatccctct tattcataat ttcctgaaaa tgaagggcta
139621 ttgttatctt tgataattta tgcttcaagt aaagaagtgt ggctctttgg catctgtatt
139681 tagcaaaatt tgctttgtat aattttaatg atgcataatg gtggtggtgt catgttttaa
139741 taatttaaaa tgttgtttat gttatcatat gtaaatagca tttatctctt aattggtggt
139801 aaaattatta atgtatactt tatggttcta gggaggtaat ggtaaaagaa aacaaggaat
139861 caaaacacaa aatgtcttat tctgggagag tgaaaaccct ctgccttcag aagaacactg
139921 ctctttggat aggaactgga ggaggccata ttttactcct ggatctttca actcgtcgac
139981 ttatacgtgt aatttacaac ttttgtaatt cggtcagagt catgatgaca gcacagctag
140041 gcaagtttct ttcctttaga tatttttcat attctctaag tcttataaaa tatgccttta
140101 ttttacgttt acattttctc tgaactttcc agtgtcatat ggatggtctt ggagggtcac
140161 acagtgaaac ataagactgg tataaattgt gaatagggtc attacagaag tggagggagt
140221 aaatgctctc agtcccacaa gagaagcaga ttactgcagc tgaacactca gtttgggtct
140281 tacttgcttt tttccttttt acctaaggca aaaatgggaa atacatggta ttgaatatat
140341 tttacttttt gagcaaagaa aataaagaaa atgtttgttt taatcatagt ctagcctccc
140401 agcttgttaa agaatctcat ttggtttttc attctataac aaatcttttt tcttgcagca
140461 atacatgctg aactgcacaa cctacaaata ttgacaaatc atattttact caaactttgt
140521 ctttttttgc ttctattttt atatttaaat atgataaaat tgtgatagca cataaaatat
140581 attttctgca taaatatatt tgcgtcttcc tttgataata atttgtttta gaaaataaca
140641 ataatagcat atatacaaaa gtttacaaaa acgacactat ggggtttaat tctgaaaaaa
140701 actagaattt atgtaacttt agcaaataat gaatgttttg aacatggtga agaaaatata
140761 ttcattgcaa gtatatgtga aagaggaaca tgtgtttttc tagcaccttc acctattttt
140821 catttataga ctttagagtt gcacaggagt tacaattaga tgctcttaat gactgtaaac
140881 tattaagata catgtccaca caagcagagc agtaggtctc tcaataggtt gtctcagtag
140941 tcttattcta ccaaagttgt tgcattctct agttgaattg tatgtacttt gggacccaaa
141001 tagcttgctt ataactgaag ttatagtgga atgtctatgg gttatagttt gattttaaaa
141061 taaagatcaa ttggaggata gcctacaagg tgctgcatga gctggcttca ctgtacctct
141121 cctgcctcca tcatctacca cattcctacc agatcttgct tgtctagatg aacatccagt
141181 tcttctcaat tactatgcta tattttgcct cagggatttg cacatgctgt ttatttcttt
141241 gcttagttaa catagctttt tttcatgctt atttaactca tatgctttga aatgttagct
141301 cctgtgtcaa aacctctgag aagccaacac tgattaggtc aaagttcccg ctttgggttc
141361 ccataacagc tttcttgcat agttttgatc atggtcatat tttcttttca ttaatgctag
141421 tctcttcact agaatataaa ctccaagacg gttgggttag tgtgtttttg tttacccctt
141481 ttttcccagg atctagccta gggcctacat agaagacttt tgatgcaaat ttgttgaata
141541 aattagtgaa tgatttgaaa agaaaatatg atattttgac atagtatcag tatatccatc
141601 catctaagtg tccatctaaa tactcatatt tgtactaaat actcatattt gtaccttacc
141661 atatccaaag aacttttcac acacattacc tcgtttaaca ttgtaacttt gggaagggta
141721 atgtataaat actgagccta ttttatagaa aggttaagct gttttctcag actcacataa
141781 ttaagaattg cagcaaggag tggatcacag attttgttat tttttaaaaa aatgctggtc
141841 tttattcaat ataattgaag ggtcacctag aaaatagaat tgtgaattca gttccaaggt
141901 atttgtgtct taaactatga acaactttac ttttttttca ggccagttta atatatagtt
141961 ttaacagaaa acttacatat tttgtttttg taaaggaagc cttaaaaatg tcatgctggt
142021 attgggctac aaccggaaaa atactgaagg tacacaaaag cagaaaggta acatttagaa
142081 ggatactgtt ttccaaacag ggcaatgatg tgaatgatgg taacatatta tgtgtttcat
142141 aaatttgtag aaaatattac atatggtata atcaggaatt ttaattggta gtttatagtg
142201 taaagaactt agacataaat tttcaaaatt acaagtgata tgaagtgtta aatatttata
142261 ttttcagctg aagtagaggt gtcaatcact agctcaacct taaacgaaat gtgaatattt
142321 tttacaactt atctatatct acataatgtc taattttgaa cagtgtttga aaaagctttt
142381 atttctttta gaatatgaaa tgttaattta ttaaatgttg atactctatt tgaaatttaa
142441 tagtttctat aatgtattat aaaacttttc caagtatagt tttttataaa taataattta
142501 gtacattagt tatagctgtg tttatattta catttatcta agtcaactaa aaatacatga
142561 gccaaactga aataaaataa gaatgtttta tgatggatct ttgaaacatg atttcatttt
142621 tttctttttc tagagataca atcttgcttg accgtttggg acatcaatct tccacatgaa
142681 gtgcaaaatt tagaaaaaca cattgaagtg agaaaagaat tagctgaaaa aatgagacga
142741 acatctgttg agtaagagag aaataggaat tgtctttgga taggaaaatt attctctcct
142801 cttgtaaata tttattttaa aaatgttcac atggaaaggg tactcacatt ttttgaaata
142861 gctcgtgtgt atgaaggaat gttattattt ttaatttaaa tatatgtaaa aatacttacc
142921 agtaaatgtg tattttaaag aactatttaa aacacaatgt tatatttctt ataaatacca
142981 gttactttcg ttcattaatt aatgaaaata aatctgtgaa gtacctaatt taagtactca
143041 tactaaaatt tataaggccg ataatttttt gttttcttgt ctgtaatgga ggtaaacttt
143101 attttaaatt ctgtgcttaa gacaggacta ttgcttgtcg atttttctag aaatctgcac
143161 ggtataatga aaatattaag acagtttccc atgtaatgta ttccttctta gattgcatcg
143221 aaatgcacta tcatatatgc ttgtaaatat tcaaatgaat ttgcactaat aaagtccttt
143281 gttggtatgt gaattctctt tgttgctgtt gcaaacagtg catcttacac aacttcactc
143341 aattcaaaag aaaactccat taaaagtact aatgaaaaaa catgacatac tgtcaaagtc
143401 ctcatatcta ggaaagacac agaaactctc tttgtcacag aaactctctg tgtctttcct
143461 agacataata gagttgtttt tcaactctat gtttgaatgt ggataccctg aattttgtat
143521 aattagtgta aatacagtgt tcagtccttc aagtgatatt tttatttttt tattcatacc
143581 actagctact tgttttctaa tctgcttcat tctaatgctt atattcatct tttccctaaa
143641 tttgtgatgc tgcagatcct acatcattca gatagaaacc tttttttttt tcagaattat
143701 agaattccac agctcctacc aagaccatga ggataaatat ctaacacttt tcagttgctg
143761 aaggagaaag gagctttagt tatgatggat aaaaatatct gccaccctag gcttccaaat
143821 tatacttaaa ttgtttacat agcttaccac aataggagta tcagggccaa atacctatgt
143881 aataatttga ggtcatttct gctttaggaa aagtactttc ggtaaattct ttggccctga
143941 ccagtattca ttatttcaga taattccctg tgataggaca actagtacat ttaatattct
144001 cagaacttat ggcattttac tatgtgaaaa ctttaaattt atttatatta agggtaatca
144061 aattcttaaa gatgaaagat tttctgtatt ttaaaggaag ctatgcttta acttgttatg
144121 taattaacaa aaaaatcata tataatagag ctctttgttc cagtgttatc tctttcattg
144181 ttactttgta tttgcaattt tttttaccaa agacaaatta aaaaaatgaa taccatattt
144241 aaatggaata ataaaggttt tttaaaaact ttaaa

For example, the nucleotide sequence corresponding to the mRNA of the human LRRK2 is depicted in SEQ ID NO: 10 (9239 bp), wherein the underscored bolded “ATG” denotes the beginning of the open reading frame. Sequence information related to LRRK2 is accessible in public databases by GenBank Accession number NM_198578.3 (nucleotide).

SEQ ID NO: 10:

1    gcgctggctg cgggcggtga gctgagctcg cccccgggga gctgtggccg gcgcccctgc
61   cggttccctg agcagcggac gttcatgctg ggagggcggc gggttggaag caggtgccac
121  catggctagt ggcagctgtc aggggtgcga agaggacgag gaaactctga agaagttgat
181  agtcaggctg aacaatgtcc aggaaggaaa acagatagaa acgctggtcc aaatcctgga
241  ggatctgctg gtgttcacgt actccgagca cgcctccaag ttatttcaag gcaaaaatat
301  ccatgtgcct ctgttgatcg tcttggactc ctatatgaga gtcgcgagtg tgcagcaggt
361  gggttggtca cttctgtgca aattaataga agtctgtcca ggtacaatgc aaagcttaat
421  gggaccccag gatgttggaa atgattggga agtccttggt gttcaccaat tgattcttaa
481  aatcctaaca gttcataatg ccagtgtaaa cttgtcagtg attggactga agaccttaga
541  tctcctccta acttcaggta aaatcacctt gctgatattg gatgaagaaa gtgatatttt
601  catgttaatt tttgatgcca tgcactcatt tccagccaat gatgaagtcc agaaacttgg
661  atgcaaagct ttacatgtgc tgtttgagag agtctcagag gagcaactga ctgaatttgt
721  tgagaacaaa gattatatga tattgttaag tgcgttaaca aattttaaag atgaagagga
781  aattgtgctt catgtgctgc attgtttaca ttccctagcg attccttgca ataatgtgga
841  agtcctcatg agtggcaatg tcaggtgtta taatattgtg gtggaagcta tgaaagcatt
901  ccctatgagt gaaagaattc aagaagtgag ttgctgtttg ctccataggc ttacattagg
961  taattttttc aatatcctgg tattaaacga agtccatgag tttgtggtga aagctgtgca
1021 gcagtaccca gagaatgcag cattgcagat ctcagcgctc agctgtttgg ccctcctcac
1081 tgagactatt ttcttaaatc aagatttaga ggaaaagaat gagaatcaag agaatgatga
1141 tgagggggaa gaagataaat tgttttggct ggaagcctgt tacaaagcat taacgtggca
1201 tagaaagaac aagcacgtgc aggaggccgc atgctgggca ctaaataatc tccttatgta
1261 ccaaaacagt ttacatgaga agattggaga tgaagatggc catttcccag ctcataggga
1321 agtcatgctc tccatgctga tgcattcttc atcaaaggaa gttttccagg catctgcgaa
1381 tgcattgtca actctcttag aacaaaatgt taatttcaga aaaatactgt tatcaaaagg
1441 aatacacctg aatgttttgg agttaatgca gaagcatata cattctcctg aagtggctga
1501 aagtggctgt aaaatgctaa atcatctttt tgaaggaagc aacacttccc tggatataat
1561 ggcaccagtg gtccccdaaa tactaacagt tatgaaacgt catgagacat cattaccagt
1621 gcagctggag gcgcttcgag ctattttaca ttttatagtg cctggcatgc cagaagaatc
1681 cagggaggat acagaatttc atcataagct aaatatggtt aaaaaacagt gtttcaagaa
1741 tgatattcac aaactggtcc tagcagcttt gaacaggttc attggaaatc ctgggattca
1801 gaaatgtgga ttaaaagtaa tttcttctat tgtacatttt cctgatgcat tagagatgtt
1861 atccctggaa ggtgctatgg attcagtgct tcacacactg cagatgtatc cagatgacca
1921 agaaattcag tgtctgggtt taagtcttat aggatacttg attacaaaga agaatgtgtt
1981 cataggaact ggacatctgc tggcaaaaat tctggtttcc agcttatacc gatttaagga
2041 tgttgctgaa atacagacta aaggatttca gacaatctta gcaatcctca aattgtcagc
2101 atctttttct aagctgctgg tgcatcattc atttgactta gtaatattcc atcaaatgtc
2161 ttccaatatc atggaacaaa aggatcaaca gtttctaaac ctctgttgca agtgttttgc
2221 aaaagtagct atggatgatt acttaaaaaa tgtgatgcta gagagagcgt gtgatcagaa
2281 taacagcatc atggttgaat gcttgcttct attgggagca gatgccaatc aagcaaagga
2341 gggatcttct ttaatttgtc aggtatgtga gaaagagagc agtcccaaat tggtggaact
2401 cttactgaat agtggatctc gtgaacaaga tgtacgaaaa gcgttgacga taagcattgg
2461 gaaaggtgac agccagatca tcagcttgct cttaaggagg ctggccctgg atgtggccaa
2521 caatagcatt tgccttggag gattttgtat aggaaaagtt gaaccttctt ggcttggtcc
2581 tttatttcca gataagactt ctaatttaag gaaacaaaca aatatagcat ctacactagc
2641 aagaatggtg atcagatatc agatgaaaag tgctgtggaa gaaggaacag cctcaggcag
2701 cgatggaaat ttttctgaag atgtgctgtc taaatttgat gaatggacct ttattcctga
2761 ctcttctatg gacagtgtgt ttgctcaaag tgatgacctg gatagtgaag gaagtgaagg
2821 ctcatttctt gtgaaaaaga aatctaattc aattagtgta ggagaatttt accgagatgc
2881 cgtattacag cgttgctcac caaatttgca aagacattcc aattccttgg ggcccatttt
2941 tgatcatgaa gatttactga agcgaaaaag aaaaatatta tcttcagatg attcactcag
3001 gtcatcaaaa cttcaatccc atatgaggca ttcagacagc atttcttctc tggcttctga
3061 gagagaatat attacatcac tagacctttc agcaaatgaa ctaagagata ttgatgccct
3121 aagccagaaa tgctgtataa gtgttcattt ggagcatctt gaaaagctgg agcttcacca
3181 gaatgcactc acgagctttc cacaacagct atgtgaaact ctgaagagtt tgacacattt
3241 ggacttgcac agtaataaat ttacatcatt tccttcttat ttgttgaaaa tgagttgtat
3301 tgctaatctt gatgtctctc gaaatgacat tggaccctca gtggttttag atcctacagt
3361 gaaatgtcca actctgaaac agtttaacct gtcatataac cagctgtctt ttgtacctga
3421 gaacctcact gatgtggtag agaaactgga gcagctcatt ttagaaggaa ataaaatatc
3481 agggatatgc tcccccttga gactgaagga actgaagatt ttaaacctta gtaagaacca
3541 catttcatcc ctatcagaga actttcttga ggcttgtcct aaagtggaga gtttcagtgc
3601 cagaatgaat tttcttgctg ctatgccttt cttgcctcct tctatgacaa tcctaaaatt
3661 atctcagaac aaattttcct gtattccaga agcaatttta aatcttccac acttgcggtc
3721 tttagatatg agcagcaatg atattcagta cctaccaggt cccgcacact ggaaatcttt
3781 gaacttaagg gaactcttat ttagccataa tcagatcagc atcttggact tgagtgaaaa
3841 agcatattta tggtctagag tagagaaact gcatctttct cacaataaac tgaaagagat
3901 tcctcctgag attggctgtc ttgaaaatct gacatctctg gatgtcagtt acaacttgga
3961 actaagatcc tttcccaatg aaatggggaa attaagcaaa atatgggatc ttcctttgga
4021 tgaactgcat cttaactttg attttaaaca tataggatgt aaagccaaag acatcataag
4081 gtttcttcaa cagcgattaa aaaaggctgt gccttataac cgaatgaaac ttatgattgt
4141 gggaaatact gggagtggta aaaccacctt attgcagcaa ttaatgaaaa ccaagaaatc
4201 agatcttgga atgcaaagtg ccacagttgg catagatgtg aaagactggc ctatccaaat
4261 aagagacaaa agaaagagag atctcgtcct aaatgtgtgg gattttgcag gtcgtgagga
4321 attctatagt actcatcccc attttatgac gcagcgagca ttgtaccttg ctgtctatga
4381 cctcagcaag ggacaggctg aagttgatgc catgaagcct tggctcttca atataaaggc
4441 tcgcgcttct tcttcccctg tgattctcgt tggcacacat ttggatgttt ctgatgagaa
4501 gcaacgcaaa gcctgcatga gtaaaatcac caaggaactc ctgaataagc gagggttccc
4561 tgccatacga gattaccact ttgtgaatgc caccgaggaa tctgatgctt tggcaaaact
4621 tcggaaaacc atcataaacg agagccttaa tttcaagatc cgagatcagc ttgttgttgg
4681 acagctgatt ccagactgct atgtagaact tgaaaaaatc attttatcgg agcgtaaaaa
4741 tgtgccaatt gaatttcccg taattgaccg gaaacgatta ttacaactag tgagagaaaa
4801 tcagctgcag ttagatgaaa atgagcttcc tcacgcagtt cactttctaa atgaatcagg
4861 agtccttctt catttttcaag accagcact gcagttaagt gacttgtact ttgtggaacc
4921 caagtggctt tgtaaaatca tggcacagat tttgacagtg aaagtggaag gttgtccaaa
4981 acaccctaag ggcattattt cgcgtagaga tgtggaaaaa tttctttcaa aaaaaaggaa
5041 atttccaaag aactacatgt cacagtattt taagctccta gaaaaattcc agattgcttt
5101 gccaatagga gaagaatatt tgctggttcc aagcagtttg tctgaccaca ggcctgtgat
5161 agagcttccc cattgtgaga actctgaaat tatcatccga ctatatgaaa tgccttattt
5221 tccaatggga ttttggtcaa gattaatcaa tcgattactt gagatttcac cttacatgct
5281 ttcagggaga gaacgagcac ttcgcccaaa cagaatgtat tggcgacaag gcatttactt
5341 aaattggtct cctgaagctt attgtctggt aggatctgaa gtcttagaca atcatccaga
5401 gagtttctta aaaattacag ttccttcttg tagaaaaggc tgtattcttt tgggccaagt
5461 tgtggaccac attgattctc tcatggaaga atggtttcct gggttgctgg agattgatat
5521 ttgtggtgaa ggagaaactc tgttgaagaa atgggcatta tatagtttta atgatggtga
5581 agaacatcaa aaaatcttac ttgatgactt gatgaagaaa gcagaggaag gagatctctt
5641 agtaaatcca gatcaaccaa ggctcaccat tccaatatct cagattgccc ctgacttgat
5701 tttggctgac ctgcctagaa atattatgtt gaataatgat gagttggaat ttgaacaagc
5761 tccagagttt ctcctaggtg atggcagttt tggatcagtt taccgagcag cctatgaagg
5821 agaagaagtg gctgtgaaga tttttaataa acatacatca ctcaggctgt taagacaaga
5881 gcttgtggtg ctttgccacc tccaccaccc cagtttgata tctttgctgg cagctgggat
5941 tcgtccccgg atgttggtga tggagttagc ctccaagggt tccttggatc gcctgcttca
6001 gcaggacaaa gccagcctca ctagaaccct acagcacagg attgcactcc acgtagctga
6061 tggtttgaga tacctccact cagccatgat tatataccga gacctgaaac cccacaatgt
6121 gctgcttttc acactgtatc ccaatgctgc catcattgca aagattgctg actacggcat
6181 tgctcagtac tgctgtagaa tggggataaa aacatcagag ggcacaccag ggtttcgtgc
6241 acctgaagtt gccagaggaa atgtcattta taaccaacag gctgatgttt attcatttgg
6301 tttactactc tatgacattt tgacaactgg aggtagaata gtagagggtt tgaagtttcc
6361 aaatgagttt gatgaattag aaatacaagg aaaattacct gatccagtta aagaatatgg
6421 ttgtgcccca tggcctatgg ttgagaaatt aattaaacag tgtttgaaag aaaatcctca
6481 agaaaggcct acttctgccc aggtctttga cattttgaat tcagctgaat tagtctgtct
6541 gacgagacgc attttattac ctaaaaacgt aattgttgaa tgcatggttg ctacacatca
6601 caacagcagg aatgcaagca tttggctggg ctgtgggcac accgacagag gacagctctc
6661 atttcttgac ttaaatactg aaggatacac ttctgaggaa gttgctgata gtagaatatt
6721 gtgcttagcc ttggtgcatc ttcctgttga aaaggaaagc tggattgtgt ctgggacaca
6781 gtctggtact ctcctggtca tcaataccga agatgggaaa aagagacata ccctagaaaa
6841 gatgactgat tctgtcactt gtttgtattg caattccttt tccaagcaaa gcaaacaaaa
6901 aaattttctt ttggttggaa ccgctgatgg caagttagca atttttgaag ataagactgt
6961 taagcttaaa ggagctgctc ctttgaagat actaaatata ggaaatgtca gtactccatt
7021 gatgtgtttg agtgaatcca caaattcaac ggaaagaaat gtaatgtggg gaggatgtgg
7081 cacaaagatt ttctcctttt ctaatgattt caccattcag aaactcattg agacaagaac
7141 aagccaactg ttttcttatg cagctttcag tgattccaac atcataacag tggtggtaga
7201 cactgctctc tatattgcta agcaaaatag ccctgttgtg gaagtgtggg ataagaaaac
7261 tgaaaaactc tgtggactaa tagactgcgt gcacttttta agggaggtaa tggtaaaaga
7321 aaacaaggaa tcaaaacaca aaatgtctta ttctgggaga gtgaaaaccc tctgccttca
7381 gaagaacact gctctttgga taggaactgg aggaggccat attttactcc tggatctttc
7441 aactcgtcga cttatacgtg taatttacaa cttttgtaat tcggtcagag tcatgatgac
7501 agcacagcta ggaagcctta aaaatgtcat gctggtattg ggctacaacc ggaaaaatac
7561 tgaaggtaca caaaagcaga aagagataca atcttgcttg accgtttggg acatcaatct
7621 tccacatgaa gtgcaaaatt tagaaaaaca cattgaagtg agaaaagaat tagctgaaaa
7681 aatgagacga acatctgttg agtaagagag aaataggaat tgtctttgga taggaaaatt
7741 attctctcct cttgtaaata tttattttaa aaatgttcac atggaaaggg tactcacatt
7801 ttttgaaata gctcgtgtgt atgaaggaat gttattattt ttaatttaaa tatatgtaaa
7861 aatacttacc agtaaatgtg tattttaaag aactatttaa aacacaatgt tatatttctt
7921 ataaatacca gttactttcg ttcattaatt aatgaaaata aatctgtgaa gtacctaatt
7981 taagtactca tactaaaatt tataaggccg ataatttttt gttttcttgt ctgtaatgga
8041 ggtaaacttt attttaaatt ctgtgcttaa gacaggacta ttgcttgtcg atttttctag
8101 aaatctgcac ggtataatga aaatattaag acagtttccc atgtaatgta ttccttctta
8161 gattgcatcg aaatgcacta tcatatatgc ttgtaaatat tcaaatgaat ttgcactaat
8221 aaagtccttt gttggtatgt gaattctctt tgttgctgtt gcaaacagtg catcttacac
8281 aacttcactc aattcaaaag aaaactccat taaaagtact aatgaaaaaa catgacatac
8341 tgtcaaagtc ctcatatcta ggaaagacac agaaactctc tttgtcacag aaactctctg
8401 tgtctttcct agacataata gagttgtttt tcaactctat gtttgaatgt ggataccctg
8461 aattttgtat aattagtgta aatacagtgt tcagtccttc aagtgatatt tttatttttt
8521 tattcatacc actagctact tgttttctaa tctgcttcat tctaatgctt atattcatct
8581 tttccctaaa tttgtgatgc tgcagatcct acatcattca gatagaaacc tttttttttt
8641 tcagaattat agaattccac agctcctacc aagaccatga ggataaatat ctaacacttt
8701 tcagttgctg aaggagaaag gagctttagt tatgatggat aaaaatatct gccaccctag
8761 gcttccaaat tatacttaaa ttgtttacat agcttaccac aataggagta tcagggccaa
8821 atacctatgt aataatttga ggtcatttct gctttaggaa aagtactttc ggtaaattct
8881 ttggccctga ccagtattca ttatttcaga taattccctg tgataggaca actagtacat
8941 ttaatattct cagaacttat ggcattttac tatgtgaaaa ctttaaattt atttatatta
9001 agggtaatca aattcttaaa gatgaaagat tttctgtatt ttaaaggaag ctatgcttta
9061 acttgttatg taattaacaa aaaaatcata tataatagag ctctttgttc cagtgttatc
9121 tctttcattg ttactttgta tttgcaattt tttttaccaa agacaaatta aaaaaatgaa
9181 taccatattt aaatggaata ataaaggttt tttaaaaact ttaaaaaaaa aaaaaaaaa

For example the polypeptide sequence corresponding to human LRRK2 is encoded by the nucleic acid sequence of SEQ ID NO: 10 and is depicted in SEQ ID NO: 11 (2527aa). Sequence information related to LRRK2 is accessible in public databases by GenBank Accession numbers NP_940980.3 (protein).

1    MASGSCQGCE EDEETLKKLI VRLNNVQEGK QIETLVQILE DLLVFTYSEH ASKLFQGKNI
61   HVPLLIVLDS YMRVASVQQV GWSLLCKLIE VCPGTMQSLM GPQDVGNDWE VLGVHQLILK
121  MLTVHNASVN LSVIGLKTLD LLLTSGKITL LILDEESDIF MIIFDAMHSF PANDEVOKLG
181  CKALHVLFER VSEEQLTEFV ENKDYMILLS ALTNFKDEEE IVLHVLHCLH SLAIPCNNVE
241  VLMSGNVRCY NIVVEAMKAF PMSERIQEVS CCLLHRLTLG NFFNILVLNE VHEFVVKAVQ
301  QYPENAALQI SALSCLALLT ETIFLNQDLE EKNENQENDD EGEEDKLFWL EACYKALTWH
361  RKNKHVQEAA CWALNNLLMY QNSLHEKIGD EDGHFPAHRE VMLSMIMHSS SKEVFQASAN
421  ALSTLLEQNV NFRKILLSKG IHLNVIELMQ KHIHSPEVAE SGCKMINHLF EGSNTSLDIM
481  AAVVPKILTV MKRHETSLPV QLEALRAILH FIVPGMPEES REDTEFHHKL NMVKKQCFKN
541  DIHKLVLAAL NRFIGNPGIQ KCGLKVISSI VHFPDALEML SLEGAMDSVL HTLQMYPDDQ
601  EIQCLGLSLI GYLITKKNVF IGTGHLLAKI LVSSLYRFKD VAEIQTKGFQ TILAILKLSA
661  SFSKLLVHHS FDLVIFHQMS SNIMEQKDOQ FLNLCCKCFA KVAMDDYLKN VMLERACDQN
721  NSIMVECLLL LGADANQAKE GSSLICQVCE KESSPKLVEL LLNSGSREQD VRKALTISIG
781  KGDSQIISLL LRRLALDVAN NSICLGGFCI GKVEPSWLGP LFPDKTSNLR KQTNIASTLA
841  RMVIRYQMKS AVEEGTASGS DGNFSEDVLS KFDEWTFIPD SSMDSVFAQS DDLDSEGSEG
901  SFLVKKKSNS ISVGEFYRDA VLQRCSPNLQ RHSNSLGPIF DHEDLLKRKR KILSSDDSLR
961  SSKLQSHMRH SDSISSLASE REYTTSLDLS ANELRDIDAL SQKCCISVHL EHLEKLELHQ
1021 NALTSFPQQL CETLKSLTHL DLHSNKFTSF PSYLLKMSCI ANLDVSRNDI GPSVVLDPTV
1081 KCPTLKQFNL SYNQLSFVPE NLTDVVEKTE QLILEGNKIS GICSPLRLKE LKILNLSKNH
1141 ISSLSENFLE ACPKVESFSA RMNFLAAMPF LPPSMTILKL SQNKFSCIPE AILNLPHLRS
1201 LDMSSNDIQY LPGPAHWKSL NLRELLFSHN QISILDLSEK AYLWSRVEKL HLSHNKLKEI
1261 PPEIGCLENL TSLDVSYNLE LRSFPNEMGK LSKIWDLPLD ELHLNFDFKH IGCKAKDIIR
1321 FLOQRLKKAV PYNRMKLMIV GNTGSGKTTL LQQLMKTKKS DLGMQSATVG IDVKDWPIQI
1381 RDKRKRDLVL NVWDFAGREE FYSTHPHFMT ORALYLAVYD LSKGQAEVDA MKPWLFNIKA
1441 RASSSPVILV GTHLDVSDEK QRKACMSKIT KELLNKRGFP AIRDYHFVNA TEESDALAKL
1501 RKTIINESLN FKIRDQLVVG QLIPDCYVEL EKIILSERKN VPIEFPVIDR KRLLQLVREN
1561 QLQLDENELP HAVHFLNESG VLLHFQDPAL QLSDLYFVEP KWLCKIMAQI LTVKVEGCPK
1621 HPKGIISRRD VEKFLSKKRK FPKNYMSQYF KILEKFQIAL PIGEEYLLVP SSLSDHRPVI
1681 ELPHCENSEI IIRLYEMPYF PMGFWSRLIN RLLEISPYML SGRERALRPN RMYWRQGIYL
1741 NWSPEAYCLV GSEVLDNHPE SFLKITVPSC RKGCILLGQV VDHIDSLMEE WFPGLLEIDI
1801 CGEGETLLKK WALYSFNDGE EHQKILLDDL MKKAEEGDLL VNPDQPRLTI PISQIAPDLI
1861 LADLPRNIML NNDELEFEQA PEFLLGDGSF GSVYRAAYEG EEVAVKIFNK HTSLRLLRQE
1921 LVVLCHLHHP SLISLLAAGI RPRMLVMELA SYGSLDRLLQ QDKASLTRTL QHRIALHVAD
1981 GLRYLHSAMI IYRDTKPHNV LLFTLYPNAA IIAKIADYGI AQYCCRMGIK TSEGTPGFRA
2041 PEVARGNVIY NQQADVYSFG LLLYDILTTG GRIVEGLKFP NEFDELEIQG KLPDPVKEYG
2101 CAPWPMVEKL IKQCLKENPQ ERPTSAQVFD ILNSAELVCL TRRILLPKNV IVECMVATHH
2161 NSRNASIWLG CGHTDRGQLS FLDLNTEGYT SEEVADSRIL CLALVHLPVE KESWIVSGTQ
2221 SGTLLVINTE DGKKRHTLEK MTDSVTCLYC MSFSKQSYQK NFLLVGTADG KLAIFEDKTV
2281 KLKGAAPLKI LNIGNVSTPL MCLSESTNST ERNVMWGGCG TKIFSFSNDF TIQKLIETRT
2341 SQLFSYAAFS DSNIITVVVD TALYIAKQNS PVVEVWDKKT EKLCGLIDCV HFLREVMVKE
2401 NKESKHKMSY SGRVKTLCLQ KNTALWIGTG GGHILLLDLS TRRLIRVIYN FCNSVRVMMT
2461 AQLGSLKNVM LVLGYNRKNT EGTQKQKEIQ SCLTVWDINL PHEVQNLEKH IEVRKFLAEK
2521 MRRTSVE

The invention provides for a nucleic acid encoding a VPS35 protein, or fragment thereof.

For example, the human genomic nucleotide sequence corresponding to the sense strand of the human VPS35 gene is depicted in SEQ ID NO: 12 (29556 bp). Sequence information related to VPS35 is accessible in public databases by GenBank Accession number NG_029970.1 (nucleotide).

