Patent Application
20130210657
The present invention relates to a method of assessing in a human subject, the risk of having Autism Spectrum Disorder (ASD) using novel biomarkers.
Autism is the prototypic form of a group of conditions, the ‘autism spectrum disorders’ (ASD), which share common characteristics (impairments in socialization, communication and repetitive interests and behaviors), but differ in developmental course, symptom pattern, cognitive and language abilities. Other ASD subtypes include Asperger disorder (less severe language and cognitive deficits) and Pervasive Developmental Disorder Not Otherwise Specified (PDD-NOS; sub-threshold symptoms and/or later onset). Sub clinical forms of ASD are often characterized as Broader Autism Phenotype (BAP). Twin and family studies provide evidence for the importance of complex genetic factors in the development of both sporadic and inherited forms of idiopathic autism. An enigma in ASD is the 4:1 male to female gender bias, which may rise to 11:1 when considering Asperger disorder.
Rare copy number variations (CNVs) and sequence-level mutations have been identified as etiologic factors in ASD. De novo CNVs are observed in 5-10% of ASD cases. A relative enrichment of CNVs disrupting synaptic complex genes is observed, with NLGN3, NLGN4, NRXN1, NRXN3, SHANK2 and SHANK3 being identified as highly-penetrant susceptibility loci for ASD and intellectual disability (ID).
In order to further understand the etiology of neurodevelopmental disorders such as ASD, it would be desirable to identify additional biomarkers of such disorders.
It has now been determined that copy number variations or other sequence variations associated with the SHANK1 gene are indicative of risk of ASD.
Accordingly, in one aspect of the invention, a method of assessing risk of ASD in a human subject is provided. The method comprises identifying in a nucleic acid-containing sample obtained from the human subject copy number variations associated with SHANK1. A determination of copy number variations associated with SHANK1 is indicative of a risk of ASD in the human subject.
This and other aspects of the invention are described in the detailed description by reference to the following figures.
(A) 46,XX.ish del(19)(q13.33q13.33)(G248P87495E12-)
(B) 46,XY.ish del(19)(q13.33q13.33)(G248P87495E12-)
(C) 46,XX.ish 5q31.3(G248P80200H8x2),19q13.33(G248P87495E12x2).
A method of assessing risk of Autism Spectrum Disorder (ASD) in a human subject is provided. The method comprises identifying in a nucleic acid-containing sample obtained from the human subject copy number variations associated with SHANK1. A determination of copy number variations associated with SHANK1 is indicative of a risk of ASD in the human subject.
The term “ASD” or “Autism Spectrum Disorder” is used herein to refer to autism, Asperger syndrome, Childhood disintegrative disorder, Rett syndrome, Pervasive Developmental Disorder Not Otherwise Specified (PDD-NOS) and Broader Autism Phenotype (BAP).
The term “SHANK1” refers to the gene that encodes a protein known as SH3 and multiple ankyrin repeat domains protein 1 (shank1), including natural variants and isoforms, e.g. isoforms 1, 2 and 3, thereof. This term encompasses the human gene sequence as set out in
In the present method of determining risk in a human subject of ASD, a biological sample obtained from the subject is utilized. A suitable biological sample may include, for example, a nucleic acid-containing sample or a protein-containing sample. Examples of suitable biological samples include saliva, urine, semen, other bodily fluids or secretions, epithelial cells, cheek cells, hair and the like. Although such non-invasively obtained biological samples are preferred for use in the present method, one of skill in the art will appreciate that invasively-obtained biological samples, may also be used in the method, including for example, blood, serum, bone marrow, cerebrospinal fluid (CSF) and tissue biopsies such as tissue from the cerebellum, spinal cord, prostate, stomach, uterus, small intestine and mammary gland samples. Techniques for the invasive process of obtaining such samples are known to those of skill in the art. The present method may also be utilized in prenatal testing for the risk of ASD using an appropriate biological sample such as amniotic fluid and chorionic villus.
