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Definitions

• the present invention relates to genetic markers for Autism Spectrum
• Autism is a heritable neurodevelopmental condition characterized by impairments in social communication and by a preference for repetitive activities. Autism is not a distinct categorical disorder but is the prototype of a group of conditions defined as Pervasive Developmental Disorders (PDDs) or Autism Spectrum Disorders (ASD), which include Asperger's Disorder, Childhood Disintegrative Disorder, Pervasive developmental disorder-not otherwise specified (PDD-NOS) and Rett Syndrome.
• PDDs Pervasive Developmental Disorders
• ASD Autism Spectrum Disorders
• ASD is diagnosed in families of all racial, ethnic and social-economic backgrounds with incidence roughly four times higher in males compared to females. Overall population prevalence of autism has increased in recent years to a current estimate of 20 in 10,000 with incidence as high as 60 in 10,000 for all autism spectrum disorders.
• autism is part of a broader recognizable disorder (e.g. fragile X syndrome, tuberous sclerosis) or is associated with cytogenetically-detectable chromosome abnormalities.
• a broader recognizable disorder e.g. fragile X syndrome, tuberous sclerosis
• cytogenetically-detectable chromosome abnormalities e.g. X syndrome, tuberous sclerosis
• co-morbidity of autism with microdeletion syndromes e.g. William-Beuren and Sotos
• other genomic disorders e.g. Prader-Willi/Angelman
• the most frequent cytogenetic anomaly is an interstitial, maternally-inherited duplication of 15qll-13 (1-3%) encompassing the Prader Willi/Angelman Syndrome critical region.
• the 22qll.2 region is associated with velo-cardio-facial Syndrome and deletions at 22ql3.3 appear to also represent a clinically definable syndrome. Both deletions are associated with the autistic phenotypes.
• Other chromosome loci associated with anomalies with a higher frequency of events observed in syndromic forms of ASD include 7q (see TCAG www.chr7.org), 2q37, 5pl4-15, 17pll.2.
• reciprocal duplications overlapping the William-Beuren deletion region have been associated with the autism phenotype.
• NLGN3 and NLGN4 genes and more recently SHANK3; however, these account for only rare causes of ASD. Other genes have been implicated, but represent rare events or have not yet been validated by other studies.
• CNVs large scale copy number variants
• a number of genetic markers have now been identified which are useful in assessing the risk of ASD in an individual, as well as being useful to diagnose the condition.
• the markers are useful both individually and in the form of a microarray to screen individuals for risk of ASD.
• a method of determining the risk of ASD in an individual comprising:
• a method of determining the risk of ASD in an individual comprising:
• nucleic acid-containing sample obtained from the individual for a mutation that modulates the expression of at least one gene selected from the group consisting of PTCHDl, SHANK3, NFIA, DPP6, DPPlO, GPR98, PQBPl, ZNF41 and FTSJl, wherein identification of a mutation that modulates the expression of at least one of said genes is indicative of a risk of ASD.
• ASD in an individual comprising:
• a biological sample obtained from the individual for abnormal levels of at least one gene product expressed by a gene selected from the group consisting of PTCHDl, SHANK3, NFIA, DPP6, DPPlO, GPR98, PQBPl, ZNF41 and FTSJl, wherein a determination that at least one of said gene products is expressed at a level that varies from the level in a healthy non-ASD individual is indicative of a risk of ASD.
• Figure 2 illustrates a genome-wide distribution of ASD-specific CNVs as described in Table 3;
• Figure 3 illustrates the chromosome 16pl 1.2 region as depicted in the
• PCR products were then fragmented with DNaseI to a size range of 250bp to 2,000bp, labelled, and hybridized to the array. After hybridization, arrays were washed on the Affymetrix fluidics stations, stained, and scanned using the Gene Chip Scanner 3000 7G and Gene Chip Operating System. Data has been submitted to the Gene Expression Omnibus database (accession GSE9222). Karyotypes were generated using standard clinical diagnostic protocols.