SEQ ID NO: 12:

1     gctagagagg gcggggcttg gaggggccgc agcgtcacat gaccgcggga ggctacgcgc
61    gggccgggtg ctgcttcctg caggctctgg ggagtcgcca tggtgagtgc tgagggggca
121   gtggcacctg ggtcgaccct ccttgtagcc cctgctctct cccaccgccc cgcactccag
181   cgagtggaga aggggcccca cagaccgttc gggattaaga ccagcccgat ttggcctgcg
241   ggatagggga cagcaggagg aaggccgcgg gcaggctgat ccgggccggg gtgggcggcg
301   gctcttggct gcggccgttg ctgagagacg gggcggcctc tctgtggggt tgacttggca
361   tgtaggcttt ggggtccatg aaggcctgcg gcctccttta agtggaatcg gtcacctgcc
421   taccacgagg ggaccggtag tcctaggtct gagcgtctgg cccccggggc gcgtggaggc
481   cctgagactc ggaggtggcg ccggcacccg cccagatgtt gcgtttctac ctttgtgcct
541   agttgtgctc ggccgtcccc acgccctcct ggaggggtcg cagtgattcc ttggcctttc
601   ttggcctcat acccgccttc ggctgcagtg tttgtcagcg agttctgggg acctgcttac
661   atgaatttcc tggaaggact caggctgtct tctaatcctg acggtcgcaa aggagactga
721   ttgtttactt tagcatttgt gcattgggcg caccttgcct cttttgtctc gccattgata
781   aaatccaagt atttgacttg ctggaagcag tacttctcct tagggcccgt ctatgacggc
841   agcaaatcgt ggtgtggctg ttcgccggta aacttgaact tcctcaaaat gtgaatcttt
901   gtgtctggtt cccacaaagg caagttgtca cttgcatttt attagcgttt aacatagcct
961   gcactgtgta aataaatttt ttgagtatat actgtatgtc cgctttaatt accttactca
1021  ctctgtgtag ataggcttct gtaaatctgt aagcctggaa acagatttca ctttaaatgt
1081  cttaatgcca gaaaggatta agtgttttac aaatactatt ttcatataac gtgttgccgt
1141  acaaggtgat tttgcctgtt tctcaggatt tttataattg ggaattgata caagaccggc
1201  gcaaaattta actttaggat ttgtgtgttt ccagcgttta tggattgaca tttatattgt
1261  tttgtaatgg aaaacactta attgaggatg tattacacac tccgattctt tgttgggtga
1321  accagttggg agcaatcagc cagacacaca gtcttgtcct catgaatttt attgggaaag
1381  gaaacttgag tattcgttct ttcacagtca ctgtctataa tttaggcagc tcgcttagtc
1441  ttcttggtgt cgcacttttg catccgaaag caagtgttgg cggccccttt ggcccttccc
1501  agccctaaaa ttccagaatc cacttcaatt catcaaacat cttgtgtgcc ttcagtgtca
1561  cggactgtgg ccctgggtac caatgtcgaa cttaagaggc tactggggga gacacgcttg
1621  tagaacttaa ctgtaataca gtgtgtggat tgctttacta acattgtcaa ctcagtgctt
1681  tggggtcatg gagggatagt tttgcctggg gattttggga aggactacag gaagtaacgt
1741  gaactgagac tataaagagg gtggaaagaa attctccagg tggaaagaaa aggtattgct
1801  aatagatgaa gccaaaacgt tcagtgtgct ccatcagagg attcttgggg atatgtattg
1861  gaaatgaagt tagagccgtt tggtgttcaa attatatttt gaagttgtca tgccctcatc
1921  atccttattt ctctctactt cagttgtcaa tgtatgccat gtcatcattt ttaaaatagt
1981  tctttcatta acgtgatatt ctgtaccttt gtttgaagaa atgagcgtta catttggttt
2041  acattgtact gtcagattac atctacagca tataagcagg gaggttgaac cacaataggt
2101  ttctaacata agtggagata attatttgta tctaaactga ataataatta tagcacttat
2161  taagtgctta ttgctaagta gtaggcacgt tttgagcttt ggtttcattt gttcttcata
2221  acagtggtgt actctcatct ctgtttcata gaagaggaaa gaaaagcaca ttgtttcagt
2281  ggagagtagt tttccaagtg atcagttcat gatctttgct ttgctctgca cattagaact
2341  gtagacttga gagggctgtt tgtgggtctc aagctaaaat gggactaatc acaaatgtta
2401  atttgattcc cattctgtag ggtttccatt ttcttctgat tcatgtagct gtgaagtacg
2461  gtcatttaca aatggaaaac cactttcatg attgaggaaa cattggttaa ttggcttgtg
2521  tttaatgata gcgtgctcat attataactg ttaaggctct atgtgacact tcatagtaag
2581  gcatcaagaa tagcccttat aatagctgtt gctagcataa ctacactgtt ttatgagtaa
2641  tatataaaaa tagattgctt tactatagct ttatgtcttc acttgtagct tactttgtaa
2701  taagtcatat tctgtaatct tcaacatttt gtatttacat ttctatttta agctgagttg
2761  agacaaaata gaagaatttt cctaaaattg cattcttttg tttacacatc tctaaatctt
2821  tctagtcatt ctatacaaat gtttttggaa ctgattgtat cctctttgta gccacagttt
2881  catttgctat ataataaaaa ttatcataca caacagccaa aaggtggaaa caacccatat
2941  gtccatggat aaacaaaatg tagtgtgtgt gtatatatat agatatataa tgaaatactt
3001  cgtttttaaa aggagggaaa ttctggcaca tactgcaaca tggatgaaca ttgaagatat
3061  tatggtcagt gaaataagcg agacataaaa gtaaaaatat tgtatgattt cacttaaatg
3121  aagtatatag agtagtcaaa ctcagagaaa agaaggtaga attgcctggg ggttcggaat
3181  gggagtttaa tggctacagt ttctatttgc atttgtaaag ataaaaagtt ttagagatag
3241  atggtggtaa ttgtattaat gtgaatgtac ctaatgccac tgaactgtac atttaaatgt
3301  taaaatggta aattttaggt acattttacc acaacaagaa gtcattttta attaaaaata
3361  tccagatgta tcataaagaa aaataaaaaa aattcagatg tatcactgtt tatctctaaa
3421  tggatcaatt gaacttaatg aaatccattg attcaaatta ttattaaatc tattgcgtcc
3481  agaggtaagg agccaaaaaa ttccaaatga tggcctggtt tcactaaagt tcagagaaga
3541  ctagcccatg atgaatagta aatttcatta agtcagagtc tttaaatgct ggtgtcatcc
3601  ttgcctctga aaccagcatt ttatggtaat agttccactg ggttaaattc atgttccctt
3661  taagtgaagt ttaaaagata cctaacttct tctttgaaat ttgtttgtgc ttctgaggaa
3721  gagtgcttgc agcagagctc agtttactag agtttttcat agggaaaaaa agggagatgc
3781  atggtgttgt tcattattca gttaatattt ttctctttcc aaagttagaa caagagaaag
3841  ttttcaattt ttataagcta tgctagttca gaagtgggtt ttatgttatc aagtttcttt
3901  gttaatctca aaatgaaatg ttgttttgct tttcttagat aatgaaacag accagatttt
3961  acttgcaggt tgatgtgtaa gtccttgcct tccacctctt caactcattg tgtgagaggc
4021  attttgtctt tagtcattgt tttaaaaaat aaaagtgaaa tgacataaca aaaaattaac
4081  cattttaaag tgaacaattt ataaagtggc atttagtatg ctcacaatac tgtacaatca
4141  ccacctttgg tttcaaaaca ttttccctac ccctaaaaga aagccagtgc ccattaagtg
4201  attattcccc tcagcctctc tcctggtaat catcagtcta ctttctgttc ctatacagaa
4261  tatttcatat tagaggaatc atacaatatg tggcttttta tgtctagctt ctttcattta
4321  gcatgatgtt ttctaggttt gtttatgtag caatacttca ttccttttca tggttgaata
4381  atattctgta attgtatgta tataccacaa cttgtttatt catccattaa ttattcagtc
4441  tttttttgtt aaatatctaa acattctaaa accagtgtat tcatttatgc agtgaacatt
4501  tgtggagcat attatgtatc aagcagtgtg ttggatccca aggatgtaaa aatgaccgtc
4561  atttataata ataatgtgat acatgctgtg gtggagatgt aaacagtgtt tatatgactt
4621  gaagaagtgg ttaattcttt ttggcatgga gattgaattc attttgcaag cagatttttg
4681  ctgatggcaa agagaagagt gtctagatat attttgtcca ctgtagctgt agatcagtat
4741  atctgaaatg gtgtaagcta tttgaggaca gtgctcttta ttgttttcta ctgtaagtca
4801  ctaacaattt tggctgtttt atttctagac tgtttagtct tttgttaaat attgccaagg
4861  aggggctggt cactgttctc gtaaactagt tccttagtct gtcttaagaa tagactgaaa
4921  tgcagatgat aagtagtcta gaggaaaaga gaggctttag agattggttt cggctatacc
4981  tatcacaaga tttcgattgg tcagatggct atgtctgggt tggattcaga gtgtgttagc
5041  agaacacagc catgaactac cactgcaagt ttctttgagg ccagcctact ttctgagaga
5101  gaggcaattc tttgtacaca tactattctc ctttgtcagt cttattctgt taacttcagc
5161  gataaggcat gactctgtgt gcagcagctg ttaataattg gtaaatgggc tgggtgcggt
5221  ggctcatgcc tataatccaa gcactttggg aggtcgaggc aggcgaatca cgaggtcagg
5281  agatggagac tatcctggct aacatggtga aaccccgtct ctactaaaaa tagaaaaaaa
5341  attagccggg tgtggtgatg ggcgcttgta gtcccagcta cttaggaggc tgaggcagga
5401  gaatggcttg aacccgggag gcggagcttg ccatgagccg agatggcacc actgcactcc
5461  agcctgggcg acagagctag actccgtctc aaaaaaaaaa tttggtaaat gggtttgagg
5521  tatagagctg gacattgttg gagaaggact atggctagaa ctatagaaat aacgtacttg
5581  ctagaagaat gtgcttgaga catcagtgga attttttatt tttcagccta caacacagca
5641  gtcccctcag gatgagcagg aaaagctctt ggatgaagcc atacaggctg tgaaggtcca
5701  gtcattccaa atgaagagat gcctggtaag aatggagatg tgggaggcac agttgcagtt
5761  cgtgtgttcc taaggaagca tgtgcagtgt cttctagagt caggtgtttc tggtaaatct
5821  aatcttcacc gtttaccagc atctatcttc agtctcatct ccctcaagca ctttgtggag
5881  caatttcaac aaagagccct gtttactcac atgtatattt atggtttggg attgtctgtc
5941  ttccctacta gaatacaagc tcataagaat aagagaccct tccttttatt tacacattac
6001  tgtattatta ccacaccagt gtctgaccag aattactagc ctccttggtt ctatacctca
6061  gacctgagga atatttaaca tataataggt actcagtaaa tatttgttga atgaatggat
6121  ttaaatgctt tgcatttgaa ttattcagct ttttttctaa atatcttgaa aactttaatt
6181  tctttgctga atagatatat ttattgtaga agctagctta aaaattatac ttaacactta
6241  tttacatatt tttatattct aaaagataaa gtaagagata atctgtgtag atacttttga
6301  ttctctggat taaaatgtaa ggaattgagc caaattggtt agtactttaa actataaatt
6361  actgtgatga agatgatgct attttacctt tgtaaaatgt cttactgtgc tttctaaagc
6421  atagtaatat gctcttgtgt cttttattgg tttaattcct aacaaattgg gaatgaaaaa
6481  taaatgtctt ggaatggaga agctgggttt gctattgctt gcttctttct cttcctgtgt
6541  atggatagtg tttcctctat ctcaaggaat tgcttgcatt tctgagttaa gtggaacata
6601  tgggcattgt gagggcttga agaatgcaag aggaaagcaa acttacatgg atagtcattt
6661  cagacagctc tgaagagtct ttaacccatg acaaagccat gtcaggatag tatcttcctt
6721  cacctgaatc agtatgccag ttctcttgat tgcaggtaaa atgtgatgaa tggagctagt
6781  ttcctagtct ctatagattg aaaagattag cattctatca agaagcttgc agtcttagct
6841  atgttaagtc ttactaagaa tcatgtatct ttttcttttt cagtagagac ggcaaggtga
6901  accgatctaa gttgtttttt taatgtggtt aaaatcattt aagtgcggta ttcttttaaa
6961  actatgtaac aagtccttga tgtaaagaat ttgtacaacc aagataaatg tttatttaaa
7021  ttaagcattc tcatctattc tcttggtatt tctgtaggac aaaaacaagc ttatggatgc
7081  tctaaaacat gcttctaata tgcttggtga actccggact tctatgttat caccaaagag
7141  ttactatgaa ctttgtatct tttgaatgtt gaagactaaa catttggacc ataccttttt
7201  cttgataagg cctattttgt ttgttcttta tgaagttttt ctggagttat cttattcttc
7261  gttatctgag tcacatggca ctccttctcc atgcagatgt gctaagtgag aaaaacactt
7321  tgagagtact cctttcctat gcttaaacat ctttaaatgt gttgtcggtg catctcaatt
7381  ttcagaccct tcatgaggat atttaggcta tgacacagtt ggttctttaa tacttagatt
7441  ttgttatgca gcagtctcaa atggacagga atttaatcat ttgccatttc aaaacccatt
7501  agcagtctga caggtaacca ttgtatttac tgctttgctt gaccacacat gctttaaaac
7561  ccttatttta aagtaagaaa agtccggcta aaattcatcc ttcgcttgaa cactttctta
7621  aaggactaaa acttaagatg tctgcccagt agttagtaat gactccaaca agtttcaaag
7681  ttttgtttag gttggcttat ttttattttt agtccttaat cataattaaa agatatggcc
7741  atttctgatg aactgcacta cttggaggtc tacctgacag atgagtttgc taaaggaagg
7801  aaagtggcag atctctacga acttgtacag tatgctggaa acattatccc aaggctgtaa
7861  gtaattacaa atcagagaac ttttgtgtct gtatttctca ctatatgtta cgtcttttat
7921  gattatcagc ttaagaaaaa gttttaaggg taacttctta acaaattgag atgaacattt
7981  tggtagatat tctcttactt gttttagagt aactagattt acgttttatg tagatatttg
8041  aggaattttg gaaatagaaa aaatggacat gcttgctatt ttttttaatg tcttgactat
8101  tagaaaaatt aatataattg ttctcttcct aatatgttta aaggtaatat ctatgttgta
8161  tatatacagt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt atagtttttt tagaggtcag
8221  tcagtggtta tattttaaat gagatatttt ccttgtcatg cgggagaaaa caacatggtt
8281  cctgtcttgt ttatttaatg ttttgttcag tgtgtttgga aataaattct tgatttgaat
8341  attttatttc taatcagcat ttcttcataa ttttcctagt taccttttga tcacagttgg
8401  agttgtatat gtcaagtcat ttcctcagtc caggaaggat attttgaaag atttggtaga
8461  aatgtgccgt ggtgtgcaac atcccttgag gggtctgttt cttcgaaatt accttcttca
8521  gtgtaccaga aatatcttac ctgatgaagg agagccaaca gagtaagtga ttttctttct
8581  taattttgtt gcaatatttc tttcattgta gaatgtataa aagtgtggaa acatatacag
8641  aaacaaagtg tgaataattc ttccacccag tcagccattt aggtagcatt tgtatataga
8701  tttcctttgt aatatagaac tcctcagtat atgtggtatc atctaaaatg tactcttatg
8761  caaattttat ctttggattg ttaggacctg cttttttcat ttaatgtaat tttttctact
8821  acattaaatc ttctttgaaa ataaaacttt tttaagagag ttgtatttgg aaattgaatt
8881  tgtaatgaaa taataaagtg tgagccagct ggatttcata attgttcctt tagtgtctat
8941  cagtttttat aatttataga ctgctagtta ccttggaata taagtgattt gaattatctg
9001  ttacgagtta gctattaact ccagagaagc aaaaataaaa gccattcaga gacactcctg
9061  tctcttgtgt tatcagtatt ctagcatcaa agtctactgt acttttatcc cacagcaggg
9121  gcagatggtc agccaactgt ggtcttcagt ggggtgagct gttcacatga caggtcccca
9181  gattaaagaa cttcattcct tttttaaaaa gtttattcat ttattttctt tcttttttta
9241  atttttaatc ttttttcagt ttgccccaac agattttgtt tttttctttt taatattttc
9301  atttatttct aaggttttta atatatcatt tatttctaat gttttttaat atatctgcat
9361  caatttcttt taaaacagta cagaaaagat aaaacattta acaatgtaga gaaattgatg
9421  aagttacttg ctttattatg ttttgagtgt ccgttttgag catttaatta ggcaatcaat
9481  aacaattttt gaaaggtact gaggtctcca tcctaggaga cgtagaaaaa taaagcagga
9541  aatccatggt ctcttccctc acaaagctta cattccaatt aaaaacaaaa tattcaacag
9601  taaaatgatg tagttagcag tacaccataa gtgttacatt ttttagcctt ttgtttttgt
9661  ttttggtttg tggggatggg gtctcattat gttgcccagg ccagttttaa actcctggcc
9721  taaagcgatc ttcctgcctt ggcctcccaa agcactggat tacggacatg agccaccatg
9781  cccatccttc tagccttttt caattaagga agttgccata agagcaagtc cagttggccc
9841  aggatgagga gttggggaaa gtaatttgcc ctttaaattt atactgtcct ctccatggta
9901  ctttttaccc tagagtctgt tccctttgaa atttaacact aagccaatga agttgaaagt
9961  gattttttat aaagcattgg tgtactatag agataagtag gaaatacaca aaggagaagg
10021 atagtagtaa gttggtcctg taaatactgt gtaaagactt ttctgtttct ttgcagtgaa
10081 gaaacaactg gtgacatcag tgattccatg gattttgtac tgctcaactt tgcagaaatg
10141 aacaagctct gggtgcgaat gcagcatcag ggacatagcc gagatagaga aaaaagagaa
10201 cgagaaagac aagaactgag aattttagtg ggaacaaatt tggtgcgcct cagtcagttg
10261 gaaggtgtaa atgtggaacg ttacaaacag gtttatatat ttttgttacc tcttcttatg
10321 ttcagagata aactgaaatc tgatttttaa aatcagaata tttttgttat acaatagtac
10381 attgaaaaac atcttaaaat ggctgttatt gaagaagact taaacggaaa gatatatatg
10441 cagtgtttgt ggattggaag acttaatatt gtcaaaatag catttaaaaa gaattgattt
10501 atagatccaa tgtaatctca gtcaaaatcc cagcagactt ttgtagaaat taagaagctg
10561 attctaaagt ttatatgaag aaacaaagaa cctggaacag ctacaacgaa tttgaaaagg
10621 aagaacaagt tgaaagaccc aagcaacctg aattaatgat ttactctaaa gctgcagtga
10681 gatcctgtgt ggtattggtg aaaaggatag acacacaaat caatggaggg gaataaaaca
10741 gggaatggca aactttacct gtgagggacc agataataaa tggtttttgg ctttgtaaga
10801 catgtgctgt acaacaagct tgtccaacct gcagcccagg acagccttga atatggccca
10861 acataagttt gtaaacttta aaacatgaga ttttttgctt tttttttttt tttttttttt
10921 tttttttaaa gctcatcagc taagtgtatt ttatgtatgg cccaagacaa ttctaattct
10981 tcttcaagcc aaaagattgg acaccctagt ctacaactaa taacagtgca gatatggtgc
11041 aaaagcaccc acaggcaata tggaagtgaa tgggcatggc tgtgttctag taaaacttta
11101 tttgtaaaaa caagcagcag ctcagtttac cgatctctga ctggacaatc cataatagac
11161 ccagatattt atgatcagtt atttttgata aaagtacaaa ggcaaccttt tcagcaagtg
11221 attctggaac aattggatgt ttatatgcaa acaaaaaacc ctgaaccttg acccatccct
11281 cataccatat agaaaaaaca cagaaatcaa tcagagacct aaatatagaa cctaataatg
11341 ttagaagaaa acacagagga aatctttatg acctaggatt agacaaagat ttctgaggat
11401 atacaagcac aagccatgaa gaaaaaagct cacttttgag aggccaaggc agatggatca
11461 cttgagtcca ggagtttgag acaggcctgg gcaacatagg gagaccccat ctctacaaaa
11521 attaccaaaa ttagctgggc atggtggaac gtacctgtag tcccagcact caggaggctt
11581 gaggtgggag gatgacttga gcctaggagg tggatgttgc attgagtgga gattgtgcca
11641 cttcactcca gcctgggcaa ccgaacaaga ccttgtctca aaaagaaaaa agcttttaaa
11701 gtttagaagt gaagtcttgg tgagaaaaat ctcaaatacg attttcaagt tagtagttca
11761 aatgcgttac tagaggaata gcttaagatt ttgaaaacag attttaaccc ttatgtgtgt
11821 tttttctctt ttagattgtt ttgactggca tattggagca agttgtaaac tgtagggatg
11881 ctttggctca agaatatctc atggagtgta ttattcaggt agctgggaac atttcatttt
11941 tttttaaacg acctatttta tctttcatta aatttaattg ttttgaaaaa attttgatgg
12001 aataggaaat aagctttcct gaataaagag ttttccttgc ggggtgtggt gactcacacc
12061 tgtaatctca gcagtttggg agttcaaggt gggaggatct cttgaggcca ggagttcaaa
12121 accagcctgg gcaacatagc acgatgccgt ttctataaaa aattaaaaaa atttttttag
12181 tgtttctttt ttttttcatg taatcttgct tcttctaaaa ataatttaaa aataggaatt
12241 ttctgtttct aacttatacc ttggtctttg tatcaatgtg gtttgttttc ctccaaaatg
12301 taggaatgag taatctgagt tttctaggtc tctgtagctt tagtttaatt gtaggtgcac
12361 tttgtttatt ggaatatttc tgtctgagct tatgtttagt agagaggttc aaaagtaatg
12421 tgtttgaatt tagttgtata agaatacagt gtttttttcc cacaaatgtg aactttacca
12481 tatgtgagtc cagaatatta cgtgaaatac ttttatttgt attgatcatt tgattttcag
12541 gttttccctg atgaatttca cctccagact ttgaatcctt ttcttcgggc ctgtgctgag
12601 ttacaccaga atgtaaatgt gaagaacata atcattgctt taattgatag gtaagacctt
12661 ccaacactgg cggataaatg ctctgacttg ggaataatga attttaaaca tttttttgaa
12721 ttatttgttt ctgttacatc tttatcatac caatgatctt aatttaatta tactataaat
12781 aatttagctt tgtgagtatg agtactaggt acttgtctag gttagacatg aaagaggctt
12841 aacttaaatg tgcaggagac gtgaagataa tgaatatctt tattctgtgt gcttaattga
12901 catttaaaga tgttgtacag acttattttt taaatcatac aaatccaaag atcatattga
12961 agaacaaaat ttgtttttta ccatgatgta agtatcttgc agtgggaact catttgattt
13021 agagtagccg taagatactg atgattgaaa atgttcaagt aatcactcta tcatcacatt
13081 ttcttaaaga aaaaatttta agtatcaaat atgtttagta catccacttt tttattttct
13141 taggtttttt tttttttttt tttttttgag acagatcctc actcttgtca cccaagctag
13201 agtgcagtga cgctgtctcg gctcactgca acctctgact cctaggttca agtgattcta
13261 gtgtctcagc ctccggagta gctgggatta cagacatgca ccaacaagcc cagctaattt
13321 ttgtattttt agtagagaca gggttttgcc aggttggcta ggctagtctc aaactcctga
13381 gctcaaatga tctgcctgcc tcagcttccc aaagtgctgg gattacagac atgagccact
13441 gcgcttggcc aatgggtggc ttttttgcag ccatgttatg tagtagtata tgatgtctgt
13501 cctacacttg taagcattgt catgaaacca gaaacctaag agaagattta tttctgcaga
13561 taccttttgt atgtttttta aaaaactaag ttattagttt taaagtctga gaatttagat
13621 aacaaatttt tccaaattgt cagctcaatc ctgggcagca aaaattccat acttattggg
13681 cccactctta aaggaagcta gtaactggat tttcctgagt tgcctgtaat gtcacttaca
13741 catctctgtc agtagtgatg cttctgggca tagcaaaatg tggatgtagt tgtgactgac
13801 aaacagataa tgataatgaa acatactatt ttgagtaatt taagatgtgg gaaataaaag
13861 ttaattttat gaattttaga cttagttgta tttcaagctt tagtaaaaat gcagtatctt
13921 aaaatagtct atgtactttt attttttaaa ggttatttat ttaaatcatg gttgttgaat
13981 acatttgtca ctttaatgca tttctgtcca tatctgctta attatgcttc aaagagttga
14041 gagaattatc ttgttgaaaa tctacttaat atggtgtgaa ataagaatgc tgatgaaaaa
14101 ggtttcattg gcaaaactgt ttagttaaaa atgaattgag gaggccgggt gcagtggctc
14161 acatctgtat tcccagcact ttgggaggcc aaggagggag gcttgcttga gtccaggtca
14221 gtaccaccct gggcaacatg gtgaaacccc atcactacag aaaacacaaa aattagctgg
14281 gtatggtggc acatgctgtt agccccagct actcaggagg ctgaggtgca aggaggatag
14341 cctgagctca gcaggtggag gtttcattga gtggagagtg cgtgactgca ctccagcctg
14401 ggcgacagag cgagactctg tctcaaaaca aaacaaaaca aaacaaaaaa aacaaaaacc
14461 ttttgggctc atacaaaata tagaaaagca ataaagaata agatgtcatc catgatctca
14521 catcccaaac cctgtatctt ttaaaataaa ggggtgtttt tttttttttt agattagctt
14581 tatttgctca ccgtgaagat ggacctggaa tcccagcgga tattaaactt tttgatatat
14641 tttcacagca ggtggctaca gtgatacagg tttgtgtagc atttctccta agttctcaaa
14701 actttgaaac ttctctgcct tccttttaca attgtttaaa ataaattgtg tggttttcta
14761 aacattccag tctagacaag acatgccttc agaggatgtt gtatctttac aagtctctct
14821 gattaatctt gccatgaaat gttaccctga tcgtgtggac tatgttgata aagttctaga
14881 aacaacagtg gagatattca ataagctcaa ccttgaacag taagtcagtt acatttttgt
14941 aaaaatcctc aagatatttt ttgtcctaga tttgcttttc tttctcaatt gttttgtgaa
15001 ctgctggcat ttgtcttgtt ttaatcatgc attaagattg tcatgcttag cactactagg
15061 ggcagaaagt agtgaccaat tacttgtttt tttatattaa ggaaattgtg gtacctatgg
15121 accataggca gtcttcaggg accagtgtct ccaatttgga tccctttctg tgtgtcaggg
15181 gcatccaatc ttttggcttc cctgggctgc actggaagaa gcattttctt gggccacaca
15241 taaaatacac taacactaac aatagctgat gagcttaaaa aaaaaatccc aaaaaaactc
15301 ataatgtttt aagaaagttt acgaatttgt gttgggccgc attcaaagcc atcctgggct
15361 gcatgcggcc tgtgggcttt gggttggaca agcttgcatg tgactgagtt tgttcttaaa
15421 ctggtaagga aactttgtca ggcagtattt atttccataa gtggtgtttt cctacgaatc
15481 agcacatggt gaaaaatgag gggctatgta tatttaaggt gcagaattaa attggtttaa
15541 atatcttttc tattttgagc tttgattttg ataccttaaa ggaaatatca acagtactat
15601 ttccaacctg aagcctcctc agctgttctg tcctagactt atggcgtcct ctagtggcca
15661 ctatgggcag ctatgatcct gttaccttcc ccagcagttc ccttcctgcc ctgttcccca
15721 ctgctctggc ttgggtcaag ccaggcctgc ctcccgccaa catattcttc agaattttac
15781 ctcatgtaat cttcctcctt tctatctccc ttccagtggt ttacctgcat caagaaaatt
15841 tcttcttttt ttcctccctt tgtgttaccc ttgttctttt ggtcattttt ggttttgtgt
15901 gtgtgcaaac tgaaaacaag tccagatgtg gaatgataag tgtgagagaa aattaaatga
19961 tgtgccaggt gtggtggctt gcacctgtaa tcccagctat tcaggaggct gaattgggag
16021 aatcacttga gtccatgagt ttgagaacag cctgggcaac atagcgagac cccgtctcta
16081 ataaaaaata aaattaaaaa taaaaaaaat ttaaattaaa aaaactaaat gatgtatctg
16141 tgtctttctc cccaagtgaa ttttaaagta aaaatagaca aagtaattag aaataacaac
16201 ctctaaagag gttgtaataa atgccccaat atgcctcaat atctacagaa tgattttact
16261 aacaactacg taaaagtcag tcagcctgct tttccttaat caccaacatc tgatgcagaa
16321 gaaatagttt atgtgttttt ctgttgtgtc aaattgctgg ttttgcatgg agtttttttc
16381 ctatttattt tcatcatgaa tatacaatac ttgttggctg gcccctggga accaaactac
16441 cacttaaaat acttccctta gaaatgtcat caaattctag acagtcatct taactccagc
16501 tataccatct gttcatgagt tggaaactgt atctagtttt gtatcaacag aaaaataata
16561 gatgaatata tatttgtgtt tagataagca tttttatcct cctgaaagga ggttgttata
16621 gtcttctgtg gtggtatgat tcacttgacc catttccttt aatgtgtaat gaaaaatttc
16681 aaattcttat ggaacaaatg ctatttgtgt atatagaaag ttaattttat tcattaagac
16741 ttctgttttt ctttttgtag tattgctacc agtagtgcag tttcaaagga actcaccaga
16801 cttttgaaaa taccagttga cacttacaac aatattttaa cagtcttgaa attaaaacat
16861 tttcacccac tctttgagta ctttgactac gagtccagaa agagcatgag ttgttatgtg
16921 cttagtaatg ttctggatta taacacagaa attgtctctc aagaccaggt aagagaatac
16981 ctacgtgcta ttttagggaa acagtgttac aattttagac tttggaccta gatacctgag
17041 atgggagggg agggtaattc aatactaaat aaaatttaca agtaactttt tcattatata
17101 aattaaaaat tggagatgta taaagaatta taaaacattt ataattccac cagatagaga
17161 ataaccactg ttaattaaca tttggtgcat atctttccag acttttgtct gtatatgtgt
17221 gtatgacata catgtgtatc gactttctca ccaaaaaaag gaatatcttg ttgatactgt
17281 attgtaattt tataactgga aacacttttg ataatggctt tgtatgccaa tggtttcacc
17341 tcagtgggtt tcttgtgcct cgcatgttac aggtggattc cataatgaat ttggtatcca
17401 cgttgattca agatcagcca gatcaacctg tagaagaccc tgatccagaa gattttgctg
17461 atgagcagag ccttgtgggc cgcttcattc atctgctgcg ctctgaggac cctgaccagc
17521 agtacttggt atgagtttac ccttagtata tccctgtatc agctcctagt gaaatcacat
17581 gttcaagtgc ttaaaatggt ttaattcact ttctggtctt agatggtttt gaaggaattg
17641 caactgaatt aaagattcac ttgaacctgg gaggcggagg ttgtagtgag ccgagattgt
17701 gccactgcac tccagcctgg gcaacacagc gagactccat ctcgaaaaaa aaaaagattc
17761 atggcatcca tgggctttta ctttatatat aaacacataa ttgtttgtaa acttctggag
17821 catgtgagta acaattcagt tgctctgatt tcttttgaag actctctgag aattacaaaa
17881 aagtctgtct tcttttgctt gagtgccgat aattattcca tgttcatttt ttctgaacta
17941 tgtattgctt ataataaact ttataagaaa tacaattctt atatttaatt ttacttttcc
18001 aaatttgcaa gtataaatta tatttgtcat attgaaaatg tgagtttttg ttttttgatg
18061 aaagatttaa aaattcattt tgcctttttc ttaacttttt tttttctgat aaagaacaat
18121 cacatgaggt tctctcttta ttattagtcc acagggaatc attgtgaaat ggataaaaca
18181 tgttgcctga gtaggtgtat cagtgaccga tactagatag atagtttatt ttagtgaagg
18241 gttagcacag ttggctgctt aattattgtt tgggcaaagt agtttaacca ttcttggatg
18301 cataaggcta ttaggctgct atgatgaaaa agacatttgc ttgaggatgt cctgactgtc
18361 tcatcccttt ctgttgactt tcttcattgt agttgacaca cctgtacttc ataatcagtg
18421 tgaaataaga ggctgacttc tgttgatagt gtgatggtct ttgtcttggt ttagtgacaa
18481 acattccagg actgtggtat tgtgctctgt gagctatgtg atctgtacag agtgactgtc
18541 ttaagtattt taactgattg ccttatgttt ctgtgtgaga ttgcttgtat ctgtgtgttt
18601 tcattttcta ttgcctacca aatatagtag ttagaaacta ttccttccgg cggggcatgg
18661 tggctcgcat ctataatcct ggcactttga gaggctgagg tggatggatc acctgaggtc
18721 aggagttcag gatcagcatg gccaacatgg tgaaacccca tctctactaa aaatgcaaaa
18781 aattagctgg gcgtggtggt gggcgcctgt aatcctagct gctcaggaga ctgaggcagg
18841 agaatcactt gaacctggga ggtggaggtt gcagtgagtg gatatcatgc cattgcactc
18901 cagcctgggc aacaagagtg aaactccgtc tcaaaaaaaa ttatgccttc tgcatgtggc
18961 tgattggtta ttcccatgta tggagatctt taatgatagg gtcattagct ctgactgccc
19021 ctaggggaaa tgcattctct tattcatcta ccatatcagg aatttcacaa aacctgaatg
19081 ccattgtgtc acatatacta aaaatatttt ataaactctg tgtttttctt gtaatttttc
19141 tgaattggct atatgttgtg ccatttcaga aaaaaaaatc caagaaaaac acagaattca
19201 tggaatattt cacaagtagc tcttttaaag tatgttagca ttttccttga cttaaatggt
19261 cttaaaattt ttttgaatga ggaggtatga tgtaccagta atatgcatat agttgttgtg
19321 tatcatagta atagttaata ttactgagct tatgccttgt gctaagtagt ggtaagcctt
19381 cacatgtgtc acttgatctt cccaacaacc ctaggagttt atagaaactt gtggctaaga
19441 gaaggtaaat aatttgccca aggccacaca tgtaataagt attagcatct gcttttaaat
19501 gtgagtctct gagtatcttc acagccttct ttttttctct tttctttttt cttttttttt
19561 ttttttgaga tggaatcttg ctctgtcacc caggctggag tgcagtggca tgatcccagc
19621 ttactgcaac ctccatctcc tgggttcaag caattctcct gcctcagcct cctcagtagc
19681 tgagattacg ggtgtgcgcc accatgccca gctaattttt gtatttttag tagatacagg
19741 gtttccctat gttggccagg ctggtctcga actcctgacc tcaactgatc tgtccacctt
19801 cggcctccca aagtgctggg attacagaca tgagtcacca cacctggcca gagcctacat
19861 tctttatcag tgcagcatac tttgcacatg tgtgtatgaa aatatattta aatatatctt
19921 tgcttctaac tcgctacctt gggcaggtta tacaacctct ctgaaactca ggcttcccca
19981 tttgtgaaat ggaatagtat ctgtctctgg gttgttgtga caacttgagg agataagaaa
20041 tatgtaaatt gcctaccata aagtatggta cattgtatat attcacaaaa tgttagcaat
20101 gatgattaga gcccacattt atttcacaaa tgattaatca gagtttggaa attttttttt
20161 ctttaatgct tttgggtcag attttgaaca cagcacgaaa acattttgga gctggtggaa
20221 atcagcggat tcgcttcaca ctgccacctt tggtatttgc agcttaccag ctggcttttc
20281 gatataaaga gaattctaaa gtggtgagtt tacttttaag tatttaggta ctttttttcc
20341 tctttcatca ctctgagtgt gtgtgtgttt gttttatatt ataaaaaatt tcaaacgtac
20401 aaaaatagac agtggtataa taaaatccca ttttcgccaa ctctctattt gttacttatc
20461 ctgtgctaag tgttcctaac ggtgatggtg gtggatcaca tactgaggga tcacgtaaaa
20521 agcacttaga aatgcaagat taaagcagtg tgaatttatg ctgaaactct ttcctaagtt
20581 ctaattcagg ttagctttaa aacctaagga gagggcctag cattgcagtc gtttctctct
20641 aaaggcatat cattgaataa tatgagttgt gggcaacttt ttatgagctt ttttcttcct
20701 caaaatggaa ccatggcttg agtcttcaca gtgtagtttt gaagaaaata cctcaagctc
20761 acgtacctga aagttggaca ttcaggttaa tgttaaggaa caacctcagt aacttaattt
20821 tgtttgtttg tttgttttga gatagggtct cattctgtcg cccgggctgg agtacagtgg
20881 cgcagtctta gctcactgca acctccaact cctgggttca agcgattctt gtgtgtcagc
20941 ctcctaagta gctgggatca caggtgtgca ccaccatgcc cagctaattt ttgtattttt
21001 agtagagaca gggtcttgcc atgttgacca gttggtctcg aacttgtggc ctcaggtgat
21061 cctgctgcct cagcctctca aagtgctagg attgtaggtg tgaaccactg catctggcct
21121 cagtatggac ttgattttct cgtaatagag aaaaaagatg tatgcagtag acctaccagc
21181 atgaaacagc agcttttggc caatttttat taggccagct tatcattcac tctttaccag
21241 cgtttatgga taggaatttg tgaatataac aataaaaata gcaaccagcc tacattacaa
21301 agccatagta attaaagcag tatggtaata tggtactggc ataaaaacag acacatagac
21361 caatggaaca gaatagagag tctagaaata aacccacaca tatgcaataa actaatcttt
21421 gataaggaca ccaagaatac acaaagggga aaagaatggt ctcttcaata aatagtattg
21481 ggaaagttgg atatccacat gcaaaagaac gcatttggac tctcatctta tgccatatat
21561 aataatgaac tcaaaatgga ttaaagacct gaaaccataa agctcctaga agaaaacata
21601 gggaaaaacc tccttgtcat tggtcaatga ttttttggat atgaaaccaa aaacctatgc
21661 aactaaagca aaaataagtt taaaaataag caaaaaataa gtttaaaata agcttaaaat
21721 aagcaaaaat aagtttaaaa taagcaaaaa ataagcaaaa ataagtttaa taaactaaaa
21781 accttctgta caacaaagga aacaatcagc agagtgaaga gacaggcaat ggaatggggg
21841 agaatatttg caaactatac atctgaaaag tggtcaatat ctaaaatata tatggaatgc
21901 aactcaatag caagcaaatg aataacttga tttaaaaatg agcaaaggat ctgaatagac
21961 atttttccaa agaagacata caggtggcca actggtatat gaacagatgt tcaacatcat
22021 ttatcaggaa aatgtaaatc aaaaccacta tgagatgtca cctcacatct gtcagaataa
22081 ctgttatcaa aaaaacagaa aatcaagtgt tggcaaggat gtagagaaat gggaaccctg
22141 tttattattg gtgggaatat aaattagtat agccattatg gaaaacagta tggaggttcc
22201 tcaaaaaact gaaactagaa ctaccatgtg accctgcagt cccacatcta gttatgcatt
22261 caaaggaaag gaaatcagta tctcaaagag atatctgcac tcccatgttt attgcagcat
22321 tattcacaat ggctgagata tggaaacaac cttagtgtcc atcgatagat gagtaaagaa
22381 attgtgttgt gtatatatgt gtgtgtatat acgtatatat gtgtgtatat gtatgtacgc
22441 acatattctc tacatagtag aataatactc agctatagaa atgaagaaaa tcttgccatt
22501 tatgacaaca gggattaatc tggaggacat tgttctaagt gaaataagcc aaacacagaa
22561 aggcaaatac tatatgactt catttatatg tagaattgtt ttttaagttg aattcatcca
22621 gcctgggtaa tatagcaaga cccaatctct attaaaaaat aaaaaggcca ggtgtggggg
22681 ctcacgcctg taatcgcagc actttgggag gccgaagcag gcggttcacc tgaggtcggg
22741 agtttgagaa cagcctgacc aacatggaga aaccccgtct ctactgaaaa tacaaaatta
22801 gctgggcgtg gtggcgcatg cctgtagtcc cagctactcg ggagtctgag gcaggagaat
22861 cacttgaacc cgggacgcag aggttgtggt gagccgagat cgtgccattg cacttcagcc
22921 tgggcaacaa gagtgaaact ccgtctaaaa aaaaaaaaaa aaaattaaaa aattagccag
22981 gcgtggtggt acatgcctat agtcctagct actcaagagg ctagggcagg gctgggtgtg
23041 gtggctcaca cctgtcatcc tagcactttg ggaggccaag gtgggtggat catttgggat
23101 caggagtttg agaccagcct ggccaacatg gtgaaaccct gtctctacta aaaatacaaa
23161 aattagccag gtgtgagggg acatgcctgt aatcccagct acttgggaag ctgaggcagg
23221 agaatcactt gaacccggga ggcagaggtt gcagtaagct gagatcgcgc cactgcactc
23281 cagcctgggt gacaaagtga gactctgtct caaaaaaaag aggctagggc aggaggacag
23341 cttgagccca ggagttggag gctgcagtga gttatgattg tgcttttgcg ctccagcctg
23401 ggtgacagag ggagacccag tcactaaaaa atggttgaac ttgtgtaagc aaagactaga
23461 acagtagttg ccaaagaata caaactttca gttataagat aaaaaaattc tggggatcaa
23521 aacgatttag ggcaaataaa taaaagtaac tagcctttac ttatttacta gcatttctta
23581 ctgtgttgtc acccactgtg ccaaggtcta tgactgccac tgtcactttt tttttttttt
23641 ttgagtcaag gtctttgttg cccagactgg gatacagtgg tatgattacg gctcactgca
23701 gcttcgaact cttaggctca agcgatcctt ccatttcagc ctcctgagta gctgggactg
23761 caagcatgtg ccaccacact ggctaatttt ttattttttg tagagacaga gtctcaccat
23821 gttgcctagg ctggtctgaa actcttgggc tcaagcgatc ctcctgcctc cttggcctcc
23881 ctaagtgttg ggattacagg catgagccac catgcccagc ctgtcgccat cttttaaaaa
23941 tgaaaagaac tgattgcttt aacaagaaga aatttggata gtcaatcatg ataaaatatt
24001 taacctcgct tgtaattaca acagcgaacc ttttaagaaa tcaaattggc aaagagaaat
24061 gaaaaataag gttcagcaat ggcgatggtg tgatgaaaat tcattctcat ctaatgttgg
24121 cagtgtgaat tactataata cttctaggaa gttggcggtg tgtaagtagg gtgttaaaat
24181 attcaataat tttacttcca agtggaattc caagaattta tactaaggga ataattaggg
24241 tctcaataaa gcttagtgta tatagaacat tcattgtaat attacagatt atgtctaaaa
24301 gggaatagtt caataaatta tgccatagcc agtctccata atattttcta gtcattaaaa
24361 tgatttcgaa ttagtatcgg gaagattgtt aggacaaaat aggaaaaatt agagctgggt
24421 gcagtggctc acgcctgtaa tcctagcact ttgggaggct gaggcaggcg gatcacctga
24481 ggtcgggagt ttgagaccag cctgaccaat atggagaaac cccgtctcta ctaaaaatac
24541 aaaattagct gggtgtggtg gcgcatgcct gtaatcccag ctattcggta ggctgaggca
24601 ggagaatcac ttgaacctgg gaggcggagg ttgtggcgag ctgagatcat gccattgcac
24661 tccagcctgg gcaacaagag ggaaactact tctcaaaaaa aaaaaaagaa aagaaaagaa
24721 aaattagata caaattactt gaagtgtgaa tcgattttaa ctctcaagaa aataaggtct
24781 agatgcagtg gctcacgcca gtaatcctag cactttggga ggctgagatg ggtggatcag
24841 ttgaggtcag cagttcaaga ctagcctggc cgataaggtg aaaccccttc tctactaaaa
24901 atacaaaaaa tagcagggcg tggtggcgcg cttgtaatcc cagctactca ggaggctgag
24961 gcagaagaat ggtttgaacc caagaggcag aggtggcagt gagccgagat cgcaccaaag
25021 agaaaaaaga aaaccacaca caaaaatgcc agttatatta cagttacata gaaaaaaaga
25081 aaggaagaca tttagcatcc gaatgttacc agtgattatc cgtgggtggt agatttaggg
25141 atgatgtgtg gatgattttg tgtatttttc taattttctc caatttggga atgtaactta
25201 caaatcagaa aaaacaatta tcagccaggt gtgatggctc atgcctgtaa tcccaacact
25261 ttgggaggct gaggcgggag gtttgctcgg ggccagtagt tcaagatcag cctgggcaac
25321 agaatgagac cctgtctcta caaaaaaaaa aaaaaaaaaa aaaaaaaaaa ttagccaggt
25381 gtggtgatgc aagcctgtag tcctagctat tcgggagtct gaggtgggag catcacttga
25441 acccaggagt tcaaggctgc aatgagctgt gatcacacca ctgcactcca gctgggtaac
25501 agagctgttg aaaaaaaaaa aaggaaagaa aaaacaggtt gagtgcagtg gctcacgcct
25561 gtgatcccag cactttggga ggctgaggcg ggcagatcac ttgaggtcag gagttaccag
25621 cctggccaac ttggtgaaac cccgccccac ttggtgaaac cccgccccta ctaaaaatac
25681 aaaattagct gggtgtggtg gtgggcacct gtaatcccag ctactcggga ggctgaggca
25741 ggagaatcac ttgaatccag gagacggagg ttgcagtgag ccaagattgt gccactgcac
25801 tccagcctgg gcaacaagag caaaactctg tctcaaaaag aaaaaacaaa taccaaatac
25861 attaacattg caaaggcaat ttaacctcaa atgatgtttt gagaagacat cctgcttgat
25921 ttacttgttt gccctataac tgaaacagag aaggaaaatg acaggaaaac tgtgcacaca
25981 acttacagta ttttgttcta ttaaaatgga tatcctggaa caagttaatt ttgaatttaa
26041 ggtaacttaa aatgtttttt cttgttttag gatgacaaat gggaaaagaa atgccagaag
26101 attttttcat ttgcccacca gactatcagt gctttgatca aagcagagct ggcagaattg
26161 cccttaagac tttttcttca aggagcacta gctgctgggg aaattggttt tgaaaatcat
26221 gagacagtcg catatgaatt catgtcccag gtgatgatct gttctttctg cgttgtcatg
26281 tcagctctgc tgggttcagt tgcttgtttg caggcatggt ggtaatgcac atgaatttac
26341 tcttctttta ctgaatgtgt aactaccacc ttcccaccat catggaacct gttaatatta
26401 ttgttgtaat tgactggtgt tgatcatttg ctgatgaaat ctaagatttc caagtgggtc
26461 atggtaaaaa tgtttcatgg aacataaaat tcgggaaatg cactcaattc ccaaaatcca
26521 gtttgggaac cctgggttaa acaaagttga aagaagtttc tttattgcaa ctttttagcg
26581 tttttaccat ctcagttgtg tcctgtggct ctcaagagag ggtgcagcat gttctgatat
26641 gaaggctgca gaagtctcac aggatggagg tttggtgaca agtactttgg aaaatgctca
26701 actagaggat ggttggtcct tgaaagtcct ttctgcttta tgttcactag gcattttctc
26761 tgtatgaaga tgaaatcagc gattccaaag cacagctagc tgccatcacc ttgatcattg
26821 gcacttttga aaggatgaag tgcttcagtc aagagaatca tgaacctctg aggactcagt
26881 gtgcccttgc tgcatccaaa cttctaaaga aacctgatca gggccgagct gtgagcacct
26941 gtgcacatct cttctggtct ggcagaaaca cggacaaaaa tggggaggag gtaaggtcat
27001 tcctgactgc atgatagcag acaggatcca taacagggat cagttgtcat ggccttgtgt
27061 tctggaggtg aaacatttgg ggtgcttgga aatctgatga acaaaattgc tttgttttgt
27121 taaaaaagag agtctcatcc tgtagtgaag cctctgcttt gaggatattg taacatagca
27181 agttcaaacc actacctgtt tttaaaaaaa tacagctgta tacttcaaaa caagaagaag
27241 gagaatgaaa aggatttaaa tttgttatgt ccctttaaaa cacgaaagag ccacggtagt
27301 gttgtgtttc tttgtatgaa aacgagatgt ttcattaatc tcttcactgt ccccctgccc
27361 ttttatttta gcttcacgga ggcaagaggg taatggagtg cctaaaaaaa gctctaaaaa
27421 tagcaaatca gtgcatggac ccctctctac aagtgcagct ttttatagaa attctgaaca
27481 gatatatcta tttttatgaa aaggaaaatg atgcggtaag tgaattagta aagtgttgtt
27541 aataaactaa tattttccct tcctactctt aggagatttg atatgtacaa aagtttatca
27601 ttctgatact ttaatcactg ttcatttgaa aaatgtaaaa taatttacag atgtcaaata
27661 ataggctaat ttgtcataat gttctagttt aagataattc ctaggctggg cgtggtggct
27721 catgcctgta atcccagcac tttgggaggc tgaggcaggc agatcacctg aggtcaggag
27781 tttgaaacca gcctggccaa cattgtgaaa ccccatctct actaaaaata caaaaattag
27841 ctaggcgtgg tggcaggcgc ctgtaatccc agctacttgg aagcctaagg caggagaatc
27901 gcttgaacct gggaggtgga ggctgcagtg agccaagact gtgccattgc actcctgcct
27961 gggcaacaag agtgaaactc cgtctcaaaa ataataataa taataattcc taaacgcagt
28021 atccttttag caatacagtt ttggtcaaga tttgtaagtt aaataaaatt ttgcttgttt
28081 ttcttttttt gacagagtct ggctctgtca cccaggctgg agtgcagtgg caatctcagc
28141 tcattgcaac ctctgcctcc caggttcaag caattctcat gcctcagcct cctgaatagc
28201 tggtattata ggcgcccggc accacgccca gctaattttt gtattattac tagagatggg
28261 gttccaccat gttggccagg ctggtctcaa aactcctgac ttcaagtgat ctgcccacct
28321 cagcctccca aagtgctggg agtacaggga tgagccactg agccgagcca attttgcttg
28381 ttttaaaggg ttgttttttt tttttttttt ttgatagtca gtaattgttc aaactaggaa
28441 ttgtatcccc atctttcttt tttcataatt actcaggtaa ttgatgagtg taacagaagc
28501 tcctcaaaac agttttatta aattgccttt cattttttgt ggtacgtgct tgatcatgaa
28561 tttgtacata ttcttttgta ggtaacaatt caggttctaa accagcttat ccaaaagatt
28621 cgagaagacc tcccgaatct tgaatccagt gaagaaacag agcagattaa caaacatttt
28681 cataacacac tggagcattt gcgcttgcgg cgggaatcac cagaatccga ggggccaatt
28741 tatgaaggtc tcatccttta aaaaggaaat agctcaccat actcctttcc atgtacatcc
28801 agtgagggtt ttattacgct aggtttccct tccatagatt gtgcctttca gaaatgctga
28861 ggtaggtttc ccatttctta cctgtgatgt gttttaccca gcacctccgg acactcacct
38921 tcaggacctt aataaaatta ttcacttggt aagtgttcaa gtctttctga tcaccccaag
28981 tagcatgact gatctgcaat ttaaaattcc tgtgatctgt aaaaaaaaaa aaaaaaaaaa
29041 aaacaaaacc cacaagcact tatcttggct actaatgaag ctctcctttt ttttgtttgt
29101 ttgtttgctt cattgttgat tgtgtatttt cttcattcct ggggagtact aacccaaaag
29161 cgtctgtctc ttgttttcta gtccagtttg agattaattt agaagaaagg aatactgtat
29221 gtgaaattca tcttgggctt tcccctaaat tgcaagataa ggccatgtgt aagattttcc
29281 ctaaaactag aatatattaa tgcatgtttg agaattttaa agcaccatgg tcaaaaccag
29341 aagctatatt ttgcatattt ggactcagcc atccattaag aacccatgtt gtcctctgga
29401 catatttatc aatataattg ggttttaagt agtataaaag aaaacttgtg atctatataa
29461 tttatgtatc accttcattg taaatttagc aggaaatgca tcacaattat gatttttttt
29521 tttgcaccag tgaaacaata aagatgctat taacaa

