Gene related to migraine in man

Migraine is a frequent paroxysmal neuro-vascular disorder, characterized by recurrent attacks of disabling headache, vomiting, photo/phonophobia, malaise, and other general symptoms (migraine without aura). Up to 20% of patients may, in addition, experience transient neurological (aura) symptoms during attacks (migraine with aura) (HCC, 1988). Up to 24% of females and 12% of males in the general population are affected, however with variable attack frequency, duration and severity (Russell et al., 1995). Knowledge about the mechanisms of the final common pathway of migraine attacks has increased substantially the last five years, resulting in improved, though still only symptomatic (and sub-optimal) acute treatment for the attack. There is, however, still very little knowledge about the etiology of migraine attacks, i.e. why and how attacks begin and recur. Accordingly, prophylactic treatment for migraine is non-specific and has only limited efficacy.

Family, twin and population-based studies suggest that genetic factors are involved In migraine, most likely as part of a multifactorial mechanism (reviewed by Haan et al., 1996). The complex genetics has hampered identification of candidate genes for migraine. Familial Hemiplegic Migraine (FHM) is a rare, autosomal dominant, subtype of migraine with aura, associated with ictal hemiparesis and, in some families cerebellar atrophy (HCC, 1988). Otherwise, the symptoms of the headache and aura phase of FHM and “normal” migraine attacks are very similar and both types of attacks may alternate within subject and co-occur within families. FHM is thus part of the migraine spectrum and can be used as a model to study the complex genetics of the more common-forms of migraine (Haan et al., 1996). A gene for FHM has been assigned to chromosome 19p13 in about half of the families tested (Joutel et al., 1993; Ophoff et al., 1994; Joutel et al., 1995). Remarkably, cerebellar atrophy was found only in families with FHM linked to chromosome 19p13, but not in unlinked families. Recently, we showed the 19p13 FHM locus to be also involved in “normal” migraine (May et al., 1995).

Episodic ataxia type 2 (EA-2) is another, autosomal dominant, paroxysmal neurological disorder, characterized by acetazolamide-responsive attacks of cerebellar ataxia and migraine-like symptoms, and interictal nystagmus and cerebellar atrophy. Recently, a gene for EA-2 was assigned to chromosome 19p13, within the same interval as for FHM (Kramer et al., 1995). This finding, as well as the clinical similarities, raise the possibility of EA-2 and FHM being allelic disorders.

Since other hereditary episodic neurological disorders responding to acetazolamide (such as hypokalaemic and hyperkalaemic periodic paralysis), as well as EA type-1 A (which, in contrast to EA-2, is associated with continuous myokymia and non-responsive to acetazolamide) have all been associated with mutations in genes encoding for ion channels (Ptacek et al., 1991; Ptacek et al., 1994; Brown et al., 1994), we specifically looked for similar genes within the FHM and EA-2 candidate region.

In view of the above, the FHM/EA-2 locus can, since FHM is part of the migraine spectrum, thus be used to study the genetic factors and biological mechanisms that are related to various episodic neurological disorders such as FHM, EA-2, common migraine and others such as epilepsy.

Calcium channels are multisubunit complexes composed of at least an α1, an α2δ, and a β subunit. The central α1 subunit is functionally the most important component, acting as a voltage sensor and forming the ion-conducting pore. The other subunits have auxiliary regulatory roles. The membrane topology of the α1 subunit consist of four hydrophobic motifs (I to IV), each containing six transmembrane α-helices (S1-S6) and one hairpin (P) between S5-S6 that spans only the outer part of the transmembrane region.

The present invention provides an isolated and/or recombinant nucleic acid, or fragments thereof, encoding a Ca

2

+-channel α1 subunit related to familial hemiplegic migraine and/or episodic ataxia type-2, derived from a gene present on chromosome 19p13.1-19p13.2; a gene encoding the α1 (ion-conducting) subunit of a P/Q-type voltage gated calcium channel. The present invention also provides access to and methods to study the genetic background and identify other subunits of the calcium channel subunit complexes and the proteins related therewith that are associated with the genetic factors and biological mechanisms that are related to various episodic neurological disorders such as FHM, EA-2, common migraine and others such as epilepsy which are related to cation channel dysfunction.

The sequence of the cDNA of the gene is highly related (≧90%) to a brain-specific rabbit and rat voltage gated P/Q-type calcium channel al subunit (Mori et al., 1991; Starr et al., 1991), and the open reading frame consists of 2261 amino acid residues. Northern blot analysis showed a brain-specific expression, especially in the cerebellum. Primary study of a cosmid contig harbouring the gene already indicated an exon distribution over at least 300 kb of genomic DNA. Recently, a neuronal Ca

2+

α1A subunit gene was localized to chromosome 19p13.1-p13.2 by FISH analysis (Diriong et al, 1995). The gene symbol is CACNL1A4 and the al subunit is classified as a P/Q-type. No sequence data for the CACNL1A4 gene have been provided by Diriong or others, but the same localization (chromosome 19p13.1) and the identical classification (P/Q-type) suggests that the Ca

2

+ channel α1 subunit we have identified is very similar to CACNL1A4. No relation with migraine has been disclosed for CACNL1A4. The genomic structures of three other human Ca

2

+ channel α1 subunit genes (CACNL1A1, CACNL1A2 and CACNL1A3) have been published to date (Hogan et al, 1994; Soldatov, 1994; Yamada et al, 1995). Both CACNL1A1 and CACNL1A2 span about 150 kb and consist of 50 and 49 exons, respectively. The smaller CACNL1A3 gene is composed of 44 exons, distributed over 90 kb.

The present invention also provides an isolated and/or recombinant nucleic acid comprising alleles of the invented gene which contain mutations relevant to the occurence of migraine and other neurological disorders which are related to cation channel dysfunction. Such mutations are for example a mutation at codon 192 resulting in the replacement of arginine by glutamine (R192Q), and/or a mutation at codon 666 resulting in the replacement of threonine by methionine, and/or a mutation at codon 714 resulting in a replacement of valine by alanine and/or a mutation at codon 1811 resulting in a replacement of isoleucine by leucine, but also other mutations of alleles of said gene which bear relationships with cation channnel dysfunction.

The present invention also provides isolated and/or recombinant nucleic acid comprising alleles of said gene which contain a polymorphic CA-repeat sequence specific for various alleles of said gene. The present invention also provides isolated and/or recombinant nucleic acids comprising alleles of said gene which contain a CAG repeat.

The present invention also provides methods and tests (such as PCR, but also other tests to detect or amplify nucleic acids are known in the art) to detect, identify and localize or distinguish genes and alleles of such genes, or fragments thereof, encoding for proteins or α, β or χ sub-units of specific cerebral cation channels, more specifically the invented gene and its various alleles encoding the α1 subunit of a P/Q-type voltage gated calcium channel and the gene encoding the β2 sub-unit, which are involved in the primary pathogenesis of neurological disorders such as FHM, migraine, EA-2 and SCA6. With such methods and tests one can study abnormalities of said gene.

The invention also provides recombinant expression vectors comprising isolated and/or recombinant nucleic acid comprising alleles of said genes or fragments therof, and provides host cells or animals that comprise such vectors or that are otherwise transformed with an isolated and/or recombinant nucleic acid according to the invention.

The invention thus also provides a rationale and methods for the testing and the development of specific prophylactic medication for migraine and other episodic neurological, in particular brain, disorders, such as epilepsy, associated with cation channel dysfunction.

The invention for example provides cells or animals that comprise recombinant vectors that comprise variants of said genes or cells or animals that are transformed with said variants. Also, the invention provides means to identify naturally occuring variants of experimental animals with changes in said gene related to FHM, EA-2, SCA7, migraine or other neurological disorders associated with cation channel dysfunction. An example of such an animal is the tottering mouse, and its variants called leaner and rolling, described in the experimental part of the invention. The invention also provides cells or animals in which changes such as deletions or mutations in said gene have been introduced by recombinant nucleic acid techniques. All such cells or animals provided by the invention can be used to study the pathophysiology of FHM, EA-2, migraine or other neurological disorders associated with cation channel dysfunction, for example to test or develop specific medication for the treatment of said disorders.

The invention also provides proteins or peptides encoded by said genes, or fragments thereof, related with cation channel dysfunction, and detection of such proteins or peptides by antibodies directed against said proteins or peptides. Such antibodies can be of natural or synthetic origin, and can be produced by methods known in the art. Such proteins and antibodies and detection methods can be used to further in vitro or in vivo studies towards the pathophysiology of FHM, EA-2, migraine or other neurological disorders associated with cation channel dysfunction, in addition such proteins, antibodies and detection methods can also be used to diagnose or identify such disorders in patients or in experimental animals.

Experimental Procedures

Subjects

Sixteen FHM patients were selected, including eight individuals from four unrelated chromosome 19-linked FHM families (NL-A, UK-B, USA-C (Ophoff et al, 1994), and USA-P (Elliot et al., 1995), two affected individuals from two small FHM families from Italy (Italy I & II) and six individuals with sporadic hemiplegic migraine (i.e. no other family member was shown to suffer from attacks of hemiplegic migraine). In families NL-A and USA-P cerebellar ataxia and/or nystagmus is associated with FHM. An additional set of four subjects from four unrelated EA-2 families linked to chromosome 19, was also included (CAN-25, -45, -191, -197. Fifty randomly collected individuals from the Dutch population (Smith et al., 1988) were used as a control to determine the allele frequencies of polymorphic sites.

Patients with migraine with or without aura were diagnosed according to the international Headache-Society (IHS) classification criteria. Patients attending the neurology outpatient clinic of Leiden University Medical Center, The Netherlands, and patients responding to calls in local newspapers or in the periodical of the Dutch Migraine Patients Association, were screened for a positive family history of migraine. Only families with migraine in at least two generations were asked to participate. Probands (n=36) and relatives (n=492) were personally examined and interviewed using semi-structured questionnaires. The questionnaire included questions about age at onset, frequency and duration of attacks, aura symptoms, premonitory signs and symptoms, triggers for attacks, medication, and additional headaches. When family members were not available for a personal interview, information on their migraine was collected by interviewing their relatives. Because of the low validity of diagnosing migraine auras through relatives, we only assessed the presence or absence of migraine headaches. Whenever possible, medical records were examined.

Genomic Structure

Ten different cosmids from the contig extending the invented gene, were subcloned separately in either M13 or pBlueScript KS vector. From each cosmid library at least 3×96 random clones with an average insert size of about 2 kb, were picked. Positive clones were identified by hybridization techniques and subsequently sequenced with vector-specific primers; intron-exon boundary sequences were completed using cDNA-based primers.

Mutation Analysis, DHPLC and SSCP

Genomic DNA was used as template to generate polymerase chain reaction (PCR) products for single-strand conformational polymorphism (SSCP) analysis and denaturing high-performance liquid chromatography (DHPLC). Amplifications were performed in standard conditions with primer pairs as listed in Table 1 or listed below. Except for the 5′ side of exon 6, primers were chosen to produce fragments that contained a single exon and at least 35 basepairs (including primer) of each flanking intron sequence. Amplification of exons 1 and 20 was performed producing two overlapping fragments and exon 19 was amplified into three overlapping fragments. In addition, the following markers;

D10S191 Primer sequence 1 (SEQ ID NO: 141) CTT TAA TTG CCC TGT CTT C

Primer sequence 2 (SEQ ID NO: 142) TTA ATT CGA CCA CTT CCC

D10S245 Primer sequence 1 (SEQ ID NO: 143) AGT GAG ACT CGT CTC TAA TG

Primer sequence 2 (SEO ID NO: 144) ACC TAC CTG AAT TCC TGA CC

DIOS89 Primer sequence 1 (SEQ ID NO: 145) AAC ACT AGT GAC ATT ATT TTC A

Primer sequence 2 (SEQ ID NO: 146) AGC TAG GCC TGA AGG CTT CT

DHPLC (Oefner et al., 1995; Hayward et al., 1996) was carried out on automated HPLC instrumentation. Crude PCR products, which had been subjected to an additional 3-minute 95° C. denaturing step followed by gradual reannealing from 95-65° C. over a period of 30 minutes prior to analysis, were eluted with a linear acetonitrile (9017-03, J. T. Baker, Phillipsburg, N.J., USA) gradient of 1.8% per minute at a flow-rate of 0.9 ml/min. The start- and end-points of the gradient were adjusted according to the size of the PCR products (Huber et al., 1995). The temperature required for successful resolution of heteroduplex molecules was determined empirically by injecting one PCR product of each exon at increasing mobile phase temperatures until a significant decrease in retention was observed.

For SSCP analysis, primary PCR products were labeled by incorporation of [α-

32

P]dCTP in a second round of PCR. Samples were diluted and denatured in formamide buffer before electrophoresis. SSCP was carried out according to published protocols (Orita et al., 1989; Glavac et al., 1994). Digestion of several exons to yield products suitable for SSCP analysis.

Sequencing of PCR products was performed with an ABI 377 automated sequencing apparatus with cycle sequencing according to the manufacturer. Furthermore, PCR products were cloned in the TA vector (Invitrogen) and subjected to manual dideoxy sequence analysis (T7 Sequencing kit, Pharmacia Biotech.).

A total of 481 blood samples were collected from patients with migraine. Genomic DNA was isolated as described by Miller et al., 1988. The analyses of the highly informative microsatellite markers D19S391, D19S394, D19S221 and D19S226, D10S191, D10S248 and D10S89 were performed by PCR; primer sequences related to these markers are available through the human Genome Data Base (GDB).

The relative frequencies of marker alleles were estimated on the entire family material, with the relevant correction for genetic relationships between individuals (Boehnke, M, 1991) with the ILINK option of the I-INKAGE package, version 5.03 (Lathrop et al., 1985). The following marker order and recombination frequencies were used in the multipoint sib-pair analysis: D19S391-5%-D19S394-3%-D19S221-5%-D19S226. Affected-sib-pair analysis was performed using the MAPMAKER/SIBS software package, simultaneously including marker information for all four DNA markers (Kruglyak, 1995). Separate analyses were performed for migraine with aura, migraine without aura, and a combination of both. Allowance was made for dominance variance. When more than two affected sibs occurred in a single sibship, weighted scores were computed according to Suarez and Hodge (1979).

In a sib-pair analysis, the occurrence of parental marker alleles is compared among sibs. Normally, 25% of sib pairs share their marker alleles from both parents, 50% share one marker allele from one of their parents, while the remaining 25% share no parental allele. Deviations from this pattern towards increased sharing, and consistent with the constraints of Holmans's (1993) possible triangle, are explained as linkage (expressed as the maximum lod score MLS). Increased sharing of marker alleles thus indicate that the marker is located closely near a genetic risk factor. The relative-risk ratio for a sib (λ

R

), defined as the ratio of the prevalence of a disease in sibs of affected individuals, divided by the prevalence of a disease in the population, can be calcutated (May et al., 1995). In other words:

λ_{p} = \frac{Affected risk for sib of a proband}{Affection risk for an individual in the general population}

Results

Genomic Structure

The combination of hybridization and PCR strategies resulted in a rapid assembly of the complete coding sequence of the human cDNA, with an open reading frame of 6783 nucleotides encoding 2261 amino acid residues (FIG.

4

). The spatial distribution of the human Ca

2

+ channel expression was assayed in rhesus monkey tissues. Total RNA was isolated from several tissues, including various brain structures, and probed with a human cDNA fragment. The probe detected a major transcript of approximately 9.8 kb in cerebellum, cerebral cortex, thalamus and hypothalamus, whereas no transcript was detected in heart, kidney, liver or muscle. There was also no hybridization signal found in RNA preparations from mouse skin tissue or from human peripheral lymphocytes. In addition, an attempt to amplify parts of the cDNA from human peripheral lymphocytes failed.

Complete alignment between the cDNA and individual exon sequences was achieved, allowing the establishment of the exon-incron structure (Table 1). The reconstruction of the exon-intron structure of the CACNL1A4 gene revealed 47 exons ranging in size from 36 bp (exon 44) to 810 bp (exon 19). The exons are distributed over about 300 kb at genomic DNA level. The result shows that the first 10 exons are located in a region of about 150 kb covered by the first 5 cosmids of the contig indicating relatively large introns at 5′ side of the gene. Sequences (

FIG. 1

) were obtained of all exons including approximately 100 bp of flanking introns, except for intron 5 adjacent to exon 6. The forward primer of exon 6 harbours the splice junction and 3 bp of exon 6. Splice sites around all exons are compatible with consensus sequence with the exception of splice donor and acceptor of the first intron.

The cosmid conzig that yielded the initial Ca

2

+ channel gene exons was extended to cover more than 300 kb. Hybridization experiments showed that the first and last cosmids of the contig were positive for 3′- and 5′-end cDNA sequences, respectively, indicating a genomic distribution of the gene over at least 300 kb (FIG.

2

). The cosmid contig has been placed into the LLNL physical map of chromosome 19 at band p13.1, between the markers D19S221 and D19S226 (FIG.

2

). We identified a new polymorphic CA-repeat sequence (D19S1150) on the cosmid contig. Oligonucleotide primers (Table 1) flanking the repeat were synthesized and amplification was performed by PCR as described. Analysis of D19S1150 in 45 random individuals from the Dutch population revealed nine alleles with an observed heterozygosity of 0.82. This highly polymorphic marker is located within the gene and is therefore very useful in genetic analysis.