In one aspect, the biological sample is screened for SHANK1 in order to detect mutations in the genome associated with ASD. It may be necessary, or preferable, to extract nucleic acid from the biological sample prior to screening the sample. Methods of nucleic acid extraction are well-known to those of skill in the art and include chemical extraction techniques utilizing phenol-chloroform (Sambrook et al., 1989), guanidine-containing solutions, or CTAB-containing buffers. As well, as a matter of convenience, commercial DNA extraction kits are also widely available from laboratory reagent supply companies, including for example, the QIAamp DNA Blood Minikit available from QIAGEN (Chatsworth, Calif.), or the Extract-N-Amp blood kit available from Sigma (St. Louis, Mo.).
Once an appropriate nucleic acid sample is obtained, it is subjected to well-established methods of screening, such as those described in the specific examples that follow, to detect genetic mutations in SHANK1 which are indicative of ASD. These mutations include genomic copy number variations (CNVs), such as gains and deletions of segments of DNA, for example, gains and deletions of segments of DNA greater than about 10 kb, such as DNA segments greater than 50 kb. Gene mutations or CNVs “associated with” ASD include CNVs in both coding and regulatory regions of the SHANK1 gene. In a preferred embodiment, gene mutations in the form of CNVs which reduce or inhibit SHANK1 expression are indicative of a risk of ASD. The CNVs may be inherited or de novo.
To determine risk of, or to diagnose ASD, in a human subject, it may be advantageous to screen for multiple CNVs that are associated with ASD applying array technology. In this regard, genomic sequencing and profiling, using well-established techniques as exemplified herein in the specific examples, may be conducted for a subject to be assessed with respect to ASD risk/diagnosis using a suitable biological sample obtained from the subject. Identification of one or more CNVs associated with ASD would be indicative of a risk of ASD, or may be indicative of a diagnosis of ASD. This analysis may be conducted in combination with an evaluation of other characteristics of the subject indicative of ASD, including for example, phenotypic characteristics.
In another aspect, a method for determining risk of ASD in a subject is also provided in which the expression or activity of the SHANK1 protein product, e.g. a SH3 and multiple ankyrin repeat domains protein 1, including isoforms thereof, is determined in a biological protein-containing sample obtained from the subject. Abnormal levels of the gene product or abnormal levels of the activity thereof, i.e. reduced or elevated levels, in comparison with levels that exist in healthy non-ASD subjects, are indicative of a risk of ASD, or may be indicative of ASD. As one of skill in the art will appreciate, standard assays may be used to identify and quantify the presence and/or activity of a selected gene product.
In a further aspect, identification of missense mutations in SHANK1 may also be indicative of risk of ASD. In this regard, a missense mutation may result in a reduction of SHANK1 expression, or in the expression of a protein product with altered activity, e.g. reduced activity, or a non-functional protein. Accordingly, in addition to detection of the mutation in a nucleic acid sample of the patient, risk of ASD may also be assessed by detection of an altered, e.g. reduced level and/or activity of the gene product of SHANK1.
Embodiments of the invention are described by reference to the following specific example which is not to be construed as limiting.
The ASD patient dataset comprised 1,158 unrelated Canadian individuals (898 males and 260 females) and 456 unrelated cases (362 males and 94 females) from Europe. The patients had clinically well characterized ASD diagnosed by expert clinicians based on the Autism Diagnostic Interview—Revised (ADI-R) and/or the Autism Diagnostic Observation Schedule (ADOS) as set out in Risi et al. J Am Acad Child Adolesc Psychiatry 2006; 45:1094-103, the relevant contents of which are incorporated herein by reference. Canadian cases were recruited from five different sites: The Hospital for Sick Children, Toronto, Ontario; McMaster University, Hamilton, Ontario; Memorial University of Newfoundland, St. John's, Newfoundland; University of Alberta, Edmonton, Alberta and the Montreal Children's Hospital of the McGill University Health Centre, Montreal, Quebec, Canada. The European ASD cases were recruited by the PARIS (Paris Autism Research International Sibpair) study and several other sites at specialized clinical centers dispersed in France, Sweden, Germany, Finland and UK. In Sweden, for some cases, the Diagnostic Interview for Social and Communication Disorders (DISCO-10) was applied instead of the ADI-R. The ID patient dataset consisted of 185 mostly French Canadians (98 males and 87 females) and 155 German non-syndromic ID cases (93 males and 62 females). Further descriptions of these datasets and the assessment procedures used are described in Hamdan et al. (Biol Psychiatry 2011; 69:898-901) and Berkel et al. (Nat Genet 2010; 42:489-91).