For example, the nucleotide sequence corresponding to the mRNA of the human VPS35 is depicted in SEQ ID NO: 13 (3298 bp), wherein the underscored bolded “ATG” denotes the beginning of the open reading frame. Sequence information related to LRRK2 VPS35 is accessible in public databases by GenBank Accession number NM_018206.4 (nucleotide).

SEQ ID NO: 13:

1    gctagagagg gcggggcttg gaggggccgc agcgtcacat gaccgcggga ggctacgcgc
61   ggggcgggtg ctgcttgctg caggctctgg ggagtcgcca tgcctacaac acagcagtcc
121  cctcaggatg agcaggaaaa gctcttggat gaagccatac aggctgtgaa ggtccagtca
181  ttccaaatga agagatgcct ggacaaaaac aagcttatgg atgctctaaa acatgcttct
241  aatatgcttg gtgaactccg gacttctatg ttatcaccaa agagttacta tgaactttat
301  atggccattt ctgatgaact gcactacttg gaggtctacc tgacagatga gtttgctaaa
361  ggaaggaaag tggcagatct ctacgaactt gtacagtatg ctggaaacat tatcccaagg
421  ctttaccttt tgatcacagt tggagttgta tatgtcaagt catttcctca gtccaggaag
481  gatattttga aagatttggt agaaatgtgc cgtggtgtgc aacatccctt gaggggtctg
541  tttcttcgaa attaccttct tcagtgtacc agaaatatct tacctgatga aggagagcca
601  acagatgaag aaacaactgg tgacatcagt gattccatgg attttgtact gctcaacttt
661  gcagaaatga acaagctctg ggtgcgaatg cagcatcagg gacatagccg agatagagaa
721  aaaagagaac gagaaagaca agaactgaga attttagtgg gaacaaattt ggtgcgcctc
781  agtcagttgg aaggtgtaaa tgtggaacgt tacaaacaga ttgttttgac tggcatattg
841  gagcaagttg taaactgtag ggatgctttg gctcaagaat atctcatgga gtgtattatt
901  caggttttcc ctgatgaatt tcacctccag actttgaatc cttttcttcg ggcctgtgct
961  gagttacacc agaatgtaaa tgtgaagaac ataatcattg ctttaattga tagattagct
1021 ttatttgctc accgtgaaga tggacctgga atcccagcgg atattaaact ttttgatata
1081 ttttcacagc aggtggctac agtgatacag tctagacaag acatgccttc agaggatgtt
1141 gtatctttac aagtctctct gattaatctt gccatgaaat gttaccctga tcgtgtggac
1201 tatgttgata aagttctaga aacaacagtg gagatattca ataagctcaa ccttgaacat
1261 attgctacca gtagtgcagt ttcaaaggaa ctcaccagac ttttgaaaat accagttgac
1321 acttacaaca atattttaac agtcttgaaa ttaaaacatt ttcacccact ctttgagtac
1381 tttgactacg agtccagaaa gagcatgagt tgttatgtgc ttagtaatgt tctggattat
1441 aacacagaaa ttgtctctca agaccaggtg gattccataa tgaatttggt atccacgttg
1501 attcaagatc agccagatca acctgtagaa gaccctgatc cagaagattt tgctgatgag
1561 cagagccttg tgggccgctt cattcatctg ctgcgctctg aggaccctga ccagcagtac
1621 ttgattttga acacagcacg aaaacatttt ggagctggtg gaaatcagcg atttcgcttc
1681 acactgccac ctttggtatt tgcagcttac cagctggctt ttcgatataa agagaattct
1741 aaagtggatg acaaatggga aaagaaatgc cagaagattt tttcatttgc ccaccagact
1801 atcagtgctt tgatcaaagc agagctggca gaattgccct taagactttt tcttcaagga
1861 gcactagctg ctggggaaat tggttttgaa aatcatgaga cagtcgcata tgaattcatg
1921 tcccaggcat tttctctgta tgaagatgaa atcagcgatt ccaaagcaca gctagctgcc
1981 atcaccttga tcattggcac ttttgaaagg atgaagtgct tcagtgaaga gaatcatgaa
2041 cctctgagga ctcagtgtgc ccttgctgca tccaaacttc taaagaaacc tgatcagggc
2101 cgagctgtga gcacctgtgc acatctcttc tggtctggca gaaacacgga caaaaatggg
2161 gaggagcttc acggaggcaa gagggtaatg gagtgcctaa aaaaagctct aaaaatagca
2221 aatcagtgca tggacccctc tctacaagtg cagcttttta tagaaattct gaacagatat
2281 atctattttt atgaaaagga aaatgatgcg gtaacaattc aggttttaaa ccagcttatc
2341 caaaagattc gagaagacct cccgaatctt gaatccagtg aagaaacaga gcagattaac
2401 aaacattttc ataacacact ggagcatttg cgcttgcggc gggaatcacc agaatccgag
2461 gggccaattt atgaaggtct catcctttaa aaaggaaata gctcaccata ctcctttcca
2521 tgtacatcca gtgagggttt tattacgcta ggtttccctt ccatagattg tgcctttcag
2581 aaatgctgag gtaggtttcc catttcttac ctgtgatgtg ttttacccag cacctccgga
2641 cactcacctt caggacctta ataaaattat tcacttggta agtgttcaag tctttctgat
2701 caccccaagt agcatgactg atctgcaatt taaaattcct gtgatctgta aaaaaaaaaa
2761 aaaaaaaaaa aacaaaaccc acaagcactt atcttggcta ctaatgaagc tctccttttt
2821 tttgtttgtt tgtttgcttc attgttgatt gtgtattttc ttcattcctg gggagtacta
2881 acccaaaagc gtctgtctct tgttttctag tccagtttga gattaattta gaagaaagga
2941 atactgtatg tgaaattcat cttgggcttt cccctaaatt gcaagataag gccatgtgta
3001 agattttccc taaaactaga atatattaat gcatgtttga gaattttaaa gcaccatggt
3061 caaaaccaga agctatattt tgcatatttg gactcagcca tccattaaga acccatgttg
3121 tcctctggac atatttatca atataattgg gttttaaata gtataaaaga aaacttgtga
3181 tctatataat ttatgtatca ccttcattgt aaatttagca ggaaatgcat cacaattatg
3241 attttttttt ttgcaccagt gaaacaataa agatgctatt aacaaaaaaa aaaaaaaa

For example, the polypeptide sequence corresponding to human VPS35 is encoded by the nucleic acid sequence of SEQ ID NO: 13 and is depicted in SEQ ID NO: 14 (796aa). Sequence information related to VPS35 is accessible in public databases by GenBank Accession numbers NP_060676.2 (protein).

1   MPTTQQSPQD EQFKLLDEAI QAVYVQSFQM KRCLDKNKLM DALKHASNML GELRTSMLSP
61  KSYYELYMAI SDELHYLEVY LTDEFAKGRK VADLYELVQY AGNIIPRLYL LITVGVVYVK
121 SFPQSRKDIL KDLVEMCRGV QHPLRGLFLR NYLLQCTRNI LPDEGEPTDE ETTGDISDSM
181 DFVLLNFAEM NKLWVRMQHQ GHSRDREKRE RERQELRILV GTNLVRLSQL EGVNVERYKQ
241 IVLTGILEQV VNCRDALAQE YLMECIIQVF PDEFHLQTLN PFLRACAELH QNVNVKNIII
301 ALIDRLALFA HREDGPGIPA DIKLFDIFSQ QVATVIQSRQ DMPSEDVVSL QVSLINLAMK
361 CYPDRVDYVD KVLETTVEIF NKLNLEHIAT SSAVSKELTR LLKIPVDTYN NILTVLKLKH
421 FHPLFEYFDY ESRKSMSCYV LSNVLDYNTE IVSQDQVDSI MNLVSTLIQD QPDQPVEDPD
481 PEDFADEQSL VGRFIHLLRS EDPDQQYLIL NTARKHFGAG GNQRIRFTLP PLVFAAYQLA
541 FRYKENSKVD DKWEKKCQKI FSFAHQTISA LIKAELAELP LRLFLQGALA AGEIGFENHE
601 TVAYEFMSQA FSLYEDEISD SKAQLAAITL IIGTFERMKC FSEENHEPLR TQCALAASKL
661 LKKPDQGRAV STCAHLFWSG RNTDKNGEEL HGGKRVMECL KKALKIANQC MDPSLQVQLF
721 IEILNRYIYF YEKENDAVTI QVLNQLIQKI REDLPNLESS EETEQINKHF HNTLEHLRLR
781 RESPESEGPI YEGLIL

For example, the polypeptide sequence corresponding to human RAB7L1 (isoform 1) has a mutation wherein the amino acid at position 67 is a lysine (L) instead of a glutamine (Q) and is depicted in SEQ ID NO: 26 (203aa).

SEQ ID NO: 26:

1   MGSRDHLFKV LVVGDAAVGK TSLVQRYSQD SFSKHYKSTV GVDFALKVLQ WSDYEIVRLQ
61  LWDIAGLERF TSMTRLYYRD ASACVIMFDV TNATTFSNSQ RWKQDLDSKL TLPNGEPVPC
121 LLLANKCDLS PWAVSRDQID RFSKENGFTG WTETSVKENK NINEAMRVLI EKMMRNSTED
181 IMSLSTQGDY INLQTKSSSW SCC

For example, the polypeptide sequence corresponding to human LRRK2 has a mutation wherein the amino acid at position 2019 is a serine (S) instead of a glycine (G) and is depicted in SEQ ID NO: 27 (2527aa).

SEQ ID NO: 27:

1    MASGSCQGCE EDEETLKKLI VRLNNVQEGK QIETLVQILE DLLVFTYSEH ASKLFQGKNI
61   HVPLLIVLDS YMRVASVQQV GWSLLCKLIE VCPGTMQSLM GPQDVGNDWE VLGVHQLILK
121  MLTVHNASVN LSVIGLKTLD LLLTSGKITL LILDEESDIF MLIFDAMHSF PANDEVQKLG
181  CKALHVLFER VSEEQLTEFV ENKDYMILLS ALTNFKDEEE IVLHVLHCLH SLAIPCNNVE
241  VLMSGNVRCY NIVVEAMKAF PMSERIQEVS CCLLHRLTLG NFFNILVLNE VHEFVVKAVQ
301  QYPENAALQI SALSCLALLT ETIFLNQDLE EKNENQENDD EGEEDKLFWL EACYKALTWH
361  RKNKHVQEAA CWALNNLLMY QNSLHEKIGD EDGHFPAHRE VMLSMLMHSS SKEVFQASAN
421  ALSTLLEQNV NFRKILLSKG IHLNVLELMQ KHIHSPEVAE SGCKMLNHLF EGSNTSLDIM
481  AAVVPKILTV MKRHETSLPV QLEALRAILH FIVPGMPEES REDTEFHHKL NMVKKQCFKN
541  DIHKLVLAAL NRFIGNPGIQ KCGLKVISSI VHFPDALEML SLEGAMDSVL HTLQMYPDDQ
601  EIQCLGLSLI GYLITKKNVF IGTGHLLAKI LVSSLYRFKD VAEIQTKGFQ TILAILKLSA
661  SFSKLLVHHS FDLVIFHQMS SNIMEQKDQQ FLNLCCKCFA KVAMDDYLKN VMLERACDQN
721  NSIMVECLLL LGADANQAKE GSSLICQVCE KESSPKLVEL LLNSGSREQD VRKALTISIG
781  KGDSQIISLL LRRLALDVAN NSICLGGFCI GKVEPSWLGP LFPDKTSNLR KQTNIASTLA
841  RMVIRYQMKS AVEEGTASGS DGNFSEDVLS KFDEWTFIPD SSMDSVFAQS DDLDSEGSEG
901  SFLVKKKSNS ISVGEFYRDA VLQRCSPNLQ RHSNSLGPIF DHEDLLKRKR KILSSDDSLR
961  SSKLQSHMRH SDSISSLASE REYITSLDLS ANELRDIDAL SQKCCISVHL EHLEKLELHQ
1021 NALTSFPQQL CETLKSLTHL DLHSNKFTSF PSYLLKMSCI ANLDVSRNDI GPSVVLDPTV
1081 KCPTLKQFNL SYNQLSFVPE NLIDVVEKLE QLILEGNKIS GICSPLRLKE LKILNLSKNH
1141 ISSLSENFLE ACPKVESFSA RMNFLAAMPF LPPSMTILKL SQNKFSCIPE AILNLPHLRS
1201 LDMSSNDIQY LPGPAHWKSL NLRELLFSHN QISILDLSEK AYLWSRVEKL HLSHNKLKEI
1261 PPEIGCLENL TSLDVSYNLE LRSFPNEMGK LSKIWDLPLD ELHLNFDFKH IGCKARDIIR
1321 FLQQRLKKAV PYNRMKLMIV GNTGSGKTTL LQQLMKTKKS DLGMQSATVG IDVKDWPIQI
1381 RDKRKRDLVL NVWDFAGREE FYSTHPHFMT QRALYLAVYD LSKGQAEVDA MKPWLFNIKA
1441 RASSSPVILV GTHLDVSDEK QRKACMSKIT KELLNKRGFP AIRDYHFVNA TEESDALAKL
1501 RKTIINESLN FYIRDQLVVG QLIPDCYVEL EKIILSERKN VPIEFPVIDR KRLLQLVREN
1561 QLQLDENELP HAVHFLNESG VLLHPQDPAL QLSDLYFVEP KWLCKIMAQI LTVKVEGCPK
1621 HPKGIISRRD VEKFLSKKRK FPKNYMSQYF KLLEKFQIAL PIGEEYLLVP SSLSDHRPVI
1681 ELPHCENSEI IIRLYEMPYF PMGFWSRLIN RLLEISPYML SGREPALRPN RMYWRQGIYL
1741 NWSPEAYCLV GSEVLDNHPE SFLKITVPSC RKGCILLGQV VDHIDSLMEE WFPGLLEIDI
1801 CGEGETLLKK WALYSFNDGE EHQKILLDDL MKKAEEGDLL VNPDQPRLTI PISQIAPDLI
1861 LADLPRNIML NNDELEFEQA PEFLLGDGSF GSVYRAAYEG EEVAVKIFNK HTSLRLLRQE
1921 LVVLCHLHHP SLISLLAAGI RPRMLVMELA SKGSLDRLLQ QDKASLTRTL QHRIALHVAD
1981 GLRYLHSAMI IYRDLKPHNV LLFTLYPNAA IIAKIADYSI AQYCCRMGIK TSEGTPGFRA
2041 PEVARGNVIY NQQADVYSFG LLLYDILTTG GRIVEGLKFP NEFDELEIQG KLPDPVKEYG
2101 CAPWPMVEKL IKQCLKENPQ ERPTSAQVFD ILNSAELVCL TRRILLPKNV IVECMVATHH
2161 NSRNASIWLG CGHTDRGQLS FLDLNTEGYT SEEVADSRIL CLALVHLPVE KESWIVSGTQ
2221 SGTLLVINTE DGKKRHTLEK MTDSVTCLYC NSFSKQSKQK NFLLVGTADG KLAIFEDKTV
2281 KLKGAAPLKI LNIGNVSTPL MCLSESTNST ERNVMWGGCG TKIFSFSNDF TIQKLIETRT
2341 SQLFSYAAFS DSNIITVVVD TALYIAKQNS PVVEVWDKKT EKLCGLIDCV HFLREVMVKE
2401 NKESKHKMSY SGRVKTLCLQ KNTALWIGTG GGHILLLDLS TRRLIRVIYN FCNSVRVMMT
2461 AQLGSLKNVM LVLGYNRKNT EGTQKQKEIQ SCLTVWDINL PHEVQNLEKH IEVRKELAEK
2521 MRRTSVE

For example, the polypeptide sequence corresponding to human LRRK2 has a mutation wherein the amino acid at position 1441 is a cysteine (C) instead of an arginine (R) and is depicted in SEQ ID NO: 28 (2527aa).

SEQ ID NO: 28:

1    MASGSCQGCE EDEETLKKLI VRLNNVQEGK QIETLVQILE DLLVFTYSEH ASKLFQGKNI
61   HVPLLIVLDS YMRVASVQQV GWSLLCKLIE VCPGTMQSLM GPQDVGNDWE VLGVHQLILK
121  MLTVHNASVN LSVIGLKTLD LLLTSGKITL LILDEESDIF MLIFDAMHSF PANDEVOKLG
181  CKALHVLFER VSEEQLTEFV ENKDYMILLS ALTNFKDEEE IVLHVLHCLH SLAIPCNNVE
241  VLMSGNVRCY NIVVEAMKAF PMSERIQEVS CCLLHRLTLG NFFNILVLNE VHEFVVKAVQ
301  QYPENAALQI SALSCLALLT ETIFLNQDLE EKNENQENDD EGEEDKLFWL EACYKALTWH
361  RKNKHVQEAA CWALNNLLMY QNSLHEKIGD EDGHFPAHRE VMLSMLMHSS SKEVFQASAN
421  ALSTLLEQNV NFRKILLSKG IHLNVLELMQ KHIHSPEVAE SGCKMLNHLF EGSNTSLDIM
481  AAVVPKILTV MKRHETSLPV QLEALRAILH FIVPGMPEES REDTEFHHKL NMVKKQCFKN
541  DIHKLVLAAL NRFIGNPGIQ KCGLKVISSI VHFPDALEML SLEGAMDSVL HTLQMYPDDQ
601  EIQCLGLSLI GYLITKKNVF IGTGHLLAKI LVSSLYRFKD VAEIQTKGFQ TILAILKLSA
661  SFSKLLVHHS FDLVIFHQMS SNIMEQKDQQ FLNLCCKCFA KVAMDDYLKN VMLERACDQN
721  NSIMVECLLL LGADANQAKE GSSLICQVCE KESSPKLVEL LLNSGSREQD VRKALTISIG
781  KGDSQIISLL LRRLALDVAN NSICLGGFCI GKVEPSWLGP LFPDKTSNLR KQTNIASTLA
841  RMVIRYQMKS AVEEGTASGS DGNFSEDVLS KFDEWTFIPD SSMDSVFAQS DDLDSEGSEG
901  SFLVKKKSNS ISVGEFYRDA VLQRCSPNLQ RHSNSLGPIF DHEDLLKRKR KILSSDDSLR
961  SSKLQSHMRH SDSISSLASE REYITSLDLS ANELRDIDAL SQKCCISVHL EHLEKLELHQ
1021 NALTSFPQQL CETIKSLTHL DLHSNKFTSF PSYLLKMSCI ANLDVSRNDI GPSVVLDPTV
1081 KCPTLKQFNL SYNQLSFVPE NLTDVVEKLE QLILEGNKIS GICSPLRLKE LKILNLSKNH
1141 ISSLSENFLE ACPKVESFSA RMNFLAAMPF LPPSMTILKL SQNKFSCIPE AILNLPHLRS
1201 LDMSSNDIQY LPGPAHWKSL NLRELLFSHN QISILDLSEK AYLWSRVEKL HLSHNKLKEI
1261 PPEIGCLENL TSLDVSYNLE LRSFPNEMGK LSKIWDLPLD ELHLNFDFKH IGCKAKDIIR
1321 FLQQRLKKAV PYNRMKLMIV GNTGSGKTTL LQQLMKTKKS DLGMQSATVG IDVKDWPIQI
1381 RDKRKRDLVL NVWDFAGREE FYSTHPHFMT QRALYLAVYD LSKGQAEVDA MKPWLFNIKA
1441 CASSSPVILV GTHLDVSDEK QRKACMSKIT KELLNKRGFP AIRDYHFVNA TEESDALAKL
1501 RKTIINESLN FKIRDQLVVG QLIPDCYVEL EKIILSERKN VPIEFPVIDR KRLLQLVREN
1561 QLQLDENELP HAVHFLNESG VLLHFQDPAL QLSDLYFVEP KWLCKIMAQI LTVKVEGCPK
1621 HPKGIISRRD VEKFLSKKRK FPKNYMSQYF KLLEKFQIAL PIGEEYLLVP SSLSDHRPVI
1681 ELPHCENSEI IIRLYEMPYF PMGFWSRLIN RLLEISPYML SGRERALRPN RMYWRQGIYL
1741 NWSPEAYCLV GSEVLDNHPE SFLKITVPSC RKGCILLGQV VDHIDSLMEE WFPGLLEIDI
1801 CGEGETLLKK WALYSFNDGE EHQKILLDDL MKKAEEGDLL VNPDQPRLTI PISQIAPDLI
1861 LADLPRNIML NNDELEFEQA PEFLIGDGSF GSVYRAAYEG EEVAVKIFNK HTSLRLLRQE
1921 LVVLCHLHHP SLISLLAAGI RPRMLVMELA SKGSLDRLLQ QDKASLTRTL QHRIALHVAD
1981 GLRYLHSAMI IYRDLKPHNV LLFTLYPNAA IIAKIADYGI AQYCCRMGIK TSEGTPGFRA
2041 PEVARGNVIY NQQADVYSFG LLLYDILTTG GRIVEGLKFP NEFDELEIQG KLPDPVKEYG
2101 CAPWPMVEKL IKQCLKENPO ERPTSAQVFD ILNSAELVCL TRRILLPKNV IVECMVATHH
2161 NSRNASIWLG CGHTDRGQLS FLDLNTEGYT SEEVADSRIL CLALVHLPVE KESWIVSGTQ
2221 SGTLLVINTE DGKKRHTLEK MTDSVTCLYC NSFSKQSKQK NFLLVGYADG KLAIFEDKTV
2281 KLKGAAPLKI LNIGNVSTPL MCLSESTNST ERNVMWGGCG TKIFSFSNDF TIQKLIETRT
2341 SQLFSYAAFS DSNIITVVVD TALYIAKQNS PVVEVWDKKT EKLCGLIDCV HFLREVMVKE
2401 NKESKHKMSY SGRVKTLCLQ KNTALWIGTG GGHILLLDLS TRRLIRVIYN FCNSVRVMMT
2461 AQLGSLKNVM LVLGYNRKNT EGTQKQKEIQ SCLTVWDINL PHEVQNLEKH IEVRKELAEK
2521 MRRTSVE

A RAB7L1, a LRRK2, or a VPS35 molecule can also encompass ortholog genes, which are genes conserved among different biological species such as humans, dogs, cats, mice, and rats, that encode proteins (for example, homologs (including splice variants), mutants, and derivatives) having biologically equivalent functions as the human-derived protein. Orthologs of a RAB7L1, a LRRK2, or a VPS35 protein include any mammalian ortholog inclusive of the ortholog in humans and other primates, experimental mammals (such as mice, rats, hamsters and guinea pigs), mammals of commercial significance (such as horses, cows, camels, pigs and sheep), and also companion mammals (such as domestic animals, e.g., rabbits, ferrets, dogs, and cats). A RAB7L1, a LRRK2, or a VPS35 molecule can comprise a protein encoded by a nucleic acid sequence homologous to the human nucleic acid, wherein the nucleic acid is found in a different species and wherein that homolog encodes a protein similar to a RAB7L1, a LRRK2, or a VPS35 protein.