Mutation Analysis

Exons and flanking intron sequences, containing the complete coding region of CACNL1A4 and part of untranslated sequences, were screened for the presence of mutations by SSCP and DHPLC analysis in 20 individuals with either FHM or EA-2. Several synonymous nucleotide substitutions and polymorphisms were identified including a highly polymorphic (CAG)n-repeat in the 3′ untranslated region of exon 47 (Table 2). Of all polymorphisms only one was identified predicting an amino acid change, an alanine to threonine substitution at codon 454 (A454T).

Four different missense mutations were found in FHM patients of which one mutation was observed in two unrelated FHM affected individuals (Table 3). The mutations were shown to segregate with the disease within the families, and were not present in about 100 control chromosomes. A G-to-A transition was identified in family Italy-II at codon 192, resulting in a substitution of arginine to glutamine (R192Q) within the first voltage sensor domain (IS4). A second missense mutation occurs at codon 666, within the P-segment of the second repeat replacing a threonine residue for methione (T666M) in family USA-P. Two other mutations are located in the 6th transmembrane spanning segment of respectively repeat II and IV. The IIS6 mutation is a T-to-C transition at codon 714, resulting in a substitution of valine to alanine (V714A), identified in FHM family UK-B. The mutation in domain IVS6 is an A-to-C transversion at codon 1811 resulting in a substitution of isoleucine to leucine (I1811L). This I1811L mutation is found in family NL-A and family USA-C, two unrelated FHM families. Comparison of haplotypes in this region, including intragenic markers, did not reveal any evidence for a common founder of family NL-A and USA-C (data not shown). No mutation was found in FHM family Italy-I nor in the six sporadic hemiplegic migraine patients. In addition to missense mutations in FHM families, we also identified mutations in two out of four EA-2 families (Table 3). In EA-2 family CAN-191, a basepair deletion occurs in exxon 22 at nucleotide position 4073 causing a frameshift and a premature stop. The second EA-2 mutation is a transition of G-to-A of the first nucleotide of intron 24, predicted to leading to an aberrant splicing in family CAN-26. The invented gene also contains a CAG repeat, of which expansions have been found in patients with autosomal dominant cerebellar ataxia (SCA6). Hence FHM, EA-2 and SCA6 are alielic ion channel disorders and different mutations are associated with different clinical symptomatologies.

Our study patients with common migraine (with or without aura) included 36 independent multigenerational Dutch families. At least some data were available on 937 family members and 212 persons who “married-in” (spouses). Of these, 442 family members (247 affected) and 86 spouses (7 affected) were personally interviewed. The distribution of the different types of migraine among the 247 affected family members are as follows: 132 family members showed migraine without aura, 93 showed migraine with aura and 22 showed months-migraine, not fulfilling all critera by IHS. Among the 7 affected spouses these figures were 4, 1 and 2, respectively. We scored the parental transmission of migraine in the 36 families (Tabel 4) to investigate if an additional X-linked dominant or mitachondrial aene was involved. An approximately 2.5:1 preponderance of females among the migraine sufferers was noted, which remained in the affected offspring. Affected fathers were found to transmit migraine to their sons in 21 cases, making X-linked dominant or mitochondrial inheritance highly unlikely.

The genetic analysis included 204 potentially affected sib pairs; after correction for more than one sib pair in a single sibship the total number of sib pairs was 108. Affected-sib-pair analysis was performed for sib pairs who were both affected with any form of migraine and, in separate analyses, for sib pairs who where both suffering from either migraine with aura or migraine without aura. The informativeness of the region between the markers D19S391, D19S394, D19S221 and D19S226 varied between 82% and 96%. The combined analysis of migraine with and without aura resulted in a maximum multipoint lod score of 1.69 (p≈0.005) with marker D19S226. For migraine with aura the maximum multipoint lod score was 1.29 corresponding with p≈0.013 with marker D19S394. The maximum lod score for migraine without aura was not significant (MLS <0.25)(data not shown). The relative risk ratio for a sib to suffer from migraine with aura (λ

p

), defined as the increase in risk of the trait attributable to the 19p13 locus, varied between λ

R

=1.5 (for marker D19S394) and λ

R

=2.4 (for marker D19S226). When combining migraine with and without aura, λ

R

was 1.25. In a selected portion of 36 Dutch families with migraine with aura and without aura, affected sib-pair analysis was performed for sib pairs who were affected with any form of migraine. The following markers, flanking the β2(CACNB2) calcium channel subunit gene on chromosome 10p12, were tested: D108191, D1OS246 and D10S89. For the combined phenotype (migraine with and without aura) a maximum pultipoint iod score of 0,95 (p<0,01) was obtained with marker; D10S191. This result gives independent evidence for a role of the P/Q type Ca

2+

channel in migraine and other neurological disorders.

Discussion

The genomic structure of the exemplified invented gene revealed 47 exons distributed over about 300 kb (Table 1; FIG.

1

). A comparison of the gene structure to already known Ca

2+

channel al subunit genes (CACNL1A1, CACNL1A2, and CACNL1A3) (Soldatov, 1994; Yamada et al., 1995; Hogan et al., 1995), reveals a similar number of exons (50, 49, and 44 respectively) but a larger genomic span (300 kb vs 90-150 kb). Remarkebly, all splice sites are according to consensus sequence except for intron 1. Splice donor as well as splice acceptor of the first intron do not contain the expected gt . . . ag intron sequence. An incorrect CDNA sequence is unlikely because the cDNA sequence containing the junction of the first two exons is identical to rabbit and rat sequence. Sequences corresponding to splice donor and acceptor are present in exon 1 and 2, suggesting an additional (yet unidentified) exon in the first intron encompassing part of sequences of exon 1 and exon 2.

To test the possible involvement of the invented gene relating to the CA

2

+-channel sub-unit in migraine FHM, SCA6 and EA-2, we performed a mutation analysis by DHPLC and SSCP and found several alterations (For example Table 2 & 3). Only one missense variation was observed also present in 2% of the normal controls (Table 2). This polymorphism is a alanine to threonine substitution at codon 454 (A454T), located in the intracellular loop between IS6 and IIS1 (FIG.

2

). This region contains a conserved alpha interaction domain (AID) that binds subunits (De Waard et al., 1996). However, A454T is located outside the AID consensus sequence and is not likely to be involved.

The identification of two mutations that disrupt the predicted translation product of the invented gene in two unrelated EA-2 patients and the segregation of these mutations with the episodic ataxia phenotype in their families provide strong evidence that the invented gene is the EA-2 gene. A basepair deletion leads to a frame-shift in the putative translation product and encounters a stop codon in the next exon. The frame-shift in this EA-2 family is predicted to yield a calcium channel al subunit polypeptide consisting of repeat I and II, and a small portion of repeat III (IIIS1). The G-to-A transition of the first nucleotide of intron 24 is affecting the nearly invariant GT dinucleotide of the intronic 5′ splice junction. The brain-specific expression of the exemplified invented gene makes it extremely difficult to test the hypothesis that this mutation produces aberrantly spliced RNAs by retaining the intron or utilizing other cryptic 5′ splice sites.

The frameshift and splice site mutations in EA-2 may suggest a dominant negative effect of the truncated proteins by overruling the (corresponding) intact al subunits.

No mutations were found in the remaining EA-2 families (CAN-25 and -197). The use of two independent techniques for mutation screening (DHPLC and SSCP) makes it unlikely that we missed a heterozygote PCR product. Mutations in the promoter region or in intron sequences, resulting in aberrant splicing, may have been the cause of EA-2 in these families. We could also have missed a mutation around the splice acceptor site of intron 5, covered by the forward primer of exon 6. However, larger deletions of e.g. complete exons with flanking intron sequence will disturb the predicted translation product, like the ΔC

4073

and splice site mutation do, but this is not detectable by a PCR-based screening method but can be seen Southern blot analysis instead.

Four different missense mutations were identified in five unrelated FHM families. These mutations all segregate with FHM within a family and are not observed in over 100 normal chromosomes. The first missense mutation that we describe in the exemplified invented gene occurs in the IS4 domain of the al subunit (Table 3; FIG.

3

). The S4 domains are postulated to be voltage sensors because they have an unusual pattern of positively charged residues at every third or fourth position separated by hydrophobic residues (Tanabe et al., 1987). In calcium channels the positively charged amino acid is an arginine residue (Stea et al., 1995). The mutation in FHM family Italy-II predicts a substitution of the first arginine in the IS4 segment with a neutral, non-polar alutamine (R192Q). The change of the net positive charge of this conserved region of the protein may influence correct functioning of the voltage sensor.

The second missense mutation in FHM family USA-P occurs in the P-segment of the second transmembrane repeat. A C-to-T transition predicts substitution of a threonine residue with methionine at codon 666 (T666M). Various observations have shown that P-segments, the hairpin between S5 and S6 that spans only the outer part of the transmembrane region, form the ion-selectivity filter of the pore and binding sites for toxins (Guy and Durell (1996). Alignment of protein sequence of different P-segments indicating that some residues occur in many different channel genes (Guy and Durell, 1996). The T666M substitution alters one of the conserved residues in the P-segment. It is conceivable that an alteration of a P-segment affects the ion-selectivity or toxin binding of a channel gene.

The remaining two missense mutations identified in FHM families alter the S6 segment of the second and the fourth repeat. A valine to alanine substitution in FHM family UK-B is found in domain IIS6 at codon 714 (V714A). Domain IVS6 is mutated in two unrelated FHM families (NL-A and USA-C), predicting a isoleucine to leucine substitution at codon 1811 (I1811L). The V714A and I1811L missense mutations do not really change the neutral-polar nature of the amino acid residues. However, both S6 mutations are located nearly at the same residue at the intracellular site of the segment and are conserved in all calcium channel al subunit genes. In addition, the A-to-C transversion leading the I1811L substitution occurred in two unrelated FHM families on different haplotypes indicating recurrent mutations rather than a founder effect. Although the exact function of the S6 domains are not known, these data strongly suggest that mutations in IIS6 and IVS6 result in FHM.

The I1811L mutation is present in two FHM families of which one (NL-A) also displays a cerebellar atrophy in (some) affected family members. The presence of cerebellar atrophy in FHM families has been reported in about 40% of chromosome 19-linked FHM families, whereas none of the unlinked families was found to have cerebellar atrophy (Terwindt et al., 1996).

The I1811L mutation excludes the possibility of allelic mutations in FHM and FHM with cerebellar atrophy. However, it is likely that FHM or FHM with cerebellar atrophy are the result of pleiotropic expression of a single defective gene.

No mutation was found in a small Italian FHM family (Italy-I). Apart from the possibilities discussed for EA-2, it should be noted that linkage to 19p13 was only suggested but never proved with significant lod scores (M. Ferrari, personal knowledge).

The four missense mutations identified indicate a mechanism for FHM in which both alleles of the α1 subunit are expressed, one harbouring an amino acid substitution which affects the function of this calcium channel α1 subunit by reducing or enhancing the electrical excitability. The relationship of FHM and other types of migraine makes it highly rewarding to investigate the involvement of the only missense variant observed (A454T) (Table 2), and to continue the search for other variants of the exemplified invented gene specific for common types of migraine.

The mutations in EA-2 and FHM demonstrate among others that the brain specific calcium channel gene CACNL1A4 is responsible for both EA-2 and FHM, and is also involved in the primary pathogenesis of the more common forms of migraine. We conducted the common migraine study in an independent sample of 36 extended Dutch families, with migraine with aura and migraine without aura. We found significant increased sharing of the marker alleles in sibs with migraine with aura (MLS=1.29 corresponding with p≈0.013). Although no such increased sharing was found for migraine without aura, a combined analysis for both migraine types resulted in an even more significant increased sharing (MLS=1.69 corresponding with p≈0.005). These results clearly indicate the involvement of the calcium cSIA-subunit gene region on 19p13 in both migraine with and without aura; the contribution to migraine with aura, however, seems strongest.

The positive findings in our study clearly demonstrate an involvement of the FHM locus region in non-hemiplegic familial migraine, notably in migraine with aura. The P/Q-type calcium channel α

1A

-subunit gene on chromosome 19p13 may be an “aura-gene” and is involved in both FHM and migraine with aura, but not in migraine without aura. This however, seems unlikely since an increased sharing of marker alleles was also found when we combined the results for migraine with and without aura. Furthermore, the increase in sharing was stronger than expected to be only due to the contribution of migraine with aura. An alternative explanation is that the gene is involved in both types of migraine, but in migraine without aura there is an additional strong effect of other, possibly environmental factors, thereby reducing the penetrance.

The latter view is also supported by the results obtained from calculating the relative risk ratios (λ

R

) for sibs from affected individuals to also have migraine. The relative risk ratio for a sib to suffer from migraine with aura was λ

R

=2.4. When combining migraine with and without aura, λ

R

was 1.25. In a population-based study the relative risk for first degree relatives of probands with migraine with aura to also have migraine with aura was λ

R

=3.8. Because of the female preponderance among migraine patients, X-linked dominant or mitochondrial inheritance has been suggested to be involved in familial migraine. Although a predominant maternal inheritance pattern was noted in our families, X-linked dominant or mitochondrial inheritance were found to be highly unlikely because affected fathers transmit migraine to their sons. Furthermore, the predominant maternal inheritance can be explained by the female preponderance among the migraine patients.

We conclude that the well-established genetic contribution to the etiology of migraine is partly, but not entirely, due to genetic factors located in the chromosomal region of the P/Q-type calcium channel α

1A

-subunit gene. Further analysis of the cerebral distribution and function of this calcium channel, as well as of the “mutated channels”, will help to unravel the pathogenetic pathway of migraine. It may also contribute to a better understanding of the mechanisms involved in related disorders such as episodic ataxia type-2, autosomal dominant cerebellar ataxia (SCA6), cerebral atrophy, and epilepsy, which all have been found to be associated with mutations in this gene. Study of FHM, EA-2 mutants and variants such as the A454T variant expressed in vitro or in mouse or other experimental animal models will ultimately lead to better understanding of the diseases, their cellular mechanisms, and the clinical relationship between FHM, EA-2, migraine, and other episodic neurological disorders such as epilepsy, and will provide rationales for the development of prophylactic therapy.

Localization and identification of the mouse gene related to the neurological mouse mutations tottering, leaning and rolling.

The tottering (tg) mutation arose spontaneously in the DBA inbred strain, and has been back-crossed into a C57BL/6J (B6) inbred strain for at least 30 generations. The genome of the tg mouse therefore is of B6 origin except for a small region around the tg gene on chromosome 8. Interestingly, the chromosome 8 region in mouse has synteny with the human chromosome 19p13.1, in which the human calcium channel alphal subunit has been identified. We therefore consider the tg locus as a possible site of the mouse homologue of the human calcium channel gene.

To determine the exact localization of the mouse homologue, PCR was carried out with primers based on human cDNA sequence selected from FIG.

1

and mouse genomic DNA aE template. In human, primers were known to be located in different flanking exons. POR amplification on human DNA yielded a 1.5kb fragment.

Forward primer (SEQ ID NO: 45)5′-caa cat cat gct ttc ctg cc-3′

Reversed primer (SEQ ID NO: 46)5′-atg atg acg gcg aca aag ag-3′

Amplification on mouse DNA yielded a 750-bp fragment. The fragment mainly consists of intronic sequences. SSCP analysis revealed several polymorphisms in the different inbred strains (each strain a specific pattern). Analysis of amplified product of the tg/tg (homozygote) and tg/+(heterozygote) mice demonstrated a DBA specific signal in the tg/tg mouse, and a heterozygous pattern of DBA and B6 inbred strains in the heterozygous tg/+mouse. These results show that the mouse homologue of the human calcium channel alphal subunit is located within the mouse tottering interval on chromosome 8.

In conclusion: the phenotypic characteristics of the tg mouse (tg/tg and tg/+) suggest involvement of ion-channels in the tg-etiology. The localization of the mouse homologue of the human calcium gene within the tottering interval show that a tottering phenotype in mouse is caused by a mutation in the mouse homologue of the CACNL1A4 gene. With various variants of the tottering mouse (the Jackson Laboratory, Bar Habor, Me., USA), such as the leaner and rolling varieties, such mutations in the mouse homologue of the CACNL1A4 gene can be found, clearly demonstrating that the gene is related to a variety of episodic neurologic disorders and using this genetic information one can engage in a variety of pathofysiological studies, as for example indicated below.

The tg mutation arose spontaneously in the DBA/2 inbred strain. tg/tg homozygotes are characterized by a wobbly gait affecting the hindquarters in particular, which begins at about 3 to 4 weeks of age, and by intermittent spontaneous seizures which resemble human epileptic absence seizures. The central nervous system of the mice appears normal by light microscopy. There is no discernible cerebellar hypoplasia. In fluorescent histochemistry studies tg/tg mice show a marked increase in number of noradrenergic fibers in the terminal fields innervated by locus ceruleus axons, the hippocampus, cerebellum, and dorsal lateral geniculate. Treatment of neonatal tg/tg mice with 6-hydroxydopamine, which selectively causes degeneration of distal noradrenergic axons from the locus ceruleus, almost completely abolishes the ataxic and seizure symptoms.