Institutional ethical review board approval was obtained for the study, and informed written consent was obtained from all participants.
To assess the presence of CNVs on a genome-wide scale, DNA from the Canadian ASD dataset was genotyped at The Centre for Applied Genomics, Toronto with one of three high-resolution microarray platforms: Affymetrix GeneChip SNP 6.0, Illumina Infinium 1M single SNP or Agilent SurePrint G3 Human CGH 1x1M. CNVs were analyzed using published methods (Pinto et al. Nat Biotechnol 2011. 29:512-20, the relevant contents of which are incorporated herein by reference). Independent validation of the deletion at the SHANK1 locus in Family 1 was performed with SYBR Green based real-time quantitative PCR (qPCR), with two independent primer pairs at the SHANK1 locus, and at the FOXP2 locus as a negative (diploid) control.
DNA from the European ASD case dataset was genotyped at the Centre National de Genotypage (CNG), at the Institut Pasteur using the Illumina Human 1M-Duo BeadChip. CNVs were analyzed as above. Validation of the array CNV calls was performed with qPCR in a similar way as described above, with two independent primer pairs at the SHANK1 locus, and at exon 18 locus of SHANK1 as a negative (diploid) control. All primers are listed in the Table that follows:
The SHANK1 locus was also examined for CNVs in published data from 2,026 healthy individuals from the Children's Hospital of Philadelphia and from 2,493 controls genotyped at the University of Washington8 and in microarray data analyzed by our group from 10,603 population based controls.4,5,9,10 This latter dataset included 1,123 controls from northern Germany,11 1,234 Canadian controls,12 1,120 population controls from Ontario,13 1,056 HapMap samples,14 4,783 controls from the Wellcome Trust Case Control Consortium (WTCCC)15 and 1,287 controls recruited by the Study of Addiction: Genetics and Environment (SAGE) consortium.16 Control samples were predominantly of European ancestry appropriate for comparison with the ASD case datasets. The Database of Genomic Variants (DGV; http://projects.tcag.ca/variation) was also examined for previously reported CNVs at the SHANK1 locus in the general population.
509 of the 1,158 ASD individuals and 340 individuals with ID were screened for mutations using Sanger-based sequencing. All coding exons and intron-exon splice sites of SHANK1 were sequenced. Primer3 software v. 0.4.0 (http://frodo.wi.mit.edu/primer3) was used to design PCR primers. PCRs were performed using standard conditions, and products were purified and sequenced directly using the BigDye Terminator sequencing (Applied Biosystems, Foster City, Calif., USA). Variant detection was performed using SeqScape software from Applied Biosystems. Novel variants detected in the cases, not previously reported in the Single Nucleotide Polymorphism Database (dbSNP) build 130, were validated by re-sequencing the proband and samples from both parents and from siblings, when available. All primers are listed in the following Table.
For the rare SHANK1 sequence missense variants identified in ASD and ID, TaqMan assays were performed to estimate their frequency in 285 control individuals of European ancestry from the Ontario Population Genomics Project control collection (138 males and 147 females) using the Applied Biosystems 7900HT real-time PCR system.
For two of the ASD patients in family 1 (III-5 and IV-3), paired-end exome sequencing was performed using Life Technologies SOLiD5500 (Life Technologies, Foster City, Calif., USA) sequencing platform. Target enrichment was performed utilizing the Agilent SureSelect 50 Mb human all exon capture kit (Agilent Technologies, Santa Clara, Calif., USA). Protocols for sequencing and target capture followed specifications from the manufacturers. BFAST (Homer et al. PLoS One 2009; 4:e7767) was used to map the generated paired end reads to the reference human genome (UCSC's hgl9). Duplicate pair end reads were removed using MarkDuplicates (Picard tools version 1.35; http://picard.sourceforge.net) and the subsequent duplicated-free alignments were refined using local realignment in colourspace implemented in SRMA version 0.1.15 (Homer et al. Genome Biol 2010; 11:R99). Calling of indels and SNPs was performed using GATK version1.0.5506 and recommended parameters (DePristo et al. Nat Genet 2011; 43:491-8). SIFT 4.0.3 (Ng et al. Nucleic Acids Res 2003; 31:3812-4) was used to annotate the variant calls to determine if amino acid substitutions were predicted to be deleterious.