The invention utilizes conventional molecular biology, microbiology, and recombinant DNA techniques available to one of ordinary skill in the art. Such techniques are well known to the skilled worker and are explained fully in the literature. See, e.g., Maniatis, Fritsch & Sambrook, “DNA Cloning: A Practical Approach.” Volumes I and II (D. N. Glover, ed., 1985); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Nucleic Acid Hybridization” (B. D. Hames & S. J. Higgins, eds., 1985); “Transcription and Translation” (B. D. Hames & S. J. Higgins, eds., 1984); “Animal Cell Culture” (R. I. Freshney, ed., 1986); “Immobilized Cells and Enzymes” (IRL Press, 1986): B. Perbal, “A Practical Guide to Molecular Cloning” (1984), and Sambrook, et al., “Molecular Cloning: a Laboratory Manual” (2001).

One skilled in the art can obtain RAB7L1, a LRRK2, or a VPS35 molecule, in several ways, which include, but are not limited to, isolating the protein via biochemical means or expressing a nucleotide sequence encoding the protein of interest by genetic engineering methods.

The invention provides for a RAB7L1, a LRRK2, or a VPS35 molecule that are encoded by nucleotide sequences. The RAB7L1. LRRK2, or VPS35molecule can be a polypeptide encoded by a nucleic acid (including genomic DNA, complementary DNA (cDNA), synthetic DNA, as well as any form of corresponding RNA). For example, a RAB7L1, a LRRK2, or a VPS35 molecule can be encoded by a recombinant nucleic acid encoding a human RAB7L1, a human LRRK2, or a human VPS35 protein, or fragment thereof. The RAB7L1, LRRK2, or VPS35 molecules of the invention can be obtained from various sources and can be produced according to various techniques known in the art. For example, a nucleic acid that encodes a RAB7L1, a LRRK2, or a VPS35 molecule can be obtained by screening DNA libraries, or by amplification from a natural source. The RAB7L1, LRRK2, or VPS35 molecule of the invention can be produced via recombinant DNA technology and such recombinant nucleic acids can be prepared by conventional techniques, including chemical synthesis, genetic engineering, enzymatic techniques, or a combination thereof. A RAB7L1, a LRRK2, or a VPS35 molecule of this invention can also encompasses variants of the human RAB7L1, LRRK2, or VPS35 proteins. The variants can comprise naturally-occurring variants due to allelic variations between individuals (e.g., polymorphisms), mutated alleles, or alternative splicing forms.

In one embodiment, a fragment of a nucleic acid sequence that comprises a RAB7L1, a LRRK2, or a VPS35 molecule can encompass any portion of at least about 8 consecutive nucleotides of SEQ ID NO: 1, 2, 3, 4, 5, 9, 10, 12, or 13. In one embodiment, the fragment can comprise at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, or at least about 30 nucleotides of SEQ ID NO: 1, 2, 3, 4, 5, 9, 10, 12, or 13. Fragments include all possible nucleotide lengths between about 8 and about 100 nucleotides, for example, lengths between about 15 and about 100 nucleotides, or between about 20 and about 100 nucleotides.

A RAB7L1, a LRRK2, or a VPS35 molecule, can be a fragment of a RAB7L1, a LRRK2, or a VPS35 protein. For example, the RAB7L1, LRRK2, or VPS35 protein fragment can encompass any portion of at least about 8 consecutive amino acids of SEQ ID NO: 6, 7, 8, 1, 14, 26, 27, or 28. The fragment can comprise at least about 10 consecutive amino acids, at least about 20 consecutive amino acids, at least about 30 consecutive amino acids, at least about 40 consecutive amino acids, a least about 50 consecutive amino acids, at least about 60 consecutive amino acids, at least about 70 consecutive amino acids, at least about 80 consecutive amino acids, at least about 90 consecutive amino acids, at least about 100 consecutive amino acids, at least about 110 consecutive amino acids, or at least about 120 consecutive amino acids of SEQ ID NOS: 6, 7, 8, 11, 14, 26, 27, or 28. Fragments include all possible amino acid lengths between about 8 and 80 about amino acids, for example, lengths between about 10 and about 80 amino acids, between about 15 and about 80 amino acids, between about 20 and about 80 amino acids, between about 35 and about 80 amino acids, between about 40 and about 80 amino acids, between about 50 and about 80 amino acids, or between about 70 and about 80 amino acids.

Methods of Treating Parkinson's Disease

In one aspect, the invention provides a method of treating Parkinson's Disease (PD) in a subject comprising: (a) determining the presence or absence of a genetic variant at the PARK16 and LRRK2 loci in a sample from a subject, wherein the presence of a PD-associated genetic variant at both the PARK16 and LRRK2 loci in the subject sample indicates the subject has an increased risk or predisposition to PD, and (b) administering a treatment if the subject has an increased risk or predisposition to PD.

In another aspect, the invention provides a method of treating Parkinson's Disease (PD) in a subject comprising: (a) determining the presence or absence of a genetic variant at the LRRK2 locus in a sample from a subject, wherein the presence of a PD-associated genetic variant at the LRRK2 locus in the subject sample indicates the subject has an increased risk or predisposition to PD, and (b) administering a treatment if the subject has an increased risk or predisposition to PD.

In another aspect, the invention provides a method of treating Parkinson's Disease (PD) in a subject comprising: (a) determining the presence or absence of a genetic variant at the PARK16 locus in a sample from a subject, wherein the presence of a PD-associated genetic variant at the PARK16 locus in the subject sample indicates the subject has an increased risk or predisposition to PD, and (b) administering a treatment if the subject has an increased risk or predisposition to PD.

As used herein. “single-nucleotide polymorphism” or “SNP” refers to variations at single-nucleotide positions in the DNA sequence among individuals. Information on SNPs can be found in publically accessible databases, such as, in the SNP database at the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/). In one embodiment, the genetic variant at the PARK 16 locus comprises single-nucleotide polymorphism (SNP) rs823114, SNP rs823154, SNP rs823128, SNP rs947211, or a combination thereof.

In one embodiment, the PARK16 locus comprises the genes SLC45A3, NUCKS1, RAB7L1, SLC41A1, and PM20D1. In one embodiment, the genetic variant at the PARK 16 locus comprises a genetic variant in the RAB7L1 gene. In another embodiment, the genetic variant at the PARK 16 locus comprises a genetic variant at the SLC45A3, NUCKS1, SLC41A1, or PM20D1 gene. In one embodiment, the genetic variant at the RAB7L1 gene is SNP rs1572931.

Without being bound by theory, genetic variants can be associated with PD. In one embodiment, the PD-associated genetic variant at the PARK16 locus comprises a guanine (G) nucleotide at SNP rs1572931.

Genetic variants can also affect the splicing of mRNA. Without being bound by theory, pre-mRNA transcribed from genomic DNA can be spliced so that introns are removed and exons are joined together. Transcribed pre-mRNA can be alternatively spliced creating a range of unique proteins (known as “isoforms”) and/or mRNAs (known as “transcript variants”) by varying the exon composition of the mRNA. In one embodiment, the PD-associated genetic variant at the PARK16 locus encodes a RAB7L1 mRNA, wherein exon 2 is excluded from the RAB7L1 mRNA sequence. In one embodiment, the PD-associated genetic variant at the PARK16 locus results in loss of expression of a RAB7L1 protein. Various mutations that affect the transcription and translation of a RAB7L1 molecule can result in loss of expression of a RAB7L1 protein.

In one embodiment, the genetic variant at the LRRK2 locus comprises SNP rs1176052. In another embodiment, the PD-associated genetic variant at the LRRK2 locus encodes the protein of SEQ ID NO: 27 or 28. In another embodiment, the protein of SEQ ID NO: 27 or 28 is associated with familial PD. In another embodiment, the genetic variant at the LRRK2 locus is associated with sporadic, or non-familial PD.

In one embodiment, the PD-associated genetic variant at the LRKK2 locus results in loss of expression of a LRKK2 protein. Various mutations that affect the transcription and translation of a LRRK2 molecule can result in loss of expression of a LRRK2 protein.

In one aspect the invention provides, a method of treating PD in a subject comprising: (a) measuring the expression levels of a set of genes in a sample from a subject, wherein the set of genes comprises at least one gene selected from the genes listed in Table 2 (b) comparing the expression levels of the set of genes in the subject sample to expression levels of the same set of genes in a reference sample or samples, wherein the reference sample or samples are from an individual who has a PD-associated SNP, and wherein similar expression levels of the set of genes in the subject sample and the set of genes in the reference sample(s) indicates the subject has an increased risk or predisposition to PD, and (c) administering a treatment if the subject has an increased risk or predisposition to PD.

In another aspect, the invention provides a method of treating PD in a subject comprising: (a) determining a level of full-length RAB7L1 in a sample from a subject, (b) comparing the level of full-length RAB7L1 from the subject sample to a full-length RAB7L1 level in a reference sample, wherein the reference sample is associated with a non-PD status, and wherein a reduced level of the full-length RAB7L in the subject sample indicates the subject has an increased risk or predisposition to PD, and (c) administering a treatment if the subject has an increased risk or predisposition to PD. In one embodiment, the level of full-length RAB7L is protein level of full-length RAB7L, or mRNA levels of the full-length RAB7L, or a combination thereof.

In another aspect, the invention provides a method of treating Parkinson's Disease (PD) in a subject comprising: (a) determining a level of isoform 3 of RAB7L1 in a sample from a subject, (b) comparing the level of isoform 3 of RAB7L1 from the subject sample to an isoform 3 of RAB7L1 level in a reference sample, wherein the reference sample is associated in non-PD status, and wherein an increased level of isoform 3 of RAB7L1 in the subject sample indicates the subject has an increased risk or predisposition to PD, and (c) administering a treatment if the subject has an increased risk or predisposition to PD. In one embodiment, the level of isoform 3 of RAB7L1 is a protein level. In one embodiment, the method further comprises determining the level of transcript variant 4, 5, or a combination thereof of RAB7L1.

A method of treating Parkinson's Disease (PD) in a subject comprising: (a) determining a level of transcript variant 4, 5, or a combination thereof of RAB7L1 in a sample from a subject, (b) comparing the level of transcript variant 4, 5, or a combination thereof of RAB7L1 from the subject sample to a transcript variant 4, 5, or a combination thereof of RAB7L1 level in a reference sample, wherein the reference sample is associated in non-PD status, and wherein an increased level of transcript variant 4, 5, or a combination thereof of RAB7L1 in the subject sample indicates the subject has an increased risk or predisposition to PD, and (c) administering a treatment if the subject has an increased risk or predisposition to PD. In one embodiment, the level of transcript variant 4, 5, or a combination thereof of RAB7L1 is a mRNA level. In another embodiment, the method further comprises determining the level of isoform 3 of RAB7L1.

In one embodiment, the invention provides for determine the level of retromer components. Without being bound buy theory, retromer is a complex of proteins which are involved in recycling between the endolysosomal compartment of a cell and the Golgi apparatus. In mammals, proteins of the retromer complex include, but are not limited to Vps26, Vps29, Vps35, SNX1, SNX2, SNX5 and SNX6. The retromer complex can act in two subcomplexes; a cargo recognition complex that comprises Vps35, Vps29 and Vps26 (Vps trimer), and SNX-BAR dimers that comprises SNX1 and SNX2 or SNX5 and SNX6.

In another aspect, the invention provides a method of treating Parkinson's Disease (PD) in a subject comprising: (a) determining a level of retromer components in a sample from a subject, (b) comparing the level of retromer components from the subject sample to a retromer component level in a reference sample, wherein the reference sample is associated with a non-PD status, and wherein a reduced level of the retromer components in the subject sample indicates the subject has an increased risk or predisposition to PD, and (c) administering a treatment if the subject has an increased risk or predisposition to PD. In one embodiment, the level of retromer component is protein level of retromer component, or mRNA levels of retromer component, or a combination thereof. In another embodiment, the retromer component is VPS35, VPS29, VPS26 or a combination thereof. In a further embodiment the retromer component is SNX1, SNX2. SNX5, SNX6, or a combination thereof. In one embodiment, the level of VPS35. VPS29, or VPS26 is protein level of VPS35, VPS29, or VPS26, or mRNA levels of VPS35, VPS29, or VPS26, or a combination thereof.

In one aspect, the invention provides a method of treating PD in a subject. In one embodiment, the treatment comprises administering to the subject an effective amount of a nucleic acid encoding a protein comprising 90% of SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 11, SEQ ID NO: 14, or a combination or fragment thereof. In another embodiment the treatment comprises administering to the subject an effective amount of a protein comprising 90% of SEQ ID NO: 6, SEQ ID NO: 26. SEQ ID NO: 11, SEQ ID NO: 14, or a combination or fragment thereof. In one embodiment, the protein comprises at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 11, or SEQ ID NO: 14.

In one embodiment, the treatment comprises administering to the subject an effective amount of a nucleic acid encoding a protein comprising 90% of SEQ ID NO: 6, SEQ ID NO: 26. SEQ ID NO: 14, or a combination or fragment thereof. In another embodiment, the treatment comprises administering to the subject an effective amount of a protein comprising 90% of SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 14, or a combination or fragment thereof. In one embodiment, the protein comprises at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of SEQ ID NO: 6, SEQ ID NO: 26, or SEQ ID NO: 14.

In one embodiment, the treatment comprises administering to the subject an effective amount of a nucleic acid encoding a protein comprising 90% of SEQ ID NO: 11, SEQ ID NO: 14, or a combination or fragment thereof. In another embodiment, the treatment comprises administering to the subject an effective amount of a protein comprising 90% of SEQ ID NO: 11, SEQ ID NO: 14, or a combination or fragment thereof. In one embodiment, the protein comprises at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 11, or SEQ ID NO: 14.

In one embodiment, the PD disease status is determined by any suitable method, including but not limited to a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. Suitable methods for determining the PD disease statuts are known to one of skill in the art.

In one embodiment, the subject is not diagnosed with PD. In another embodiment, the subject is diagnosed with PD. In another embodiment, the subject is diagnosed with a pre-disease prodromal state.

In one embodiment, the method further comprises a physical examination of the subject, a neurological examination of the subject, a brain scan, or a combination thereof. Methods and types of physical examinations are known to one of skill in the art.

In one embodiment, the method further comprises a step of sequencing nucleic acids isolated from the subject's sample to determine the presence or absence of a PD-risk associated SNP, wherein the presence of a PD-risk associated SNP is further indicative that the subject is at risk of developing PD or is suffering from PD.

In one embodiment, the subject is suspected of having PD or is at risk of developing PD based on the presence of any one of parkinsonism symptoms. Determination of parkinsonism symptoms are known to one of skill in the art.

In one embodiment, the sample is a CSF sample, blood sample, plasma sample, serum sample or any combination thereof. Methods of sample collection are known to one of skill in the art.

Expression Systems and Purification of Recombinant Proteins One skilled in the art understands that polypeptides (for example RAB7L1, LRRK2, or VPS35, and the like) can be obtained in several ways, which include but are not limited to, expressing a nucleotide sequence encoding the protein of interest, or fragment thereof, by genetic engineering methods.

In one embodiment, the nucleic acid is expressed in an expression cassette, for example, to achieve overexpression in a cell. The nucleic acids of the invention can be an RNA, cDNA, cDNA-like, or a DNA of interest in an expressible format, such as an expression cassette, which can be expressed from the natural promoter or an entirely heterologous promoter. The nucleic acid of interest can encode a protein, and may or may not include introns. Any recombinant expression system can be used, including, but not limited to, bacterial, mammalian, yeast, insect, or plant cell expression systems.

Host cells transformed with a nucleic acid sequence encoding a RAB7L1, a LRRK2, or a VPS35 molecule can be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The polypeptide produced by a transformed cell can be secreted or contained intracellularly depending on the sequence and/or the vector used. Expression vectors containing a nucleic acid sequence encoding a RAB7L1, a LRRK2, or a VPS35 molecule can be designed to contain signal sequences which direct secretion of soluble polypeptide molecules encoded by a RAB7L1, a LRRK2, or a VPS35 molecule, through a prokaryotic or eukaryotic cell membrane.

Nucleic acid sequences comprising a RAB7L1, a LRRK2, or a VPS35 molecule that encode a polypeptide can be synthesized, in whole or in part, using chemical methods known in the art. Alternatively, a RAB7L1, a LRRK2, or a VPS35 molecule can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques. Protein synthesis can either be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer). Optionally, fragments of a RAB7L1, a LRRK2, or a VPS35 molecule can be separately synthesized and combined using chemical methods to produce a full-length molecule.

A synthetic peptide can be substantially purified via high performance liquid chromatography (HPLC). The composition of a synthetic RAB7L1, LRRK2, or VPS35 molecule can be confirmed by amino acid analysis or sequencing. Additionally, any portion of an amino acid sequence comprising a protein encoded by a RAB7L1, a LRRK2, or a VPS35 molecule can be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins to produce a variant polypeptide or a fusion protein.

The invention further encompasses methods for using a protein or polypeptide encoded by a nucleic acid sequence of a RAB7L1, a LRRK2, or a VPS35 molecule, such as the sequences shown in SEQ ID NOS: 6, 7, 8, 11, 14, 26, 27, or 28. In another embodiment, the polypeptide can be modified, such as by glycosylations and/or acetylations and/or chemical reaction or coupling, and can contain one or several non-natural or synthetic amino acids. An example of a RAB7L1, a LRRK2, or a VPS35 molecule has the amino acid sequence shown in either SEQ ID NO: 6, 7, 8, 11, 14, 26, 27, or 28. In certain embodiments, the invention encompasses variants of a human protein encoded by a RAB7L1, a LRRK2, or a VPS35 molecule.

One skilled in the art understands that expression of desired protein products can be carried out in prokaryotes (e.g. E. coli and B. subtilis), in plant cell systems infected with recombinant virus expression vectors (e.g., tobacco mosaic virus, TMV: cauliflower mosaic virus, CaMV), in insect cells (e.g. Autographa californica nuclear polyhedrosis virus (AcNPV) can be used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae), in yeast cells (e.g. Saccharomyces sp., Pichia sp.), or in mammalian cells (e.g. BHK cells, VERO cells, CHO cells and the like).

Expression vectors (also known in the art as fusion-vectors) can be designed to add a number of amino acid residues, usually to the N-terminus of the expressed recombinant protein. Such fusion vectors can serve three functions: 1) to increase the solubility of the desired recombinant protein; 2) to increase expression of the recombinant protein of interest; and 3) to aid in recombinant protein purification by acting as a ligand in affinity purification.

An exogenous nucleic acid can be introduced into a cell via a variety of techniques known in the art, such as lipofection, microinjection, calcium phosphate or calcium chloride precipitation, DEAE-dextrin-mediated transfection, or electroporation. Electroporation is carried out at approximate voltage and capacitance to result in entry of the DNA construct(s) into cells of interest. Other methods used to transfect cells can also include modified calcium phosphate precipitation, polybrene precipitation, liposome fusion, and receptor-mediated gene delivery.

Various culturing parameters can be used with respect to the host cell being cultured. Appropriate culture conditions for mammalian cells are well known in the art (Cleveland W L, et al., J Immunol Methods, 1983, 56(2): 221-234) or can be determined by the skilled artisan (see, for example, Animal Cell Culture: A Practical Approach 2nd Ed., Rickwood, D. and Hames, B. D., eds. (Oxford University Press: New York, 1992)). Cell culturing conditions can vary according to the type of host cell selected. Commercially available medium can be utilized.

A RAB7L1, a LRRK2, or a VPS35 molecule can be purified from any human or non-human cell which expresses the polypeptide, including those which have been transfected with expression constructs that express a RAB7L1, a LRRK2, or a VPS35 molecule. A purified RAB7L1, LRRK2, or VPS35 molecule can be separated from other compounds which normally associate with the RAB7L1, LRRK2, or VPS35 molecules, in the cell, such as certain proteins, carbohydrates, or lipids, using methods practiced in the art. The desired polypeptide molecule (for example, a RAB7L1, a LRRK2, or a VPS35 molecule) is isolated or purified away from contaminating soluble proteins and polypeptides by suitable purification techniques. Non-limiting purification methods for proteins include: size exclusion chromatography; affinity chromatography; ion exchange chromatography; ethanol precipitation; reverse phase HPLC; chromatography on a resin, such as silica, or cation exchange resin, e.g., DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, e.g., Sephadex G-75, Sepharose; protein A sepharose chromatography for removal of immunoglobulin contaminants; and the like. Other additives, such as protease inhibitors (e.g., PMSF or proteinase K) can be used to inhibit proteolytic degradation during purification. Purification procedures that can select for carbohydrates can also be used, e.g., ion-exchange soft gel chromatography, or HPLC using cation- or anion-exchange resins, in which the more acidic fraction(s) is/are collected.

Methods of Administration

Nucleic Acid Delivery Methods.

The invention provides methods for treating Parkinson's Disease (PD) in a subject. In one embodiment, the method can comprise administering to the subject a RAB7L1, a LRRK2, or a VPS35 molecule (e.g, a RAB7L1, a LRRK2, or a VPS35 polypeptide or a RAB7L1, a LRRK2, or a VPS35 polynucleotide).

Various approaches can be carried out to restore the activity or function of a RAB7L1, a LRRK2, or a VPS35 molecule in a subject, such as those carrying an genetic variant in a RAB7L1, a LRRK2, or a VPS35 gene locus. For example, supplying wild-type RAB7L1. LRRK2, or VPS35 gene function to such subjects can treat Parkinson's Disease. Increasing a RAB7L1, a LRRK2, or a VPS35 gene expression level or activity can be accomplished through gene or protein therapy.

A nucleic acid encoding a RAB7L11, a LRRK2, or a VPS35 molecule can be introduced into the cells of a subject. For example, the wild-type gene (or fragment thereof) can also be introduced into the cells of the subject in need thereof using a vector as described herein. The vector can be a viral vector or a plasmid. The gene can also be introduced as naked DNA. The gene can be provided so as to integrate into the genome of the recipient host cells, or to remain extra-chromosomal. Integration can occur randomly or at precisely defined sites, such as through homologous recombination. For example, a functional copy of a RAB7L1, a LRRK2, or a VPS35 molecule can be inserted in replacement of an altered version in a cell, through homologous recombination. Further techniques include gene gun, liposome-mediated transfection, or cationic lipid-mediated transfection. Gene therapy can be accomplished by direct gene injection, or by administering ex vivo prepared genetically modified cells expressing a functional polypeptide.

Delivery of nucleic acids into viable cells can be effected ex vivo, in situ, or in vivo by use of vectors, and more particularly viral vectors (e.g., lentivirus, adenovirus, adeno-associated virus, or a retrovirus), or ex vivo by use of physical DNA transfer methods (e.g., liposomes or chemical treatments). Non-limiting techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, and the calcium phosphate precipitation method (see, for example. Anderson, Nature, supplement to vol. 392, no. 6679, pp. 25-20 (1998)). Introduction of a nucleic acid or a gene encoding a polypeptide of the invention can also be accomplished with extrachromosomal substrates (transient expression) or artificial chromosomes (stable expression). Cells may also be cultured ex vivo in the presence of therapeutic compositions of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic purposes.

Nucleic acids can be inserted into vectors and used as gene therapy vectors. A number of viruses have been used as gene transfer vectors, including papovaviruses, e.g., SV40 (Madzak et al., (1992)J Gen Virol. 73(Pt 6):1533-6), adenovirus (Berkner (1992) Curr Top Microbiol Immunol. 158:39-66; Berkner (1988) Biotechniques, 6(7):616-29; Gorziglia and Kapikian (1992). J Virol. 66(7):4407-12; Quantin et al., (1992) Proc Natl Acad Sci USA. 89(7):2581-4; Rosenfeld et al., (1992) Cell. 68(1):143-55; Wilkinson et al., (1992) Nucleic Acids Res. 20(9):2233-9; Stratford-Perricaudet et al., (1990) Hum Gene Ther. 1(3):241-56), vaccinia virus (Moss (1992) Curr Opin Biotechnol. 3(5):518-22), adeno-associated virus (Muzyczka, (1992) Curr Top Microbiol Immunol. 158:97-129; Ohi et al., (1990) Gene. 89(2):279-82), herpesviruses including HSV and EBV (Margolskee (1992) Curr Top Microbiol Immunol. 158:67-95; Johnson et al., (1992) Brain Res Mol Brain Res.12(1-3):95-102; Fink et al., (1992) Hum Gene Ther. 3(1):11-9; Breakefield and Geller (1987) Mol Neurobiol. 1(4):339-71; Freese et al., (1990) Biochem Pharmacol. 40(10):2189-99), and retroviruses of avian (Bandyopadhyay and Temin (1984) Mol Cell Biol. 4(4):749-54; Petropoulos et al., (1992) J Virol. 66(6):3391-7), murine (Miller et al. (1992) Mol Cell Biol. 12(7):3262-72; Miller et al., (1985) J Virol. 55(3):521-6; Sorge et al., (1984) Mol Cell Biol. 4(9):1730-7; Mann and Baltimore (1985)J Virol. 54(2):401-7; Miller et al., (1988)J Virol. 62(11):4337-45), and human origin (Shimada et al., (1991) J Clin Invest. 88(3):1043-7; Helseth et al., (1990) J Virol. 64(12):6314-8; Page et al., (1990)J Virol. 64(11):5270-6; Buchschacher and Panganiban (1992) J Virol. 66(5):2731-9).

Non-limiting examples of in vivo gene transfer techniques include transfection with viral (typically retroviral) vectors (see U.S. Pat. No. 5,252,479, which is incorporated by reference in its entirety) and viral coat protein-liposome mediated transfection (Dzau et al., Trends in Biotechnology 11:205-210 (1993), incorporated entirely by reference). For example, naked DNA vaccines are generally known in the art; see Brower, Nature Biotechnology, 16:1304-1305 (1998), which is incorporated by reference in its entirety. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see, e.g., U.S. Pat. No. 5,328,470) or by stereotactic injection (see, e.g., Chen, et al., 1994. Proc. Natl. Acad. Sci. USA 91: 3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the gene delivery system.

For reviews of gene therapy protocols and methods see Anderson et al., Science 256:808-813 (1992); U.S. Pat. Nos. 5,252,479, 5,747,469, 6,017,524, 6,143,290, 6,410,010 6,511,847; 8,398,968; and 8,404,653 which are all hereby incorporated by reference in their entireties. For an example of gene therapy treatment in humans see Porter et al., NEJM 2011 365:725-733 and Kalos et al. Sci. Transl. Med. 2011. 201 3(95):95. For additional reviews of gene therapy technology, see Friedmann, Science, 244:1275-1281 (1989); Verma, Scientific American: 68-84 (1990); Miller, Nature, 357: 455-460 (1992); Kikuchi et al., J Dermatol Sci. 2008 May; 50(2):87-98; Isaka et al., Expert Opin Drug Deliv. 2007 September; 4(5):561-71; Jager et al., Curr Gene Ther. 2007 August; 7(4):272-83; Waehler et al., Nat Rev Genet. 2007 August; 8(8):573-87; Jensen et al., Ann Med. 2007; 39(2):108-15; Herweijer et al., Gene Ther. 2007 January; 14(2):99-107; Eliyahu et al., Molecules, 2005 Jan. 31; 10(1):34-64; and Altaras et al., Adv Biochem Eng Biotechnol. 2005; 99:193-260, all of which are hereby incorporated by reference in their entireties.

These methods described herein are by no means all-inclusive, and further methods to suit the specific application is understood by the ordinary skilled artisan. Moreover, the effective amount of the compositions can be further approximated through analogy to compounds known to exert the desired effect.

Protein Delivery Methods.

Protein replacement therapy can increase the amount of protein by exogenously introducing wild-type or biologically functional protein by way of infusion. A replacement polypeptide can be synthesized according to known chemical techniques or may be produced and purified via known molecular biological techniques. Protein replacement therapy has been developed for various disorders. For example, a wild-type protein can be purified from a recombinant cellular expression system (e.g., mammalian cells or insect cells-see U.S. Pat. No. 5,580,757 to Desnick et al.; U.S. Pat. Nos. 6,395,884 and 6,458,574 to Selden et al.; U.S. Pat. No. 6,461,609 to Calhoun et al.; U.S. Pat. No. 6,210,666 to Miyamura et al.; U.S. Pat. No. 6,083,725 to Selden et al.; U.S. Pat. No. 6,451,600 to Rasmussen et al.; U.S. Pat. No. 5,236,838 to Rasmussen et al. and U.S. Pat. No. 5,879,680 to Ginns et al.), human placenta, or animal milk (see U.S. Pat. No. 6,188,045 to Reuser et al.), or other sources known in the art. After the infusion, the exogenous protein can be taken up by tissues through non-specific or receptor-mediated mechanism.

A RAB7L1, a LRRK2, or a VPS35 molecule can also be delivered in a controlled release system. For example, the RAB7L1, LRRK2, or VPS35 molecule can be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In one embodiment, a pump can be used (see Sefton (1987) Biomed. Eng. 14:201; Buchwald et al. (1980) Surgery 88:507; Saudek et al. (1989) N. Engl. J. Med. 321:574). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, (1983) J. Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al. (1985) Science 228:190; During et al. (1989) Ann. Neurol. 25:351; Howard et al. (1989) J. Neurosurg. 71:105). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled release systems are discussed in the review by Langer (Science (1990) 249:1527-1533).

Pharmaceutical Compositions and Methods of Administration

In some embodiments, a RAB7L1, a LRRK2, or a VPS35 molecule can be supplied in the form of a pharmaceutical composition, comprising an isotonic excipient prepared under sufficiently sterile conditions for human administration. Choice of the excipient and any accompanying elements of the composition comprising a RAB7L1, a LRRK2, or a VPS35 molecule will be adapted in accordance with the route and device used for administration. In some embodiments, a composition comprising a RAB7L1, a LRRK2, or a VPS35 molecule can also comprise, or be accompanied with, one or more other ingredients that facilitate the delivery or functional mobilization of the RAB7L1, LRRK2, or VPS35 molecule.

These methods described herein are by no means all-inclusive, and further methods to suit the specific application is understood by the ordinary skilled artisan. Moreover, the effective amount of the compositions can be further approximated through analogy to compounds known to exert the desired effect.

According to the invention, a pharmaceutically acceptable carrier can comprise any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Any conventional media or agent that is compatible with the active compound can be used. Supplementary active compounds can also be incorporated into the compositions.

A RAB7L1, a LRRK2, or a VPS35 molecule can be administered to the subject one time (e.g., as a single injection or deposition). Alternatively, a RAB7L1, a LRRK2, or a VPS35 molecule can be administered once or twice daily to a subject in need thereof for a period of from about 2 to about 28 days, or from about 7 to about 10 days, or from about 7 to about 15 days. It can also be administered once or twice daily to a subject for a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 times per year, or a combination thereof. Furthermore, a RAB7L1, a LRRK2, or a VPS35 molecule can be co-administrated with another therapeutic.

In one embodiment, a RAB7L1, a LRRK2, or a VPS35 molecule can be co-administrated with a Parkinson's Disease drug. Some non-limiting examples of conventional PD drugs include: levodopa, carbidopa/levodopa (co-careldopa), benserazide/levodopadopamine (co-beneldopa), dopamine agonists (e.g., bromocriptine, pergolide, pramipexole, ropinirole, piribedil, cabergoline, apomorphine, and lisuride), MAO-B inhibitors (e.g. selegiline, and rasagiline), amantadine, and anticholingerics.

A RAB7L1, a LRRK2, or a VPS35 molecule may also be used in combination with surgical or other interventional treatment regimens used for the treatment of PD.

A RAB7L1, a LRRK2, or a VPS35 molecule can be administered to a subject by any means suitable for delivering the protein, nucleic acid or compound to cells of the subject. For example, it can be administered by methods suitable to transfect cells. Transfection methods for eukaryotic cells are well known in the art, and include direct injection of the nucleic acid into the nucleus or pronucleus of a cell; electroporation; liposome transfer or transfer mediated by lipophilic materials; receptor mediated nucleic acid delivery, bioballistic or particle acceleration; calcium phosphate precipitation, and transfection mediated by viral vectors.