The leaner mutation of the tottering mouse arose spontaneously in the AKR/J strain. Homozygotes are recognized at 8 to 10 days of age by ataxia, stiffness, and retarded motor activity. Adults are characterized by instability of the trunk, and hypertonia of trunk and limb muscles. The cerebellum is reduced in size, particularly in the anterior region, in tg<la>/tg<la>mice, as is the case with a certain number of FHM patients. There is loss of granule cells beginning at 10 days of age and loss of Purkinje and Golgi cells beginning after 1 month. Cell loss later slows but continues throughout life. Granule and Purkinje cells are more severely affected than Golgi cells and the anterior folia more severely affected than other parts of the cerebellum. The cerebellum of tg<la>/tg mice shows shrinkage and degenerative changes of the Purkinje cells. The loss in cerebellar volume in tg<la>/tg and in tg/tg mice is specific to the molecular layer, with no change in volume of the granule cell layer or the white matter layer. Allelism of aleaner with tottering was shown in complementation and linkage tests.

A third variety of the tottering mouse is (tg<rol>) called the rolling Nagoya. Found among descendants of a cross between the SIII and C57BL/6 strains, the tg<rol>mutation apparently occurred in the SIII strain. Homozygotes show poor motor coordination of hindlimbs that may lead to falling and rolling, and sometimes show stiffness of the hindlimbs and tail. No seizures have been observed. Symptoms are recognizable at 10 to 14 days old. They appear a little earlier than those of tg/tg mice and are somewhat more severe. The cerebellum is grossly normal until 10 days of age, but after that grows more slowly than normal. The size of the anterior part of the central lobe of the cerebellum is reduced with reduction in the numbers of granule, basket, and stellate cells but normal numbers of Purkinje cells. There is a reduced concentration of glutamate and an increased concentration of glycine and taurine in the cerebellum and decreased activity of tyrosine hydroxylase in the cerebellum and other areas.

LEGENDS TO FIGURES

FIG. 1

Nucleic acid sequences of 47 exons and flanking intron sequences containing the complete coding region of the invented gene and part of untranslated sequences SEQ ID NO: 41-SEQ ID NO: 42.

FIG. 2

Genetic map, cosmid contig and global exon distribution of the invented gene om chromosome 19p13.1. The cosmid contog is shown with EcoRI restriction sites, available via Lawrence Livermore National Laboratory; exon positions are indicated schematically, regardless of exon or intron sizes (Table 1). D19S1150 is a highly polymorpmic intragenic (Ca)

n-repeat

.

FIG. 3

Membrane topology of α1 subunit of the P/Q-type Ca

2+

-channel. The location and amino acid substitutions are indicated for mutations that cause FHM or EA-2.

FIG. 4

The coding sequence SEQ ID NO:43 of human cDNA of the invented gene with an open reading frame encoding 2261 amino acid residues SEQ ID NO: 44.

REFERENCES

1. Browne, D. L., Gancher S. T, Nutt, J. G., Brunt, E. R., Smith E. A., Kramer P., and Litt M. (1994). Episodic ataxia/myokymia syndrome is associated with point mutations in the human potassium channel gene, KCNA1

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2. Diriong S., Lory P., Williams M. E., Ellis S. B., Harpold M. M., and Taviaux S. (1995). Chromosomal localization of the human genes for α1A, α1B, and α1E voltage-dependent Ca

2

+ channel subunits.

Genomics

30: 605-609.

3. Headache Classification Committee (HCC) of the International Headache Society (1988). Classification and diagnostic criteria for headache disorders, cranial neuralgias and facial pain

Cephalalgia

8: 19-28.

4. Hogan, K.,Powers, P. A., and Gregg, R. G. (1994). Cloning of the human skeletal muscle alpha 1 subunit of the dihydropyridine-sensitive L-type calcium channel (CACNL1A3).

Genomics

24: 608-609.

5. Joutel A., Bousser M-G., Biousse V., Labauge P., Chabriat H., Nibbio A.,Maciazek J., Meyer B., Bach M-A., Weissenbach J., Lathrop G. M., and Tournier-Lasserve E. (1993). A gene for familial hemiplegic migraine maps to chromosome 19

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6. Joutel A., Ducros A., Vahedi K., Labauge P., Delrieu O., Pinsard N., Mancini J., Ponsat G., Gaoftiere F., Gasant J. L., Maziaceck J. Weissenbach J., Bousser M. G., and Tournier-Lasserve E. (1994). Genetic heterogeneity of familial hemiplegic migraine.

Am. J. Hum. Genet

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7. Hayward-Lester, A., Chilton, B. S., Underhill, P. A., Oefner, P. J., Doris, P. A. (1996). Quantification of specific nucleic acids, regulated RNA processing and genomic polymorphisms using reversed-phase HPLC.

In: F. Ferr (Ed.), Gene Quantification, Birkhuser Verlag, Basel, Switzerland.

8. Huber, C. G., Oefner, P. J., Bonn, G. K. (1995) Rapid and accurate sizing of DNA fragments by ion-pair chromatography on alkylated nonporous poly(styrene-divinylbenzene) particles.

Anal. Chem

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9. Kramer P. L., Yue Q., Gancher S. T., Nutt J. G., Baloh R., Smith E., Browne D., Bussey K., Lovrien E., Nelson S, and Litt M. (1995). A locus for the nystagmus-associated form of episodic ataxia maps to an 11-cM region on chromosome 19p.

Am. J. Hum. Genet

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10. May A, Ophoff R. A., Terwindt G. M., Urban C., Van Eijk R., Haan J., Diener H. C., Lindhout D., Frants R. R., Sandkuiji L. A., and Ferrari M. D. (1995). Familial hemiplegic migraine locus on 19p13 is involved in the common forms of migraine with and without aura.

Hum. Genet

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11. Mori Y, Friedrich T., Kim M. S., Mikami A., Nakai J., Ruth P., Bosse E., Hofmann F., Flockerzi V., Furuichi T., Mikoshiba K. Imoto K., Tanabe T., and Numa S. (1991). Primary structure and functional expression from complementary DNA of a brain calcium channel.

Nature

350: 398-402.

12. Oefner, P. J., Underhill, P. A. (1995) Comparative DNA sequencing by denaturing high-performance liquid chromatography (DHPLC).

Am. J. Hum. Genet

. 57 [Suppl.], A266.

13. Ophoff R. A., Van Eijk R., Sandkuijl L. A., Terwindt G. M., Grubben C. P. M., Haan J., Lindhout D., Ferrari M. D., and Frants R. R. (1994). Genetic heterogeneity of familial hemiplegic migraine.

Genomics

22: 21-26.

14. Orita, M., Suzuki, Y., Sekiya, T., and Hayashi, K. (1989). Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction.

Genomics

5: 874-879.

15. Ptacek L. J., George A. L., Griggs R. C., Tawil R., Kallen R. G., Barchi R. L., Robertson M., and Leppert M. F. (1991). IdentifLication of a mutation in the gene causing hyperkalemic periodic paralysis.

Cell

67: 1021-1027.

16. Ptacek L. J., Tawil R., Griggs R. C., Engel A. G., Layzer R. B., Kwiecinski H., McManis P. G., Santiago L., Moore M., Fouad G., Bradley P., and Leppert M. F. (1994). Dihydropyridine receptor mutations cause hypokalemic periodic paralysis.

Cell

77: 863-868.

17. Ravnik-Glavac, M., Glavac D., and Dean, M. (1994). Sensitivity of single-strand conformation polymorphism and heteroduplex method for mutation detection in the cystic fibrosis gene. Hum.

Mol. Genet

. 3: 801-807.

18. Russell, M. B., Rasmussen B K., Thorvaldsen P., and Olesen J. (1995). Prevalence and sex-ratio of the subtypes of migraine.

Int. J Epidemiol

. 24: 612-618.

19. Starr T. V. B., Prystay W., and Snutch T. (1991). Primary structure of a calcium channel that is highly expressed in the rat cerebellum.

Proc. Natl. Acad. Sci

. 88: 5621-5625.

20. Soldatov N. M. (1994) Genomic structure of Human L-type Ca

2

+ channel. Genomics 22: 77-87.

21. Teh B. T., Silburn P., Lindblad K., Betz R., Boyle R., Schalling M., and Larsson C. (1995). Familial periodic cerebellar ataxia without myokymia maps to a 19-cM region on 19p13

. Am. J. Hum. Genet

. 56: 1443-1449.

22. Terwindt G. M., Ophoff R. A., Haan J., Frants R. R., and Ferrari M. D. (1996). Familial hemiplegic migraine: a clinical comparison of families linked and unlinked to chromosome 19

. Cephalagia

16: 153-155.

23. Von Brederlow, B., Hahn, A. F., Koopman, W. J., Ebers, G. C., and Bulman, D. (1995). Mapping the gene for acetozolamide responsive hereditary paroxysmal cerebellar ataxia to chromosome 19p.

Hum. Mol. Genet

. 2: 279-284.

24. Yamada Y., Masuda K., Li Q., Ihara Y, Kubota A., Miura T., Nakamura K., Fujii Y., Seino S., and Seino Y. (1995) The structures of the Human Calcium channel α1 subunit (CACNL1A2) and β-subunit (CACNLB3) genes.

Genomics

27: 312-319.

TABLE 1

Exon/intron organization of the human invented gene and exon-specific primer pairs

Exon

cDNA

Size

Domain

Cosmid(s)

Primer Forward

Primer Reversed

Size

1

UTR-568

500

25960/30151

tct ccg cag tcg tag ctc ca 53

ggt tgt aga gtg cca tgg tc 87

320

cgc aaa gga tgt aca agc ag 54

att ccc aag cct cca ggg tag 88

370

2

569-674

106

I S1

30151

cac ctc caa cac cct tct tt 55

tct gtg ccc tgc tcc act c 89

240

3

675-814

140

I S2, I S3

30151

acg ctg acc ttg cct tct ct 56

caa cca aaa gcc tcg taa tc 90

230

4

815-906

92

I S3, I S4

28913

aaa acc cac cct ctg ttc tc 57

ttg tca ggg tcg gaa act ca 91

160

5

907-1059

153

28913/27415

ctt ggt ggc ggg gtt t 58

ctg cct aat cct ccc aag ag 92

290

6

1060-1253

194

27415

tcc ctt ccc ttt tgt aga tg 59

gtg ggg ctg tgt tgt ctt t 93

350

7

1254-1357

104

I S6

27415

gac aga gcc aca aga gaa cc 60

agc aaa gag gag tga gtg gg 44

250

8

1358-1473

116

34077/27415

ata ctc tgg ctt ttc tat gc 61

gca tga ctc tct ttg tac tc 95

230

9

1474-1530

57

34077

gca gag aat ggg ggt gg 62

ctg agg tgg gtt tag agc ac 96

180

10

1531-1623

93

34077

ggg taa cgt ctt ttt ctc ttg c 63

atg tct ctt ggg cga tag gt 97

200

11

1624-1833

210

II S1

16894/32236

att tct tct gaa gga aca gc 64

gga ggg atc agg gag ttc gc 98

310

12

1834-1946

113

II S2, II S3

16894

caa gcc taa cct cct ctc tg 65

tca ttc cag gca aga gct g 99

200

13

1947-2051

105

II S3, II S4

16894

att tgg agg gag gag ttt gg 66

tca ctt tcc caa ctt tct gg 100

310

14

2052-2191

140

II S4, II S5

16894

cag aaa gtt ggg aaa gta gc 67

ttg aat tcc tgt gaa gga c 101

250

15

2192-2264

73

16894

ctt gga gat gag ata ctg agc 68

cag gca ctt tca tct gtg ac 102

200

16

2265-2382

118

II S6

16894

tcc aca gct gca tct cca ag 69

acc ctc cct tga gcc cc 103

270

17

2383-2450

68

II S6

16894

cag tgg ttg ctt ttc ctg ac 70

ttg cca gag aaa cat tct cc 104

130

18

2451-2557

107

16894

tga aca aag att cca cgt gc 71

ttc agg agc cag ggt agc atc 105

170

19

2558-3367

810

16894

tag caa tgc tct aag tcc tc 72

tgt ttc ctg agg aag tcc tc 106

320

cgc agg aga acc gca aca a 73

gcg atg acg tcg atg ctc 107

450

gc agc agg gag agc cgc agc 74

tac cgt cat tct gcg gat tc 108

300

20

3368-3831

464

16894

ggt tct ttt tca ttc act tgc 75

ttt cct ggc agt ctt agc tc 109

430

gag aat agc ctt atc gtc ac 76

cag tga tgt gag agc aga 110

200

21

3832-3973

142

16894/34275

tgg gaa att gtg gag gga gc 77

tga ctt ccg cca ccc tgg tg 111

250

22

3974-4103

130

III S1

16894/34275

agc ctg tgg tct gag tgg ac 78

tag gaa ggg gtg tgc tct gtc 112

210

23

4104-4163

60

III S2, III S3

16894/34275

atc cac tgc tct ctt gct tt 79

gtg gtt ctc act tat aat ctg 113

170

24

4164-4270

107

III S3

34275

tgg cct cat tgg ctt ccc tgc 80

aag agg aaa ccc ttg cga ag 114

250

25

4271-4370

100

III S4

34275

cta ccc aac ctg acc tct gc 81

aca tga taa ccc tga cag tc 115

220

26

4371-4531

161

III S5

34275

ctc atg ctc tct gtc aac tc 82

tgg ttc caa tgg gaa tgt gc 116

250

27

4532-4669

138

34275

ctg ctt ccc aag cag tct ag 83

tcc tgg ata gat ttc cag tc 117

300

28

4670-4871

202

III S6

34275

agt ttt taa agg aca gat gg 84

ttt ccc tgc ccc att cct ttg c 118

280

29

4872-5036

165

IV S1

34275

ctc tgc cgc tct cac cac tg 85

ttt atc agg tag agg cag g 119

250

30

5037-5147

111

IV S1, IV S2

34275

ttc caa gcc cat agc tgt agc 86

tga ccc tgc tac tcc tgc ttc 120

180

31

5148-5231

84

IV S3

15496

act gtg cct cta aca tgc ac 121

aag tgc tgg ctc aag cag 138

250

32

5232-5348

117

IV S4

15496

tct gtg agt ggt gac agc tc 122

gtc acc tgt ctt ctc agc 139

240

33

5349-5414

66

IV S5

15496

tgg aag gac tct ggc acg tg 123

gga ggc tct ggg aac ctt ag 140

250

34

5415-5530

116

15496

aga agc cac tgg agg aat ggc 124

att atc aga gca ggt ccc ctt c 141

250

35

5531-5681

151

IV S6

15496

tcc gag tct ctg att tct cc 125

aga cgg ccc tca cag tgt c 142

210

36

5682-5809

128

IV S6

15496

ttc att ccc tcg gtc tct gc 126

ctg act gaa cct gtg aga c 143

350

37

5810-5906

97

15496

tgt gaa ccc att gcc tgc a 127

tgg gaa tga ctg cgc ttg c 144

200

38

5907-6012

106

15496

atg cct ggg aat gac tgc 128

tgt cac gcc tgt ctg tgc 145

200

39

6013-6120

108

15496

tga cac cca ggc agg cag 129

tct gtc ctg gtg gat tgg atc 146

200

40

6121-6221

101

15496

ttg gtg agc tca ccg tgt 130

ttc ccg tgg tga cat gca agc 147

200

41

6222-6331

110

15496

gtc cac aca ctg ctc tct gc 131

aca ctc cac ctc cct ggc 148

320

42

6332-6470

139

15496

gcc agg gag gtg gag tgt 132

ggt tcc ttc cac cgc aac 149

550

43

6471-6584

114

15496/30762

caa ctc ccc aat ggc tc 133

cct acc cag tgc aga gtg agg 150

350

44

6585-6620

36

15496/30762

tct gtg tgc acc atc cca tg 134

aag gat tgg gct cca tgg ag 151

200

45

6621-6807

187

15496/30762

gtt ggt gct agc tgc tga c 135

ctt tct tct tcc tta gtg tc 152

330

46

6808-7061

254

15496/30762

gtg tgc tgt ctg acc ctc ac 136

agc ctg ggg tca ctt gca gc 153

320

47

7062-UTR

≧350

/30762

cct tgg ttt caa ttt tcg tgt ag 137

tgg ggc ctg ggt acc tcc ta 154

280

Note.

Sizes of exons and PCR products are given in basepairs; domains of protein are indicated according to Stea et al., 1995.

( ) The sequence identification number (SEQ ID NO:) is in parenthesis immediately following each sequence.

TABLE 2

Polymorphisms in coding sequence of the invented gene

Location

Nucleotide change

Frequency

Consequence

exon 4

nt 854

G - A

Thr

193

0.02

—

exon 6

nt 1151

A - G

Glu

292

0.07

—

exon 8

nt 1457

G - A

Glu

394

0.38

—

exon 11

nt 1635

G - A

Ala

454

0.02

Ala

454

- Thr

(A454T)

exon 16

nt 2369

G - A

Thr

698

0.12

—

exon 19

nt 3029

G - A

Glu

918

0.07

—

exon 23

nt 4142

T - C

Phe

1289

0.22

—

exon 46

nt 6938

T - C

His

222′

0.46

—

exon 47

nt 7213

(CAG)

n

3′UTR

#

—

Note

Frequency as observed in 100 control chromosome: # Seven alleles of (CAG)

n

were observed in the range between n = 4 to n = 14, with a heterozygosity value of 0.75.