• Structural variants found in ASD cases were initially prioritized to possibly be etiologic if they were not in controls and, (i) de novo in origin (25 cases) (see Table 5 below), (ii) overlapping (27 cases at 13 loci) in two or more unrelated samples (see Table 7 below), (iii) recurrent (same breakpoints) in two or more unrelated samples (four cases at two loci), (iv) or inherited (the remainder).
• CNVs were found at known ASD loci: NLGN4 and 22q, 15q, SHANK3 and NRXNl in categories i, ii, iii, and iv, respectively.
• ASD structural variants found in controls eg. NRXNl
• New ASD candidates identified were those with a structural change
• Additional positional candidate genes identified were those found interrupted by balanced cytogenetic breakpoints including NEGRI, PIP 5 Kl B, GABRGl, KLHLi, STK3, ST7, SATB2 (Table 1). Moreover, 77 CNVs in the stringent dataset overlapped with the Autism Chromosome Rearrangement Database providing a second line of evidence for involvement ( Figure 2). For example, a 4.6 Mb de novo duplication at XpI 1.23-11.22 was detected in a female SK0306-004 (Table 5) and a male in the database.
• DPP6 and DPPlO emerge as being positional and functional candidates.
• DPP6 (-1.5 Mb in size at 2ql4.1) and DPPlO (-1.3 Mb at 7q36.2) code for accessory trans-membrane dipeptidyl peptidase-like subunits that affect the expression and gating of Kv4.2 channels (KCND2).
• Kv4.2 channels function in regulation of neurotransmitter release and neuronal excitability in the glutamatergic synapse at the same sites where SHANK3 and the NLGN gene products are found.
• autism balanced breakpoints have been mapped near KCND2 at 7q31.
• Structural variants overlapping loci involved in medical genetic conditions including Waardenburg Type HA (3pl4.1), speech and language disorder (7q31), mental retardation (MR)(15q23-q24, 16pll.2) and velocardialfacial syndrome (VCFS) (22ql3) were identified (Table 5), amongst others. Identification of the structural variant at these loci led to clinical re-assessment and either identification or refinement of the diagnosis, for additional syndromic features. Other instances (eg. SK0186-/TCHD7 deletion) ( Figure 4c) prompted re-testing of the entire family and eventually a diagnosis of mild-ASD in a previously undiagnosed sibling. This family was then redesignated multiplex as opposed to simplex.
• the deletion was determined to be an ⁇ 156kb deletion on Xp22.11 on a male proband.
• the physical position of this CNV is chrX:22,962,800-23,l 19,000 (UCSC 2004 Assembly).
• the deletion is flanked by SNP probes rs7055928 and rsl918560 (at 22.956 and 23.133 Mb from the Xp terminus, respectively).
• the most proximal and distal SNPs (from the Affymetrix SNP microarrays) within the deleted region, as determined by the SNP microarray analysis, are rs7879064 (23.119Mb) and rs4828958(22.972 Mb).
• PCR amplicons from within the deleted region were used to confirm the deletion by Qpcr (PCR primers and locations are given below). This deletion spans the entire exon 1 of the PTCHDl gene (NM l 73495). Analysis of both Sty and Nsp chips data identified this event and was further validated using PCR and QPCR techniques. The following primers were used:
• DDV Genomic Variants
• the mutation screening revealed an I173V mutation.
• Controls (439) were tested for the I173V and V195I mutations, 500 controls for ML336-337II, and 282 controls for L73F and E479G. None of these mutations were present in controls. Furthermore, the fact that these mutations were all maternally inherited to male probands, and were not observed in our control populations, indicates that the mutations are associated with ASD. In turn, it is reasonable to assume that these mutations contribute to the etiology of autism, and perhaps in-combination with other disease-related loci, give rise to the ASD phenotype.
• DPD dihydropyrimidine dehydrogenase

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