The compositions of this invention can be formulated and administered to reduce the symptoms associated with PD by any means that produce contact of the active ingredient with the agent's site of action in the body of a human or non-human subject. They can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic active ingredients or in a combination of therapeutic active ingredients. They can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.

Pharmaceutical compositions for use in accordance with the invention can be formulated in conventional manner using one or more physiologically acceptable carriers or excipients. The therapeutic compositions of the invention can be formulated for a variety of routes of administration, including systemic and topical or localized administration. Techniques and formulations generally can be found in Remmington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa. (20^th ed., 2000), the entire disclosure of which is herein incorporated by reference. For systemic administration, an injection is useful, including intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, the therapeutic compositions of the invention can be formulated in liquid solutions, for example in physiologically compatible buffers, such as PBS, Hank's solution, or Ringer's solution. In addition, the therapeutic compositions can be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included. Pharmaceutical compositions of the present invention are characterized as being at least sterile and pyrogen-free. These pharmaceutical formulations include formulations for human and veterinary use.

Any of the therapeutic applications described herein can be applied to any subject in need of such therapy, including, for example, a mammal such as a dog, a cat, a cow, a horse, a rabbit, a monkey, a pig, a sheep, a goat, or a human.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EM™(BASF, Parsippany. N.J.) or phosphate buffered saline (PBS). The composition must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a pharmaceutically acceptable polyol like glycerol, propylene glycol, liquid polyetheylene glycol, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it can be useful to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the RAB7L1, LRRK2, or VPS35 molecule in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated herein. In the case of sterile powders for the preparation of sterile injectable solutions, examples of useful preparation methods are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.

Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as known in the art

A composition of the invention can be administered to a subject in need thereof. Subjects in need thereof can include but are not limited to, for example, a mammal such as a dog, a cat, a cow, a horse, a rabbit, a monkey, a pig, a sheep, a goat, or a human. A composition of the invention can also be formulated as a sustained and/or timed release formulation. Such sustained and/or timed release formulations can be made by sustained release means or delivery devices that are well known to those of ordinary skill in the art, such as those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 4,710,384; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5.073.543; 5,639,476; 5,354,556; and 5,733,566, the disclosures of which are each incorporated herein by reference. The pharmaceutical compositions of the invention (e.g., that have a therapeutic effect) can be used to provide slow or sustained release of one or more of the active ingredients using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or the like, or a combination thereof to provide the desired release profile in varying proportions. Suitable sustained release formulations known to those of ordinary skill in the art, including those described herein, can be readily selected for use with the pharmaceutical compositions of the invention. Single unit dosage forms suitable for oral administration, such as, but not limited to, tablets, capsules, gel-caps, caplets, or powders, that are adapted for sustained release are encompassed by the invention.

In the methods described herein, a RAB7L1, a LRRK2, or a VPS35 molecule, can be administered to the subject either as RNA, in conjunction with a delivery reagent, or as a nucleic acid (e.g., a recombinant plasmid or viral vector) comprising sequences which express the gene product. Suitable delivery reagents for administration of the a RAB7L1, a LRRK2, or a VPS35 molecule, include the Mirus Transit TKO lipophilic reagent; lipofectin; lipofectamine; cellfectin; or polycations (e.g., polylysine), or liposomes.

The dosage administered can be a therapeutically effective amount of the composition sufficient to result in treatment of PD, and can vary depending upon known factors such as the pharmacodynamic characteristics of the active ingredient and its mode and route of administration; time of administration of active ingredient; age, sex, health and weight of the recipient; nature and extent of symptoms; kind of concurrent treatment, frequency of treatment and the effect desired; and rate of excretion.

In some embodiments, the effective amount of the administered RAB7L1, LRRK2, or VPS35 molecule is at least about 0.01 μg/kg body weight, at least about 0.025 μg/kg body weight, at least about 0.05 μg/kg body weight, at least about 0.075 μg/kg body weight, at least about 0.1 μg/kg body weight, at least about 0.25 μg/kg body weight, at least about 0.5 μg/kg body weight, at least about 0.75 μg/kg body weight, at least about 1 μg/kg body weight, at least about 5 μg/kg body weight, at least about 10 μg/kg body weight, at least about 25 μg/kg body weight, at least about 50 μg/kg body weight, at least about 75 μg/kg body weight, at least about 100 μg/kg body weight, at least about 150 μg/kg body weight, at least about 200 μg/kg body weight, at least about 250 μg/kg body weight, at least about 300 μg/kg body weight, at least about 350 μg/kg body weight, at least about 400 μg/kg body weight, at least about 450 μg/kg body weight, at least about 500 μg/kg body weight, at least about 550 μg/kg body weight, at least about 600 μg/kg body weight, at least about 650 μg/kg body weight, at least about 700 μg/kg body weight, at least about 750 μg/kg body weight, at least about 800 μg/kg body weight, at least about 850 μg/kg body weight, at least about 900 μg/kg body weight, at least about 950 μg/kg body weight, at least about 1000 μg/kg body weight, at least about 1500 μg/kg body weight, at least about 2000 μg/kg body weight, at least about 2500 μg/kg body weight, at least about 3000 μg/kg body weight, at least about 3500 μg/kg body weight, at least about 4000 μg/kg body weight, at least about 4500 μg/kg body weight, at least about 5000 μg/kg body weight, at least about 5500 μg/kg body weight, at least about 6000 μg/kg body weight, at least about 6500 μg/kg body weight, at least about 7000 μg/kg body weight, at least about 7500 μg/kg body weight, at least about 8000 μg/kg body weight, at least about 8500 μg/kg body weight, at least about 9000 μg/kg body weight, at least about 9500 μg/kg body weight, or at least about 10000 μg/kg body weight.

In one embodiment, a RAB7L1, a LRRK2, or a VPS35 molecule is administered at least once daily. In another embodiment, a RAB7L1, a LRRK2, or a VPS35 molecule is administered at least twice daily. In some embodiments, a RAB7L1, a LRRK2, or a VPS35 molecule is administered for at least 1 week, for at least 2 weeks, for at least 3 weeks, for at least 4 weeks, for at least 5 weeks, for at least 6 weeks, for at least 8 weeks, for at least 10 weeks, for at least 12 weeks, for at least 18 weeks, for at least 24 weeks, for at least 36 weeks, for at least 48 weeks, or for at least 60 weeks. In further embodiments, a RAB7L1, a LRRK2, or a VPS35 molecule is administered in combination with a second therapeutic agent.

Toxicity and therapeutic efficacy of therapeutic compositions of the present invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀. Therapeutic agents that exhibit large therapeutic indices are useful. Therapeutic compositions that exhibit some toxic side effects can be used.

Administration of a RAB7L1, a LRRK2, or a VPS35 molecule is not restricted to a single route, but may encompass administration by multiple routes. Multiple administrations may be sequential or concurrent. Other modes of application by multiple routes will be apparent to one of skill in the art.

Methods of Detection

Embodiments of the invention provide for detecting expression of a RAB7L1, a LRRK2, or a VPS35 molecule. In one embodiment, a gene alteration can result in increased or reduced protein expression and/or activity. The alteration can be determined at the level of the DNA, RNA, or polypeptide.

In some embodiments, the detecting comprises detecting in a biological sample whether there is a reduction in an mRNA encoding a RAB7L1, a LRRK2, or a VPS35 protein, or a reduction in a RAB7L1, a LRRK2, or a VPS35 protein, or a combination thereof. In further embodiments, the detecting comprises detecting in a biological sample whether there is a reduction in an mRNA encoding a RAB7L1, a LRRK2, or a VPS35 protein, or a reduction in a RAB7L1, a LRRK2, or a VPS35 protein, or a combination thereof. The presence of such an alteration is indicative of the presence or predisposition to PD.

Methods for detecting and quantifying RAB7L1, LRRK2, or VPS35 molecules in biological samples are known the art. For example, protocols for detecting and measuring the expression of a polypeptide encoded by a RAB7L1, a LRRK2, or a VPS35 molecule, using either polyclonal or monoclonal antibodies specific for the polypeptide are well established. Non-limiting examples include Western blot, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).

In one embodiment, a biological sample comprises, a blood sample, serum, cells (including whole cells, cell fractions, cell extracts, and cultured cells or cell lines), tissues (including tissues obtained by biopsy), body fluids (e.g., urine, sputum, amniotic fluid, synovial fluid), or from media (from cultured cells or cell lines). In one embodiment, the sample is a CSF sample, a blood sample, a plasma sample, a serum sample, or any combination thereof. The methods of detecting or quantifying RAB7L1, LRRK2, or VPS35 molecules include, but are not limited to, amplification-based assays with (signal amplification) hybridization based assays and combination amplification-hybridization assays.

Any suitable biological sample can be used in the instant methods. The biological sample can be taken from body fluid, such as urine, saliva, bone marrow, blood, and derivative blood products (sera, plasma, PBMC, circulating cells, circulating RNA). The biological sample can be taken from a human subject, from an animal, or from a cell culture. The biological sample can be obtained in vivo, in vitro or ex vivo. Non-limiting examples of biological samples include blood, serum, plasma, cerebrospinal fluid, mucus, tissue, cells, and the like, or any combination thereof. In a non-limiting embodiment the biological sample is blood. In a non-limiting embodiment the biological sample is serum. In a non-limiting embodiment the biological sample is plasma. Any suitable method to isolate nucleic acids from biological samples are contemplated for use in the invention. Biological samples for analysis are stored under suitable conditions. In non-limiting examples biological samples are kept at about 4° C. In non-limiting examples biological samples are kept at about −20° C. In non-limiting examples biological samples are kept at about −70-80° C.

A RAB7L1, a LRRK2, or a VPS35 molecule can be determined at the nucleic acid level. Optionally, detection can be determined by performing an oligonucleotide ligation assay, a confirmation based assay, a hybridization assay, a sequencing assay, an allele-specific amplification assay, a microsequencing assay, a melting curve analysis, a denaturing high performance liquid chromatography (DHPLC) assay (for example, see Jones et al, (2000) Hum Genet., 106(6):663-8), or a combination thereof. In one embodiment, the detection or determination comprises nucleic acid sequencing, selective hybridization, selective amplification, gene expression analysis, or a combination thereof. In one embodiment, the detection is performed by sequencing all or part of a RAB7L1, a LRRK2, or a VPS35 molecule, or by selective hybridization or amplification of all or part of the RAB7L1 LRRK2, or VPS35 molecule. A nucleic acid specific amplification can be carried out before the quantification step. In one embodiment, the detecting comprises using a northern blot; real time PCR and primers directed to SEQ ID NO: 1, 2, 3, 4, 5, 9, 10, 12, or 13; a ribonuclease protection assay; a hybridization, amplification, or sequencing technique to detect a RAB7L1, a LRRK2, or a VPS35 molecule; or a combination thereof. In another embodiment, the PCR primers comprise at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 consecutive nucleotides comprising SEQ ID NO: 1, 2, 3, 4, 5, 9, 10, 12, 13, 15, 16, 17, 18, 19, 24, 25, or a combination of the primers.

Hybridization detection methods are based on the formation of specific hybrids between complementary nucleic acid. A detection technique involves the use of a nucleic acid probe specific for the presence of a RAB7L1, a LRRK2, or a VPS35 molecule, followed by the detection of the presence of a hybrid. The probe can be in suspension or immobilized on a substrate or support (for example, as in nucleic acid array or chips technologies). The probe can be labeled to facilitate detection of hybrids. In one embodiment, the probe according to the invention can comprise a nucleic acid directed to SEQ ID NO: 1, 2, 3, 4, 5, 9, 10, 12, or 13. In another embodiment, the probe that detects the presence of a RAB7L1, a LRRK2, or a VPS35 molecule comprises SEQ ID NO: 15, 16, 17, 18, 19, 24, or 25.

A guide to the hybridization of nucleic acids is found in e.g., Sambrook, ed., Molecular Cloning: A Laboratory Manual (3^rd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, 1989; Current Protocols In Molecular Biology, Ausubel, ed. John Wiley & Sons, Inc., New York, 2001; Laboratory Techniques In Biochemistry And Molecular Biology: Hybridization With Nucleic Acid Probes, Part I. Theory and Nucleic Acid Preparation, Tijssen, ed. Elsevier, N. Y., 1993.

Sequencing can be carried out using techniques well known in the art, using automatic sequencers. The sequencing can be performed on a RAB7L1, a LRRK2, or a VPS35 molecule. In another embodiment, the sequencing can be performed using SEQ ID NO: 24, or 25.

Amplification is based on the formation of specific hybrids between complementary nucleic acid sequences that serve to initiate nucleic acid reproduction. Amplification can be performed according to various techniques known in the art, such as by polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA). These techniques can be performed using commercially available reagents and protocols. Useful techniques in the art encompass real-time PCR, allele-specific PCR, or PCR based single-strand conformational polymorphism (SSCP). Amplification usually requires the use of specific nucleic acid primers, to initiate the reaction. In one embodiment, amplification comprises using forward and reverse PCR primers directed to SEQ ID NO: 1, 2, 3, 4, 5, 9, 10, 12, or 13. In certain subjects, the downregulation of a RAB7L1, a LRRK2, or a VPS35 molecule corresponds to a subject with PD. In one embodiment, amplification can comprise using forward and reverse PCR primers comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 consecutive nucleotides comprising SEQ ID NO: 15, 16, 17, 18, 19, 24, or 25.

Non-limiting amplification methods include, e.g., polymerase chain reaction. PCR (PCR Protocols. A Guide To Methods And Applications, ed. Innis, Academic Press, N. Y., 1990 and PCR Strategies, 1995, ed. Innis, Academic Press. Inc., N.Y.); ligase chain reaction (LCR) (Wu (1989) Genomics 4:560; Landegren (1988) Science 241:1077; Barringer (1990) Gene 89:117); transcription amplification (Kwoh (1989) PNAS 86:1173); and, self-sustained sequence replication (Guatelli (1990) PNAS 87:1874); Q Beta replicase amplification (Smith (1997) J. Clin. Microbiol. 35:1477-1491), automated Q-beta replicase amplification assay (Burg (1996) Mol. Cell. Probes 10:257-271) and other RNA polymerase mediated techniques (e.g., NASBA, Cangene, Mississauga, Ontario; see also Berger (1987) Methods Enzymol. 152:307-316; U.S. Pat. Nos. 4,683,195 and 4,683,202; and Sooknanan (1995) Biotechnology 13:563-564). All the references stated above are incorporated by reference in their entireties.

The invention provides for a nucleic acid primer, wherein the primer can be complementary to and hybridize specifically to a portion of a RAB7L1, a LRRK2, or a VPS35 molecule. Primers can be specific for a RAB7L1, a LRRK2, or a VPS35 molecule. By using such primers, the detection of an amplification product indicates the presence of a a RAB7L1, a LRRK2, or a VPS35 molecule. Examples of primers of this invention can be single-stranded nucleic acid molecules of about 8 to about 15 nucleotides in length. Perfect complementarity is useful to ensure high specificity; however, certain mismatch can be tolerated. For example, a nucleic acid primer or a pair of nucleic acid primers as described above can be used in a method for detecting the presence of a genetic variant in a subject. In one embodiment, primers can be used to detect the absence of reduced level of a RAB7L1, a LRRK2, or a VPS35 molecule. In some embodiments, the primers are directed to SEQ ID NO: 1, 2, 3, 4, 5, 9, 10, 12, or 13. In another embodiment, the PCR primers comprise at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 consecutive nucleotides comprising SEQ ID NO: 15, 16, 17, 18, 19, 24, or 25.

Compositions and Kits of the Invention

In one aspect, the invention provides a composition for evaluating the existence of, or predisposition to, PD in a subject, said composition comprising polynucleotides or oligonucleotides, wherein each polynucleotide or oligonucleotide hybridizes to a gene, gene fragment, or gene transcript of at least two different markers in a subject sample, wherein the markers comprise LRRK2, RAB7L1 and VPS35.

In another aspect, the invention provides a composition for evaluating the existence of, or predisposition to, PD in a subject, said composition comprising polynucleotides or oligonucleotides, wherein each polynucleotide or oligonucleotide hybridizes to a gene, gene fragment, or gene transcript of a different marker in a subject sample, each marker being one of the genes listed in Table 2.

In one embodiment, the composition comprises a microarray, a microfluidics card, a chip, or a chamber. In another aspect, the invention provides a diagnostic kit comprising the microarray, microfluidics card, chip, or chamber.

In another aspect, the invention provides a diagnostic kit for determining the levels of RAB7L1, LRRK2, VPS35, or a combination thereof, the kit comprising at least one oligonucleotide or polynucleotide to selectively quantify the levels of RAB7L1, LRRK2, VPS35, or a combination thereof. In one embodiment, the oligonucleotide or polynucleotide comprises SEQ ID NO: 15, 16, 17, or 18. In another embodiment, the oligonucleotide or polynucleotide comprises at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of SEQ ID NO: 15, 16, 17, or 18.

In another aspect, the invention provides for a diagnostic kit for determining whether a sample from a subject exhibits a presence or absence of a PD-associated genetic variant, the kit comprising at least one oligonucleotide or polynucleotide for sequencing nucleic acids isolated from the subject's sample to determine the presence or absence of a PD-risk associated SNP, wherein the presence of a PD-risk associated SNP is further indicative that the subject is at risk of developing PD or is suffering from PD. In one embodiment, the oligonucleotide or polynucleotide comprises SEQ ID NO: 24, or 25. In another embodiment, the oligonucleotide or polynucleotide comprises at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of SEQ ID NO: 24, or 25.

The kits of the invention may also include reagents necessary or useful for the amplification of target nucleic acids, which may include, but is not limited to, DNA polymerase enzymes, primer extension deoxynucleotide triphosphates, and any buffer or other solutions generally used in PCR amplification reactions and kits.

In one embodiment, the kit can further comprise reagents and/or protocols for performing a hybridization, or amplification. In one embodiment, the kit can comprise nucleic acid primers that specifically hybridize to and can prime a polymerase reaction from a RAB7L1, a LRRK2, or a VPS35 molecule comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 consecutive nucleotides comprising SEQ ID NOS: 15, 16, 17, 18, 19, 24, or 25, or a combination of the primers. In one embodiment, primers can be used to detect the absence or reduction of a RAB7L1, a LRRK2, or a VPS35 molecule, such as a primer directed to SEQ ID NOS: 1, 2, 3, 4, 5, 9, 10, 12, or 13. In another embodiment, the PCR primer comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 consecutive nucleotides comprising SEQ ID NOS: 15, 16, 17, 18, 19, 24, or 25. In some embodiments, the kit comprises a probe for detecting a RAB7L1, a LRRK2, or a VPS35 molecule.

The diagnosis methods can be performed in vitro, ex vivo, or in vivo. These methods utilize a sample from the subject in order to assess the status of a RAB7L1, a LRRK2, or a VPS35 molecule. The sample can be any biological sample derived from a subject, which contains nucleic acids or polypeptides. Examples of such samples include, but are not limited to, fluids, tissues, cell samples, organs, and tissue biopsies. Non-limiting examples of samples include blood, liver, plasma, serum, saliva, urine, or seminal fluid. The sample can be collected according to conventional techniques and used directly for diagnosis or stored. The sample can be treated prior to performing the method, in order to render or improve availability of nucleic acids or polypeptides for testing. Treatments include, for instance, lysis (e.g., mechanical, physical, or chemical), centrifugation. The nucleic acids and/or polypeptides can be pre-purified or enriched by conventional techniques, and/or reduced in complexity. Nucleic acids and polypeptides can also be treated with enzymes or other chemical or physical treatments to produce fragments thereof. In one embodiment, the sample is contacted with reagents, such as probes or primers, in order to assess the absence or presence of a RAB7L1, a LRRK2, or a VPS35 molecule. Contacting can be performed in any suitable device, such as a plate, tube, well, or glass. In some embodiments, the contacting is performed on a substrate coated with the reagent, such as a nucleic acid array or a specific ligand array. The substrate can be a solid or semi-solid substrate such as any support comprising glass, plastic, nylon, paper, metal, or polymers. The substrate can be of various forms and sizes, such as a slide, a membrane, a bead, a column, or a gel. The contacting can be made under any condition suitable for a complex to be formed between the reagent and the nucleic acids or polypeptides of the sample.

These methods described herein are by no means all-inclusive, and further methods to suit the specific application will be apparent to the ordinary skilled artisan. Moreover, the effective amount of the compositions can be further approximated through analogy to compounds known to exert the desired effect.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention.

All publications and other references mentioned herein are incorporated by reference in their entirety, as if each individual publication or reference were specifically and individually indicated to be incorporated by reference. Publications and references cited herein are not admitted to be prior art.

Examples

A number of Examples are provided below to facilitate a more complete understanding of the present invention. The following examples illustrate the exemplary modes of making and practicing the present invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.

Example 1

LRRK2 and PARK16 PD Risk Variants Impart a Common Brain Transcriptome Impact

An unbiased and systematic approach was sought to assess the phenotypic impacts of common genetic variants associated with PD risk, particularly in brain tissue from yet unaffected carriers (FIG. 1A), in order to circumvent the limitations of the analysis of diseased patient autopsy tissue. To this end, the transcriptome-wide gene expression profiles of brain tissue samples from cohorts of unaffected individuals who share either a risk or a protective allele at any given PD risk SNP were compared (FIG. 1B). Such a Global Phenotypic Impact (GPI) quantifies the effect of disease risk variants onto the transcriptome-wide gene expression profile in brain. A key aspect of the GPI analysis herein is that tissue from unaffected individuals was tested, in hope of avoiding secondary effects of disease pathology such as cell loss.

The transcriptome-wide GPI at 7 PD-associated loci was assessed (SNCA, LRRK2, MAPT, HLA-DRA, PARK16, LAMP3, STK39, Table 1) (Simon-Sanchez et al., 2009) in a publically available gene expression dataset from cerebral cortex autopsy brain tissue of 185 individuals not apparently affected by a neurodegenerative disease (GSE15222).

TABLE 1
SNPs used for the GPI analysis and linkage with PD associated
SNPs identified by GWAS.
		PD-	Dis-
		associated	tance
Locus	GPI SNPs	SNPs	(bp)	D′	R	Source
LRRK2	rs7306944	rs2708453	42545	0.936	1	PDGene
		rs7304279	29835	0.878	0.937	PDGene
MAPT	rs17563787	rs1981997	243580	1	1	PDGene
		rs393152	94097	1	1	PDGene
PARK16	rs823128	rs1620334	15533	1	1	PDGene
		rs823123	11966	1	1	PDGene
SNCA	rs356168	rs356168	0	1	1	PDGene
STK39	rs10176669	rs10208207	9887	0.824	0.936	PDGene
		rs2102808	32166	0.365	0.869	PDGene
LAMP3	rs9822789	rs11711441	18401	0.948	1	PDGene
HLA-DR	rs2076530	rs3129882	45714	0.484	0.67	T. R.
						Hamza
						et al.

The GPIs of the 7 loci revealed a high degree of overlap in terms of the identity of transcripts altered in expression level and the valence of such alterations: genes were coordinately altered in their expression by each of the 7 PD-associated loci (over 15-fold greater than expected by chance: p=1.5E-5 by resampling statistics; FIGS. 8A-8B. Table 2).