The nonsense variant in PCDHGA11 was validated by Sanger sequencing. All primers are listed in the table below.
Chromosome metaphases were prepared according to standard protocols from primary blood samples. Metaphase FISH were performed using two fosmid probes (hg18 co-ordinates): G248P80200H8 (chr5:140,769,097-140,810,244, SpectrumOrange) overlapping the Y313X nonsense mutation in PCDHGA11 and G248P87495E12 (chr19: 55,874,318-55,921,609, SpectrumGreen) residing within the 63.8 kb deletion disrupting SHANK1.
Eleven individuals from Family 1 (I-1, I-2, II-2, II-4, II-5, III-1, III-2, III-3, III-5, III-6 and IV-3) were genotyped using the Illumina Omni 2.5M-quad BeadChip microarray platform. Genotype information from 5,629 SNPs was used for linkage analysis. These markers were selected to have high call rate, high MAF, no Mendelian errors, low pairwise LD between them, and genotype proportions consistent with Hardy Weinberg equilibrium. Markers with ambiguous alleles were also removed. Additionally, only markers which were present in the HapMap3 release 28 CEU population were included, so that allele frequencies could be properly estimated. Parametric linkage analysis with the MMLS (maximized maximum LOD score) method was performed using the program Merlin (Abecasis et al. Nat Genet 2002; 30:97-101). This analysis is appropriate when the correct method of inheritance is not known and is more powerful than non-parametric analysis, since it uses all individuals in the pedigree and not just the affected ones. In the MMLS method, the pedigree is analyzed for linkage under several different inheritance models (dominant and recessive with varying penetrance) and the model with the maximum LOD score was chosen.
Initially, 1,158 Canadian individuals with ASD were examined using high-resolution microarray scanning and a hemizygous microdeletion at chromosome 19q13.33 in ASD proband III-5 in Family 1 was identified. The deletion was determined to be 63.8 kilobases (kb) eliminating exon 1 to 20 of SHANK1, and the neighboring CLEC11A gene coding for a growth factor for primitive hematopoietic progenitor cells (
In separate experiments, 456 individuals from Europe with ASD were examined using microarrays and a 63.4 kb hemizygous CNV was identified in individual F2-II-1 from Family 2 deleting the last three exons of SHANK1 and the entire centromeric synaptotagmin-3 (SYT3) gene, with a role in Ca(2+)-dependent exocytosis of secretory vesicles (
aClassification based on Tsuchiya et al.23
aClassification based on Tsuchiya et al.23
To test for sequence-level mutations in SHANK1, Sanger sequencing was used to examine all 23 exons and splice sites in 509 unrelated ASD (384 male and 125 female) and 340 ID (191 males and 149 females). Detected were 26 rare missense variants in 23 ASD and 7 ID cases, which were not found in the Single Nucleotide Polymorphism Database (dbSNP) build 130 or in 285 control individuals from the Ontario general population (as shown in Table 7 below and
Whole-exome sequencing was conducted in subjects III-5 and IV-3 from Family 1 to search for potential mutations in other genes (see Table 4 below of the Supplementary Appendix). A non-sense mutation predicted to introduce a stop codon (Y313X) in the PCDHGA11 prodocadherin gene on chromosome 5q31.3 was identified. PCDHGA11 is a member of the protocadherin gamma gene cluster thought to have an important role in establishing connections in the brain. The mutation was found to segregate precisely with the SHANK1 deletion. Since SHANK1 and PCDHGA11 reside on different autosomes, translocation or transposition was tested for, and such linkage was ruled out (
Individuals with deletions involving SHANK1, including four male cases with higher-functioning ASD or the BAP from a multi-generation family carrying inherited gene deletions (
The proband III-5 from family 1 (
IV-1 was clinically diagnosed with Asperger disorder at age 8. He was born 10 days overdue by cesarean section. Early developmental milestones were within normal limits. His parents appreciated developmental differences at age 3 when it was noted that he was not interested in other children and was preoccupied with objects. He had an encyclopedic knowledge of cars. He would approach other children, but tended to play beside them and became upset with changes in routine. He exhibited difficulties with eye contact and understanding social cues and rules. Additional assessment was conducted at age 10. He met all the cut-offs for autistic disorder on the ADI-R except for the nonverbal total. The ADOS, scores were below cut-off for a diagnosis of ASD due to strengths in the communication domain. Descriptive gestures, were present, although they were vague and infrequent, accounting for his communication score of 1 (cut-off is 2). Impairments in reciprocal social interaction continued to be evident. On psychometric testing, he had a significant verbal-performance discrepancy with lower performance than verbal scores (114 vs. 86). IV-1 qualified for a diagnosis of Asperger disorder.