TABLE 3

Mutations of the invented gene in families with FHM or EA-2

Disease

Family

Location

Domain

Nucleotide change

Consequence

FHM

It-II

exon 4

I S4

nt 850

G - A

Arg

192 - Gln

R192Q

(gain of Sfcl site)

FHM

US-P

exon 16

P-segment

nt 2272

C - T

Thr

666

- Met

T666M

FHM

UK-B

exon 17

II S6

nt 2416

T - C

Val

214

- Ala

V714A

(gain of Bovl site)

FHM

NL-A/US-C

exon 36

IV S6

nt 5706

A - C

Ile

181

- Leu

I1811L

(gain of MnII site)

EA-2

CAN-191

exon 22

III S1

nt 4073

deletion C

frameshift

STOP

1294

(loss of NiaIV site)

EA-2

CAN-26

intron 24

space site

nt 4270-1

G - A

AC/gt - AC/at

aberrant

(loss of BsaAl site)

splicing

TABLE 4

Parental transmission of migraine for 36 unrelated Dutch families.

affected

parents

N

offspring

N

N (%)

ratio*

heathy father x migraine

51

daughters

72

48 (66.7%)

2.3:1

mother

sons

72

21 (29.2%)

migraine father x healthy

daughters

26

17 (65.4%)

2.5:1

mother

18

sons

15

4 (26.7%)

*ratio of proportion affected sons/proportion affected daughters

146

1

953

DNA

human

1
tttttttacg ttctcttttt tttcgagtgg tgactggatg ctgattcttc ctcgtatttt 60
tgctgcttct ctctccctcc cctccttccc gggcccgggc ccgccccgca ccctccttcc 120
gcccctcctt ctccggggtc agccaggaag atgtcccgag ctgctatccc cggctcggcc 180
cgggcagccg ccttctgagc ccccgacccg agcgccgagc cgccgcgcga tgggctgggc 240
cgtggagcgt ctccgcagtc gtagctccag ccgccgcgct cccagccccg gcagcctcag 300
catcagcggc ggcggcggcg gcggcggcgt cttccgcatc gttcgccgca gcgtaaccgg 360
agccctttgc tctttgcaga atggcccgct tcggagacga gatgccggcc cgctacgggg 420
gaggaggctc cggggcagcc gccggggtgg tcgtgggcag cggaggcggg cgaggagccg 480
ggggcagccg gcagggcggg cagcccgggg cgcaaaggat gtacaagcag tcaatggcgc 540
agagagcgcg gaccatggca ctctacaacc ccatccccgt ccgacagaac tgcctcacgg 600
ttaaccggtc tctcttcctc ttcagcgaag acaacgtggt gagaaaatac gccaaaagat 660
caccgaatgg ccatatcctt ttgcccgaac cccagcagca gctgcgcctc cccctcctcc 720
ctccgcctcc cctcttccag gctgggagag agacccgggg gttgatggga ggtggggagg 780
aggggggtct tccaggggct gggagagggg gcaccgggag gagtgtgaaa gaatctctcc 840
accccgagct gggttgagct accctggagg cttgggaatg ggtttttcgg gggctggggg 900
ccggccagcc ggagagtgga tccttcccaa ggaccgactc tagaatgaga tct 953

2

527

DNA

human

2
gatctttycc actggggtca gtgggggtgg gtgcacctcc aacacccttc ttttctttga 60
acaagatttt tccttaattc cccaatactc cctttgaata tatgatttta gccaccatca 120
tagcgaattg catcgtcctc gcactggagc agcatctgcc tgatgatgac aagaccccga 180
tgtctgaacg gctggtgagt gatgtctttt ctcagggtct tctccttggc tttagcagga 240
cattaatttt tgggggagtg gagcagggca cagaggaggc tctcagtcct ggagcccaga 300
gccagatcat gggaagccta aatttccttt tcattttttc ttgaaccaga gtctcgctct 360
gtcacccagg ctggagtgca gtggttcagt catagctcac tgcagcctcc acctcctggg 420
ctcaagccat cctcccactg cagcctcctg agtagcaggg actaacaggt gccaccatgc 480
ccagttaatt ttcttatttt tatctttttt tgtaagaaga tggggat 527

3

441

DNA

human

3
gatcttgtca acatctgccc agcccaagac gctgaccttg ccttctctcc cttccaggat 60
gacacagaac catacttcat tggaattttt tgtttcgagg ctggaattaa aatcattgcc 120
cttgggtttg ccttccacaa aggctcctac ttgaggaatg gctggaatgt catggacttt 180
gtggtggtgc taacggggta agtggcgcgt gctatacgct ttggatttaa ctagctgaag 240
gattacgagg cttttggttg gtgtggtccg ggccaggctc aggaaggctg agcccttgtg 300
ttctccctcc ccttgttatg cgcctgcctc ctttctgcca acaccccacc tccatgtctc 360
agctgtatat tacagcagat gctttctgtt acaattaaaa taatagctca ttattgttgg 420
ctgcttccag agtgctttat g 441

4

259

DNA

human

4
aaaactgagg ccagtggtgt cgagtcacct gcctgtggtc acccaaccaa tacaggacag 60
cttggaatcc caagcacccc cgccctgctg tctgaccccc aaaacccacc ctctgttctc 120
cattctggct tctttctttc agcatcttgg cgacagttgg gacggagttt gacctacgga 180
cgctgagggc agttcgagtg ctgcggccgc tcaagctggt gtctggaatc ccaagtgcgt 240
gagtttccga ccctgacaa 259

5

399

DNA

human

5
cttaatattc cctcaggaac acacctgctt tgtctgggag agacctgggc gtcttggtgg 60
cggggttttg ggggtacttg ctcatgggct tatggggcct ctctctgtgt ccccccaggt 120
ttacaagtcg tcctgaagtc gatcatgaag gcgatgatcc ctttgctgca gatcggcctc 180
ctcctatttt ttgcaatcct tatttttgca atcatagggt tagaatttta tatgggaaaa 240
tttcatacca cctgctttga agaggggaca ggtaggtcca cggagcatga tgcatctttc 300
cagttttctc cttcagggac aagctcttgg gaggattagg caggggtgtg cttctttctc 360
ctggcagctg ggaggaccgt ctccttcaga gagcactac 399

6

586

DNA

human

6
ttttttccct tcccttttgt agatgacatt cagggtgagt ctccggctcc atgtgggaca 60
gaagagcccg cccgcacctg ccccaatggg accaaatgtc agccctactg ggaagggccc 120
aacaacggga tcactcagtt cgacaacatc ctgtttgcag tgctgactgt tttccagtgc 180
ataaccatgg aagggtggac tgatctcctc tacaatgtaa gtgatgctgg gacagtgtgt 240
gtggacaatc agagtctcag ggaggtggcc tcctgggacc agtgagactc caaggctgca 300
atggagggac cctgagctgg gaaaggcagc ccaaggacaa cacagcccca ctgaagctgg 360
cctgaggctc aggcttttga agattacagg ggctcatgag cagaactcta actatagggc 420
atagaagtct ggagggcccc cagatgcaac atcatttttc attgtgcaag tgtttagata 480
taattttaga tttttgaata cggaaaggtt atgtgatcca aaatccaaca cagataaaag 540
atagagtaat atctttggac gtaggcgagg ggtccctgcc ctgagg 586

7

387

DNA

human

7
tttcttcaga aaacggttcc ttcctccatt tccccctctg ggatgccaga gccccagaac 60
tccacaagcc aagaacattt aagacagagc cacaagagaa ccgagcttcc ccttccctca 120
cctgtcaggt tctatctgag tcccagtcaa ctctcacctg ctttccctcc tcacacccta 180
cagagcaacg atgcctcagg gaacacttgg aactggttgt acttcatccc cctcatcatc 240
atcggctcct tttttatgct gaaccttgtg ctgggtgtgc tgtcagggta agtttctgct 300
actccccacc ccatcccact cactcctctt tgctaacttc tttccaagta gaggccattg 360
aagctttgtt ttcattcact agacaga 387

8

412

DNA

human

8
cccagtcttt tcccagaagt cctgactcct cctgttgaaa actcctgacc tccagggact 60
tctgaatccc caaacacaca cacacacaaa cacacacaca cacacacaca cacacacaca 120
caaacacaca cacaaacgtt tcctaacatt ttcaaaacag ccatactctg gcttttctat 180
gcttctccag ggagtttgcc aaagaaaggg aacgggtgga gaaccggcgg gcttttctga 240
agctgaggcg gcaacaacag attgaacgtg agctcaatgg gtacatggaa tggatctcaa 300
aagcaggtga ggccctttca tcctggggcc cagggatgga gatcccaggc cacagagtac 360
aaagagagtc atgcagtttg gagaaggcta agctgggagg gttatgatgg ga 412

9

611

DNA

human

9
gagtaggaag ttagaggcag ggtggtcagg gaaggcttct ctaaggaagt accctctgag 60
cagagagacc tgaaggacgt gaagaaggaa gctgtgggga tgtcaaggga aggggcattc 120
caggcagaga cagcaagtgc aaaggccctg agctaggaac gtatttgaga cacagcaagg 180
aagccagtgc agctgaaaca gagtgagagg tggggacagc tggaggagag gaagacagga 240
aggtgatgga gatcagatca agcaggggct tataggctgt ggtgtggaca ttggttttta 300
ttttgcgcga ggtggggaga atgttggcta ttgctactgt tgcggaggtg gggcttgaag 360
tcacaaacca cccagcagca tgttttttgg tcggttgagc tgtcaccatc agtcagcaga 420
gaatgggggt ggccgggcag acccttcttc ctggtccaag ggagaactca tcctccaaat 480
gcaggagctt aactctgtgc tcttcctctt cagaagaggt gatcctcgcc gaggatgaaa 540
ctgacgggga gcagaggcat ccctttgatg gtaactgctc taaacccacc tcaggggtgg 600
gtcccagggg a 611

10

656

DNA

human

Unsure

(638)..(638)

n = g, a, c, t or u

10
ttaatccaag acacactgtg tgtcctatat ggtctgtgtt cgaaaaaggg taacgtcttt 60
ttctcttgcc atgtttccat tgttaggagc tctgcggaga accaccataa agaaaagcaa 120
gacagatttg ctcaaccccg aagaggctga ggatcagctg gctgatatag cctctgtggg 180
tgagtccctt cctctgccac ctatcagttg ttcatcacct atcgcccaag agacatggtg 240
gggtgggggc agagggcttg caaaccgtgc tgcctggatt tgggtctcag ctccaccctt 300
tcccacctgt gcgtgtgtcc tgggcagatt acatcattat gggaataaca tccgtgccta 360
gcttctcatt attttgtggg aattcaacta aatgatcccc atgaagcatg gcaaaccagc 420
acctggcagg gacgaagctc ccagtcaagt tggtgaatgt ttgtgactca ttcgggaagt 480
atcatggggg acctgcttat attaggtgct tggttgcaaa caaacaaggc agtcacgagg 540
ctgagctggg aggatcactt gagcctggga agtggaggct gcaataagcc attattgtgt 600
tactgcactc cagcctggca cagaaaaaaa aaaaaaanac aaactgagcc agcaca 656

11

778

DNA

human

11
gatcacttct aaagttaaat gtccatggga aaacagtctc atccacatct ctttctggag 60
gccttccaag cgtgctccat gcagctctgt tgcctgcccc tgcatcaggg aatggaggct 120
ctgctttatc ctgccctgtg gtgtgactcc cagaggcatc agatgtggct gggagtggga 180
gacatggaaa attggctcct gcaacagaga actatcagcc ttcccatcaa ttggttactt 240
ctaattctgt tatttttcag gggcactgtc ttctcataag ctccatctat gcaaaactaa 300
gcccatgggt catgatggtt ccctcaggcc agaggcttgc tggagagact aatggatccc 360
ctggctaaaa tctgtgcttg ggctgcacat tggttaattt cttctgaagg aacagcctga 420
gcctgacatt ctccatcttt tccctggcag gttctccctt cgcccgagcc agcattaaaa 480
gtgccaagct ggagaactcg accttttttc acaaaaagga gaggaggatg cgtttctaca 540
tccgccgcat ggtcaaaact caggccttct actggactgt actcagtttg gtagctctca 600
acacgctgtg tgttgctatt gttcactaca accagcccga gtggctctcc gacttccttt 660
gtgagtatca cccagcccca cccctgccaa ctccctgatc cctccctcac accctttttc 720
cacttctctt tctctggtag tatgtgtatc ttctttggtc ctcattgaat ctgccctt 778

12

626

DNA

human

Unsure

(27)..(27)

n = g, a, c, t or u

12
gatcacttgt ggccaggagt tcaagancag ccagggcaac atagtgagga cccccatctc 60
cacattaaaa attttaaaaa gaaaaaagat aagtcagaag ttgggtgtgg tgacacatgc 120
ctgtagttct agcatgttgg aggccaaatc agggaaactg tttgaggcca ggagtttgaa 180
accagcctaa cagcatagca agacctcatc tctacaaaaa ataaaaagtt taaaaatgat 240
aataaaagga aagtcagagc cacctggaac ccctaccctc agcaagccta acctcctctc 300
tgtttcctcc ttctcccttc tagactatgc agaattcatt ttcttaggac tctttatgtc 360
cgaaatgttt ataaaaatgt acgggcttgg gacgcggcct tacttccact cttccttcaa 420
ctgctttgac tgtggggtaa gtgctcttgt ttctaagagt tcatttctcc agctcttgcc 480
tggaatgaca gatacctgga cacattaaag ggagaaaggt aaagtcaccc ctgaatatga 540
gagactcaga tggatgcaga aggaatgaga aaacaatcca aacactggca aggatacagt 600
gtacccagaa ccctcaacca ccgcca 626

13

976

DNA

human

Unsure

(5)..(7)

n = g, a, c, t or u

13
gatcngncat gcacaccagc ctgggtgata agagcaagac tcctctcaaa ataaatgaat 60
aaataaaaat aaataaataa ataagaggcc gggtgcagtg gctcaatgct ttggaaagtg 120
gaggccaaca gttggagaga ccaaagcagg aggatggctt cagcccagaa gtttgaggcc 180
mgcctgggca atactagcga gacactatct ctataaaaat gttttaaaat tagccagatg 240
tggtggggca cacctgtaat cccagctact caagaggctg aggtgggagg atcacttaag 300
cccaggagga cagtgctgca gtgagctatg attgcgccca ctgcactcca gcctgggtga 360
cacagtgaga cccggtctct atagataaat gaatggatga atgagggggt caaggatcct 420
cacccggctt ccatttggag ggaggagttt ggttgagttc ttgcaaggtt ggtacctagg 480
aaatgcttgc cagttctgga gcccagacac tgtccctgga catgagacca ggttctctgc 540
cctaggttat cattgggagc atcttcgagg tcatctgggc tgtcataaaa cctggcacat 600
cctttggaat cagcgtgtta cgagccctca ggttattgcg tattttcaaa ctcacaaagt 660
aagtctttgg ggttcctgga catttgtaca gggggtgggg atgggggaca tggtggggcc 720
gcctccagaa agttgggaaa gtgagcctcg tgtttcgagg gctgactccg gggcctgcct 780
wccccgcctg gcctgagtcc tcgcctggsc tctgtcggca ggtactgggc atctctcaga 840
aacctggtcg tctctctcct caactccatg aagtccatca tcagcctgtt gtttctcctt 900
ttcctgttca ttgtcgtctt cgcccttttg ggaatgcaac tcttcggcgg ccagtaagtc 960
cttcacagga attcaa 976

14

1110

DNA

human

Unsure

(620)..(620)

n = g, a, c, t or u

14
ccctccacgt gcaggctgcc ttcctcgtag cccagacacc catttgcggt cacccaaatg 60
ggcagggccc tgggtaccac tcagggtttc ctggggacag agatgatgga aacgttcgtt 120
tccttggaga tgagatactg agccacaccc tcagagcacc ccgggtgggg ccaacgtgaa 180
atgtctgtgt cctccctgca ggtttaattt cgatgaaggg actcctccca ccaacttcga 240
tacttttcca gcagcaataa tgacggtgtt tcaggtacag cctccacctg gccccacggg 300
ccaacacctc tcagtgtcac agatgaaagt gcctgctcca catccaaggg gcttccctga 360
actcctcctt ctctacctgg ccttttcaca ccactttgaa acacagattt tatggttatc 420
attattcaat tatggtgagg ccaacagatc aggagatgaa tgtcattgga aagatagttt 480
gtggctgggc acggtggctc acacccataa tcccagcact ttggccaggt acggtggctc 540
acacctgtaa tcccaacgct ttgggaagcc caggtgggcg gatcacttga gatcaggaat 600
tcgagaccag cctggccaan atggtgaaac cccatctcta ctaaaaatac aaaaattagc 660
cgggcgtggt agcacatgcc tgtaatccca gctactcggg agatgaggca caagaattgc 720
ttgaacctgg gaggcagagg ttgcagtgag ccaagatcgc gccactgcac tcmagcctgg 780
gcaacagagt gagactccat ctcaaaaaag caaaagaaaa aaaaaaccac tttgggaggt 840
caagatggga ggactacttg aggccaggag tttgagacaa gtctgggcaa catagtgaga 900
ctccgtctct gcaaaaaaat wataataata attagctggg catggtgata catacctcct 960
agctactagg gcagctgaag tggaaggatt gcttaagccc aggaggttga ggctgcagta 1020
agctacaatc acaccactat actccagcct gggcgagaga gcaaagccct gtctcaaaaa 1080
cgaaaagaaa gtttgttata ctcacagatc 1110