TABLE 2
Individual gene transcripts commonly impacted by the high-risk
allele at 7 PD loci. Presented are the gene transcript-level GPI sub-components: the list of
genes whose expression levels correlate with the PD high-risk allele, and in the same
direction for each of the 7 PD loci studied (“SNCA”, “LRRK2”, “MAPT”, “PARK16”,
“HLA-DRA”, “STK39”, “LAMP3”). Genes are identified by their Illumina probesets
(“Probe”) and their Gene Symbol. Positive values correspond with a relative increase in gene
expression level in the presence of a high-risk allele, negative values with a decrease. The
average correlation across the 7 loci is indicated (“Average”) for each gene.
	Gene
Probe	Symbol	SNCA	LRRK2	MAPT	PARK16	HLA-DRA	STK39	LAMP3	Avg.
GI_16945968-S	LRP15	0.10	0.17	0.09	0.17	0.00	0.16	0.14	0.12
GI_42657118-S	LOC389203	0.04	0.17	0.13	0.09	0.07	0.17	0.11	0.11
GI_30089996-A	BAF53A	0.01	0.12	0.17	0.11	0.08	0.07	0.21	0.11
GI_39777591-S	SLC2A10	0.09	0.25	0.11	0.01	0.10	0.16	0.03	0.11
GI_16554595-A	IER3	0.03	0.14	0.10	0.11	0.20	0.02	0.14	0.11
GI_38788371-S	AQR	0.11	0.13	0.15	0.06	0.04	0.02	0.22	0.10
GI_23238230-A	HMGN3	0.04	0.12	0.18	0.07	0.03	0.10	0.20	0.10
GI_7705400-S	HDCMA18P	0.05	0.17	0.21	0.06	0.08	0.06	0.12	0.10
GI_4503932-S	GATM	0.04	0.15	0.19	0.03	0.02	0.11	0.19	0.10
GI_21450827-S	MGC7036	0.01	0.10	0.13	0.13	0.08	0.16	0.10	0.10
GI_22748758-S	MGC40157	0.04	0.08	0.13	0.06	0.04	0.15	0.20	0.10
GI_13376994-S	ME2	0.05	0.12	0.18	0.05	0.02	0.10	0.18	0.10
GI_18860915-S	XRN2	0.08	0.13	0.14	0.00	0.07	0.08	0.20	0.10
GI_8922630-S	C14orf114	0.07	0.22	0.17	0.03	0.01	0.10	0.10	0.10
GI_34335150-S	RPS15A	0.10	0.06	0.13	0.05	0.08	0.07	0.22	0.10
GI_38502322-S	C9orf10OS	0.04	0.17	0.16	0.05	0.00	0.05	0.22	0.10
GI_42734361-S	DOCK7	0.10	0.10	0.18	0.05	0.01	0.18	0.06	0.10
GI_6006027-S	NRAS	0.05	0.19	0.12	0.04	0.13	0.07	0.08	0.10
GI_4507668-S	TPT1	0.02	0.06	0.08	0.11	0.08	0.09	0.24	0.10
GI_31543202-S	MGC8974	0.03	0.15	0.22	0.07	0.06	0.08	0.06	0.10
GI_41393567-S	HEBP2	0.02	0.25	0.09	0.06	0.05	0.11	0.08	0.09
GI_37551941-S	LOC284347	0.09	0.13	0.08	0.04	0.03	0.14	0.13	0.09
GI_28872862-S	KIAA1194	0.10	0.08	0.12	0.05	0.13	0.04	0.12	0.09
GI_4809273-S	ANXA5	0.03	0.11	0.03	0.10	0.16	0.06	0.15	0.09
GI_23065549-S	GSTM2	0.02	0.07	0.17	0.10	0.07	0.11	0.11	0.09
GI_4507894-S	VIM	0.01	0.17	0.07	0.08	0.04	0.10	0.16	0.09
GI_4557730-S	LTBP1	0.03	0.08	0.10	0.08	0.17	0.08	0.09	0.09
GI_34222292-S	GYS1	0.01	0.10	0.10	0.17	0.14	0.07	0.05	0.09
GI_37540659-S	KIAA1345	0.15	0.12	0.14	0.01	0.08	0.08	0.05	0.09
GI_34485729-S	PRKY	0.02	0.21	0.14	0.07	0.03	0.05	0.09	0.09
GI_5031632-S	FARP1	0.04	0.07	0.16	0.13	0.11	0.04	0.07	0.09
GI_33469973-A	ATF4	0.02	0.10	0.20	0.04	0.04	0.03	0.20	0.09
GI_22095362-S	C14orf135	0.02	0.21	0.23	0.00	0.03	0.03	0.09	0.09
GI_21361081-S	CRLF1	0.10	0.15	0.09	0.09	0.04	0.12	0.03	0.09
GI_4557354-I	BCL2	0.01	0.20	0.10	0.08	0.05	0.09	0.08	0.09
GI_45439305-S	DARS	0.01	0.06	0.10	0.08	0.17	0.11	0.08	0.09
GI_31982935-S	SGPL1	0.00	0.16	0.11	0.08	0.04	0.12	0.09	0.09
GI_8923891-S	PXMP2	0.14	0.09	0.07	0.03	0.00	0.15	0.12	0.09
GI_7661645-S	DKFZP566E144	0.08	0.13	0.13	0.02	0.10	0.03	0.12	0.09
GI_4503182-S	CYB5	0.04	0.06	0.08	0.09	0.01	0.06	0.25	0.09
GI_15431291-S	RPL12	0.01	0.09	0.08	0.04	0.01	0.13	0.24	0.09
GI_40255312-S	P38IP	0.00	0.13	0.13	0.09	0.03	0.07	0.14	0.09
GI_31542585-S	EIF4EBP2	0.04	0.11	0.06	0.04	0.12	0.16	0.05	0.08
GI_31343475-S	GNA13	0.00	0.12	0.14	0.07	0.04	0.10	0.10	0.08
GI_24475891-S	CSPG6	0.01	0.08	0.17	0.04	0.01	0.05	0.24	0.08
GI_42716292-S	EMP2	0.02	0.16	0.05	0.12	0.11	0.02	0.11	0.08
GI_31543652-S	SRP14	0.01	0.17	0.08	0.04	0.04	0.11	0.13	0.08
GI_25453469-S	EEF1A1	0.04	0.08	0.08	0.07	0.02	0.09	0.19	0.08
GI_27436919-I	SPAG9	0.03	0.11	0.13	0.04	0.07	0.06	0.14	0.08
GI_21359839-S	SNRPG	0.01	0.06	0.10	0.08	0.01	0.12	0.19	0.08
GI_15451787-S	PDGFRA	0.00	0.11	0.06	0.13	0.02	0.16	0.10	0.08
GI_19923602-S	CYBRD1	0.01	0.17	0.13	0.03	0.07	0.10	0.05	0.08
GI_19743895-A	TADA3L	0.10	0.04	0.11	0.06	0.01	0.12	0.12	0.08
GI_5730084-S	TCTEL1	0.04	0.10	0.07	0.13	0.04	0.11	0.08	0.08
GI_15193293-S	PGR1	0.13	0.00	0.12	0.07	0.01	0.02	0.22	0.08
GI_21704284-S	JAM2	0.07	0.09	0.10	0.07	0.10	0.03	0.09	0.08
GI_4502370-S	BCAR3	0.06	0.15	0.19	0.00	0.05	0.09	0.01	0.08
GI_37541013-S	LOC374395	0.01	0.00	0.08	0.08	0.08	0.10	0.20	0.08
GI_34304355-A	SCAPIN1	0.02	0.13	0.10	0.13	0.07	0.04	0.07	0.08
GI_21735593-I	PDCD2	0.12	0.06	0.12	0.03	0.01	0.14	0.07	0.08
GI_23510344-I	FYN	0.04	0.11	0.05	0.13	0.05	0.12	0.04	0.08
GI_7661537-S	BRI3	0.12	0.14	0.07	0.06	0.05	0.03	0.08	0.08
GI_6806894-S	PKP4	0.02	0.08	0.15	0.08	0.07	0.01	0.13	0.08
GI_41872597-S	CPNE3	0.00	0.19	0.17	0.08	0.00	0.10	0.01	0.08
GI_41350211-S	BRD7	0.00	0.07	0.11	0.05	0.00	0.08	0.22	0.08
GI_37542859-S	DKFZp313M0720	0.06	0.13	0.07	0.08	0.03	0.08	0.10	0.08
GI_34304116-S	UBC	0.05	0.03	0.07	0.06	0.03	0.05	0.23	0.08
GI_5174588-S	MTF1	0.08	0.02	0.06	0.14	0.10	0.05	0.08	0.08
GI_21361584-S	KIAA0992	0.01	0.17	0.08	0.09	0.04	0.12	0.01	0.08
GI_4503532-S	EIF4B	0.03	0.09	0.10	0.11	0.02	0.01	0.17	0.08
GI_19263339-S	GPT2	0.04	0.13	0.04	0.08	0.07	0.14	0.01	0.07
GI_40255140-S	ChGn	0.20	0.10	0.06	0.01	0.03	0.10	0.02	0.07
GI_41406065-A	H2AV	0.01	0.03	0.10	0.21	0.03	0.09	0.04	0.07
GI_30795205-S	PPP2R5A	0.05	0.07	0.07	0.08	0.08	0.08	0.08	0.07
GI_31542744-S	FLJ23091	0.06	0.14	0.04	0.09	0.02	0.12	0.03	0.07
GI_22538424-S	ATPAF2	0.09	0.08	0.08	0.12	0.03	0.08	0.02	0.07
GI_24308042-S	KIAA0828	0.04	0.08	0.09	0.07	0.08	0.11	0.02	0.07
GI_28316809-S	MGC31967	0.02	0.07	0.08	0.06	0.06	0.02	0.19	0.07
GI_16905527-A	DAP3	0.03	0.10	0.07	0.09	0.01	0.04	0.17	0.07
GI_22035601-A	MAP4K4	0.01	0.07	0.06	0.14	0.08	0.02	0.12	0.07
GI_24497491-S	SLC22A5	0.02	0.09	0.12	0.11	0.09	0.04	0.03	0.07
GI_21536323-A	HNRPUL1	0.02	0.04	0.03	0.05	0.14	0.09	0.13	0.07
GI_40254815-S	HSPCA	0.04	0.09	0.14	0.01	0.00	0.04	0.18	0.07
GI_32481212-S	MK-STYX	0.05	0.03	0.15	0.02	0.05	0.05	0.16	0.07
GI_17402905-S	RPL22	0.04	0.03	0.05	0.02	0.05	0.13	0.17	0.07
GI_28466988-S	ATP10D	0.08	0.15	0.10	0.05	0.05	0.07	0.00	0.07
GI_10346134-S	MAPRE2	0.03	0.04	0.13	0.04	0.06	0.03	0.16	0.07
GI_33946332-I	ZC3HAV1	0.05	0.19	0.02	0.03	0.07	0.10	0.04	0.07
GI_41393560-S	LAP3	0.03	0.07	0.08	0.11	0.03	0.05	0.11	0.07
GI_10863994-S	ZNF410	0.03	0.11	0.10	0.07	0.07	0.05	0.08	0.07
GI_6715608-S	MAPK4	0.07	0.12	0.07	0.08	0.03	0.06	0.05	0.07
GI_4505336-S	NUBP1	0.06	0.09	0.04	0.06	0.10	0.05	0.08	0.07
GI_40353732-S	NPM1	0.01	0.05	0.13	0.01	0.05	0.03	0.19	0.07
GI_5579480-S	ARHN	0.06	0.05	0.09	0.01	0.05	0.00	0.21	0.07
GI_4503606-S	ETFA	0.04	0.06	0.09	0.06	0.01	0.15	0.05	0.07
GI_34147357-S	MGC2747	0.07	0.03	0.01	0.04	0.11	0.11	0.11	0.07
GI_4503174-S	CXCR4	0.05	0.06	0.12	0.08	0.08	0.02	0.06	0.07
GI_37552472-S	LOC286088	0.02	0.04	0.10	0.06	0.03	0.00	0.22	0.07
GI_24432092-S	PHF13	0.01	0.08	0.12	0.02	0.07	0.09	0.08	0.07
GI_38683837-S	CD47	0.01	0.06	0.12	0.11	0.04	0.02	0.11	0.07
GI_31543149-S	MGC11308	0.01	0.05	0.10	0.13	0.06	0.02	0.09	0.07
GI_4501882-S	ACTA2	0.02	0.05	0.01	0.11	0.16	0.06	0.05	0.07
GI_4557394-S	CA2	0.04	0.10	0.10	0.01	0.03	0.04	0.14	0.07
GI_28872718-S	BTG2	0.04	0.08	0.01	0.12	0.22	0.00	0.00	0.07
GI_45359860-S	POLR2L	0.05	0.03	0.03	0.09	0.00	0.14	0.12	0.07
GI_17864091-S	DNAH7	0.06	0.08	0.11	0.07	0.05	0.08	0.01	0.07
GI_20986484-S	YAP1	0.01	0.15	0.06	0.03	0.03	0.14	0.04	0.07
GI_34147334-S	FLJ20811	0.06	0.00	0.06	0.02	0.05	0.07	0.19	0.07
GI_22538460-S	NCOR1	0.05	0.09	0.08	0.00	0.01	0.01	0.22	0.07
GI_15451934-I	CDC14B	0.02	0.13	0.07	0.06	0.07	0.04	0.06	0.07
GI_4557378-S	SERPING1	0.07	0.07	0.00	0.08	0.11	0.06	0.07	0.06
GI_29789284-S	COMMD7	0.07	0.00	0.11	0.10	0.12	0.04	0.01	0.06
GI_4506342-S	PXMP3	0.02	0.14	0.10	0.03	0.01	0.11	0.03	0.06
GI_24308106-S	DKFZp566C0424	0.00	0.12	0.03	0.07	0.11	0.05	0.07	0.06
GI_34328906-I	DNAJB6	0.04	0.07	0.10	0.07	0.06	0.03	0.08	0.06
GI_38373686-S	AP1G1	0.05	0.02	0.12	0.06	0.08	0.07	0.05	0.06
GI_13654234-A	RGS20	0.06	0.11	0.05	0.05	0.02	0.14	0.02	0.06
GI_4502100-S	ANXA1	0.03	0.07	0.02	0.08	0.08	0.01	0.14	0.06
GI_34222274-S	SSA2	0.05	0.11	0.08	0.06	0.11	0.01	0.01	0.06
GI_15011920-S	NOLA3	0.12	0.02	0.06	0.05	0.04	0.06	0.08	0.06
GI_23510452-S	COTL1	0.03	0.06	0.02	0.01	0.10	0.10	0.10	0.06
GI_23111020-A	RGN	0.01	0.10	0.08	0.01	0.01	0.17	0.05	0.06
GI_32483398-S	PAK2	0.07	0.04	0.13	0.12	0.01	0.03	0.04	0.06
GI_10835186-S	SOD2	0.06	0.02	0.03	0.16	0.05	0.06	0.05	0.06
GI_23346408-S	C20orf111	0.06	0.02	0.07	0.06	0.03	0.06	0.12	0.06
GI_5032214-S	UK114	0.01	0.09	0.10	0.04	0.01	0.15	0.03	0.06
GI_39725690-S	SPUF	0.02	0.13	0.05	0.05	0.05	0.02	0.11	0.06
GI_40255040-S	TMP21	0.01	0.04	0.09	0.02	0.01	0.07	0.18	0.06
GI_39725692-S	FLJ10420	0.01	0.07	0.03	0.05	0.07	0.08	0.12	0.06
GI_31542848-S	GMPR	0.11	0.05	0.04	0.08	0.08	0.01	0.05	0.06
GI_24475893-S	GNB2L1	0.02	0.03	0.02	0.05	0.02	0.08	0.19	0.06
GI_31581523-S	COBL	0.02	0.15	0.11	0.06	0.02	0.03	0.03	0.06
GI_18375506-S	APEX2	0.01	0.11	0.06	0.01	0.10	0.11	0.02	0.06
GI_34147490-S	APG3	0.05	0.01	0.11	0.00	0.02	0.04	0.18	0.06
GI_39930611-I	KLHL5	0.01	0.10	0.07	0.08	0.10	0.05	0.01	0.06
GI_42658769-S	LOC401457	0.09	0.08	0.03	0.03	0.05	0.00	0.13	0.06
GI_40254977-S	FIP1L1	0.01	0.06	0.05	0.06	0.10	0.03	0.10	0.06
GI_23510357-A	RIOK1	0.04	0.06	0.14	0.05	0.02	0.02	0.07	0.06
GI_33946290-S	FLJ12443	0.00	0.03	0.03	0.19	0.03	0.09	0.03	0.06
GI_21361092-S	TPST1	0.15	0.05	0.00	0.02	0.01	0.08	0.10	0.06
GI_40254896-S	DKFZp434K1210	0.05	0.10	0.02	0.08	0.05	0.05	0.05	0.06
GI_8922853-S	FLJ11078	0.07	0.10	0.04	0.06	0.04	0.07	0.01	0.06
GI_8051633-S	RARRES3	0.05	0.03	0.02	0.12	0.03	0.08	0.08	0.06
GI_31543826-I	TSC22	0.03	0.09	0.12	0.01	0.03	0.00	0.12	0.06
GI_37694061-I	AQP1	0.04	0.06	0.02	0.11	0.03	0.08	0.06	0.06
GI_19747274-A	PHF10	0.02	0.16	0.03	0.07	0.08	0.02	0.01	0.06
GI_4557342-S	ALDH7A1	0.02	0.06	0.04	0.09	0.01	0.15	0.02	0.06
GI_14043023-S	BAG3	0.01	0.13	0.01	0.07	0.14	0.02	0.02	0.06
GI_18105013-A	3-Apr	0.00	0.04	0.02	0.03	0.07	0.10	0.13	0.06
GI_21618360-S	FXYD5	0.04	0.06	0.02	0.08	0.09	0.02	0.09	0.06
GI_4503100-S	CSRP2	0.03	0.09	0.06	0.05	0.07	0.02	0.08	0.06
GI_23308566-S	ASRGL1	0.05	0.10	0.01	0.11	0.02	0.08	0.02	0.06
GI_42658538-S	DKFZP434A0225	0.03	0.03	0.06	0.13	0.09	0.02	0.03	0.06
GI_40255086-S	LOC118491	0.04	0.05	0.10	0.02	0.07	0.06	0.06	0.06
GI_7661691-S	DKFZP586N0721	0.09	0.13	0.00	0.07	0.02	0.01	0.05	0.06
GI_4757755-S	ANXA2	0.01	0.07	0.04	0.08	0.10	0.01	0.06	0.05
GI_14249551-S	DIRC2	0.06	0.09	0.05	0.05	0.08	0.02	0.04	0.05
GI_20070179-S	EIF4EBP1	0.02	0.07	0.05	0.14	0.04	0.01	0.05	0.05
GI_4503680-S	FCGBP	0.01	0.06	0.05	0.03	0.08	0.03	0.12	0.05
GI_41188450-S	LOC388727	0.01	0.04	0.01	0.15	0.08	0.08	0.01	0.05
GI_42717993-A	DTNA	0.04	0.08	0.06	0.06	0.02	0.05	0.08	0.05
GI_4505488-S	ODC1	0.10	0.10	0.03	0.00	0.04	0.05	0.05	0.05
GI_19923436-S	AK3L1	0.04	0.06	0.01	0.02	0.10	0.13	0.00	0.05
GI_31341159-S	MGC21416	0.15	0.02	0.06	0.00	0.11	0.01	0.00	0.05
GI_8922937-S	FLJ11200	0.00	0.07	0.10	0.05	0.05	0.06	0.03	0.05
GI_34222296-S	FCGRT	0.01	0.08	0.02	0.07	0.07	0.09	0.02	0.05
GI_32307151-S	OXTR	0.11	0.04	0.02	0.04	0.03	0.04	0.07	0.05
GI_34222132-S	TXNDC	0.04	0.07	0.06	0.04	0.04	0.09	0.02	0.05
GI_22035637-A	MGST1	0.01	0.09	0.01	0.06	0.02	0.13	0.03	0.05
GI_34147669-S	SLC39A1	0.02	0.04	0.04	0.04	0.13	0.02	0.04	0.05
GI_21071079-S	FBXL7	0.00	0.09	0.03	0.06	0.06	0.08	0.01	0.05
GI_31543361-S	ORF1-FL49	0.05	0.08	0.02	0.07	0.01	0.10	0.00	0.05
GI_42716312-S	ANG	0.01	0.02	0.05	0.06	0.08	0.09	0.02	0.05
GI_6006016-S	LGALS3BP	0.01	0.05	0.03	0.10	0.06	0.02	0.06	0.05
GI_40549455-S	NHS	0.01	0.13	0.05	0.03	0.04	0.04	0.03	0.05
GI_7019466-S	CNOT4	0.02	0.12	0.09	0.05	0.00	0.03	0.02	0.05
GI_24797094-A	PYCR1	0.01	0.13	0.06	0.07	0.03	0.01	0.02	0.05
GI_7657057-S	EIF2B2	0.02	0.06	0.05	0.02	0.03	0.10	0.05	0.05
GI_20127485-S	M6PRBP1	0.00	0.01	0.00	0.10	0.11	0.04	0.06	0.05
GI_4755145-S	AEBP1	0.01	0.02	0.02	0.12	0.08	0.01	0.05	0.04
GI_33943097-S	RAB5B	0.07	0.00	0.06	0.07	0.09	0.01	0.00	0.04
GI_16445423-S	WDR12	0.04	0.08	0.04	0.02	0.02	0.02	0.08	0.04
GI_42476129-S	RAP80	0.04	0.03	0.07	0.07	0.06	0.03	0.01	0.04
GI_34147349-S	MGC2601	0.03	0.03	0.03	0.03	0.08	0.08	0.02	0.04
GI_29789057-S	KIBRA	0.04	0.06	0.01	0.00	0.04	0.11	0.03	0.04
GI_14670391-A	BAZ1B	0.04	0.00	0.07	0.00	0.12	0.01	0.04	0.04
GI_4557800-S	NP	0.04	0.01	0.02	0.10	0.02	0.06	0.03	0.04
GI_34147469-S	MGC15396	0.02	0.08	0.03	0.03	0.07	0.02	0.05	0.04
GI_13129101-S	MGC955	0.04	0.00	0.02	0.10	0.02	0.06	0.03	0.04
GI_39753966-S	CSPG5	0.00	0.02	0.04	0.03	0.00	0.08	0.11	0.04
GI_33636718-S	TIMM44	0.04	0.03	0.05	0.02	0.07	0.04	0.02	0.04
GI_40354215-S	SIX5	0.00	0.10	0.02	0.07	0.04	0.05	0.00	0.04
GI_18702322-S	DNCL2B	0.02	0.10	0.06	0.02	0.04	0.02	0.01	0.04
GI_20127480-S	EPM2A	0.08	0.02	0.00	0.04	0.06	0.05	0.02	0.04
GI_18104963-A	CAPS	0.00	0.05	0.01	0.05	0.10	0.03	0.02	0.04
GI_4507432-S	TEGT	0.04	0.04	0.01	0.04	0.08	0.06	0.00	0.04
GI_24586683-A	DMN	0.02	0.07	0.06	0.00	0.07	0.02	0.02	0.04
GI_20149303-S	KIAA1160	0.09	0.03	0.03	0.03	0.00	0.02	0.05	0.04
GI_4557586-S	FAH	0.06	0.01	0.03	0.05	0.04	0.07	0.01	0.04
GI_11496992-S	ADPRTL3	0.07	0.06	0.03	0.04	0.04	0.01	0.00	0.04
GI_37550187-S	LOC375468	0.01	0.12	0.01	0.02	0.00	0.01	0.07	0.04
GI_34222312-S	GPSM2	0.01	0.09	0.02	0.04	0.01	0.01	0.05	0.03
GI_34147344-S	KCTD14	0.04	0.08	0.08	0.03	0.00	0.00	0.01	0.03
GI_7706271-S	CGI-30	0.07	0.06	0.02	0.00	0.01	0.07	0.01	0.03
GI_34147623-S	SORD	0.02	0.04	0.09	0.02	0.02	0.04	0.01	0.03
GI_40255159-S	MGC20446	0.04	0.06	0.06	0.01	0.00	0.04	0.01	0.03
GI_19311011-S	BRIX	0.00	0.04	0.04	0.01	0.01	0.02	0.10	0.03
GI_24797087-A	PEX10	0.01	0.02	0.00	0.00	0.02	0.12	0.05	0.03
GI_20336263-A	GGA2	0.06	0.03	0.01	0.02	0.02	0.06	0.02	0.03
GI_21361710-S	HCNGP	0.02	0.00	0.02	0.00	0.02	0.04	0.03	0.02
GI_31543911-S	USP20	−0.02	−0.11	−0.16	−0.12	−0.14	−0.04	−0.16	−0.11
GI_5902039-S	RABL2B	−0.01	−0.04	−0.05	−0.25	−0.15	−0.06	−0.14	−0.10
GI_32454736-A	TRIM3	−0.12	−0.02	−0.13	−0.12	−0.13	−0.02	−0.14	−0.10
GI_18598508-S	CDR2	−0.02	−0.07	−0.09	−0.18	−0.14	−0.10	−0.08	−0.10
GI_7549818-A	RABL2A	−0.04	−0.02	−0.05	−0.23	−0.16	−0.05	−0.13	−0.10
GI_14149994-S	DKFZp434N035	−0.02	−0.13	−0.18	−0.07	−0.11	−0.06	−0.11	−0.10
GI_37577147-A	NCKIPSD	−0.07	−0.06	−0.11	−0.11	−0.11	−0.04	−0.17	−0.10
GI_34147578-S	PISD	−0.06	−0.02	−0.14	−0.14	−0.13	−0.08	−0.09	−0.10
GI_4505328-S	NAPA	−0.09	−0.07	−0.21	−0.04	−0.03	−0.10	−0.11	−0.09
GI_13162281-S	STS	−0.02	−0.15	−0.10	−0.13	−0.04	−0.09	−0.11	−0.09
GI_21450766-S	C6orf136	−0.05	−0.12	−0.10	−0.10	−0.10	−0.07	−0.09	−0.09
GI_11641403-S	CKMT1	−0.05	−0.17	−0.08	−0.08	−0.10	−0.07	−0.06	−0.09
GI_33636749-S	E2-230K	0.00	−0.12	−0.12	−0.08	0.00	−0.16	−0.11	−0.09
GI_37577121-I	UBE2J1	−0.07	−0.07	−0.11	−0.11	−0.07	−0.01	−0.17	−0.09
GI_31377702-S	TTC13	−0.06	−0.13	−0.05	−0.09	−0.12	−0.06	−0.08	−0.09
GI_32528302-S	INSM2	−0.05	−0.07	−0.18	−0.17	−0.02	−0.02	−0.09	−0.08
GI_14149741-S	KIAA1536	−0.08	−0.11	−0.10	−0.06	−0.09	−0.04	−0.10	−0.08
GI_34222158-S	FLJ10925	−0.07	−0.14	−0.09	−0.15	−0.03	0.00	−0.09	−0.08
GI_8923764-S	CACNA2D3	−0.01	−0.17	−0.06	−0.04	−0.05	−0.08	−0.15	−0.08
GI_28372510-S	ZDHHC22	−0.02	−0.14	−0.12	−0.14	−0.11	−0.03	0.00	−0.08
GI_21735551-S	MAP3K12	−0.08	−0.12	−0.12	−0.02	−0.03	−0.01	−0.16	−0.08
GI_21361926-S	C6orf31	−0.06	−0.03	−0.11	−0.05	−0.14	−0.05	−0.11	−0.08
GI_22095352-S	BCAS3	−0.14	−0.11	−0.17	0.00	−0.01	0.00	−0.10	−0.08
GI_24430154-A	PSMC4	−0.06	−0.10	−0.14	−0.08	−0.10	0.00	−0.06	−0.08
GI_24307956-S	PIB5PA	−0.04	−0.03	−0.09	−0.14	−0.04	−0.05	−0.14	−0.08
GI_29540546-A	TRO	−0.10	−0.08	−0.03	−0.10	−0.04	−0.05	−0.13	−0.08
GI_32454751-S	ORC2L	−0.07	−0.11	−0.06	−0.12	−0.04	−0.05	−0.08	−0.08
GI_37588868-S	RNF123	−0.06	−0.01	−0.15	−0.06	−0.03	−0.09	−0.13	−0.08
GI_22538494-S	WBSCR17	−0.07	−0.09	−0.12	−0.01	−0.11	−0.06	−0.07	−0.08
GI_14149656-S	KIAA1049	−0.01	−0.15	−0.13	−0.06	−0.05	−0.06	−0.06	−0.07
GI_23618866-S	SFXN1	−0.10	−0.01	−0.04	−0.08	−0.07	−0.01	−0.22	−0.07
GI_31342415-S	LOC90529	−0.01	−0.06	−0.08	−0.09	−0.09	−0.08	−0.12	−0.07
GI_17017983-S	CDK9	−0.10	−0.10	−0.05	−0.05	−0.04	−0.03	−0.15	−0.07
GI_21536352-S	ACTL6	−0.01	−0.12	−0.13	−0.08	−0.06	−0.01	−0.09	−0.07
GI_38158008-A	CIDEA	−0.01	−0.17	−0.02	−0.05	−0.10	−0.04	−0.13	−0.07
GI_20336241-S	PCSK1	−0.04	−0.09	−0.09	−0.12	−0.07	−0.07	−0.03	−0.07
GI_19557635-A	PPIL3	−0.02	−0.01	−0.11	−0.12	−0.01	−0.11	−0.13	−0.07
GI_13376446-S	C20orf98	−0.02	−0.07	−0.17	−0.02	−0.08	0.00	−0.14	−0.07
GI_14042940-S	eIF2A	−0.01	−0.14	−0.04	−0.14	−0.01	−0.08	−0.09	−0.07
GI_27436965-A	KCNAB1	−0.10	−0.05	−0.09	−0.07	−0.09	−0.03	−0.08	−0.07
GI_4757805-S	C16orf7	−0.06	−0.10	−0.15	−0.05	−0.02	−0.02	−0.10	−0.07
GI_7662423-S	KIAA0972	−0.08	−0.08	−0.02	−0.18	−0.02	−0.07	−0.06	−0.07
GI_34303930-S	MGC20262	−0.10	−0.01	−0.11	−0.09	−0.01	−0.02	−0.16	−0.07
GI_4758897-A	PEX16	−0.07	−0.11	−0.20	−0.04	−0.02	0.00	−0.06	−0.07
GI_19923722-S	RPS6KC1	−0.01	−0.09	−0.10	−0.09	−0.07	−0.05	−0.10	−0.07
GI_40255224-S	FLJ12892	−0.05	−0.01	−0.02	−0.15	−0.10	−0.04	−0.10	−0.07
GI_34222360-S	ATP1A1	−0.02	−0.11	−0.11	−0.02	−0.07	−0.09	−0.05	−0.07
GI_5454157-S	VARS2	−0.04	−0.14	−0.14	−0.04	−0.02	−0.09	−0.02	−0.07
GI_31657108-S	ZNF282	−0.04	−0.13	−0.04	−0.03	−0.02	−0.18	−0.04	−0.07
GI_16933538-A	GLMN	−0.03	−0.07	−0.02	−0.10	−0.11	−0.01	−0.15	−0.07
GI_41327772-S	DDX46	−0.13	−0.07	−0.09	−0.05	−0.05	−0.03	−0.06	−0.07
GI_24431993-S	MGC3234	−0.06	−0.07	−0.06	−0.07	−0.03	−0.05	−0.13	−0.07
GI_19718776-S	FEN1	−0.01	−0.12	−0.07	−0.07	−0.08	−0.07	−0.05	−0.07
GI_38026914-A	ARHGEF11	0.00	−0.04	−0.10	−0.05	−0.08	−0.15	−0.04	−0.07
GI_38570137-S	MGC15677	−0.11	−0.09	−0.11	−0.04	0.00	−0.06	−0.06	−0.07
GI_34222197-S	C10orf22	−0.07	−0.03	−0.01	−0.07	−0.07	−0.09	−0.12	−0.07
GI_12232402-S	FLJ13868	−0.07	−0.02	−0.05	−0.09	−0.10	−0.01	−0.12	−0.07
GI_13899304-S	CD99L2	−0.06	−0.05	−0.13	−0.01	−0.05	−0.07	−0.09	−0.07
GI_31543933-S	VMP	−0.05	−0.07	−0.12	−0.06	−0.06	−0.03	−0.08	−0.07
GI_13376430-S	FLJ13397	−0.02	−0.04	−0.04	−0.09	−0.10	−0.03	−0.13	−0.07
GI_7669496-S	JWA	−0.04	−0.04	−0.07	−0.04	−0.11	−0.02	−0.14	−0.07
GI_34147471-S	MGC20781	−0.01	−0.10	−0.11	−0.12	−0.05	−0.02	−0.06	−0.06
GI_41146823-S	LOC389197	−0.10	−0.06	−0.07	−0.06	−0.08	−0.02	−0.06	−0.06
GI_42734386-S	LOC199692	−0.03	0.00	−0.04	−0.04	−0.12	−0.03	−0.20	−0.06
GI_21362099-S	ELOVL4	−0.03	−0.10	−0.01	−0.08	−0.06	−0.05	−0.13	−0.06
GI_18105052-S	RAE1	−0.05	−0.08	−0.07	−0.02	−0.08	−0.11	−0.03	−0.06
GI_34222118-S	SYT4	−0.10	−0.06	0.00	−0.05	−0.12	−0.04	−0.08	−0.06
GI_34147419-S	ACBD6	0.00	−0.18	−0.08	−0.06	−0.06	−0.02	−0.05	−0.06
GI_19923214-S	MEF2C	−0.02	−0.06	−0.07	−0.07	−0.08	−0.02	−0.11	−0.06
GI_4758181-S	DNM1	−0.08	−0.09	−0.12	−0.01	−0.05	0.00	−0.08	−0.06
GI_22748930-S	FBXL14	−0.05	−0.02	−0.03	−0.07	−0.05	−0.02	−0.20	−0.06
GI_7706644-S	PME-1	−0.04	−0.12	−0.08	−0.02	−0.08	−0.06	−0.03	−0.06
GI_6466453-S	SNCB	−0.10	−0.09	−0.07	−0.01	−0.11	−0.05	−0.02	−0.06
GI_21361484-S	DKFZP434P1750	−0.06	−0.13	−0.12	−0.02	−0.05	−0.04	−0.03	−0.06
GI_7657503-S	RBM9	−0.02	−0.11	−0.03	−0.07	−0.04	−0.07	−0.09	−0.06
GI_19718745-A	OSBPL1A	−0.03	−0.04	−0.10	−0.07	−0.04	−0.08	−0.07	−0.06
GI_18373307-S	RAB40C	0.00	−0.06	−0.11	−0.04	−0.02	−0.10	−0.10	−0.06
GI_33598918-A	SCAMP1	−0.04	−0.02	−0.06	−0.06	−0.02	−0.08	−0.14	−0.06
GI_21361102-S	SLC25A12	0.00	−0.09	−0.02	−0.14	−0.07	−0.03	−0.07	−0.06
GI_8922070-S	LOC55565	−0.04	−0.06	−0.10	0.00	−0.05	0.00	−0.16	−0.06
GI_4507212-S	SRP19	−0.05	−0.06	−0.05	−0.08	−0.03	−0.01	−0.14	−0.06
GI_40068504-S	BSCL2	−0.01	−0.12	−0.08	−0.01	−0.08	−0.04	−0.08	−0.06
GI_13027629-S	DGCR14	−0.09	−0.11	−0.14	−0.04	0.00	−0.01	−0.01	−0.06
GI_9257239-A	SDFR1	−0.05	−0.03	−0.02	−0.03	−0.13	−0.02	−0.14	−0.06
GI_25121973-S	LOC151835	−0.06	0.00	−0.04	−0.17	−0.03	−0.08	−0.02	−0.06
GI_5453861-S	PDE4A	−0.03	−0.09	−0.09	−0.12	−0.03	−0.02	−0.02	−0.06
GI_34222152-S	VSNL1	−0.14	−0.03	−0.02	−0.02	−0.09	−0.02	−0.08	−0.06
GI_32490571-S	EPB41L3	−0.06	−0.10	−0.03	−0.04	−0.03	−0.12	−0.03	−0.06
GI_13325063-S	CELSR2	−0.02	−0.04	−0.06	−0.11	−0.06	−0.09	−0.02	−0.06
GI_27764866-S	SYP	−0.01	−0.11	−0.08	−0.04	−0.11	−0.03	−0.01	−0.06
GI_37622344-A	ZNF42	−0.06	−0.05	−0.05	−0.05	0.00	−0.05	−0.12	−0.06
GI_10092616-S	PCBP3	−0.02	−0.06	−0.06	−0.06	−0.10	−0.01	−0.06	−0.06
GI_31343339-S	FLJ33996	0.00	−0.12	−0.03	−0.07	−0.05	−0.08	−0.04	−0.06
GI_13375816-S	NEIL1	−0.04	−0.01	−0.08	−0.16	−0.01	−0.01	−0.08	−0.05
GI_18550284-S	KIAA1912	−0.03	−0.01	−0.03	−0.05	−0.11	−0.03	−0.13	−0.05
GI_4507104-S	SNAPC3	−0.01	−0.04	−0.05	−0.11	−0.09	−0.05	−0.02	−0.05
GI_20127649-S	KIAA0157	−0.04	−0.02	0.00	−0.06	−0.07	−0.06	−0.12	−0.05
GI_35493938-S	ProSAPiP1	−0.01	−0.05	−0.09	−0.03	−0.01	−0.08	−0.12	−0.05
GI_33667083-S	DNAJC9	−0.01	−0.02	−0.04	−0.10	−0.12	−0.05	−0.05	−0.05
GI_37555997-S	LOC375663	−0.09	−0.01	−0.07	−0.08	−0.03	0.00	−0.09	−0.05
GI_22748944-S	MGC26690	−0.04	−0.07	−0.08	−0.04	0.00	−0.01	−0.13	−0.05
GI_39725643-A	MR-1	−0.01	−0.11	−0.07	−0.06	−0.03	−0.07	−0.03	−0.05
GI_42734431-S	NLK	−0.04	−0.03	−0.05	−0.08	−0.03	−0.09	−0.04	−0.05
GI_4502286-S	ATP2B1	−0.09	−0.02	−0.08	−0.05	−0.01	−0.04	−0.07	−0.05
GI_33391149-S	NPM2	−0.03	−0.06	−0.07	−0.09	−0.06	−0.01	−0.04	−0.05
GI_44955932-I	UBQLN1	−0.06	0.00	0.00	−0.08	−0.06	−0.10	−0.07	−0.05
GI_24308367-S	FLJ38944	−0.01	−0.09	−0.10	−0.05	−0.04	0.00	−0.07	−0.05
GI_37059735-S	CWF19L1	−0.03	−0.07	−0.01	−0.08	0.00	−0.08	−0.08	−0.05
GI_24797146-S	SEPHS2	−0.05	−0.07	−0.05	−0.04	−0.06	−0.04	−0.05	−0.05
GI_42476122-S	RUSC1	−0.01	−0.04	−0.03	−0.04	−0.08	−0.04	−0.10	−0.05
GI_24307960-S	KIAA0406	−0.01	−0.04	−0.05	−0.06	−0.02	−0.02	−0.14	−0.05
GI_40354211-S	PIP3-E	−0.06	−0.08	−0.01	−0.09	−0.03	−0.02	−0.05	−0.05
GI_34147620-A	AMPD2	−0.03	−0.03	−0.09	−0.03	−0.06	−0.02	−0.08	−0.05
GI_21071040-S	CNTNAP2	−0.02	−0.06	−0.04	−0.07	−0.05	−0.05	−0.04	−0.05
GI_27436982-S	KCND2	−0.03	−0.04	−0.04	−0.04	−0.09	−0.02	−0.09	−0.05
GI_29029532-A	SULT4A1	−0.01	−0.13	−0.03	−0.05	−0.06	−0.06	0.00	−0.05
GI_4507830-S	ULK1	0.00	−0.03	−0.10	−0.04	−0.03	0.00	−0.13	−0.05
GI_32698821-S	LOC90637	−0.10	−0.04	−0.02	−0.05	−0.04	−0.04	−0.05	−0.05
GI_11968046-S	PAF53	−0.03	−0.05	−0.04	0.00	−0.08	−0.01	−0.12	−0.05
GI_45359844-S	G3BP2	−0.04	−0.06	−0.03	−0.03	−0.12	−0.01	−0.04	−0.05
GI_34101281-S	SCNN1D	−0.01	−0.06	−0.03	−0.07	−0.03	−0.05	−0.06	−0.05
GI_34147584-S	DMAP1	−0.03	−0.07	−0.06	−0.04	−0.01	−0.03	−0.08	−0.04
GI_37537698-S	LOC147965	−0.03	−0.05	−0.07	−0.05	−0.01	−0.02	−0.06	−0.04
GI_24432025-S	FLJ14360	0.00	−0.02	−0.10	−0.01	−0.01	−0.02	−0.14	−0.04
GI_8922197-S	FLJ10038	0.00	−0.13	−0.03	−0.06	−0.01	−0.02	−0.05	−0.04
GI_10938009-A	TSC2	−0.07	−0.05	−0.07	−0.08	−0.01	−0.01	0.00	−0.04
GI_24308110-S	DKFZp564O1863	−0.05	−0.03	−0.04	−0.07	−0.07	−0.01	−0.02	−0.04
GI_4826693-S	DGCR2	−0.04	−0.06	−0.10	0.00	−0.03	−0.02	−0.02	−0.04
GI_22035549-S	APBA2	−0.05	−0.08	−0.01	−0.02	−0.09	−0.01	−0.01	−0.04
GI_19923443-S	CGI-141	−0.03	0.00	−0.04	−0.09	−0.08	−0.02	−0.01	−0.04
GI_30795206-S	PPP2R5B	−0.05	−0.05	−0.08	−0.05	−0.04	0.00	0.00	−0.04
GI_22027545-S	CACNG3	−0.05	−0.01	−0.03	−0.02	−0.07	−0.04	−0.04	−0.04
GI_23065565-S	GPR24	−0.01	−0.03	−0.05	−0.04	−0.02	−0.09	−0.03	−0.04
GI_45439315-I	PPIE	−0.02	−0.09	−0.03	−0.01	−0.04	−0.05	−0.01	−0.04
GI_40255102-S	ZNF488	−0.05	−0.09	−0.03	−0.01	0.00	−0.05	−0.01	−0.03
GI_4505460-S	ENC1	−0.03	−0.04	0.00	−0.03	−0.07	−0.03	−0.04	−0.03
GI_30795230-S	BASP1	−0.01	−0.02	−0.01	−0.02	−0.06	−0.05	−0.06	−0.03
GI_4758403-S	FRG1	−0.03	−0.03	−0.02	−0.02	−0.04	−0.03	−0.03	−0.03
GI_45439343-I	PPIL5	−0.02	−0.02	−0.04	−0.06	0.00	−0.02	−0.03	−0.03
GI_16445028-S	IGSF8	−0.05	0.00	−0.07	−0.01	0.00	−0.02	−0.04	−0.03
GI_19913415-A	AP2A1	0.00	−0.02	−0.03	−0.02	−0.02	−0.06	−0.04	−0.03
GI_29126235-S	PGSF1	−0.02	−0.01	−0.03	−0.01	0.00	−0.05	−0.01	−0.02

This observation of an overlapping GPI for these 7 PD-associated loci was moreover confirmed in an additional independent dataset of cerebral frontal cortex autopsy brain tissue of 143 individuals (p=1.6 E-3 by resampling statistics: derived from GEO GSE15745).

Function annotation was performed on the gene expression changes that underlie the common GPIs among PD risk variants. Strikingly, among the annotated gene sets most significantly reduced in expression are “mitochondria” functions (FIGS. 8C-8D), consistent with the well-described association of defects in mitochondria with PD pathology (Zheng et al., 2010). Furthermore, the common overlapping transcriptomic signature of gene expression changes associated with these 7 PD risk variants revealed a pattern most similar to the transcriptome changes observed in the context of PD patient brain tissue (relative to unaffected brain tissue; FIG. 8C), rather than to other CNS disorders such as Alzheimer's disease or schizophrenia.

LRRK2 and PARK16 Variants Cooperatively Determine PD Risk

Among the 7 analyzed PD risk locus GPIs, those at the PARK16 and LRRK2 loci were found to be the most similar. Furthermore, variants at these two loci impacted the transcriptome in a non-additive manner, signifying a genetic interaction (as determined by analysis of carriers of both risk variants; FIG. 1D). It was investigated whether these loci similarly genetically interact in terms of their impact on PD risk: namely, whether harboring either a risk (or protective) allele at one of these loci would modify the association of the second locus with PD risk. In an initial study on an Ashkenazi Jewish (AJ) population, the effect of a risk-associated variant at the LRRK2 locus was in fact highly dependent on the presence of the risk variant at the PARK16 locus, and vice versa (FIG. 1E). Such ‘epistasis’ between the LRRK2 and PARK16 loci regarding PD risk was replicated by reanalysis of 3 other independent GWAS, strongly supporting a common mechanism of action (FIG. 1E). Although prior studies have not reported genetic interactions with the common sporadic PD risk-associated variants at the LRRK2 locus, a GWAS of patients who harbor rare familial LRRK2 mutations identified a broad 15 Mb region of Chromosome 1 as harboring a genetic modifier of age of PD onset (Latourelle et al., 2011). It is noted that this region encompassed the PARK16 locus. Meta-analysis in 4 independent sporadic PD GWAS datasets (as above) of the 74 identified SNP variants within this Chromosome 1 region for epistasis with the common LRRK2 SNP variant regarding PD risk identified the PARK16-associated variant as by far the most significantly interacting variant (combined p-value for epistasis: 4.6E-6; FIG. 1F, Table 3).