Individual IV-3 was first evaluated at age 3. At 18 months, his parents became concerned because he was not talking He developed single words at 24 months. He communicated by leading his parents by the hand and exhibited repetitive behaviors. He did not offer comfort or empathy and did not initiate social interaction, although he would play with his parents. Certain noises bothered him such as the washing machine or the toilet flushing; he became upset if his mother had her hair down or a jacket unzipped. Assessment at age 5 years 8 months indicated he was positive on the ADI-R for autism and for ASD on the ADOS. He had made good progress in social interaction and language. His expressive language consisted of short sentences and phrases with some echoed speech and mild articulation difficulties. His IQ and expressive and receptive language scores were in the low average range, leading to a best-estimate diagnosis of Asperger disorder.
aAutism Spectrum Diagnosis based on Autism Diagnostic Interview-Revised (ADI-R) and Autism Diagnostic Observation Schedule (ADOS; one of 4 possible modules administered based on age and language level). In some cases the Social Responsiveness Scale (SRS) was administered and reported T-scores represent Average skills (≦59T), Mild to Moderate Concerns (60T to 75T), Severe range (76T or higher). Also the diagnosis for II-1 in Family 2 was based on the Childhood Autism Rating Scale (CARS).
bIQ measured using age appropriate Weschler scale (WPPSI-Wechsler Preschool and Primary Scale of Intelligence; WISC-Intelligence Scale for Children; WASI-Wechsler Abbreviated Scale of Intelligence). Standard scores and percentiles (% ile) presented for full scale IQ (FSIQ), verbal IQ (VIQ) and/or performance IQ (PIQ). FSIQ is not a valid estimate of IQ when significant discrepancy exists between VIQ and PIQ. Leiter International Performance Scale-Revised (Leiter-R) is a measure of non-verbal IQ (NVIQ) only. Percentile classifications: Very Superior (VS; >98th % ile), Superior (S; 91st-97th % ile), High Average (HA; 75th-90th % ile), Average (A; 25th-74th % ile), Low Average (LA; 9th-24th % ile), Borderline (B; 2nd-8th % ile), and Extremely Low (EL; <2nd % ile).
cLanguage measured using the Oral and Written Language Scales (OWLS). Standard scores and percentiles presented for total language (TL), receptive language (RL), and/or expressive language (EL). Language was rated as nonverbal, average, or delayed (≦16th % ile). The Peabody Picture Vocabulary Test (PPVT-4th edition) measured receptive vocabulary (RV).
dAdaptive Behavior measured using the Vineland Adaptive Behavior Scales (VABS which edition. Standard score and percentiles presented for Adaptive Behavior Composite (ABC); Communication (COM); Daily Living Skills (DLS); Socialization (SOC); Motor (MOT; only for children aged 7 years or less).
Male individual F2-II-1 (
This is the first description of hemizygous deletions of the SHANK1 gene in ASD. The striking segregation of ASD in only male SHANK1 deletion carriers in Family 1 represents the first example of autosomal sex-limited expression in ASD. The finding of an unrelated male with ASD carrying an independent de novo deletion of SHANK/supports the interpretation that the SHANK1 CNV segregating in Family 1 is indeed the primary etiologic event leading to ASD.
The data indicate SHANK1 deletions are associated with higher-functioning ASD in males. Insofar as all affected males have IQ in the typical range and have good verbal ability (with a lack of clinically significant language delay), they would also qualifiy for a diagnosis of Asperger disorder.
It is noted that the neuronal genes PCDHGA11 and SYT3, could also contribute to aspects of the ASD phenotype in Family 1 and Family 2, respectively.
This application claims the benefit of U.S. Provisional Patent Application No. 61/590,591, filed on Jan. 25, 2012, and incorporates such provisional patent application in its entirety by reference.
| Number | Date | Country | |
|---|---|---|---|
| 61590591 | Jan 2012 | US |