15

982

DNA

human

15
gatcctccca ccttggcctc ccaaagtgct gggattacag gcatgagcca tggcatgcgg 60
tctcttcctg ttcttataag ggcactaata ccatcatgaa gtcccccatg acctcatcta 120
accctagtta cctcttaaag gccccatctc caaataccat cccatcatag gttagggctt 180
caactcatga atttggaggc gggcacaatt tagtccataa caaatcccct taatcacatc 240
aagtaagaca gagttacagg agggtctgtg actcctccag ggtcccattt tcctagaagc 300
caggctaaga gccccacgac gcaggaacgg ccctttctac tcgcaaacaa agagaaaagc 360
caaggagaag ccaacacgga gtctggctct gcaaaccggg caggattgtt aaagacctcc 420
tgggctcggg gatggggtgg gcggattccg gctccacagc tgcatctcca aggggcccgt 480
ggctgagagg ggggttggct gtgtgtttct tcctcccctt tcagatcctg acgggcgaag 540
actggaacga ggtcatgtac gacgggatca agtctcaggg gggcgtgcag ggcggcatgg 600
tgttctccat ctatttcatt gtactgacgc tctttgggaa ctgtatcctt catggagaga 660
gagaagggga caggcctgga cctctggcag aggagaggtt gcaggggctc aagggagggt 720
actgagagac ccagataccc agggcccaag tggtgtccca ccagtggttg cttttcctga 780
ctcagacatt tgcagacacc ctcctgaatg tgttcttggc catcgctgtg gacaatctgg 840
ccaacgccca ggagctcacc aaggtggagg cggtgggaga atgtttctct ggcaaagtta 900
ccacctgccc atggcagatc aagcactttt ttggattaac tgagccacag gaaataacat 960
tttcaaatag atkaaaaaga tc 982

16

314

DNA

human

16
ccttggttct gattggtcga aatatttcaa atgttgcccc tggtcagcaa cagggtcaga 60
agtgagtccc caaggcctag ttcatgtttt gtgaacaaag attccacgtg ccttttcagg 120
acgagcaaga ggaagaagaa gcagcgaacc agaaacttgc cctacagaaa gccaaggagg 180
tggcagaagt gagtcctctg tccgcggcca acatgtctat agctgtgtaa gtcccctaat 240
ccctgggatg ctaccctggc tcctgaacgt gtccgaccac tatccaggca cagatttggg 300
aagcagtggg ggtg 314

17

1113

DNA

human

17
gcccctagcc aggtgggagc catggagggt tcttgagcag aggaggctgg gacctgactc 60
agatgctcac agactcctag cattcaggtg gggagtagag ggtggagagc aggagtggga 120
ggctgagatg tgggttggtt cgcctgggtc atccatccaa gctacagtgc ctagcaatgc 180
tctaagctcc tgtgaccatg ccactgcagg aaagagcaac agaagaatca aaagccagcc 240
aagtccgtgt gggagcagcg gaccagtgag atgcgaaagc agaacttgct ggccagccgg 300
gaggccctgt ataacgaaat ggacccggac gagcgctgga aggctgccta cacgcggcac 360
ctgcggccag acatgaagac gcacttggac cggccgctgg tggtggaccc gcaggagaac 420
cgcaacaaca acaccaacaa gagccgggcg gccgagccca ccgtggacca gcgcctcggc 480
cagcagcgcg ccgaggactt cctcaggaaa caggcccgct accacgatcg ggcccgggac 540
cccagcggct cggcgggcct ggacgcacgg aggccctggg cgggaagcca ggaggccgag 600
ctgagccggg aggaccccta cggccgcgag tcggaccacc acgcccggga gggcagcctg 660
gagcaacccg ggttctggga gggcgaggcc gagcgaggca aggccgggga cccccaccgg 720
aggcacgtgc accggcaggg gggcagcagg gagagccgca gcgggtcccc gcgcacgggc 780
gcggacgggg agcatcgacg tcatcgcgcg caccgcaggc ccggggagga gggtccggag 840
gacaaggcgg agcggagggc gcggcaccgc gagggcagcc ggccggcccg gggcggcgag 900
ggcgagggcg agggtcccga cgggggcgag cgcaggagaa ggcaccggca tggcgctcca 960
gccacgtacg agggggacgc gcggagggag gacaaggagc ggaggcatcg gaggaggaag 1020
taagtggagg tgacctcgaa tccgcagaat gacggtaaca ttaataatac aacagccaaa 1080
gtagcacgtg ctgtgtattt gttataaaat ata 1113

18

590

DNA

human

18
gtcctgaaac tttgcctttt aatcctaaat cattgttggt tctttttcat tcacttgcct 60
tcctcagaga gaaccagggc tccggggtcc ctgtgtcggg ccccaacctg tcaaccaccc 120
ggccaatcca gcaggacctg ggccgccaag acccacccct ggcagaggat attgacaaca 180
tgaagaacaa caagctggcc accgcggagt cggccgctcc ccacggcagc cttggccacg 240
ccggcctgcc ccagagccca gccaagatgg gaaacagcac cgaccccggc cccatgctgg 300
ccatccctgc catggccacc aacccccaga acgccgccag ccgccggacg cccaacaacc 360
cggggaaccc atccaatccc ggccccccca agacccccga gaatagcctt atcgtcacca 420
accccagcgg cacccagacc aattcagcta agactgccag gaaacccgac cacaccacag 480
tggacatccc cccagcctgc ccaccccccc tcaaccacac cgtcgtacaa ggtgagaccc 540
tctgctctca catcactggg caggggacct ggcgtcctgg agccagaggt 590

19

340

DNA

human

Unsure

(1)..(340)

n = g, a, c, t or u

19
ggagtacacc gaggagttcc cagagacttg tgggaaattg tggagggagc cctgtgttgg 60
ttcttgtccc aacagtgaac aaaaacgcca acccagaccc actgccaaaa aaagaggaag 120
agaagaagga ggaggaggaa gaagacgacc gtggggaaga cggccctaag ccaatgcctc 180
cctatagctc catgttcatc ctgtccacga ccaacccgtg agtatggccc ccgagcagag 240
ggcagggggg gctgggtctc ccaccagggt ggcggaannn nnnnnnnnnn nnnnnnnctc 300
ccaccagggt ggcggaagtc aggccagatt agagggcaat 340

20

477

DNA

human

20
gatctcagta gtggtaggta acatgagatt atggaagaaa agggtttgtg agcctgtggt 60
ctgagtggac ctctgcacgc ccatctgtct ccaacagcct tcgccgcctg tgccattaca 120
tcctgaacct gcgctacttt gagatgtgca tcctcatggt cattgccatg agcagcatcg 180
ccctggccgc cgaggaccct gtgcagccca acgcacctcg gaacaacgtg agtcccacag 240
agcacacccc ttcctagcct ggctgctctg cctcaggcca ctttctcctg catccaaaat 300
gctcataggt agggtgggat gttggggtca cccctaggca tagcccttat ggctgctggt 360
tgagagggga agctctgatt ccttggggat gctcttggga gcaagacatt ccttgaggca 420
gtttctctgt gagcctggtg gggtggaggt ggcccagagt gactggggct gaaaatt 477

21

168

DNA

human

21
gatccactgc tctcttgctt ttatccctta caggtgctgc gatactttga ctacgttttt 60
acaggcgtct ttacctttga gatggtgatc aaggtgagtg cagattataa gtgagaacac 120
acggtaattt ttttttttaa gcaagtgcag ggctgggcac agtggatc 168

22

368

DNA

human

22
gatctaagag ccggcaagcc agagctggct tccatcaggc aaaggggggc cgcctcatgg 60
ggcaggggct ccccactcct ccctgggagt cctctggcca ctgcccatcc ctgcaagatg 120
aggtggcctc attggcttcc ctgcctctcc ccgagaggct agagagtggg tggcagcacc 180
ccagggtggg gatcaggtgg gggttctgag caccctctct tctcccccac agatgattga 240
cctggggctc gtcctgcatc agggtgccta cttccgtgac ctctggaata ttctcgactt 300
catagtggtc agtggggccc tggtagcctt tgccttcacg taagtctctt cgcaagggtt 360
tcctcttg 368

23

515

DNA

human

23
gatcttaacc ccaagacact tcatctaaag gaaaaactgc cataatacac agattatttt 60
aggtcagctc actttactgc catctgctgg gaagttgtaa taatacaaat atccatacac 120
gatggctagg atgttatcag cacctccttt aatgtgttgt ccttgagcag tgtacaacct 180
gctcagctgt acatgataac cctgacagtc ccccccaccg caccccacca tctcccaatc 240
tcaccttgag ctttggcagc cgcttgatgg ttttaagagg tcgtagcacc cggaggactc 300
ggagggattt aatcgtgttg atgtcttttc ctttgctatt gccactgtgg aggaatgttt 360
aggtgggaag aagggaagag aggaagcaga ggtcaggttg ggtagggggc agcccacagc 420
tccatgggac cctacccttc ccaggcctag aagtctgggg tgagcttggc acaagcctgc 480
cctttcctgg tgaagagtgg tccattttac cctgt 515

24

406

DNA

human

24
ggccactgga ggcagaaggt tggcaggtcc ccagcccctc atgctctctg tcaactccac 60
cccacaggct gtgtttgact gtgtggtgaa ctcacttaaa aacgtcttca acatcctcat 120
cgtctacatg ctattcatgt tcatcttcgc cgtggtggct gtgcagctct tcaaggggaa 180
attcttccac tgcactgacg agtccaaaga gtttgagaaa gattgtcggt gggtctccgc 240
tttccagcac attcccattg gaaccagcag gtgggcaggg gggaagtggc tagaggcatt 300
ggccacttgg gctcagagac tggagaagtg atgagccttg gaagtgactc agttgcaacc 360
agcttggatc aagggtagaa agaaaaccgg ttttagaatt tgagtc 406

25

516

DNA

human

Unsure

(421)..(516)

n = g, a, c, t or u

25
gatctcaaac tcctggcctc aagtgataca tctgccttgg cctcctaaag tgttgggatt 60
acaggcgtga gcaccatgcc cggcctccaa gacctttctt attgctaagc tctcaggccc 120
tttatcctcc tgctccccag ggctcctcct ggatagattt ccagtcgggc cacttactgt 180
ggccagcctt ctcccgtgga cacggtgaag agggtcagca gagcccacag cacattgtcg 240
taatggaatt catacttctt ccactcccgg tctcgcgcct tcacctcatt cttctcgtag 300
aggaggtatt tgcctctgcc acagagagtg gggactgtta gtaaatggga aagaggggct 360
gtcttgcact tgtctttggt tatcagagac agggggaggg aaaggaagag tggtccacca 420
ncctagactg cttgggaagc agtgacttcc catcctgcca ccatgtgttc ctgtgcttca 480
taggggatgn cgtgtgcaat ctacttttna ggataa 516

26

489

DNA

human

26
accttcctca tcacccttgg gtccctgtct ctctccttcc tgccccttcc ctctccctgc 60
cccattcctt gcagggtcct caagcattcg gtggacgcca cctttgagaa ccagggcccc 120
agccccgggt accgcatgga gatgtccatt ttctacgtcg tctactttgt ggtgttcccc 180
ttcttctttg tcaatatctt tgtggccttg atcatcatca ccttccagga gcaaggggac 240
aagatgatgg aggaatacag cctggagaaa aatgaggtgc cacttccaat tccatctgtc 300
ctttaaaaac tggggacaca cacaaacttt aaaacacaca caacacccag gaaccccttt 360
ctaggggtac ctgggggagg gaacagaagc attgtcccaa ccgaatccag tcttcagggc 420
agcccttcat ggagtttcag aggaaacaca tcatatagtg tatgtatcag tcagtttaga 480
ctaggttat 489

27

512

DNA

human

Unsure

(1)..(512)

n = g, a, c, t or u

27
tagcccatgc aanaatgggg aaatgncagt gcaagttttg gcagttgntg acatctcaag 60
caactgtanc tgttgggata agaaagcaat ggtgagaagg aanagaganc ccaggaatcc 120
tggctggggg caananaggc agagactcaa gcagaagcac ttgagaaccg cgacgagtta 180
gacagagggt gcccggtgta cagccacctt cctcctgcct ctgccgctct caccactggc 240
ctctctcccg cagagggcct gcattgattt cgccatcagt gccaagccgc tgacccgaca 300
catgccgcag aacaagcaga gcttccagta ccgcatgtgg cagttcgtgg tgtctccgcc 360
tttcgagtac acgatcatgg ccatgatcgc cctcaacacc atcgtgctta tgatgaaggt 420
aagtgcccca caccagcccc cagcactant taacccccac ctcgttcctg cctctaccct 480
gataaaatga aaccatttgc agatttccca ga 512

28

411

DNA

human

Unsure

(306)..(309)

n = g, a, c, t or u

28
gggtctttcc tgaactgtgc ctcctaccag tgaggttgct cagaccttgc ctggggctgg 60
agtgttgcct ggagaacagc catgaagctg acctccccac ttcccacttc ccacccctgc 120
tcgctgaccc ctgctactcc tgcttctttc ccctagttct atggggcttc tgtggcttat 180
gaaaatgccc tgcgggtgtt caacatcgcc ttcacctccc tcttctctct ggaatgtgtg 240
ctgaaagcca tggcttttgg gattctggta agtaccacct tggggctaca gctatgggct 300
tggaanaanc ccaaggggga acaatgggtc ctggatgatg gtctcccaac gtggccccaa 360
gaaccccaac ctcaagggtg gcttcagtat cctgcccagt ggccacagat c 411

29

420

DNA

human

29
ctgtcccggg cactccgctg atgggcaact gtgcctctaa catgcaccgg ccagcctagg 60
gggccgggaa ccaagccctc tgttggcatc tctgtcttgt gggtccccat tctagaatta 120
tttccgcgat gcctggaaca tcttcgactt tgtgactgtt ctgggcagca tcaccgatat 180
cctcgtgact gagtttgggg taagtctccc tccagcttct ctctgggtga ctctgggctg 240
gacgaggcag gcggcagggg gcgggggagc ggtcccagag gcagtgtgtc ccggaagcca 300
tagctgcttg agccagcact tggccatgac cagagaggga gaactggggc cccggggaca 360
agggcagccc ctcaggaggg cattgtgggg agatgggggt aacaaagctt ggctgtaggg 420

30

342

DNA

human

30
ttaatagtgc tttctctctc cctccttatt tggggtctgg cttgcttttt tcctgttggt 60
tggcttcatg taggggcctc tgtgagtggt gacagctctg agcctttggg gtgggtggat 120
ggtcacccct cttcctccat ctccccagaa taacttcatc aacctgagct ttctccgcct 180
cttccgagct gcccggctca tcaaacttct ccgtcagggt tacaccatcc gcattcttct 240
ctggaccttt gtgcagtcct tcaaggtgag tcctcgtccc tgctgctggc ccaggggctg 300
agaagacagg tgaccctcat gctctggctg aatgtagaag tc 342

31

559

DNA

human

Unsure

(536)..(536)

n = g, a, c, t or u

31
cccccaagaa gaatgcccac caagccctgg aaggactctg gcacgtggca tatggccacc 60
caacccagtg gggcagagca ctgggacaag ggaggaagac tgcagtgcgg ctgagggacc 120
cccagcactc ttcttcattg ccttttttcc caccaggccc tgccttatgt ctgtctgctg 180
atcgccatgc tcttcttcat ctatgccatc attgggatgc aggtgagtgt cgtgtcccta 240
aggttcccag agcctcccaa ggagggcagc cacccttaga aaggggtggg tcagaggagc 300
ctggttcaca gaagcagcca tggaggttga gctgggtttc ccagaagcca ctggaggaat 360
ggcagcccct ggtcgtcacc cwmcaattcc acaggtgttt ggtaacattg gcatcgacgt 420
ggaggacgag gacagtgatg aagatgagtt ccaaatcact gagcacaata acttccggac 480
cttcttccag gccctcatgc tctcttccgg tcagaagggg acctgctctg ataatnctgt 540
ttccgtgggg tggggtgcc 559

32

316

DNA

human

32
tcagagccat gctcactgtg tgctccactc ctgaggaggc gttggtacca gtcagggctg 60
ggtgtccgag tctctgattt ctccctgtcc tcaggagtgc caccggggaa gcttggcaca 120
acatcatgct ttcctgcctc agcgggaaac cgtgtgataa gaactctggc atcctgactc 180
gagagtgtgg caatgaattt gcttattttt actttgtttc cttcatcttc ctctgctcgt 240
ttctggtgag tctgtggaca ctgtgagggc cgtctgggct ccctaagcct ggcttccttt 300
cagggagtgg ttctgt 316