TABLE 3
LRRK2-PARK16 epistasis meta-analysis.
	NGRC	NINDS
SNP	Chr1 pos.	SNP	Int. OR	p	SNP	Int. OR	p
rs12063329	148269060	rs32063329	0.95	0.6179	rs12063329	0.82	0.4924
rs6684514	154522030	rs6684514	1.04	0.6500	rs6684514	0.97	0.8855
rs10908495	154530564	rs10908495	1.04	0.6220	rs10908495	0.96	0.8442
rs10908496	154530624	rs10908496	1.04	0.6428	rs10908496	0.96	0.8601
rs10908498	154544799	rs10908498	1.04	0.6140	rs10908498	0.96	0.8442
rs10908502	154566706	rs10908502	1.04	0.6528	rs10908502	0.96	0.8442
rs2789425	158216275	rs2789424	1.09	0.2855	rs2789425	0.96	0.8509
rs2737703	158322556	rs2737703	1.02	0.8052	rs2737703	1.69	0.0218
rs2369406	158331042	rs2369406	0.92	0.2705	rs2369406	0.72	0.1424
rs12057296	163609587	rs12057296	1.02	0.7676	rs12057296	1.37	0.1456
rs3767443	165787088	rs3767443	1.14	0.0841	rs3767443	0.84	0.4414
rs10489248	169912212	rs10489248	0.95	0.6108	rs10489248	1.22	0.5113
rs2014613	169914660	rs10489248	0.95	0.6108	rs2014613	1.19	0.5730
rs3753539	169982808	rs10753181	0.93	0.3902	rs3753539	1.10	0.7107
rs10912977	173565736	rs12565878	0.99	0.9015	rs10912977	0.76	0.2614
rs4652143	174250617	rs4652143	1.04	0.7926	rs4652143	1.17	0.7223
rs11587254	174420189	rs11579161	1.11	0.5384	rs4652143	1.17	0.7223
rs2294254	175445331	rs989536	0.99	0.9413	rs2294254	1.08	0.7621
rs946817	176264332	rs946817	1.06	0.4805	rs946817	1.19	0.4904
rs1281323	180368363	rs1281336	1.02	0.7906	rs1281323	0.91	0.6719
rs2254327	180490378	rs6662373	1.00	0.9916	rs2254327	1.57	0.0487
rs10911659	183171884	rs10911659	0.93	0.3234	rs10911659	0.91	0.6539
rs234092	183186797	rs234092	0.92	0.3495	rs234092	1.09	0.7840
rs234095	183197652	rs234096	0.95	0.5392	rs234095	1.03	0.9132
rs6684195	183293436	rs2378957	1.09	0.2687	rs6684195	1.01	0.9704
rs1200610	183418001	rs1200610	0.96	0.6659	rs1200610	1.14	0.6486
rs1208517	183539106	rs1208517	1.00	0.9810	rs1208517	1.06	0.8244
rs10489485	183605173	rs10489486	1.04	0.6589	rs10489486	0.88	0.6173
rs2186024	190696708	rs2186024	1.07	0.3788	rs1286024	0.84	0.3315
rs2494354	192721321	rs4427392	0.96	0.6875	rs7515494	1.25	0.5291
rs2494312	192763213	rs4427392	0.96	0.6875	rs2494312	1.21	0.5771
rs927724	194441413	rs927724	1.08	0.5087	rs2094026	2.04	0.0601
rs599779	197604771	rs576141	0.91	0.1984	rs599779	1.12	0.5986
rs1898240	197613703	rs1898240	0.93	0.4634	rs1898240	0.96	0.8838
rs487359	197648234	rs571754	0.91	0.2021	rs577752	0.95	0.8005
rs1890133	197721501	rs1890133	0.96	0.6363	rs1890133	1.42	0.1824
rs1400875	200088066	rs2820295	0.97	0.6978	rs1400875	1.26	0.3257
rs2820312	200135880	rs2820312	0.97	0.7299	rs2820312	1.23	0.3759
rs2050935	201598170	rs1977812	0.90	0.2352	rs2050935	0.78	0.3073
rs4950978	203291196	rs1470537	1.07	0.4049	rs4950978	0.97	0.8878
rs1470637	203299906	rs3851287	1.08	0.4049	rs1470637	1.13	0.5737
rs823114	203986155	rs823114	0.04	0.0137	rs823114	0.01	0.0019
rs2802221	205643068	rs2651361	1.18	0.0289	rs2802221	0.74	0.1727
rs643930	206224964	rs560311	0.97	0.6541	rs643930	0.93	0.7161
rs10729481	206755035	rs11119079	1.08	0.6451	rs10779481	1.15	0.8361
rs11119078	206758345	rs11119079	1.08	0.6451	rs11119078	1.23	0.7245
rs1933564	207098611	rs1933564	1.06	0.4926	rs1933564	1.21	0.3966
rs11119439	208447880	rs591594	0.97	0.7184	rs11119439	0.96	0.8673
rs590152	208460541	rs591594	0.97	0.7184	rs590152	0.96	0.8673
rs12124008	209710495	rs12124008	1.17	0.0333	rs12124008	0.79	0.2615
rs3104209	211172801	rs3124669	1.18	0.0225	rs104209	1.04	0.8567
rs487208	213209901	rs1452632	0.90	0.1258	rs487208	0.76	0.1663
rs7548730	213911770	rs7548730	1.05	0.4447	rs7548730	1.09	0.7149
rs7547186	215003504	rs7547186	1.05	0.4928	rs7547186	1.02	0.9243
rs10495064	215896812	rs10495064	1.16	0.1339	rs10495064	1.63	0.1077
rs6673733	215982980	rs6673733	0.95	0.4659	rs6673733	0.75	0.1760
rs2377281	216350745	rs2377781	1.07	0.3383	rs2377781	1.25	0.3612
rs9441867	220044136	rs4433403	1.07	0.4005	rs9441867	0.74	0.2178
rs1341331	224702683	rs12118824	0.98	0.7591	rs1341331	1.17	0.4648
rs750426	224715286	rs4653740	1.04	0.6729	rs250426	0.78	0.3220
rs898833	224737622	rs898833	1.03	0.7660	rs898833	0.81	0.4035
rs11122571	228888383	rs11122571	0.92	0.2962	rs11122571	1.24	0.3892
rs7540252	228949072	rs7540252	1.04	0.6123	rs7540252	0.76	0.2078
rs1316408	228952336	rs1316408	1.01	0.3658	rs1316408	1.20	0.4449
rs7542797	229620410	rs7542797	1.19	0.0763	rs7542797	0.93	0.7858
rs487770	232843716	rs621901	0.86	0.0944	rs487770	0.95	0.8613
rs12746334	233775161	rs12746334	1.27	0.0768	rs12746334	1.20	0.6337
rs12135445	237809880	rs12135445	0.99	0.9037	rs12135445	0.89	0.6888
rs879081	238706181	rs879081	1.05	0.5440	rs879081	1.35	0.2019
rs9287247	238713161	rs9287247	0.97	0.6916	rs9287247	0.74	0.1831
rs2066380	238968131	rs2066380	0.86	0.0854	rs2066380	0.82	0.3853
rs9661248	243001296	rs9661248	0.96	0.8497	rs9661248	1.33	0.3027
rs4654274	244735167	4654274	1.02	0.8553	rs4654274	0.98	0.9330
AJ	MAYO	Combined
SNP	Int. OR	p	SNP	Int. OR	p	p
rs12063329	0.94	0.8662	rs2039800	1.02	0.9430	0.5194
rs6684514	0.67	0.1444	rs11264467	0.86	0.5694	0.3901
rs10908495	0.67	0.1444	rs11264467	0.86	0.5694	0.3865
rs10908496	0.67	0.1444	rs11264467	0.86	0.5694	0.3840
rs10908498	0.67	0.1444	rs11264467	0.86	0.5694	0.3896
rs10908502	0.67	0.1444	rs11264467	0.86	0.5694	0.3748
rs2789425	1.10	0.7236	rs2789425	0.78	0.4531	0.8090
rs2737703	1.09	0.7647	rs7512587	0.83	0.5479	0.2630
rs2369406	1.27	0.3857	rs2369406	1.09	0.7563	0.4867
rs12057296	1.00	0.9895	rs6675585	0.75	0.3243	0.7069
rs3767443	1.05	0.8429	rs1229430	1.09	0.7504	0.4612
rs10489248	0.92	0.7340	rs10913508	1.67	0.0889	0.4505
rs2014613	0.91	0.7030	rs10913508	1.67	0.0889	0.4919
rs3753539	0.89	0.6261	rs11808099	1.27	0.5313	0.8613
rs10912977	0.75	0.3376	rs6656777	0.71	0.3228	0.1102
rs4652143	1.03	0.9589	rs2502841	0.81	0.6137	0.9342
rs11587254	1.13	0.8516	rs17351808	1.13	0.7798	0.4763
rs2294254	0.91	0.7554	rs12758344	1.28	0.4058	0.7081
rs946817	0.96	0.9041	rs7519563	0.96	0.8925	0.5668
rs1281323	1.43	0.1514	rs1281338	0.68	0.1825	0.9775
rs2254327	1.20	0.4666	rs2254702	0.77	0.3262	0.3934
rs10911659	0.67	0.1191	rs234122	1.22	0.4752	0.2542
rs234092	0.88	0.6704	rs4650678	0.69	0.3239	0.2998
rs234095	0.74	0.3455	rs234122	1.22	0.4752	0.7135
rs6684195	1.07	0.7890	rs12030554	0.95	0.8735	0.5315
rs1200610	1.33	0.3384	rs7413268	NA	NA	0.5710
rs1208517	1.57	0.1727	rs6424975	1.16	0.5814	0.2907
rs10489486	1.17	0.5726	rs6689206	1.20	0.4825	0.5581
rs2186024	0.91	0.7059	rs12119534	0.87	0.6666	0.6530
rs2494354	1.38	0.6329	rs2494155	NA	NA	0.6841
rs2494312	1.53	0.5164	rs2494315	NA	NA	0.6424
rs927724	1.38	0.3749	rs7518775	0.66	0.3943	0.1977
rs599779	0.88	0.6135	rs556744	1.13	0.7071	0.6567
rs1898240	0.94	0.8383	rs16844836	1.10	0.7876	0.6840
rs487359	0.65	0.0974	rs590448	0.88	0.6975	0.0696
rs1890133	1.45	0.3036	rs7538527	0.95	0.8832	0.3837
rs1400875	0.93	0.7980	rs2644112	1.40	0.2747	0.4743
rs2820312	0.97	0.9146	rs2820312	1.13	0.7156	0.6901
rs2050935	0.83	0.5526	rs12406229	0.71	0.3761	0.0653
rs4950978	0.77	0.3270	rs3862948	0.71	0.3380	0.5629
rs1470637	0.75	0.2661	rs9787334	0.71	0.2640	0.6768
rs423114	0.14	0.0414	rs911154	0.41	0.0779	4.64E−06
rs280221	0.63	0.0947	rs966256	1.53	0.1499	0.7680
rs643930	1.52	0.1195	rs658347	1.07	0.7857	0.6112
rs10779481	0.85	0.7714	rs11119076	NA	NA	0.8277
rs11119078	1.24	0.7355	rs11119079	NA	NA	0.5064
rs1933564	0.83	0.4452	rs12043779	1.05	0.8821	0.6460
rs11119439	1.15	0.5851	rs11119426	1.08	0.8554	0.9201
rs590152	1.15	0.5851	rs845451	1.29	0.3782	0.6529
rs12124008	1.46	0.1492	rs11580728	0.88	0.7044	0.3009
rs3104209	1.77	0.0290	rs3104212	0.69	0.2494	0.0807
rs487208	1.24	0.4067	rs1890007	0.77	0.4210	0.1485
rs7548730	1.64	0.0655	rs7518358	1.09	0.7963	0.1064
rs7547186	1.94	0.0244	rs11572775	1.47	0.2693	0.0387
rs10495064	0.79	0.6309	rs10495065	1.10	0.7549	0.1417
rs6673733	0.62	0.0705	rs17046838	1.41	0.2314	0.1779
rs2377781	1.71	0.0428	rs10863375	1.29	0.3272	0.0148
rs9441857	0.73	0.2379	rs4846353	0.96	0.3905	0.3927
rs1341331	0.60	0.0442	rs16845973	0.76	0.3276	0.1993
rs750426	0.67	0.2191	rs750426	1.01	0.9805	0.3754
rs898833	0.67	0.2344	rs250426	1.01	0.9805	0.3946
rs11122571	1.37	0.2126	rs2296800	0.98	0.9581	0.6134
rs7540252	0.88	0.6022	rs2282319	0.91	0.7398	0.4219
rs1316408	1.30	0.3477	rs12082061	1.03	0.9301	0.3272
rs7542797	0.77	0.4822	rs10864669	0.93	0.7780	0.7962
rs487770	0.64	0.3261	rs607368	1.40	0.3102	0.3642
rs12746334	0.61	0.2846	rs11577962	NA	NA	0.4971
rs12135445	0.84	0.6462	rs16838380	0.85	0.6353	0.4670
rs879081	1.12	0.6495	rs4659570	1.02	0.9501	0.2301
rs9287247	0.69	0.1627	rs882869	0.99	0.9792	0.1152
rs2066380	0.80	0.3605	rs11802581	1.22	0.5513	0.1461
rs9661248	1.10	0.7731	rs4658608	1.49	0.3460	0.3002
rs4654274	1.02	0.9341	rs2184975	0.84	0.5095	0.8109

Taken together, these data strongly support a genetic interaction between LRRK2 and PARK16 that initially impacts human CNS tissue physiology, as reflected by the transcriptome signature in unaffected carriers, and ultimately favors PD pathology in a small subset of individuals at risk.

Evidence of a LRRK2-RAB7L) Pathway

As 5 candidate genes are present within the PARK16 locus (SLC45A3, NUCKS, RAB7L1, SLC41A1, and PM20D1), each of the genes were experimentally screened for a functional interaction with LRRK2 (FIG. 2A). A previously-described primary rat neuron in vitro culture model was used, in which transient expression of familial PD-associated LRRK2 G2019S or R1441C mutant alleles leads to a marked reduction in neurite process length (MacLeod et al., 2006). Overexpression of RAB7L1, but not other genes in the PARK16 locus, significantly suppressed the LRRK2 mutation-induced neurite length phenotype (FIG. 2B). RAB7L1 did not modify neurite length in the context of overexpression of wild-type LRRK2 (FIG. 2A). RAB7L1 is a small cytosolic GTPase, structurally distinct from RAB7 despite its name (also known as RAB29) (Shimizu et al., 1997). One of ˜60 small GTPases in the human genome, RAB7L1 has previously been shown to localize primarily to the Golgi apparatus and implicated in vesicular sorting in the context of Salmonella or Hepatitis C infection (Berger et al., 2009; Spano et al., 2011). But the function of RAB7L1 in CNS neurons remains unknown. Orthologues of RAB7L1 in other organisms, including C. elegans Glo-1 and Drosophila melanogaster Lighloid, have been implicated in trafficking to lysosome-related organelles (Hermann et al., 2005) and in the regulation of neurite process length (Grill et al., 2007), reminiscent of LRRK2 mutant phenotypes (MacLeod et al., 2006). Thus this gene was of particular interest.

Because GTPases such as RAB7L1 are typically only active in the GTP-bound state, mutant forms were generated that are constitutively active (CA; Q67L; this mutation is deficient in GTPase activity) or inactive (IN; T2 IN; a mutation within the presumptive GTP binding site). As expected, overexpression of the CA RAB7L1, but not IN RAB7L1, significantly suppressed the LRRK2 mutation-induced neurite length phenotype. Of other Rab family members, expression of RAB3A or RAB5A failed to rescue the phenotype, whereas RAB7 CA was effective in suppressing the process length shortening induced by LRRK2 mutation (FIG. 2B). In contrast to RAB7L1 overexpression, knockdown of RAB7L1 alone led to a significant reduction in neurite process length, similar to the effect of the LRRK2 G2019S mutant expression (FIG. 2B, 9B).

Next more direct evidence of a physical interaction between LRRK2 and RAB7L1 was sought and thus co-immunoprecipitation studies were performed. Epitope-tagged forms of LRRK2 and RAB7L1 (3×Flag-LRRK2 and GFP-RAB7L1) were co-transfected into HEK293T cells, and after 48 hrs, cell lysates were immunoprecipitated with an anti-Flag antibody and then probed for RAB7L1. Flag-immunoprecipitation of LRRK2 effectively co-precipitated RAB7L1 (FIG. 3A). The interaction did not appear to be altered by the presence of the G2019S mutant, or using a kinase-dead variant K1906M of LRRK2 (MacLeod et al., 2006). Similarly, immunoprecipitation of RAB7L1 with an antibody to the GFP tag co-precipitated LRRK2 only in the presence of RAB7L1-GFP (FIG. 3B). To probe for an interaction between LRRK2 and RAB7L1 in a more physiological context, RAB7L1 protein was examined in brain lysates from transgenic mice that harbor human wild-type LRRK2 or a familial PD mutant form of LRRK2, R1441C, within a large bacterial artificial chromosome (BAC) construct. Transgenic LRRK2 is broadly expressed throughout the CNS of these mice, although at relatively low levels (FIG. 10A). Brain tissue lysates were immunoprecipitated for LRRK2 protein with a rabbit monoclonal antibody. Western blotting of the lysates for RAB7L1 demonstrated co-immunoprecipitation of RAB7L1 (FIG. 3C).

In vitro fluorescence microscopy studies were consistent with the presence of RAB7L1 and LRRK2 in common subcellular compartments. GFP-tagged RAB7L1, transfected into SH-SY5Y cells, localized primarily to the Golgi apparatus (as identified with the Golph4 marker), as well as along tubular structures emerging from Golgi apparatus, consistent with prior reports (Spano et al., 2011). LRRK2 staining appeared more diffuse than RAB7L1 but there was significant overlap (FIG. 3D). In contrast to the wild-type form, the RAB7L1 CA or IN mutant forms appeared more diffusely localized through the cytoplasm, as did a RAB7L1 alternative transcript (AT) deficient in the predicted GTP-binding region (FIG. 3D); accumulation of the IN and AT mutant proteins was significantly reduced (FIGS. 3D and 10B).

In Vivo Analysis of a LRRK2-RAB7L1 Pathway in Drosophila Dopamine Neurons

To pursue potential mechanisms of LRRK2 pathology in vivo, a Drosophila model was established. Although transgenic mouse models expressing mutant LRRK2 have been widely described (Andres-Mateos et al., 2009; Li et al., 2009; Piccoli et al., 2011; Tong et al., 2009), these do not show consistent neurodegenerative phenotypes. Dopamine neuron-selective expression of human familial PD-associated G2019S-mutant LRRK2—using either a tyrosine hydroxylase (TH) (Friggi-Grelin et al., 2003) or dopa decarboxylase (DDC) promoter-Gal4 driver (Fischer et al., 1988)—induced premature mortality of young adult animals (FIG. 4A; nontransgenic mean lifespan 37.1 days+/−1; G2019S mean lifespan 4.8 days+/−0.2), akin to previous reports (Ng et al., 2009). In contrast, transgenic expression of wild-type human LRRK2 did not lead to a discernible phenotype. Furthermore, expression of the mutant G2019S LRRK2 transgene in several other cell types, including motor neurons, eye tissues, or muscles (using a variety of promoter-Gal4 driver constructs), failed to lead to a discernible effect on survival or otherwise.

Subsequently a targeted screen for potential genetic modifiers of the LRRK2 G2019S mutant phenotype was performed, based on the idea that LRRK2 may modify a specific intracellular trafficking process, and focused on RAB7L1. A series of 16 Drosophila Rab genes, (see Table 4; out of 33 identified in Drosophila), or CA or IN forms of these (Zhang et al., 2007), were investigated.

TABLE 4
Rab GTPase genes screen for a rescue of the LRRK2 G2019S
phenotype in Drosophila.
			Average
			adult
	TH-driven	Transgene	lifespan
	Rab GTPase	mutation	(days)	SEM	n
	Rab1CA	Q70	5.9	0.43	23
	Rab2CA	Q65	4.7	0.44	27
	Rab3CA	Q80	7	0.5	22
	Rab4CA	Q67	6	0.36	21
	Rab5CA	Q88	5.9	0.37	20
	Rab6CA	Q71	5.6	0.44	22
	Rab7WT	n/a	10.2	0.59	21
	Rab7L1 DN	T33	5.6	0.53	22
	Rab7L1WT	n/a	23.3	1.09	52
	Rab7L1CA	Q79	24	1.11	45
	Rab8CA	Q67	6.8	0.39	21
	Rab9CA	Q71	5.3	0.4	23
	Rab10WT	n/a	5.6	0.39	22
	Rab14CA	Q94	6.6	0.37	20
	Rab18CA	A64	4.6	0.42	20
	Rab23CA	Q96	6.7	0.49	20
	RabX2CA	D66	4.8	0.4	20
	RabX4CA	Q67	5.9	0.52	22

Briefly, LRRK2 G2019S mutants were mated with a panel of previously described transgenic Drosophila strains that allow for overexpression of wild-type (WT) or constitutively active (CA), forms of the Rab genes (Zhang et al., 2007), using a standard balancer chromosome-based mating scheme. Co-expression of a majority of these Rab transgenes with LRRK2 within dopamine neurons produced no effect on the survival of animals co-expressing LRRK2 G2019S (FIG. 4A; Table 4). In contrast, overexpression of wild-type and CA forms of the Drosophila RAB7L1 orthologue (termed lightoid) afforded a dramatic rescue of the LRRK2 G2019S premature mortality phenotype (mean lifespan 24.0 days+/−1 for the CA; FIG. 4A). Of note, among the other Rabs screened, only Rab7 led to a statistically significant—albeit much weaker—survival benefit (mean lifespan 14.3 days+/−0.6). Rab1, which was previously found to rescue a defect in vesicular trafficking to the Golgi apparatus in alpha-Synuclein overexpression models of PD (Cooper et al., 2006), did not rescue the LRRK2 defect, suggesting distinct mechanisms.

Next, dopamine neuron survival at the dorsomedial posterior protocerebral (PPM1) and dorsolateral posterior protocerebral (PPL1) clusters of Drosophila CNS mushroom bodies was quantified in terms of the loss of expression of a dopamine neuron-specific nuclear localization signal (NLS)-GFP marker protein, using fluorescent confocal microscopy analysis of whole mounted tissue. Expression of LRRK2 G2019S, but not the WT form, led to the preferential loss of neurons in the dorsomedial cluster, reminiscent of the phenotype in other Drosophila models of PD (Feany and Bender. 2000). Co-expression of CA RAB7L1 rescued the LRRK2 G2019S dopamine neuron loss phenotype (FIG. 4B). Deficiency of the RAB7L1 orthologue (in lightoid homozygous mutants) selectively in dopamine neurons by expression of an siRNA construct (Dietzl et al., 2007), led to a significant loss of dopamine neurons (FIG. 4B).

PARK16 Risk Variants Modify RAB7L1 Splicing and Expression

The combination of human brain transcriptomic, human genetic, and model system studies support a role for PARK16, and specifically the PARK16 locus gene RAB7L1, in a pathway with LRRK2. Next possible molecular mechanisms at play at the PARK16 locus that may be responsible for a link between common genetic variants, RAB7L1 function, and PD risk were investigated. A challenge to this is that typically many variants at a given chromosomal location are so closely associated (in ‘linkage dysequilibrium’) so as to make impossible the identification of which is truly ‘causal’ rather than just coincidental. On reanalysis of existing genome-wide splicing data from human lymphoblasts (Montgomery et al., 2010), the PD-associated PARK16 haplotype was found to be associated with alternative splicing of RAB7L1, characterized by the skipping of exons 2 and 3. It is noted that a common SNP variant within the PARK16 locus, rs1572931, that is in linkage dysequilibrium with SNP rs947211 (Hamza et al., 2010) and thus similarly linked to PD risk, falls precisely within regulatory sequences for splicing at the Intron1-exon2 boundary (FIG. 5A). Akin to the lymphoblast transcriptome splicing data, analysis of a set of human cortical brain samples revealed that the rs1572931 genotype is similarly associated with modified splicing of RAB7L in human forebrain (FIGS. 5B, 12A; see Table 6), where the protective PARK16 haplotype is associated with increased exon 2 inclusion in RAB7L1 mRNA. Based strictly on human gene expression data, a causal role for SNP rs1572931 in altered splicing of RAB7L1 cannot be directly assigned (as other SNPs in linkage disequilibrium could be responsible for the observed association). Thus the causal effect of rs1572931 was evaluated using minigene reporter vectors that harbor either the risk-associated or protective allele at rs1572931, but are otherwise identical (FIGS. 5Cii, 12B). Upon transfection into SH-SY5Y human neuroblastoma cells, the rs1572931 risk allele led to increased RAB7L1 exon 2 skipping relative to the protective allele (FIGS. 5D, 12C-E).

TABLE 6
Human Brain Cortical Samples.
	Brain ID	Age	Sex	Status	rs1572931
	B1	80	F	Unaff.	AA
	B2	82	M	LBD	AA
	B3	56	F	ALS	GA
	B4	62	F	ALS	GA
	B5	67	M	ALS	GA
	B6	72	M	Unaff.	GA
	B7	87	M	Unaff.	GA
	B8	57	F	Unaff.	GA
	B9	84	M	Unaff.	GA
	B10	58	M	FTD	GA
				MND
	B11	85	M	FTD	GA
				MND
	B12	87	F	PD	GA
	B13	84	M	PD/D	GA
	B14	80	M	PD/D	GA
	B15	68	F	PSP	GA
	B16	83	M	AD	GG
	B17	89	F	AD	GG
	B18	79	F	AD	GG
	B19	89	F	AD	GG
	B20	89	F	AD	GG
	B21	83	M	AD	GG
	B22	73	M	AD	GG
	B23	86	F	AD	GG
	B24	75	F	AD	GG
	B25	62	F	AD	GG
	B26	89	F	AD	GG
	B27	89	F	AD	GG
	B28	89	M	AD	GG
	B29	88	M	AD	GG
	B30	76	M	AD	GG
	B31	80	M	ALS	GG
	B32	64	M	ALS	GG
	B33	60	M	ALS	GG
	B34	79	F	ALS	GG
	B35	66	M	ALS	GG
	B36	88	F	ALS	GG
	B37	71	F	ALS	GG
	B38	76	F	Unaff.	GG
	B39	57	M	Unaff.	GG
	B40	80	M	Unaff.	GG
	B41	65	F	Unaff.	GG
	B42	62	M	Unaff.	GG
	B43	89	M	Unaff.	GG
	B44	89	M	Unaff.	GG
	B45	57	F	Unaff.	GG
	B46	52	F	Unaff.	GG
	B47	80	M	FTD	GG
	B48	61	M	FTD	GG
	B49	74	M	FTD	GG
	B50	50	M	FTD	GG
				MND
	B51	77	M	FTD	GG
	B52	73	M	FTD	GG
	B53	84	M	LBD	GG
	B54	80	M	LBD	GG
	B55	76	F	PD	GG
	B56	83	M	PD	GG
	B57	74	M	PD	GG
	B58	80	F	PD	GG
	B59	77	F	PD	GG
	B60	80	F	PD	GG
	B61	85	M	PD	GG
	B62	77	M	PD	GG
	B63	77	F	PD/D	GG
	B64	84	F	PD/D	GG
	B65	81	F	PD/D	GG
	B66	69	F	PD/D	GG
	B67	65	M	PD/D	GG
	B68	72	F	PD	GG

Exon skipping is predicted to lead to the formation of a truncated form of RAB7L1 protein that lacks the predicted GTP-binding domain in the amino-terminal region (FIG. 12C). Overexpression of this truncated form leads to low level accumulation of a shortened protein product (FIG. 10B), and reduced localization to the Golgi apparatus (FIG. 3D); although the shortened product can bind with LRRK2 protein (FIG. 3B), expression of this truncation mutant in primary neurons failed to rescue the reduced neurite length phenotype associated with G2019S mutant LRRK2 (FIG. 12F), whereas expression of the wild-typeRAB7L1 effectively rescued the phenotype. Consistent with these in vitro findings, a significant reduction in full-length RAB7L1 protein was observed in cerebral cortex tissue from unaffected individuals who carry the PARK16 risk allele, when compared to non-carrier individuals (FIG. 5E). It is noted that a similar reduction is seen in PD patient cerebral cortex tissue regardless of the PARK16 genotype. This appears specific to PD, as no such decrease is observed in tissue from patients suffering from other neurodegenerative disorders examined (frontotemporal dementia or amyotrophic lateral sclerosis) who do not carry the PARK16 risk allele (FIG. 5E). Taken together, these findings argue in favor of a post-transcriptional (splicing) mechanism of action for the PARK16 PD risk variant's impact on RAB7L1 levels. However, given the linkage disequilibrium structure of the region, additional transcriptional regulatory effects may exist (Gan-Or et al., 2012).

Lysosomal Changes and Retromer-Associated Sorting Defects in LRRK2 and RAB7L1 Mutant Neurons

A cellular role for the LRRK2-RAB7L1 pathway was investigated. Prior studies have broadly implicated both of these gene products in intracellular sorting (Sakaguchi-Nakashima et al., 2007; Spano et al., 2011). Expression of the LRRK2 G2019S clinical mutation in rat primary neurons induced lysosomal swelling, as quantified by immunostaining for the lyosomal marker LAMP2 or using the lysosomotropic dye Lysotracker, consistent with prior work and other studies (Dodson et al., 2012; MacLeod et al., 2006; Stafa et al., 2012) (FIG. 6A). In addition to lysosomal enlargement, there was significant reduction in lysosomal accumulation of the cation-independent mannose 6-phosphate receptor (MPR) in terms of the fraction of LAMP2-positive structures stained with MPR. As MPR is required also for the recruitment of lysosomal hydrolases, its deficiency is predicted to lead to functional lysosomal deficits. Knockdown of RAB7L1 was similarly associated with swollen lysosomes and reduced lysosomal MPR, whereas overexpression of RAB7L1 suppressed the lysosomal phenotypes in the context of LRRK2 G2019S expression (FIG. 6A).

MPR is typically recycled between the endolysosome compartment and the Golgi apparatus by the retromer complex (Arighi et al., 2004; Bonifacino and Hurley, 2008; Seaman, 2009; St. George-Hyslop et al., 2009). Given the primary apparent localization of RAB7L1 to the Golgi apparatus (FIG. 3D), as well as the enrichment of LRRK2 at this organelle (FIG. 3D)(Stafa et al., 2012), without being bound by theory, the lysosomal compartment defects described above may be secondary to altered retromer mediated trafficking machinery between these organelles (Bonifacino and Hurley, 2008; Seaman, 2004). Analysis of Golgi structures by immunostaining with the Golph4 marker in primary neurons transfected with either LRRK2 G2019S or shRNA for RAB7L1 did not reveal evidence of gross structural changes, but MPR co-localization at the Golph4-positive Golgi apparatus structures was significantly reduced (FIG. 6B). Accumulation of MPR at early endosomes, assessed by co-staining with the marker early endosomal antigen-1 (EEA1; FIG. 6C), did not appear altered, whereas accumulation at the cell surface appeared increased. The total areas of Golph4, MPR, or EEA staining were unaffected by G2019S LRRK2 expression or RAB7L1 knockdown (FIGS. 6A-C).

The retromer complex is required for retrograde transport of selective cargo—including MPR—between lysosomes and the Golgi apparatus, through endosomal intermediates, in mammalian cells (FIG. 6D) (Bonifacino and Hurley, 2008; St. George-Hyslop et al., 2009), and defects can lead to lysosomal swelling (Arighi et al., 2004). Furthermore, rare mutations in a retromer component, VPS35, were recently linked to rare familial forms of PD (Vilarino-Guell et al., 2011; Zimprich et al., 2011). Knockdown of VPS35 in primary neuron cultures led to reduced MPR co-localization with the Golgi apparatus and with late endosomes/lysosome markers (FIGS. 6A-B), as previously described (Seaman, 2009). Similarly, expression of a familial PD-associated mutation in VPS35, D620N (Vilarino-Guell et al., 2011; Zimprich et al., 2011), phenocopied the MPR missorting phenotype of G2019S mutant LRRK2 expression or VPS35 knockdown (FIGS. 6A-B), suggesting a dominant negative mode of action which is consistent with a predicted structural alteration of a retromer complex interaction motif (Vilarino-Guell et al., 2011; Zimprich et al., 2011). In contrast, overexpression of wild-type VPS35, which promotes trafficking through the retromer pathway, suppressed the altered MPR localization seen with G2019S mutant LRRK2 expression (FIGS. 6A-B). Thus, although it is likely that the LRRK2-RAB7L1 pathway impacts intracellular sorting processes in addition to retromer complex function, suppression of retromer dysfunction is sufficient to rescue the deficits associated with defects in the LRRK2-RAB7L1 pathway.

The functional relationship of VPS35 with the LRRK2-RAB7L1 pathway was further investigated in the context of neurite process maintenance. In rat primary neurons, overexpression of VPS35 alone did not directly modify neurite process length, but effectively suppressed the loss of neurite processes in the context of LRRK2 G2019S expression or RAB7L1 knockdown (FIG. 7A). In contrast, knockdown of VPS35 with an shRNA vector, or expression of the VPS35 D620N mutant form, led to neurite process length reduction that phenocopied the effect of LRRK2 G2019S expression. In vivo analysis in the Drosophila CNS further supported a role for retromer dysfunction in the context of LRRK2-RAB7L1 pathway defects. Overexpression of Drosophila VPS35 in Drosophila CNS dopamine neurons rescued the LRRK2 G2019S dopamine neuron loss phenotype (FIG. 7B), and similarly extended the lifespan of G2019S LRRK2 mutant-expressing flies. In contrast, knockdown of VPS35 selectively in Drosophila TH-positive dopamine neurons led to significant cell loss and a reduced lifespan (FIG. 7B).

Reduction in Retromer Complex Component Levels in the Context of LRRK2-RAB7L1 Pathway Defects.

Next potential molecular mechanisms for the apparent defects in retromer pathway function in the context of LRRK2 G2019S mutation or RAB7L1 knockdown were investigated. In mouse N2A neuroblastoma cells, expression of LRRK2 G2019S or knockdown of RAB7L1 led to a significant reduction in the levels of accumulated VPS35 as well as VPS29, a second component of the retromer complex (FIG. 7C). Levels of retromer complex components are dependent on the formation of the entire complex, which also includes VPS29, and thus loss of any complex component is predicted to impact levels of others (Kim et al., 2010). Analysis of transgenic mouse total brain tissue overexpressing the R1441C mutant form of LRRK2 also led to a significant reduction in the accumulation of VPS35 and VPS29, and VPS26 (FIG. 7D).

Although the precise mechanism by which the LRRK2-RAB7L1 pathway impacts retromer complex function and levels remains to be determined, co-immunoprecipitation studies of LRRK2 with VPS35 support a direct interaction between these proteins: Lysates from SH-SY5Y cells co-expressing epitope-tagged V5-LRRK2 (or vector) and eGFP-VPS35 forms, were immunprecipitated for the eGFP tag. Subsequent Western blotting revealed co-purification of LRRK2 with eGFP-VPS35 (FIG. 7E). Similarly, immunoprecipitation of LRRK2 from LRRK2 transgenic mouse brain tissue led to the co-precipitation of endogenous VPS35 (FIG. 7F). It is possible the interactions of LRRK2 with VPS3S and RAB7L1 are within a single complex or multiple complexes.