33

694

DNA

human

Unsure

(413)..(413)

n = g, a, c, t or u

33
gtgtagtgag aactcacctc tccattcccc agtctctttc tgtctctgtc tcatttcctt 60
tccccatctt ctctctatcc ctctctccat ctggggcctc tgtgtctgtc tttgggtctg 120
tctgtccgtc tgactgtctg tatccttctc acttcactca ttcattccct cggtctctgc 180
cccattctct cttggtcccg ggtccccaca gatgctgaat ctctttgtcg ccgtcatcat 240
ggacaacttt gagtacctca cccgagactc ctccatcctg ggcccccacc acctggatga 300
gtacgtgcgt gtctgggccg agtatgaccc cgcagcttgg taagaagtca ccccgaatcc 360
tccagccaca atactcacct ctccctggaa ctggaacacg ggctaggcta ggnccccaga 420
ctctggagca ctgaactcct ggggctccta gcaggggtct cacaggttca gtcaggagag 480
aagatataag aatcatcacc cttgcatacc ccagattaaa cacgtagggt gccaacctgc 540
ccaaaccctg gaggactttc tgggaaatga ggagggcgtc aaccatgaga tgtctgaaga 600
gccctctcct cctacgagtc tctcctgtct ctcactgtga agtctccaga tggtgaggat 660
cgattagcca ggctccagga gaaaccaaca gact 694

34

474

DNA

human

34
aagggaggtg cctgcagtcc cgaactcgac tgacatccta cacccctggg tctccccagt 60
gtctgggaat gtactgggaa ttcacttgtc cccagtctct cccactcctt gaagccaggg 120
acaccccagc ctcgggcatc atgacctcgt tgtgtgccca gggagcccgt gtgaacccat 180
tgcctgcact aacccccttt cttctccttt cagcggtcgg attcattata aggatatgta 240
cagtttatta cgagtaatat ctccccctct cggcttaggc aagaaatgtc ctcatagggt 300
tgcttgcaag gtttgacttc cactaaaacc tgctagcatc catggaatga gtgtggcttg 360
gggttcttca atatatatat ttcatatata tatatatata tatctctctc tctctaaaaa 420
aacagagcca tctctctttc ttgcattaaa ctagaaaact ctcttagcca acag 474

35

413

DNA

human

Unsure

(323)..(413)

n = g, a, c, t or u

35
cctgggtagg ggcgggcgcg gctcacggga gacccaggag ggatgcctgg gaatgactgc 60
gcttgccttg ggttttctgt agcggcttct gcggatggac ctgcccgtcg cagatgacaa 120
caccgtccac ttcaattcca ccctcatggc tctgatccgc acagccctgg acatcaagat 180
tgccaagggt aaggaaggga caggggcggg cacagacagg cgtgacaggg tggaactggg 240
gatctcctcc ctaccccaaa ctagaggatc tgctgtcacc acccggatct tcattcactc 300
ttccattcat tcgttccaca ggnntttttg gnnnttggnn ntttggtgtt tttttttttt 360
ttttgagaca gagtcttgct ctgttgccca ggcagcagtg cggtgacatg atc 413

36

636

DNA

human

Unsure

(332)..(332)

n = g, a, c, t or u

36
gggtctcgtt ctcgggagcc tatggctttg cagctgaccc agagtccagc tgacacccag 60
gcaggcagtc agggtctgtc tacaccccca ttgcaggagg agccgacaaa cagcagatgg 120
acgctgagct gcggaaggag atgatggcga tttggcccaa tctgtcccag aagacgctag 180
acctgctggt cacacctcac aagtgtaaga gctgagccca gccctgggat ccaatccacc 240
aggacagatg gagggggagg gaaaggggag gcctggggag agtgttggct gggctggtat 300
acacagggac ccaggacaag gtccccaaag angcctgccc ttggtgagct caccgtgtgt 360
gtcccccagc cacggacctc accgtgggga agatctacgc agccatgatg atcatggagt 420
actaccggca gagcaaggcc aagaagctgc aggccatgcg cgaggagcag gtgcgctgtt 480
cgccgctctg gggacatctg ggctggggac agtggcttgc atgtcaccac gggaaccaac 540
tggaatatga gggtggctga gccccagggc aggtccctga aaagtagggg ctggtgcaca 600
gcagctcaca cctgcaatct cagtgctttg agaggc 636

37

829

DNA

human

37
gatcttcagg gccatgggag ctgcaggaag gactctggct ttttccccaa gcaagtggga 60
gccatggagg gttctaagca aaggagggat aggacctgac tcaagtgctc atgggcgccc 120
tctggtggct cttgtggaac agtggggttg aaggtaggag cgggagacct gggagaaggt 180
gcctgcagtg agagatgagg acgcgggacc aggctggggc tatgacttgg gtggaggagt 240
gagaagtggt ccagttctgc gtggaattgg aagggtctag atggatgaga cctgagagag 300
tgtgtgtgtg tgtgtgtgtg tatactgggg atgtcgcaat gccttctggg taccaccgtc 360
caccacccca cccttgtcca cacactgctc tctgccccat tccccaggac cggacacccc 420
tcatgttcca gcgcatggag cccccgtccc caacgcagga agggggacct ggccagaacg 480
ccctcccctc cacccagctg gacccaggag gagccctgtg agtgtcaccc ctgccaggga 540
ggtggagtgt gggggtgccg tggtccccac gttctggaag ctgcccaagc gcccactgct 600
accccggcct ctgtccccca tgcaggatgg ctcacgaaag cggcctcaag gagagcccgt 660
cctgggtgac ccagcgtgcc caggagatgt tccagaagac gggcacatgg agtccggaac 720
aaggcccccc taccgacatg cccaacagcc agcctaactc tcaggtgcct ctgtccccca 780
actccccaat ggctcccagg gcccgggtgg ttgcggtgga aggaaccat 829

38

801

DNA

human

Unsure

(161)..(161)

n = g, a, c, t or u

38
tcactgcaac ctccaccttc cagtctcaag tgattcctcc tgcctcagcc tcccaagtca 60
ctggattaca ggcgcccacc accatgctca ggtatttttt tttgtatttt tagtagagac 120
ggggtttcac aatgttggtc aggctggtct cgaactgctg nccattgtga tctggaggtc 180
aggccccaga gctcatctgg ctttgccatt cgtccgcagt ccgtggagat gcgagagatg 240
ggcagagatg gctactccga cagcgagcac tacctcccca tggaaggcca gggccgggct 300
gcctccatgc cccgcctccc tgcagagaac caggtgaggg ctttcaccac tgccctgggg 360
ctggacccct cactctgcac tgggtagggc caggcccccc cacaagcagc ccagtgcatc 420
ccctcctgcc ggactcaggc ctgggtaggg actccttcag tctctgaagc agtctgcagg 480
ccccacccac cacctggtca cacctggagc acctgcagac cctcctccct cacagaggac 540
agagaggaaa gtgctccccc tggggcagag ggcagtggcc actgcaaaat ggtctctggc 600
tgccctggtt ggaggctgca gacaggggag gttgtggaar atttgtgggt gcagcagggt 660
tcaacagggc cagctgagac ctgccacgaa gawcctttga ggccaggagt ttgagaccag 720
gttgggcaac atagcaaaac cctgtctctt tttttttttt gagacggagt ttcactcttg 780
ttgccccagg ctggagtgac a 801

39

329

DNA

human

Unsure

(177)..(177)

n = g, a, c, t or u

39
cctcctcact cttccctctt gcctttatat ttattttctt ctttctgttt tttctgtgtg 60
caccatccat ggggctgtga cagaggagaa ggggccggcc acgtgggaat aacctcagtg 120
tatgtacggc ctgcccaggg cccagcaggc tccggccccc tcttcctccc caccccncct 180
ccagggagtc ccgtaatctc taccggtccc cggaccccac cctttctttg gcaatcgcac 240
cctctcccct ccatggagcc caatccttgt gtgtggtgtc ctgtgtgtgc cctgacccat 300
aagcctggtg gggcggccat ccccatcct 329

40

554

DNA

human

40
gatcaggggg agccaaggcc ccatggcatc ccctggcccc tgccccagga tggtcacacc 60
gcagtcaccg aaggccacca ccaggctgcc acaatggggc aggaaggacc gggaccactt 120
ggtgctagct gctgacccca gcccaccggc ctgtcccctc ccccagacca tctcagacac 180
cagccccatg aagcgttcag cctccgtgct gggccccaag gcccgacgcc tggacgatta 240
ctcgctggag cgggtcccgc ccgaggagaa ccagcggcac caccagcggc gccgcgaccg 300
cagccaccgc gcctctgagc gctccctggg ccgctacacc gatgtggaca caggtgggca 360
gccctgtggt gctcagggac aagcagaaca gaggagagga gaggggagga gaaggcaggg 420
cggaggagac actaaggaag aagaaaggga gaggcctcca tggagagggg acagagcggg 480
ccaggcagcg gctgcaggaa cctgggtact accccctccc cccaacccac tgacctgcct 540
cggttcaggg gatc 554

41

461

DNA

human

41
ctgtgtgctg tctgaccctc acccggccca ggcttgggga cagacctgag catgaccacc 60
caatccgggg acctgccgtc gaaggagcgg gaccaggagc ggggccggcc caaggatcgg 120
aagcatcgac agcaccacca ccaccaccac caccaccacc atcccccgcc ccccgacaag 180
gaccgctatg cccaggaacg gccggaccac ggccgggcac gggctcggga ccagcgctgg 240
tcccgctcgc ccagcgaggg ccgagagcac atggcgcacc ggcaggtggg tgcggctgca 300
agtgacccca ggctgggctc ggccgggagg cggggaggag agaaggggat accccatcca 360
acagccactc taggcaaagg tccccggatc ccggctgtga ccacctccca tcctgccccc 420
aagccaccgg ggtgcccggc ggccggagcg gagcacggat c 461

42

664

DNA

HUMAN

42
tttctcattt ctcttttcac ttttgttgtg ttggtttccg actcctcccc tccctgtctc 60
actccccctc ctcccctccc tcctccctgt ggctgttgct tttttccatt caatgtcctg 120
tgtcccccct ctcctcctcc tcctcctcct ccccctcctc cctctcctcc cggcccctct 180
cccttcgctc ccctcatctt cctcccaatc ccgtgtctcc tttgattttg ttgtatcttt 240
ttttttgatt tcctttgttt caattttcgt gtagggcagt agttccgtaa gtggaagccc 300
agccccctca acatctggta ccagcactcc gcggcggggc cgccgccagc tcccccagac 360
cccctccacc ccccggccac acgtgtccta ttcccctgtg atccgtaagg ccggcggctc 420
ggggcccccg cagcagcagc agcagcagca gcagcagcag caggcggtgg ccaggccggc 480
cgggcggcca ccagcggccc tcggaggtac ccaggcccca cggccgagcc tctggccgga 540
gatcggcgcc cacggggggc cacagcagcg gccgcacgcc caggatggag aggcgggtcc 600
aggcccggcc cggagcgagt ctccagggcc tggtcgacac ggcggggccc ggctggcggc 660
agtc 664

43

6789

DNA

HUMAN

43
atggcccgct tcggagacga gatgccggcc cgctacgggg gaggaggctc cggggcagcc 60
gccggggtgg tcgtgggcag cggaggcggg cgaggagccg ggggcagccg gcagggcggg 120
cagcccgggg cgcaaaggat gtacaagcag tcaatggcgc agagagcgcg gaccatggca 180
ctctacaacc ccatccccgt ccgacagaac tgcctcacgg ttaaccggtc tctcttcctc 240
ttcagcgaag acaacgtggt gagaaaatac gccaaaaaga tcaccgaatg gcctcccttt 300
gaatatatga ttttagccac catcatagcg aattgcatcg tcctcgcact ggagcagcat 360
ctgcctgatg atgacaagac cccgatgtct gaacggctgg atgacacaga accatacttc 420
attggaattt tttgtttcga ggctggaatt aaaatcattg cccttgggtt tgccttccac 480
aaaggctcct acttgaggaa tggctggaat gtcatggact ttgtggtggt gctaacgggc 540
atcttggcga cagttgggac ggagtttgac ctacggacgc tgagggcagt tcgagtgctg 600
cggccgctca agctggtgtc tggaatccca agtttacaag tcgtcctgaa gtcgatcatg 660
aaggcgatga tccctttgct gcagatcggc ctcctcctat tttttgcaat ccttattttt 720
gcaatcatag ggttagaatt ttatatggga aaatttcata ccacctgctt tgaagagggg 780
acagatgaca ttcagggtga gtctccggct ccatgtggga cagaagagcc cgcccgcacc 840
tgccccaatg ggaccaaatg tcagccctac tgggaagggc ccaacaacgg gatcactcag 900
ttcgacaaca tcctgtttgc agtgctgact gttttccagt gcataaccat ggaagggtgg 960
actgatctcc tctacaatag caacgatgcc tcagggaaca cttggaactg gttgtacttc 1020
atccccctca tcatcatcgg ctcctttttt atgctgaacc ttgtgctggg tgtgctgtca 1080
ggggagtttg ccaaagaaag ggaacgggtg gagaaccggc gggcttttct gaagctgagg 1140
cggcaacaac agattgaacg tgagctcaat gggtacatgg aatggatctc aaaagcagaa 1200
gaggtgatcc tcgccgagga tgaaactgac ggggagcaga ggcatccctt tgatggagct 1260
ctgcggagaa ccaccataaa gaaaagcaag acagatttgc tcaaccccga agaggctgag 1320
gatcagctgg ctgatatagc ctctgtgggt tctcccttcg cccgagccag cattaaaagt 1380
gccaagctgg agaactcgac cttttttcac aaaaaggaga ggaggatgcg tttctacatc 1440
cgccgcatgg tcaaaactca ggccttctac tggactgtac tcagtttggt agctctcaac 1500
acgctgtgtg ttgctattgt tcactacaac cagcccgagt ggctctccga cttcctttac 1560
tatgcagaat tcattttctt aggactcttt atgtccgaaa tgtttataaa aatgtacggg 1620
cttgggacgc ggccttactt ccactcttcc ttcaactgct ttgactgtgg ggttatcatt 1680
gggagcatct tcgaggtcat ctgggctgtc ataaaacctg gcacatcctt tggaatcagc 1740
gtgttacgag ccctcaggtt attgcgtatt ttcaaagtca caaagtactg ggcatctctc 1800
agaaacctgg tcgtctctct cctcaactcc atgaagtcca tcatcagcct gttgtttctc 1860
cttttcctgt tcattgtcgt cttcgccctt ttgggaatgc aactcttcgg cggccagttt 1920
aatttcgatg aagggactcc tcccaccaac ttcgatactt ttccagcagc aataatgacg 1980
gtgtttcaga tcctgacggg cgaagactgg aacgaggtca tgtacgacgg gatcaagtct 2040
caggggggcg tgcagggcgg catggtgttc tccatctatt tcattgtact gacgctcttt 2100
gggaactaca ccctcctgaa tgtgttcttg gccatcgctg tggacaatct ggccaacgcc 2160
caggagctca ccaaggacga gcaagaggaa gaagaagcag cgaaccagaa acttgcccta 2220
cagaaagcca aggaggtggc agaagtgagt cctctgtccg cggccaacat gtctatagct 2280
gtgaaagagc aacagaagaa tcaaaagcca gccaagtccg tgtgggagca gcggaccagt 2340
gagatgcgaa agcagaactt gctggccagc cgggaggccc tgtataacga aatggacccg 2400
gacgagcgct ggaaggctgc ctacacgcgg cacctgcggc cagacatgaa gacgcacttg 2460
gaccggccgc tggtggtgga cccgcaggag aaccgcaaca acaacaccaa caagagccgg 2520
gcggccgagc ccaccgtgga ccagcgcctc ggccagcagc gcgccgagga cttcctcagg 2580
aaacaggccc gctaccacga tcgggcccgg gaccccagcg gctcggcggg cctggacgca 2640
cggaggccct gggcgggaag ccaggaggcc gagctgagcc gggaggaccc ctacggccgc 2700
gagtcggacc accacgcccg ggagggcagc ctggagcaac ccgggttctg ggagggcgag 2760
gccgagcgag gcaaggccgg ggacccccac cggaggcacg tgcaccggca ggggggcagc 2820
agggagagcc gcagcgggtc cccgcgcacg ggcgcggacg gggagcatcg acgtcatcgc 2880
gcgcaccgca ggcccgggga ggagggtccg gaggacaagg cggagcggag ggcgcggcac 2940
cgcgagggca gccggccggc ccggggcggc gagggcgagg gcgagggtcc cgacgggggc 3000
gagcgcagga gaaggcaccg gcatggcgct ccagccacgt acgaggggga cgcgcggagg 3060
gaggacaagg agcggaggca tcggaggagg aaagagaacc agggctccgg ggtccctgtg 3120
tcgggcccca acctgtcaac cacccggcca atccagcagg acctgggccg ccaagaccca 3180
cccctggcag aggatattga caacatgaag aacaacaagc tggccaccgc ggagtcggcc 3240
gctccccacg gcagccttgg ccacgccggc ctgccccaga gcccagccaa gatgggaaac 3300
agcaccgacc ccggccccat gctggccatc cctgccatgg ccaccaaccc ccagaacgcc 3360
gccagccgcc ggacgcccaa caacccgggg aacccatcca atcccggccc ccccaagacc 3420
cccgagaata gccttatcgt caccaacccc agcggcaccc agaccaattc agctaagact 3480
gccaggaaac ccgaccacac cacagtggac atccccccag cctgcccacc ccccctcaac 3540
cacaccgtcg tacaagtgaa caaaaacgcc aacccagacc cactgccaaa aaaagaggaa 3600
gagaagaagg aggaggagga agaagacgac cgtggggaag acggccctaa gccaatgcct 3660
ccctatagct ccatgttcat cctgtccacg accaaccccc ttcgccgcct gtgccattac 3720
atcctgaacc tgcgctactt tgagatgtgc atcctcatgg tcattgccat gagcagcatc 3780
gccctggccg ccgaggaccc tgtgcagccc aacgcacctc ggaacaacgt gctgcgatac 3840
tttgactacg tttttacagg cgtctttacc tttgagatgg tgatcaagat gattgacctg 3900
gggctcgtcc tgcatcaggg tgcctacttc cgtgacctct ggaatattct cgacttcata 3960
gtggtcagtg gggccctggt agcctttgcc ttcactggca atagcaaagg aaaagacatc 4020
aacacgatta aatccctccg agtcctccgg gtgctacgac ctcttaaaac catcaagcgg 4080
ctgccaaagc tcaaggctgt gtttgactgt gtggtgaact cacttaaaaa cgtcttcaac 4140
atcctcatcg tctacatgct attcatgttc atcttcgccg tggtggctgt gcagctcttc 4200
aaggggaaat tcttccactg cactgacgag tccaaagagt ttgagaaaga ttgtcgaggc 4260
aaatacctcc tctacgagaa gaatgaggtg aaggcgcgag accgggagtg gaagaagtat 4320
gaattccatt acgacaatgt gctgtgggct ctgctgaccc tcttcaccgt gtccacggca 4380
gaaggctggc cacaggtcct caagcattcg gtggacgcca cctttgagaa ccagggcccc 4440
agccccgggt accgcatgga gatgtccatt ttctacgtcg tctactttgt ggtgttcccc 4500
ttcttctttg tcaatatctt tgtggccttg atcatcatca ccttccagga gcaaggggac 4560
aagatgatgg aggaatacag cctggagaaa aatgagaggg cctgcattga tttcgccatc 4620
agtgccaagc cgctgacccg acacatgccg cagaacaagc agagcttcca gtaccgcatg 4680
tggcagttcg tggtgtctcc gcctttcgag tacacgatca tggccatgat cgccctcaac 4740
accatcgtgc ttatgatgaa gttctatggg gcttctgtgg cttatgaaaa tgccctgcgg 4800
gtgttcaaca tcgccttcac ctccctcttc tctctggaat gtgtgctgaa agccatggct 4860
tttgggattc tgaattattt ccgcgatgcc tggaacatct tcgactttgt gactgttctg 4920
ggcagcatca ccgatatcct cgtgactgag tttgggaata acttcatcaa cctgagcttt 4980
ctccgcctct tccgagctgc ccggctcatc aaacttctcc gtcagggtta caccatccgc 5040
attcttctct ggacctttgt gcagtccttc aaggccctgc cttatgtctg tctgctgatc 5100
gccatgctct tcttcatcta tgccatcatt gggatgcagg tgtttggtaa cattggcatc 5160
gacgtggagg acgaggacag tgatgaagat gagttccaaa tcactgagca caataacttc 5220
cggaccttct tccaggccct catgcttctc ttccggagtg ccaccgggga agcttggcac 5280
aacatcatgc tttcctgcct cagcgggaaa ccgtgtgata agaactctgg catcctgact 5340
cgagagtgtg gcaatgaatt tgcttatttt tactttgttt ccttcatctt cctctgctcg 5400
tttctgatgc tgaatctctt tgtcgccgtc atcatggaca actttgagta cctcacccga 5460
gactcctcca tcctgggccc ccaccacctg gatgagtacg tgcgtgtctg ggccgagtat 5520
gaccccgcag cttgcggtcg gattcattat aaggatatgt acagtttatt acgagtaata 5580
tctccccctc tcggcttagg caagaaatgt cctcataggg ttgcttgcaa gcggcttctg 5640
cggatggacc tgcccgtcgc agatgacaac accgtccact tcaattccac cctcatggct 5700
ctgatccgca cagccctgga catcaagatt gccaagggag gagccgacaa acagcagatg 5760
gacgctgagc tgcggaagga gatgatggcg atttggccca atctgtccca gaagacgcta 5820
gacctgctgg tcacacctca caagtccacg gacctcaccg tggggaagat ctacgcagcc 5880
atgatgatca tggagtacta ccggcagagc aaggccaaga agctgcaggc catgcgcgag 5940
gagcaggacc ggacacccct catgttccag cgcatggagc ccccgtcccc aacgcaggaa 6000
gggggacctg gccagaacgc cctcccctcc acccagctgg acccaggagg agccctgatg 6060
gctcacgaaa gcggcctcaa ggagagcccg tcctgggtga cccagcgtgc ccaggagatg 6120
ttccagaaga cgggcacatg gagtccggaa caaggccccc ctaccgacat gcccaacagc 6180
cagcctaact ctcagtccgt ggagatgcga gagatgggca gagatggcta ctccgacagc 6240
gagcactacc tccccatgga aggccagggc cgggctgcct ccatgccccg cctccctgca 6300
gagaaccaga ggagaagggg ccggccacgt gggaataacc tcagtaccat ctcagacacc 6360
agccccatga agcgttcagc ctccgtgctg ggccccaagg cccgacgcct ggacgattac 6420
tcgctggagc gggtcccgcc cgaggagaac cagcggcacc accagcggcg ccgcgaccgc 6480
agccaccgcg cctctgagcg ctccctgggc cgctacaccg atgtggacac aggcttgggg 6540
acagacctga gcatgaccac ccaatccggg gacctgccgt cgaaggagcg ggaccaggag 6600
cggggccggc ccaaggatcg gaagcatcga cagcaccacc accaccacca ccaccaccac 6660
catcccccgc cccccgacaa ggaccgctat gcccaggaac ggccggacca cggccgggca 6720
cgggctcggg accagcgctg gtcccgctcg cccagcgagg gccgagagca catggcgcac 6780
cggcagtag 6789

44

2262

PRT

human

44
Met Ala Arg Phe Gly Asp Glu Met Pro Ala Arg Tyr Gly Gly Gly Gly
1 5 10 15
Ser Gly Ala Ala Ala Gly Val Val Val Gly Ser Gly Gly Gly Arg Gly
20 25 30
Ala Gly Gly Ser Arg Gln Gly Gly Gln Pro Gly Ala Gln Arg Met Tyr
35 40 45
Lys Gln Ser Met Ala Gln Arg Ala Arg Thr Met Ala Leu Tyr Asn Pro
50 55 60
Ile Pro Val Arg Gln Asn Cys Leu Thr Val Asn Arg Ser Leu Phe Leu
65 70 75 80
Phe Ser Glu Asp Asn Val Val Arg Lys Tyr Ala Lys Lys Ile Thr Glu
85 90 95
Trp Pro Pro Phe Glu Tyr Met Ile Leu Ala Thr Ile Ile Ala Asn Cys
100 105 110
Ile Val Leu Ala Leu Glu Gln His Leu Pro Asp Asp Asp Lys Thr Pro
115 120 125
Met Ser Glu Arg Leu Asp Asp Thr Glu Pro Tyr Phe Ile Gly Ile Phe
130 135 140
Cys Phe Glu Ala Gly Ile Lys Ile Ile Ala Leu Gly Phe Ala Gly His
145 150 155 160
Lys Gly Ser Tyr Leu Arg Asn Gly Trp Asn Val Met Asp Phe Val Val
165 170 175
Val Leu Thr Gly Ile Leu Ala Thr Val Gly Thr Glu Phe Asp Leu Arg
180 185 190
Thr Leu Arg Ala Val Arg Val Leu Arg Pro Leu Lys Leu Val Ser Gly
195 200 205
Ile Pro Ser Leu Gln Val Val Leu Lys Ser Ile Met Lys Ala Met Ile
210 215 220
Pro Leu Leu Gln Ile Gly Leu Leu Leu Phe Phe Ala Ile Leu Ile Phe
225 230 235 240
Ala Ile Ile Gly Leu Glu Phe Tyr Met Gly Lys Phe His Thr Thr Cys
245 250 255
Phe Glu Glu Gly Thr Asp Asp Ile Gln Gly Glu Ser Pro Ala Pro Cys
260 265 270
Gly Thr Glu Glu Pro Ala Arg Thr Cys Pro Asn Gly Thr Lys Cys Gln
275 280 285
Pro Tyr Trp Glu Gly Pro Asn Asn Gly Ile Thr Gln Phe Asp Asn Ile
290 295 300
Leu Phe Ala Val Leu Thr Val Phe Gln Cys Ile Thr Met Glu Gly Trp
305 310 315 320
Thr Asp Leu Leu Tyr Asn Ser Asn Asp Ala Ser Gly Asn Thr Trp Asn
325 330 335
Trp Leu Tyr Phe Ile Pro Leu Ile Ile Ile Gly Ser Phe Phe Met Leu
340 345 350
Asn Leu Val Leu Gly Val Leu Ser Gly Glu Phe Ala Lys Glu Phe Glu
355 360 365
Arg Val Glu Asn Arg Arg Ala Phe Leu Lys Leu Arg Arg Gln Gln Gln
370 375 380
Ile Glu Arg Glu Leu Asn Gly Tyr Met Glu Trp Ile Ser Lys Ala Glu
385 390 395 400
Glu Val Ile Leu Ala Glu Asp Glu Thr Asp Gly Glu Gln Arg His Pro
405 410 415
Phe Asp Gly Ala Leu Arg Arg Thr Thr Ile Lys Lys Ser Lys Thr Asp
420 425 430
Leu Leu Asn Pro Glu Glu Ala Glu Asp Gln Leu Ala Asp Ile Ala Ser
435 440 445
Val Gly Ser Pro Phe Ala Arg Ala Ser Ile Lys Ser Ala Lys Leu Glu
450 455 460
Asn Ser Thr Phe Phe His Lys Lys Glu Arg Arg Met Arg Phe Tyr Ile
465 470 475 480
Arg Arg Met Val Lys Thr Gln Ala Phe Tyr Trp Thr Val Leu Ser Leu
485 490 495
Val Ala Leu Asn Thr Leu Cys Val Ala Ile Val His Tyr Asn Gln Pro
500 505 510
Glu Trp Leu Ser Asp Phe Leu Tyr Tyr Ala Glu Phe Ile Phe Leu Gly
515 520 525
Leu Phe Met Ser Glu Met Phe Ile Lys Met Tyr Gly Leu Gly Thr Arg
530 535 540
Pro Tyr Phe His Ser Ser Phe Asn Cys Phe Asp Cys Gly Val Ile Ile
545 550 555 560
Gly Ser Ile Phe Glu Val Ile Trp Ala Val Ile Lys Pro Gly Thr Ser
565 570 575
Phe Gly Ile Ser Val Leu Arg Ala Leu Arg Leu Leu Arg Ile Phe Lys
580 585 590
Val Thr Lys Tyr Trp Ala Ser Leu Arg Asn Leu Val Val Ser Leu Leu
595 600 605
Asn Ser Met Lys Ser Ile Ile Ser Leu Leu Phe Leu Leu Phe Leu Phe
610 615 620
Ile Val Val Phe Ala Leu Leu Gly Met Gln Leu Phe Gly Gly Gln Phe
625 630 635 640
Asn Phe Asp Glu Gly Thr Pro Pro Thr Asn Phe Asp Thr Phe Pro Ala
645 650 655
Ala Ile Met Thr Val Phe Gln Ile Leu Thr Gly Glu Asp Trp Asn Glu
660 665 670
Val Met Tyr Asp Gly Ile Lys Ser Gln Gly Gly Val Gln Gly Gly Met
675 680 685
Val Phe Ser Ile Tyr Phe Ile Val Leu Thr Leu Phe Gly Asn Tyr Thr
690 695 700
Leu Leu Asn Val Phe Leu Ala Ile Ala Val Asp Asn Leu Ala Asn Ala
705 710 715 720
Gln Glu Leu Thr Lys Asp Glu Gln Glu Glu Glu Glu Ala Ala Asn Gln
725 730 735
Lys Leu Ala Leu Gln Lys Ala Lys Glu Val Ala Glu Val Ser Pro Leu
740 745 750
Ser Ala Ala Asn Met Ser Ile Ala Val Lys Glu Gln Gln Lys Asn Gln
755 760 765
Lys Pro Ala Lys Ser Val Trp Glu Gln Arg Thr Ser Glu Met Arg Lys
770 775 780
Gln Asn Leu Leu Ala Ser Arg Glu Ala Leu Tyr Asn Glu Met Asp Pro
785 790 795 800
Asp Glu Arg Trp Lys Ala Ala Tyr Thr Arg His Leu Arg Pro Asp Met
805 810 815
Lys Thr His Leu Asp Arg Pro Leu Val Val Asp Pro Gln Glu Asn Arg
820 825 830
Asn Asn Asn Thr Asn Lys Ser Arg Ala Ala Glu Pro Thr Val Asp Gln
835 840 845
Arg Leu Gly Gln Gln Arg Ala Glu Asp Phe Leu Arg Lys Gln Ala Arg
850 855 860
Tyr His Asp Arg Ala Arg Asp Pro Ser Gly Ser Ala Gly Leu Asp Ala
865 870 875 880
Arg Arg Pro Trp Ala Gly Ser Gln Glu Ala Glu Leu Ser Arg Glu Asp
885 890 895
Pro Tyr Gly Arg Glu Ser Asp His His Ala Arg Glu Gly Ser Leu Glu
900 905 910
Gln Pro Gly Phe Trp Glu Gly Glu Ala Glu Arg Gly Lys Ala Gly Asp
915 920 925
Pro His Arg Arg His Val His Arg Gln Gly Gly Ser Arg Glu Ser Arg
930 935 940
Ser Gly Ser Pro Arg Thr Gly Ala Asp Gly Glu His Arg Arg His Arg
945 950 955 960
Ala His Arg Arg Pro Gly Glu Glu Gly Pro Glu Asp Lys Ala Glu Arg
965 970 975
Arg Ala Arg His Arg Glu Gly Ser Arg Pro Ala Arg Gly Gly Glu Gly
980 985 990
Glu Gly Glu Gly Pro Asp Gly Gly Glu Arg Arg Arg Arg His Arg His
995 1000 1005
Gly Ala Pro Ala Thr Tyr Glu Gly Asp Ala Arg Arg Glu Asp Lys
1010 1015 1020
Glu Arg Arg His Arg Arg Arg Lys Glu Asn Gln Gly Ser Gly Val
1025 1030 1035
Pro Val Ser Gly Pro Asn Leu Ser Thr Thr Arg Pro Ile Gln Gln
1040 1045 1050
Asp Leu Gly Arg Gln Asp Pro Pro Leu Ala Glu Asp Ile Asp Asn
1055 1060 1065
Met Lys Asn Asn Lys Leu Ala Thr Ala Glu Ser Ala Ala Pro His
1070 1075 1080
Gly Ser Leu Gly His Ala Gly Leu Pro Gln Ser Pro Ala Lys Met
1085 1090 1095
Gly Asn Ser Thr Asp Pro Gly Pro Met Leu Ala Ile Pro Ala Met
1100 1105 1110
Ala Thr Asn Pro Gln Asn Ala Ala Ser Arg Arg Thr Pro Asn Asn
1115 1120 1125
Pro Gly Asn Pro Ser Asn Pro Gly Pro Pro Lys Thr Pro Glu Asn
1130 1135 1140
Ser Leu Ile Val Thr Asn Pro Ser Gly Thr Gln Thr Asn Ser Ala
1145 1150 1155
Lys Thr Ala Arg Lys Pro Asp His Thr Thr Val Asp Ile Pro Pro
1160 1165 1170
Ala Cys Pro Pro Pro Leu Asn His Thr Val Val Gln Val Asn Lys
1175 1180 1185
Asn Ala Asn Pro Asp Pro Leu Pro Lys Lys Glu Glu Glu Lys Lys
1190 1195 1200
Glu Glu Glu Glu Glu Asp Asp Arg Gly Glu Asp Gly Pro Lys Pro
1205 1210 1215
Met Pro Pro Tyr Ser Ser Met Phe Ile Leu Ser Thr Thr Asn Pro
1220 1225 1230
Leu Arg Arg Leu Cys His Tyr Ile Leu Asn Leu Arg Tyr Phe Glu
1235 1240 1245
Met Cys Ile Leu Met Val Ile Ala Met Ser Ser Ile Ala Leu Ala
1250 1255 1260
Ala Glu Asp Pro Val Gln Pro Asn Ala Pro Arg Asn Asn Val Leu
1265 1270 1275
Arg Tyr Phe Asp Tyr Val Phe Thr Gly Val Phe Thr Phe Glu Met
1280 1285 1290
Val Ile Lys Met Ile Asp Leu Gly Leu Val Leu His Gln Gly Ala
1295 1300 1305
Tyr Phe Arg Asp Leu Trp Asn Ile Leu Asp Phe Ile Val Val Ser
1310 1315 1320
Gly Ala Leu Val Ala Phe Ala Phe Thr Gly Asn Ser Lys Gly Lys
1325 1330 1335
Asp Ile Asn Thr Ile Lys Ser Leu Arg Val Leu Arg Val Leu Arg
1340 1345 1350
Pro Leu Lys Thr Ile Lys Arg Leu Pro Lys Leu Lys Ala Val Phe
1355 1360 1365
Asp Cys Val Val Asn Ser Leu Lys Asn Val Phe Asn Ile Leu Ile
1370 1375 1380
Val Tyr Met Leu Phe Met Phe Ile Phe Ala Val Val Ala Val Gln
1385 1390 1395
Leu Phe Lys Gly Lys Phe Phe His Cys Thr Asp Glu Ser Lys Glu
1400 1405 1410
Phe Glu Lys Asp Cys Arg Gly Lys Tyr Leu Leu Tyr Glu Lys Asn
1415 1420 1425
Glu Val Lys Ala Arg Asp Arg Glu Trp Lys Lys Tyr Glu Phe His
1430 1435 1440
Tyr Asp Asn Val Leu Trp Ala Leu Leu Thr Leu Phe Thr Val Ser
1445 1450 1455
Thr Ala Glu Gly Trp Pro Gln Val Leu Lys His Ser Val Asp Ala
1460 1465 1470
Thr Phe Glu Asn Gln Gly Pro Ser Pro Gly Tyr Arg Met Glu Met
1475 1480 1485
Ser Ile Phe Tyr Val Val Tyr Phe Val Val Phe Pro Phe Phe Phe
1490 1495 1500
Val Asn Ile Phe Val Ala Leu Ile Ile Ile Thr Phe Gln Glu Gln
1505 1510 1515
Gly Asp Lys Met Met Glu Glu Tyr Ser Leu Glu Lys Asn Glu Arg
1520 1525 1530
Ala Cys Ile Asp Phe Ala Ile Ser Ala Lys Pro Leu Thr Arg His
1535 1540 1545
Met Pro Gln Asn Lys Gln Ser Phe Gln Tyr Arg Met Trp Gln Phe
1550 1555 1560
Val Val Ser Pro Pro Phe Glu Tyr Thr Ile Met Ala Met Ile Ala
1565 1570 1575
Leu Asn Thr Ile Val Leu Met Met Lys Phe Tyr Gly Ala Ser Val
1580 1585 1590
Ala Tyr Glu Asn Ala Leu Arg Val Phe Asn Ile Ala Phe Thr Ser
1595 1600 1605
Leu Phe Ser Leu Glu Cys Val Leu Lys Ala Met Ala Phe Gly Ile
1610 1615 1620
Leu Asn Tyr Phe Arg Asp Ala Trp Asn Ile Phe Asp Phe Val Thr
1625 1630 1635
Val Leu Gly Ser Ile Thr Asp Ile Leu Val Thr Glu Phe Gly Asn
1640 1645 1650
Asn Phe Ile Asn Leu Ser Phe Leu Arg Leu Phe Arg Ala Ala Arg
1655 1660 1665
Leu Ile Lys Leu Leu Arg Gln Gly Tyr Thr Ile Arg Ile Leu Leu
1670 1675 1680
Trp Thr Phe Val Gln Ser Phe Lys Ala Leu Pro Tyr Val Cys Leu
1685 1690 1695
Leu Ile Ala Met Leu Phe Phe Ile Tyr Ala Ile Ile Gly Met Gln
1700 1705 1710
Val Phe Gly Asn Ile Gly Ile Asp Val Glu Asp Glu Asp Ser Asp
1715 1720 1725
Glu Asp Glu Phe Gln Ile Thr Glu His Asn Asn Phe Arg Thr Phe
1730 1735 1740
Phe Gln Ala Leu Met Leu Leu Phe Arg Ser Ala Thr Gly Glu Ala
1745 1750 1755
Trp His Asn Ile Met Leu Ser Cys Leu Ser Gly Lys Pro Cys Asp
1760 1765 1770
Lys Asn Ser Gly Ile Leu Thr Arg Glu Cys Gly Asn Glu Phe Ala
1775 1780 1785
Tyr Phe Tyr Phe Val Ser Phe Ile Phe Leu Cys Ser Phe Leu Met
1790 1795 1800
Leu Asn Leu Phe Val Ala Val Ile Met Asp Asn Phe Glu Tyr Leu
1805 1810 1815
Thr Arg Asp Ser Ser Ile Leu Gly Pro His His Leu Asp Glu Tyr
1820 1825 1830
Val Arg Val Trp Ala Glu Tyr Asp Pro Ala Ala Cys Gly Arg Ile
1835 1840 1845
His Tyr Lys Asp Met Tyr Ser Leu Leu Arg Val Ile Ser Pro Pro
1850 1855 1860
Leu Gly Leu Gly Lys Lys Cys Pro His Arg Val Ala Cys Lys Arg
1865 1870 1875
Leu Leu Arg Met Asp Leu Pro Val Ala Asp Asp Asn Thr Val His
1880 1885 1890
Phe Asn Ser Thr Leu Met Ala Leu Ile Arg Thr Ala Leu Asp Ile
1895 1900 1905
Lys Ile Ala Lys Gly Gly Ala Asp Lys Gln Gln Met Asp Ala Glu
1910 1915 1920
Leu Arg Lys Glu Met Met Ala Ile Trp Pro Asn Leu Ser Gln Lys
1925 1930 1935
Thr Leu Asp Leu Leu Val Thr Pro His Lys Ser Thr Asp Leu Thr
1940 1945 1950
Val Gly Lys Ile Tyr Ala Ala Met Met Ile Met Glu Tyr Tyr Arg
1955 1960 1965
Gln Ser Lys Ala Lys Lys Leu Gln Ala Met Arg Glu Glu Gln Asp
1970 1975 1980
Arg Thr Pro Leu Met Phe Gln Arg Met Glu Pro Pro Ser Pro Thr
1985 1990 1995
Gln Glu Gly Gly Pro Gly Gln Asn Ala Leu Pro Ser Thr Gln Leu
2000 2005 2010
Asp Pro Gly Gly Ala Leu Met Ala His Glu Ser Gly Leu Lys Glu
2015 2020 2025
Ser Pro Ser Trp Val Thr Gln Arg Ala Gln Glu Met Phe Gln Lys
2030 2035 2040
Thr Gly Thr Trp Ser Pro Glu Gln Gly Pro Pro Thr Asp Met Pro
2045 2050 2055
Asn Ser Gln Pro Asn Ser Gln Ser Val Glu Met Arg Glu Met Gly
2060 2065 2070
Arg Asp Gly Tyr Ser Asp Ser Glu His Tyr Leu Pro Met Glu Gly
2075 2080 2085
Gln Gly Arg Ala Ala Ser Met Pro Arg Leu Pro Ala Glu Asn Gln
2090 2095 2100
Arg Arg Arg Gly Arg Pro Arg Gly Asn Asn Leu Ser Thr Ile Ser
2105 2110 2115
Asp Thr Ser Pro Met Lys Arg Ser Ala Ser Val Leu Gly Pro Lys
2120 2125 2130
Ala Arg Arg Leu Asp Asp Tyr Ser Leu Glu Arg Val Pro Pro Glu
2135 2140 2145
Glu Asn Gln Arg His His Gln Arg Arg Arg Asp Arg Ser His Arg
2150 2155 2160
Ala Ser Glu Arg Ser Leu Gly Arg Tyr Thr Asp Val Asp Thr Gly
2165 2170 2175
Leu Gly Thr Asp Leu Ser Met Thr Thr Gln Ser Gly Asp Leu Pro
2180 2185 2190
Ser Lys Glu Arg Asp Gln Glu Arg Gly Arg Pro Lys Asp Arg Lys
2195 2200 2205
His Arg Gln His His His His His His His His His His Pro Pro
2210 2215 2220
Pro Pro Asp Lys Asp Arg Tyr Ala Gln Glu Arg Pro Asp His Gly
2225 2230 2235
Arg Ala Arg Ala Arg Asp Gln Arg Trp Ser Arg Ser Pro Ser Glu
2240 2245 2250
Gly Arg Glu His Met Ala His Arg Gln
2255 2260