To relate those findings to sporadic PD, VPS35 levels in PD or unaffected human brain tissue were analyzed. Firsta meta-analysis of substantia nigra (SN) mRNA expression levels in 5 publically available microarray gene expression datasets from patients and controls was carried out (Table 5: totally 144 individuals, 63 unaffected individuals and 81 PD patients), and a highly significant decreased in VPS35 mRNA levels (FIG. 7G) was observed.

TABLE 5
GEO datasets used for the meta-analysis of VPS35 mRNA levels
in SN. Foldchanges and p-values for each individual dataset are indicated.
		Re-	Dis-	Fold
Dabaset	Probe	gion	ease	change	n	p-vaue
GSE26927	ILMN_21093	SN	PD	−27%	20	4.90E−03
GSE8397	217727_x_at	SN	PD	−10%	28	1.47E−01
GSE7621	217727_x_at	SN	PD	−14%	25	1.18E−01
GSE20292	217727_x_at	SN	PD	−29%	29	2.80E−04
GSE202923	217727_x_at	SN	PD	−9%	26	3.40E−01
GSE20159	ILMN_1761721	SN	LBD	−29%	33	3.00E−02

Such a decrease was also observed in gene expression data from laser-microdissected PD SN dopamine neurons, when compared to similar cells isolated from unaffected patients (FIG. 7G), as well as in PD cerebral cortex tissue (FIGS. 7H-7I). Taken together, these results support a role for retromer deficiency in the impact of PD-associated genetic risk variants on brain neurons.

Discussion

Using a brain transcriptomic approach as a starting point, evidence is provided that the impacts of several distinct PD risk-associated common genetic variants are overlapping, even in unaffected PD-free carrier tissue. This points to a convergent pathway of action for such variants. Focusing subsequently on LRRK2 and the PARK16 locus gene RAB7L1, in vitro and in vivo studies support a functional interaction: these gene products bound together and functionally interacted in the regulation of neurite process length in vilro, as well as in the context of dopamine neuron survival in vivo. The impact of LRRK2 and PARK16 variants on brain gene expression was observed even in unaffected carriers of the PARK16 or LRRK2 locus risk variants suggesting the existence of a pre-disease prodromal state in such carriers, that favors subsequent progression.

The most prominent neuronal sorting phenotypes observed in the context of PD-associated LRRK2-RAB7L1 pathway changes were at lysosomes and the Golgi apparatus. Without being bound by theory, the proximal site of action for these proteins may be in defective retromer function at the Golgi apparatus, given the enrichment of both proteins at this structure. Trafficking of MPR to the Golgi apparatus—a function of the retromer complex—is defective, and associated with lysosomal swelling. Although the precise mechanism of retromer dysfunction is unclear, retromer pathway components including VPS35 appear reduced in the context of LRRK2 mutation or RAB711 knockdown. Recently described familial PD-associated clinical mutations in VPS35 phenocopy the deficits associated with LRRK2-RAB7L1 pathway dysfunction, whereas overexpression of VPS35 can rescue such deficits. It is also noted that RAB7 was identified in both the in vitro and in vivo screens of RAB proteins as suppressing the phenotype of LRRK2 mutant pathology, albeit less robustly than RAB7L1. RAB7 is the only RAB protein previously implicated in the regulation of retromer function (Rojas et al., 2008).

Prior studies have supported a role for LRRK2 in vesicular trafficking (Biskup et al., 2006; Dodson et al., 2012; Higashi et al., 2009; MacLeod et al., 2006; Stafa et al., 2012). However, cellular mechanisms of LRRK2 relevant in human brain—and in the context of PD or PD risk variants—have remained unclear. The studies herein are unusual in pursuing a PD genetic pathway using both human brain and model system analyses. A genetic interaction between LRRK2 and RAB7L1 was identified in the context of PD risk, and variants at the loci of these genes impact the brain transcriptome in an overlapping manner. Subsequent cell and animal model studies support a model where LRRK2 and RAB7L1 defects may modify intracellular sorting and retromer pathway function.

It is possible that PD-related defects in LRRK2 and RAB7L1 adversely impact aspects of vesicular trafficking unrelated to retromer function. Nonetheless, inducing retromer function appears sufficient to rescue cellular defects and neuronal survival in these models, suggesting a therapeutic venue in PD patients. It is interesting to note that VPS35 deficits, as well as genetic variants at retromer complex receptor loci such as SORLA (Rogaeva et al., 2007), have also been associated with a second major neurodegenerative disorder, Alzheimer's disease (Muhammad et al., 2008); this suggests a broader role for retromer dysfunction in neurodegeneration. Without being bound by theory, different cargos may be involved in the association of the retromer pathway with distinct pathological processes in Alzheimer's and Parkinson's. To this end, it is of interest to investigate the impact of such retromer dysfunction on aSyn and other proteins associated with PD pathology.

EXPERIMENTAL PROCEDURE

Drosophila Methods

Drosophila were cultured by standard methods on yeast-cornmeal-agar medium at 25° C. Wild-type and mutant G2019S LRRK2 transgenes were expressed specifically in catecholaminergic neurons, including dopamine neurons, using the Gal4-UAS system described (Fischer et al., 1988). Driver lines used include OK6 (motor neuron), Gmr (eye), G14 (muscle), TH (dopaminergic neuron), and DDC (dopaminergic neuron). A UAS-GFP::nuclear localization sequence (NLS) marker was used to visualize nucleii of cells in which trangenes were expressed (stock 4775 (w 1118; P{UAS-GFP.nls}14), Drosophila Stock Center, Bloomington, Ind.). For the RAB screen, UAS-LRRK2 (G2019S) transgenic Drosophila, crossed with the TH-Gal4 driver, were screened against a UAS-Rab transgenic library (Zhang et al., 2007). Crossings were typically performed using standard balancer chromosome techniques. To generate strains in which the homozygous LRRK2 transgene and another (Driver or marker) transgene lay on the same chromosome (III), genetic recombination was using standard techniques. Adult Drosophila mushroom bodies were dissected as in (Wu and Luo, 2006) and imaged immediately, without fixation, using a Zeiss LSM510 Meta confocal fluorescent microscope. For mortality curves, transgenic Drosophila surviving through adult metamorphosis were counted daily, from the day of pupal eclosion onward. Locomotion deficits were assessed by methods know in the art.

Primary Neurons Culture

Sprague-Dawley rat or mouse PI primary dissociated cortical cultures were prepared and transfected essentially as described (Xia et al., 1996) with the following modifications: cells were plated at high density, approximately 250,000 cells/cm2, in 24-well plates with 500 ul medium/well. Culture medium used for plating cells was Neurobasal-A supplemented with 2% B-27 and 10% FBS. At 1 day after plating, medium was changed to reduced serum (1% FBS+added antimitotic agents: 70 μM uridine and 25 μM 5-fluorodeoxyuridine) and replaced weekly thereafter; for transfections no DMSO was added to the transfection mixture, cells were not subjected to glycerol shock, and a total of 3 μg plasmid DNA was used per well. Cells were fixed in 4% PFA and immunostained with mouse α-RAB7L1 (Santa Cruz, 1:100), and rabbit monoclonal α-LRRK2 (Michal J. Fox Foundation MJFF4, 1:100), then with appropriate fluorescent secondaries (Jackson, 1:1000-2000). Neurite length and neurite puncta (defined as swellings greater than 2 um in diameter) were counted for for at least 20 neurons per condition. Mean puncta number per neuron was normalized to total average neurite length versus wild-type LRRK2 transfected cells. Fluorescent microscopy was performed using a Nikon TE 2000-S microscope and a Zeiss LSM510 Meta confocal microscope. Images were analyzed using Image-Pro Plus (Mediacybernetics) software version 5.1.0.20.

Colocalization Analysis

Primary rat cortical neurons were cultured on glass coverslips, transfected, and fixed as previously described in this methods section. Cells were immunostained for MRP (Abcam #ab2733, 1:400), Golph4(Abcamab #28049, 1:500), Lamp2 (Sigma L0668, 1:500). Fluorescent microscopy was performed using a Zeiss LSM510 Meta confocal microscope. Images were analysed using NIH ImageJ software version 1.45.

Cell Culture, Transfection and Cytochemistry

HEK293T and SH-SY5Y cells were maintained in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37° C. in a 5% CO₂ atmosphere. Transient expression was performed by transfecting the plasmids using Lipofectamine2000 (Invitrogen) according to the manufacturer's instructions. The transfected SH-SY5Y cells grown on glass coverslips for 24 hours were fixed with 4% paraformaldehyde in PBS for 30 minutes, washed three times with PBS and subjected to the observation of fluorescence. For immunostaining of golgi, fixed cells were blocked and permeabilized with PBS containing 0.1% TritonX-100 and 3% bovine serum albumin followed by incubation with anti-Golph4 polyclonal antibody (abcam) and Alexa Fluor 555 goat anti-rabbit IgG (Invitrogen). Staining of nuclei was performed by using SYTOX Orange nucleic acid stain (Invitrogen). Fluorescence was detected using Zeiss LSM 510 confocal microscope.

Immunohistochemistry and Signal Quantification

LRRK2 R1441C or Wt BAC transgenic mice (Li et al., 2009) (Jackson Laboratory) were sacrificed and perfused immediately with 4% PFA for 20 min. Brains were cut by vibratome into sections 60 μm thick. Sections were blocked in 5% NDS overnight at 4 C, then incubated with primary antibodies overnight at 4 C. Antibodies used were sheep monoclonal α-TH (Pelfreeze. 1:500), mouse a-RAB7L1 (Santa Cruz, 1:100), and rabbit monoclonal α-LRRK2 (Michal J. Fox Foundation MJFF4, 1:100). Sections were incubated at room temperature for 2 hours with appropriate fluorescent secondaries (Jackson Laboratories. 1:1000). Microscopy was performed with a Zeiss LSM510 Meta confocal. Fluorescence signal intensity was quantified using NIH ImageJ.

Human Autopsied Brain Samples

Cortical BA9 area brain samples were obtained from the New York Brain Bank and are detailed in Table 5. Anonymous, de-identified tissue from the brain bank was used.

Quantitative Real Time RT-PCR

RT-qPCR was done as described in (Rhinn et al., 2008) RAB7L1 ratio was quantified using ΔΔCt method using primers pairs: RAB7L1_Ex2_fw

(SEQ ID NO: 15)
(CAGCAAACACTACAAGTCCACG)

and RAB7L1_Ex3_rv

(SEQ ID NO: 16)
(CAGCTGAAGCCGCACTATCTCG);

RAB7L1_Ex4_fw

(SEQ ID NO: 17)
(GACAGCAAGCTCACACTACCCA);

RAB7L1_Ex5_rv

(SEQ ID NO: 18)
(TCTGTCCAACCTGTGAAACCGT)

for human brain samples.

Minigene Splicing Assay

The human SH-SY5Y neuroblastoma cell line (ATCC) was cultured following ATCC's instructions, plated at densities of 4.10e5 cells per well (48-well plates) in wells coated with 0.1% gelatin (Specialty Media, Millipore) 24 hours prior to transfections. Transfections were performed with Lipofectamine 2000 reagent (Invitrogen) following manufacturer's instructions. After transfection with plasmids encoding the reporter contruct, RNA was extracted using miRNeasy kit (Qiagen) and reverse transcribed using Superscript III reverse transcriptase (Invitrogen) following manufacturer's instructions. The eDNA was amplified by PCR using the following primers:

(SEQ ID NO: 19)
GGAGGGCGTCTAGGGAATCGAG

(Fw, complementary to exon1 of RAB7L1) and

(SEQ ID NO: 20)
CTTCAGGGTCAGCTTGCCGTAG

(Rev., complementary to GFP CDS) and Accuprime high-fidelity polymerase (Invitrogen) following manufacturer's instruction with an hybridization at 55 C and an elongation step of 1 min. Pictures from an ethidhium bromide stained agarose gels of the migrated PCR products was analyzed using ImageJ software.

Supplementary Experimental Procedures

Western Blots

Mouse brain protein fractions were prepared as follows. Mouse striata were dissected and homogenized by motorized dounce in Krebs-Ringer buffer with 0.32 M sucrose, then centrifuged at 3000×g for 10 min. Supernatant was collected and centrifuged at 10,000×g for 30 min. Pellet was resuspended in NuPage loading buffer (Invitrogen). Human brain proteins were prepared from frozen blocks using RIPA reagent (Pierce) following manufacturer's instruction. SDS-Page and Western Blot were performed according to manufacturer's protocols with NuPage Bis-Tris Mini Gel and Xcell II Blot Module (Invitrogen). Antibodies used include: LRRK2 (MJFF #1 & #2, 1:200), Rab5 (Abcam ab18211, 1:500), RAB7L1 (clone 2B8, Sigma, 1:400, clone 31-E, Santa Cruz sc-81924, 1:400), anti-Flag M2 (Sigma, 1:1000), anti-GFP (Covance, 1:1000), anti-alpha-tubulin (DM1A, 1:2000), SNAP25 (Abcam ab41455, 1:500), VAMP2 (Abcam ab3347, 1:500), beta-actin (clone C4) (Abcam ab3280, 1:800) and appropriate HRP-conjugated secondaries (Jackson, 1:2000). Blots were visualized using Supersignal luminol substrate (ThermoScientific #34075).

Immunoprecipitation

For cultured cells, HEK293T cells transfected for 48 hours were lysed with lysis buffer containing 0.5% Triton-X. 1 mM EDTA and protease inhibitor cocktail (Sigma). The lysates were rotated at 4° C. for 1 hr followed by centrifugation at 20,000 g for 5 min. The supernatant was added to 30 ul (slurry volume) of Dynabeads protein G (Invitrogen) preincubated without (preclear) and with an anti-flag M2 monoclonal antibody (Sigma) and the mixture was rotated for 30 min at 4° C. The beads were washed three times with ice-cold PBS and subjected to immunoblotting.

For mouse tissue, whole brains were dissected and homogenized by motorized dounce at 0° C. in Invitrosol detergent (Invitrogen) and Ix Protease inhibitor cocktail (Pierce) according to manufacturer's protocol Lysate was incubated shaking for 30 min at 4° C. with gel beads (Pierce Co-IP kit #26149) covalently conjugated to LRRK2 antibody (either MJFF #2 or #4) or a control IgG antibody. Beads were washed 4×10 min each, and then eluted. Eluant was analyzed by Western Blot, probed for LRRK2 (MIFF #1, 1:200), Rab7L1 (Santa Cruz sc-81924, 1:400), Rab11 (Abcam ab3612, 1:400), and beta-actin (clone C4) (Abcamab3280, 1:600).

DNA Constructs

The plasmid encoding rat RAB7L1 cDNA sequence was purchased from Open Biosystems, and the sequence was digested and ligated into BgIII-EcoRI site of pEGFP-C1 expression vector (Clontech) to generate N-terminally GFP-tagged RAB7L1. As the purchased RAB7L1 sequence contained 286 bp insertion in the middle of cDNA resulting in the generation of stop codon, this insertion was removed by a long-PCR protocol. The plasmids encoding constitutive active (Q67L) and dominant negative (T2 IN) rat RAB7L1 were generated by using site-directed PCR-mutagenesis kit (Stratagene) from the plasmid encoding N-terminally GFP-tagged wild-type RAB7L. All sequences were verified by DNA sequencing. Plasmids encoding wild-type and mutant Rab7 constructs were from Addgene; Rab3 and Rab5 constructs were also used. Plasmids encoding full-length human LRRK2 (wild-type, G2019S, K1906M) tagged with 3×FLAG at the N-terminus were used. Splice reporter minigene bearing plasmid was created by insertion of a synthesized sequence corresponding to the first exon, the first intron and the second exon and 200 bp of the second intron of human RAB7L1 gene in a pEGFP-N1 vector (Clontech) between its Xhol and HindIII restriction sites. Rab7L1 shRNA plasmid came from Sigma (MISSION shRNA clone NM_144875). LRRK2 plasmids used were those published (MacLeod et al., 2006), and confirmed.

polyA-RNAseq

Library generation and sequencing: First-strand cDNA was synthesized from 1 μg of RNA per biological sample using SuperScript III (Invitrogen) following manufacturer's instructions and using the pdT-FS oligonucleotide to prime the reverse transcription. Barcoded first-strand samples from different samples were then pooled and treated with RNase H (Invitrogen) at 37° C. for 20 minutes followed by 15 minutes at 75° C. to degrade RNA template. First-stand cDNA was then purified using QIAquick PCR Purification kit (Qiagen) in a total volume of 30 uL. Second-strand cDNA was synthesized from 25 uL of first-strand cDNA template by adding 10 μl 10× buffer 2 (NEB), 5 μl 10 mM dNTPs, 20 U Klenow Fragment (3′→5′exo-; NEB), 10 μl of 100 μM tagged 2nd strand primer (R-SS oligonucleotide:

5′-TCCGATCTGANNNNNNN-3′

with N=A,C,T,G mix (SEQ ID NO: 21)) and 46 μl water. The reaction mix was incubated at 37° C. for 30 minutes, followed by 10 minutes at 75° C. then cooled down at 4° C. Double-stranded cDNA was purified using PureLink PCR micro columns (Invitrogen) in a 30 uL volume. Illumina-compatible libraries were then generated by PCR from 25 uL of double-stranded cDNA template using Accuprime Pfx polymerase (Invitrogen) following manufacturer's instruction with NNSR forward

(SEQ ID NO: 22)
(5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACG
CTCTTCCGATCTC-3′)

and NNSR reverse

(SEQ ID NO: 23)
(5′-CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAA
CCGCTCTTCCGATCTGA-3′)

primers. Thermo-cycling conditions were 2 min at 94° C. followed by 2 cycles of 94° C. for 10 s, 40° C. for 2 min, 68° C. for 1 min; 8 cycles of 94° C. for 10 s, 60° C. for 30 s, 68° C. for 1 min; 15 cycles of 94° C. for 15 s, 60° C. for 30 s, 68° C. for 1 min with an additional 10 s added at each cycle; and 68° C. for 5 min before cooling to 4° C. Amplified libraries were purified using PureLink PCR micro columns (Invitrogen) and directly used to generate clusters for sequencing-by-synthesis using the Illumina HiSeq 2000 platform. 100 bp single-end reads were obtained by sequencing to generate more than 300 million reads for the 34 samples.

Data was analyzed using Galaxy (Goecks et al., 2010): Illumina reads were converted to FASTQ Sanger format using FASTQ Groomer, the first 27 bp at their 5′ends of the reads were trimmed using FASTQ Trimmer to remove the polyA and adapters sequences and mapped to human hg19 genome using Burrows-Wheeler Alignment tools (Langmead et al., 2009) with Galaxy's default settings allowing 4% of missing alignments. All those tools are included in the Galaxy NGS toolset.

Human Sample Genotyping

DNA was extracted from brain samples using DNeasy kit (Qiagen) and amplified by PCR using primers RAB7L1_Genot_fw

(SEQ ID NO: 24)
(GGTGAGCCTCCGCACTCG)

and RAB7L1_Genot_rv

(SEQ ID NO: 25)
(TTCCCACCCACCGCCTGT)

and Accuprime polymerase (Invitrogen) following manufacturer's instruction with an hybridization at 55° C. and an elongation step of 1 min. PCR products were purified using PureLink PCR columns (Invitrogen) submitted to Sanger sequencing (GeneWiz, NJ) using RAB7L1_Genot_fw primer and analyzed using SeqScanner (Applied Biosystems).

GWAS Epistasis Analysis

The AJ GWAS dataset included a total of 278 cases and 178 controls that were genotyped using the Illumina Human 610-quad bead arrays (Cases n=91 and controls n-96) or the Illumina Human 660-quad bead arrays (Cases n=191 and controls n=84). Details of the genotyping and quality control assessments are provided in Liu et al (2011). Subjects were participants in two studies the Genetic Epidemiology of Parkinson Disease and the AJ study. Ascertainment and a description of the study participants is provided in detail in Marder et al (Marder et al., 2003) and Liu et (Liu et al., 2011). All subjects in the GWAS AJ dataset were genotyped for the LRRK2 G2019S mutation and for GBA mutations common in the AJ population (N370S, L444P, 84insGG, IVS2+1 G>A, V394L, R496H, D409H, A456P and V460V). For the epistasis analysis LRRK2 and GBA mutation carriers were removed from the dataset. The final dataset is comprised of 239 PD cases and 178 controls.

In addition to the AJ dataset, 3 additional publicly available GWAS datasets were studied and downloaded from NCBI's dbGap repository (Mailman et al., 2007): NGRC (CIDR/NGRC Genes and Environment, dbGap phs000196.v2.p1, (Hamza et al., 2010)), that comprises 2013 cases and 1995 controls, NINDS (NINDS-Genome-Wide Genotyping in Parkinson's Disease, dbGap phs000089.v1.p1, (Fung et al., 2006)) that comprises 267 cases and 270 controls and Mayo (Mayo-Perlegen LEAPS (Linked Efforts to Accelerate Parkinson's Solutions) Collaboration, dbGap phs000048.v1.p1, (Maraganore et al., 2005)) that comprises 443 cases and 443 controls. All subsequent genetic analysis were done using gplink (Purcell et al., 2007) software. Epistasis between PARK16 (rs823114, as identified to modify PD age of onset in LRRK2 mutations carriers (Latourelle et al., 2011)) and LRRK2 (rs11176052 as identified by PD GWAS meta-analysis (Lill et al., 2012)) in each dataset was evaluated using epi function. As those SNPs were not present in the lower density Perlgen array used in the Mayo datasets, their best proxies were determined using SNAP in the 1000 genomes CEU population (see below); rs823154 and rs11174928 were used for the analysis in this dataset for PARK16 and LRRK2 respectively. Odd-ratios and their confidence intervals were evaluated using asso function. Meta-analysis and combination of p-values were done using Metal (http://www.sph.umich.edu/csg/abecasis/metal/).

SNP Selection

SNP-SNP pairwise linkage disequilibrium was assessed by SNAP phase (Johnson et al., 2008) using the CEU population panel from the 1000 genomes and HapMap dataset. PD associated SNPs were evaluated based on PDGene meta-analysis results (Lill et al., 2012).

GPI Analysis

Genome-wide SNP variant and gene expression data for 364 individuals were previously described (Myers et al., 2007). Normalized data corrected for covariates such as age, sex and batch effects were processed using R for gene expression analysis and gplink (Purcell et al., 2007) for genotypes. Subsequently for a given SNP, Pearson's correlation coefficient is calculated between the expression level of each gene (within the whole transcriptome dataset) and the allele load across the panel of samples. Associations were arbitrarily described with the high-risk variant at any given disease-associated SNP with positive values, and with the protective low-risk variant with negative values. As a consequence, a gene whose expression is consistently higher in samples from individuals who carry the disease-associated high-risk variant (relatively to the expression in the context of the protective low-risk variant) across the entire sample set will show a positive correlation coefficient (such as Gene 1 in FIG. 12B). By assessing the correlation coefficients across the entire transcriptome for a given variant, the GPI can be obtained.

Formally, the GPI for a SNP is a n-vector of numerical values between −1 and 1, where n is a number of genes whose expression levels is available, and corresponds to the collection of the expression level correlation with the allelic load for each individual gene. The GPI of SNPx was thus calculated as

$\mathrm{GPISNPx}=\left[\begin{array}{c}r\left(A_{G_i},L_{{\mathrm{SNP}}_x}\right)\\ \dots \\ \dots \\ \begin{array}{c}\dots \\ r\left(A_{G_n},L_{{\mathrm{SNP}}_x}\right)\end{array}\end{array}\right]$

with

$A_{G_i}=\left[\begin{array}{ccc}{\left(a_{G_i}\right)}_1& \cdots & {\left(a_{G_i}\right)}_p\end{array}\right],$

expression level of gene i across all samples, (aGi)_j being the expression level of gene i in sample j

$L_{{\mathrm{SNP}}_x}=\left[\begin{array}{ccc}{\left(l_{{\mathrm{SNP}}_x}\right)}_1& \cdots & {\left(l_{{\mathrm{SNP}}_x}\right)}_p\end{array}\right],$

high-risk allele load of SNP x across all samples (I_SNPx) being the high-risk allele load of SNP x in sample j.

${\left(l_{{\mathrm{SNP}}_x}\right)}_1=\{\begin{array}{cc}0& \mathrm{if}j\mathrm{is}\mathrm{homozygous}\mathrm{for}\mathrm{the}\mathrm{low}\mathrm{risk}\mathrm{allele}\mathrm{of}\mathrm{SNPx}\\ 1& \mathrm{if}j\mathrm{is}\mathrm{heterozyguous}\mathrm{for}\mathrm{SNPx}\\ 2& \mathrm{if}j\mathrm{is}\mathrm{homozygous}\mathrm{for}\mathrm{the}\mathrm{high}\mathrm{risk}\mathrm{allele}\mathrm{of}\mathrm{SNPx}\end{array}$

(See FIG. 12A). (AG1, LSNPx) is the Pearson correlation coefficient between the expression level of gene 1 and the disease-associated allelic load of SNPx across all samples. (A, LSNPx) is positive for genes whose expression levels are increased in the presence the risk allele and negative for genes whose expression levels are decreased (Genes 1 and 3 respectively in FIG. 12B).

The intersection between the GPIs calculated for two SNPs is evaluated in a threshold-free approach by considering all the genes which show a correlation in the same direction for different GPIs. Formally, with GPI_{SNPxX SNPy}=GPI_SNPx∩GPI_SNPy:

${\mathrm{GPI}}_{{\mathrm{SNP}}_x\times {\mathrm{SNP}}_y}\left[k\right]=\{\begin{array}{cc}\frac{{\mathrm{GPI}}_{{\mathrm{SNP}}_x}\left[k\right]+{\mathrm{GPI}}_{{\mathrm{SNP}}_y}\left[k\right]}{2}& \mathrm{if}{\mathrm{GPI}}_{{\mathrm{SNP}}_x}\left[k\right],{\mathrm{GPI}}_{{\mathrm{SNP}}_y}\left[k\right]>0\\ 0& \mathrm{if}{\mathrm{GPI}}_{{\mathrm{SNP}}_x}\left[k\right],{\mathrm{GPI}}_{{\mathrm{SNP}}_y}\left[k\right]<0\end{array}$

In order to study potential interactions between two SNPs, a quantitative trait was defined for which classical genetic interaction analysis could be applied.

A genetic interaction is broadly defined as when the combined phenotypic effect of two mutations (in distinct genes) is not equal to the sum of the two individual phenotypic effects. Thus, such a non-additive interaction can either represent synergy (the combined effect is greater than the sum of its parts) or occlusion (the combined effect is less than the sum of its parts). The prediction for an occlusive genetic interaction is that the transcriptome effect of a risk allele at either one of the 2 genes will preclude the effect of a second risk allele.

A quantitative trait phenotype was defined for the classical genetic interaction analysis. This is most simply done by examining gene expression values that are highly impacted in common by the 2 SNPs individually (as identified above by the GPI intersection genes), and then querying the effect of their combination. Without being bound by theory, any of the gene expression values from the GPI intersection could be queried for a genetic interaction. Rather than querying individual genes expression phenotypes, a single scalar value was generated that represents the combined effect on the expression patterns of all of the relevant genes (as defined by the GPI analysis above; we used the genes most significantly impacted with p<0.01, empirically assessed by resampling). To compute this scalar value, termed the expression quantitative trait (eQT), a standard linear algebra manipulation was used: the combined quantitative trait is the sum of the expression levels of the selected genes, weighted by their GPI intersection coefficients (which reflects how consistently and strongly they are affected by both SNPs).

Formally, the complex expression phenotype for sample j will be:

${\left({\mathrm{CEP}}_{\mathrm{xy}}\right)}_j={\sum }_{m=1}^M{\mathrm{GPI}}_{{\mathrm{SNP}}_x}\times {\mathrm{SNP}}_y\left[t_m\right]·{\left(a_{G_{t_m}}\right)}_j$

with a selection of M genes from GPI_{sNPxX SNPy} and tm the position in the GPIs vector of the m^th highest absolute value in GPI_{sNPxX SNPy}.

To actually perform the interaction analysis, we first determine the genotypes for the two SNPs of interest and then proceeded to linear model regression for the quantitative trait across all samples. Computationally, the effect of both SNPs on the quantitative phenotype was assessed using R lm function, by fitting of the linear model CEP_xy˜x0+x1·L_SNPx+x2·L_SNPy+x3·L_SNPxL_SNPy. The test for interaction is based on the significance associated with the coefficient x₃.

Disease Association Expression Profiles

For a given disease dataset, Pearson's correlation coefficient is calculated between the expression level of each gene (within the whole transcriptome dataset) and the disease phenotype across the panel of samples. Associations with the disease were arbitrarily described as positive values, consistently with the orientation assigned in the GPI calculation to the high-risk allele load of a disease-associated SNP. As a consequence, a gene whose expression is consistently higher in disease samples than in unaffected will show a positive correlation coefficient. By assessing the correlation coefficients across the entire transcriptome for a given variant, a global disease profile (GDP) can be obtained.

Formally, the GDP produces an object of the same class as the GPI, a n-vector of numerical values between −1 and 1, where n is a number of genes whose expression levels is available, and corresponds to the collection of the expression level correlation with the disease phenotype (0 for unaffected, 1 for disease) across the samples. Formally,

$GDPD=\hspace{1em}\left[\begin{array}{c}r\left(A_{G_1},P_D\right)\\ \dots \\ \dots \\ \begin{array}{c}\dots \\ r\left(A_{G_n},P_D\right)\end{array}\end{array}\right]$

with PD=[(P_D)1 . . . (P_D)p], binary phenotype associated to disease D across all samples, (PD)j being the disease-associated binary phenotype in sample j.

${\left(l_{\mathrm{SNPx}}\right)}_j=\hspace{1em}\{\begin{array}{cc}0& \mathrm{if}j\mathrm{is}\mathrm{unaffected}\\ 1& \mathrm{if}j\mathrm{is}\mathrm{affected}\end{array}$

and r(A_G1, P_D) the Pearson correlation coefficient between the expression level of gene i and the disease-phenotype across all samples. As a consequence, a gene whose expression level is increase in the course of the disease will be associated to a positive correlation in the disease expression profile. When multiple Affymetrix probesets were available for a given gene, the probeset showing the highest expression level was systematically selected. For resampling procedures, the values of the phenotype vector are randomly reattributed to the different samples (label switch).

Datasets of normalized gene expression (accession numbers GSE20168, GSE7621, GSE1297, GSE3790, GSE12654) were downloaded from the Gene Expression Omnibus website (http://www.ncbi.nlm.nih.govigeo/).

Similarity between a disease expression profile and a GPI intersection is evaluated by the Pearson correlation between the GPI intersection and the GDP across the subset of genes that show the highest absolute value in the GPI intersection. (In the case of the 7 PD SNPs intersection, the top 135 genes out of the 352 non-null were selected as a core set, with a FDR<5% based on resampling analysis of a 7 SNPs GPI intersection size). All data manipulations and analysis were done using R.

SNP within Probes

Common SNPs within the target sequences of microarray probes have indeed emerged as a potential technical issue for eQTL analysis in recent years (Alberts et al., 2007; Chen et al., 2009), due to the mis-hybridization caused by the allelic variant of such a SNP that does not match the designed target sequence; as a consequence of the poorer hybridization of the probe to its target sequence, the amount of target sequence might be under-evaluated. Classical eQTL studies aim at identifying relationships between the allelic load of a given variant (herein called “studied variant”) and the level of expression of a given gene. In the case of such eQTL studies, results can be biased if the allele leading to a poorer hybridization segregates with one allele of the studied SNP. This most often happens for cis-eQTL, as the local physical structure of the chromosome can lead to a systemic segregation between the studied variant and the one within the probe if those are in linkage disequilibrium. The GPI analysis can be seen as a globalization at a transcriptome-wide scale of eQTL studies, where the effect of a studied variant is considered on transcriptome-wide gene expression levels in a single measurement. As all the genes are considered with the same weight, there are two direct implications to that 1) the global measurement should be robust to potential technical issues (such as SNP-in-Probe) affecting a single probe, as this will only affect a fraction of a percent of the total GPI signal 2) the GPI is mostly based on trans-effect measurements, as more than 99% of the genes will be considered as “trans” by reference to any studied variant.

To empirically support the robustness of the GPI to SNPs in probes, all the probes sequences used in the study were obtained from Illumina, and were mapped on the human genome using Burrows-Wheeler Alignment (Langmead et al., 2009) to identify those that target sequences containing a SNP whose minor allele frequency is superior to 5% in HapMap Caucasian populations (Consortium, 2003). The whole analysis was then reproduced by excluding the 272 probes satisfying those criteria, and the same results were obtained as the one presented in the manuscript in FIGS. 1C and D, establishing the robustness of the GPI procedure, based on its transcriptome-wide design and ruling out any significant effect caused by the presence of SNPs within the probes.

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Claims

1-46. (canceled)

47. A method of treating Parkinson's Disease in a subject comprising administering an effective amount of a composition that stabilizes retromer complex to a subject in need thereof.

48. A method of treating Parkinson's Disease in a subject comprising administering an effective amount of a composition that suppresses retromer complex dysfunction to a subject in need thereof.

49. A method of treating Parkinson's Disease in a subject comprising administering an effective amount of a composition that induces retromer complex function to a subject in need thereof.

50. A method of treating Parkinson's Disease in a subject comprising administering an effective amount of a composition that stabilizes retromer complex component interactions.

51. A method of treating Parkinson's Disease in a subject comprising administering an effective amount of a composition that reduces retromer complex-associated sorting defects.

52. A method of treating Parkinson's Disease in a subject comprising administering an effective amount of a composition that inhibits reduction in retromer complex-component levels.

53. A method of treating Parkinson's Disease in a subject comprising administering an effective amount of a composition that increases retromer complex-component levels.

54. A method of treating Parkinson's Disease in a subject comprising administering an effective amount of a composition that increases retromer complex activity.

55. The method of claims 47-54, wherein retromer complex comprises VPS35, VPS29, VPS26 or a combination thereof.

56. The method of claims 47-54, wherein the composition comprises a nucleic acid encoding a protein comprising 90% identity to SEQ ID NO: 14 or a fragment thereof.

57. The method of claims 47-54, wherein the composition comprises a protein comprising 90% identity to SEQ ID NO: 14 or a fragment thereof.

58. The method of claims 47-54, wherein the composition further comprises a pharmaceutically acceptable excipient.

59. The method of claims 47-54, wherein the subject has one or more mutations in the polypeptide VPS35, VPS29, or VPS26.

60. The method of claims 47-54, wherein the subject has a genetic variant in the LRRK2 or RAB7L1 gene.

61. The method of claim 60, wherein the genetic variant in the LRRK2 gene encodes a LRRK2 protein comprising a G2019S mutation.

62. The method of claim 60, wherein the genetic variant in the RAB7L1 gene comprises a guanine (G) nucleotide at SNP rs1572931.

63. The method of claim 60, wherein the genetic variant in the LRRK2 gene comprises SNP rs11176052.

64. The method of claims 47-54, wherein the subject has sporadic non-familial Parkinson's Disease.