45

20

DNA

human

45
caacatcatg ctttcctgcc 20

46

20

DNA

human

46
atgatgacgg cgacaaagag 20

47

40

DNA

human

47
tctccgcagt cgtagctcca cgcaaaggat gtacaagcag 40

48

41

DNA

human

48
ggttgtagag tgccatggtc attcccaagc ctccagggta g 41

49

20

DNA

HUMAN

49
acctccaac acccttcttt 20

50

19

DNA

HUMAN

50
tctgtgccct gctccactc 19

51

20

DNA

HUMAN

51
acgctgacct tgccttctct 20

52

20

DNA

HUMAN

52
caaccaaaag cctcgtaatc 20

53

20

DNA

HUMAN

53
aaaacccacc ctctgttctc 20

54

20

DNA

HUMAN

54
ttgtcagggt cggaaactca 20

55

16

DNA

HUMAN

55
cttggtggcg gggttt 16

56

20

DNA

HUMAN

56
ctgcctaatc ctcccaagag 20

57

20

DNA

HUMAN

57
tcccttccct tttgtagatg 20

58

19

DNA

HUMAN

58
gtggggctgt gttgtcctt 19

59

20

DNA

HUMAN

59
gacagagcca caagagaacc 20

60

20

DNA

HUMAN

60
agcaaagagg agtgagtggg 20

61

20

DNA

HUMAN

61
atactctggc ttttctatgc 20

62

20

DNA

HUMAN

62
gcatgactct ctttgtactc 20

63

17

DNA

HUMAN

63
gcagagaatg ggggtgg 17

64

20

DNA

HUMAN

64
ctgaggtggg tttagagcag 20

65

22

DNA

HUMAN

65
gggtaacgtc tttttctctt gc 22

66

20

DNA

HUMAN

66
atgtctcttg ggcgataggt 20

67

20

DNA

HUMAN

67
atttcttctg aaggaacagc 20

68

20

DNA

HUMAN

68
ggagggatca gggagttggc 20

69

20

DNA

HUMAN

69
caagcctaac ctcctctctg 20

70

19

DNA

HUMAN

70
tcattccagg caagagctg 19

71

20

DNA

HUMAN

71
atttggaggg aggagtttgg 20

72

20

DNA

HUMAN

72
tcactttccc aactttctgg 20

73

20

DNA

HUMAN

73
cagaaagttg ggaaagtagc 20

74

19

DNA

HUMAN

74
ttgaattcct gtgaaggac 19

75

21

DNA

HUMAN

75
cttggagatg agatactgag c 21

76

20

DNA

HUMAN

76
caggcacttt catctgtgac 20

77

20

DNA

HUMAN

77
tccacagctg catctccaag 20

78

18

DNA

HUMAN

78
accctccctt gagcccct 18

79

20

DNA

HUMAN

79
cagtggttgc ttttcctgac 20

80

20

DNA

HUMAN

80
ttgccagaga aacattctcc 20

81

20

DNA

HUMAN

81
tgaacaaaga ttccacgtgc 20

82

21

DNA

HUMAN

82
ttcaggagcc agggtagcat c 21

83

59

DNA

HUMAN

83
tagcaatgct ctaagtcctg cgcaggagaa ccgcaacaag cagcagggag agccgcagc 59

84

58

DNA

HUMAN

84
tgtttcctga ggaagtcctc gcgatgacgt cgatgctcta ccgtcattct gcggattc 58

85

41

DNA

HUMAN

85
ggttcttttt cattcacttg cgagaatagc cttatcgtca c 41

86

39

DNA

HUMAN

86
tttcctggca gtcttagctg cagtgatgtg agagcagag 39

87

20

DNA

HUMAN

87
tgggaaattg tggagggagc 20

88

20

DNA

HUMAN

88
tgacttccgc caccctggtg 20

89

21

DNA

HUMAN

89
taggaagggg tgtgctctgt g 21

90

20

DNA

HUMAN

90
agcctgtggt ctgagtggac 20

91

20

DNA

HUMAN

91
atccactgct ctcttgcttt 20

92

22

DNA

HUMAN

92
gtggttctca cttataatct gc 22

93

21

DNA

HUMAN

93
tggcctcatt ggcttccctg c 21

94

20

DNA

HUMAN

94
aagaggaaac ccttgcgaag 20

95

20

DNA

HUMAN

95
ctacccaacc tgacctctgc 20

96

20

DNA

HUMAN

96
acatgataac cctgacagtc 20

97

20

DNA

HUMAN

97
ctcatgctct ctgtcaactc 20

98

20

DNA

HUMAN

98
tggttccaat gggaatgtgc 20

99

20

DNA

HUMAN

99
ctgcttccca agcagtctag 20

100

20

DNA

HUMAN

100
tcctggatag atttccagtc 20

101

20

DNA

HUMAN

101
agtttttaaa ggacagatgg 20

102

22

DNA

HUMAN

102
tttccctgcc ccattccttt gc 22

103

20

DNA

HUMAN

103
ctctgccgct ctcaccactg 20

104

19

DNA

HUMAN

104
tttatcaggt agaggcagg 19

105

21

DNA

HUMAN

105
ttccaagccc atagctgtag c 21

106

21

DNA

HUMAN

106
tgaccctgct actcctgctt c 21

107

20

DNA

HUMAN

107
actgtgcctc taacatgcac 20

108

18

DNA

HUMAN

108
aagtgctggc tcaagcag 18

109

20

DNA

HUMAN

109
tctgtgagtg gtgacagctc 20

110

18

DNA

HUMAN

110
gtcacctgtc ttctcagc 18

111

20

DNA

HUMAN

111
tggaaggact ctggcacgtg 20

112

20

DNA

HUMAN

112
ggaggctctg ggaaccttag 20

113

21

DNA

HUMAN

113
agaagccact ggaggaatgg c 21

114

22

DNA

HUMAN

114
attatcagag caggtcccct tc 22

115

20

DNA

HUMAN

115
tccgagtctc tgatttctcc 20

116

19

DNA

HUMAN

116
agacggccct cacagtgtc 19

117

20

DNA

HUMAN

117
ttcattccct cggtctctgc 20

118

19

DNA

HUMAN

118
ctgactgaac ctgtgagac 19

119

19

DNA

HUMAN

119
tgtgaaccca ttgcctgca 19

120

19

DNA

HUMAN

120
tgggaatgac tgcgcttgc 19

121

18

DNA

HUMAN

121
atgcctggga atgactgc 18

122

18

DNA

HUMAN

122
tgtcacgcct gtctgtgc 18

123

18

DNA

HUMAN

123
tgacacccag gcaggcag 18

124

18

DNA

HUMAN

124
tctgacgcct gtctgtgc 18

125

18

DNA

HUMAN

125
ttggtgagct caccgtgt 18

126

21

DNA

HUMAN

126
ttcccgtggt gacatgcaag c 21

127

20

DNA

HUMAN

127
gtccacacac tgctctctgc 20

128

18

DNA

HUMAN

128
acactccacc tccctggc 18

129

18

DNA

HUMAN

129
gccagggagg tggagtgt 18

130

18

DNA

HUMAN

130
ggttccttcc accgcaac 18

131

17

DNA

HUMAN

131
caactcccca atggctc 17

132

21

DNA

HUMAN

132
cctacccagt gcagagtgag g 21

133

20

DNA

HUMAN

133
tctgtgtgca ccatcccatg 20

134

20

DNA

HUMAN

134
aaggattggg ctccatggag 20

135

19

DNA

HUMAN

135
gttggtgcta gctgctgac 19

136

20

DNA

HUMAN

136
ctttcttctt ccttagtgtc 20

137

20

DNA

HUMAN

137
gtgtgctgtc tgaccctcac 20

138

20

DNA

HUMAN

138
agcctggggt cacttgcagc 20

139

23

DNA

HUMAN

139
cctttgtttc aattttcgtg tag 23

140

20

DNA

HUMAN

140
tggggcctgg gtacctccga 20

141

19

DNA

HUMAN

141
ctttaattgc cctgtcttc 19

142

18

DNA

HUMAN

142
ttaattcgac cacttccc 18

143

20

DNA

HUMAN

143
agtgagactc gtctctaatg 20

144

20

DNA

HUMAN

144
acctacctga attcctgacc 20

145

22

DNA

HUMAN

145
aacactagtg acattatttt ca 22

146

20

DNA

HUMAN

146
agctaggcct gaaggcttct 20

Gene related to migraine in man

Information

Patent Number

Date Filed

Date Issued

Inventors

Original Assignees

Examiners

Agents

CPC

US Classifications

Field of Search

US

International Classifications

Abstract

Description

Claims

Priority Claims (1)

PCT Information

Foreign Referenced Citations (1)

Non-Patent Literature Citations (22)

Entry
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Ellis et. al.; GenBank Seq. No. AAQ84660, 1995,5-9.*
Skolnick et. al.; From genes to protein struture and function: novel applications of computational approaches in the genomic era, 2000, TIBETECH, vol. 18: 34-39.*
Kaye et. al.; A single amino acid substitution results in a retinoblastoma protein defective in phosphorylation and oncoprotein binding, 1990, Proc. Natl. Acad. Sci., vol. 87: 6922-6926.*
Rudinger; Characteristics of the amino acids as components of a peptide hormone sequence, 1976, Peptide Hormones: 1-7.*
Ellis et al., AC Q84659, Feb. 16, 1995, Gene seq. 36.*
Opoff, R.A., AC Z80116, Nov. 01, 1996, Gene Embl.*
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