Abstract

Autism is a neurodevelopmental disorder of complex etiology in which genetic factors play a major role. We have implicated the neurexin 1 (NRXN1) gene in two independent subjects who display an autism spectrum disorder (ASD) in association with a balanced chromosomal abnormality involving 2p16.3. In the first, with karyotype 46,XX,ins(16;2)(q22.1;p16.1p16.3)pat, NRXN1 is directly disrupted within intron 5. Importantly, the father possesses the same chromosomal abnormality in the absence of ASD, indicating that the interruption of α-NRXN1 is not fully penetrant and must interact with other factors to produce ASD. The breakpoint in the second subject, with 46,XY,t(1;2)(q31.3;p16.3)dn, occurs ∼750 kb 5′ to NRXN1 within a 2.6 Mb genomic segment that harbors no currently annotated genes. A scan of the NRXN1 coding sequence in a cohort of ASD subjects, relative to non-ASD controls, revealed that amino acid alterations in neurexin 1 are not present at high frequency in ASD. However, a number of rare sequence variants in the coding region, including two missense changes in conserved residues of the α-neurexin 1 leader sequence and of an epidermal growth factor (EGF)-like domain, respectively, suggest that even subtle changes in NRXN1 might contribute to susceptibility to ASD. Autism is a neurodevelopmental disorder of complex etiology in which genetic factors play a major role. We have implicated the neurexin 1 (NRXN1) gene in two independent subjects who display an autism spectrum disorder (ASD) in association with a balanced chromosomal abnormality involving 2p16.3. In the first, with karyotype 46,XX,ins(16;2)(q22.1;p16.1p16.3)pat, NRXN1 is directly disrupted within intron 5. Importantly, the father possesses the same chromosomal abnormality in the absence of ASD, indicating that the interruption of α-NRXN1 is not fully penetrant and must interact with other factors to produce ASD. The breakpoint in the second subject, with 46,XY,t(1;2)(q31.3;p16.3)dn, occurs ∼750 kb 5′ to NRXN1 within a 2.6 Mb genomic segment that harbors no currently annotated genes. A scan of the NRXN1 coding sequence in a cohort of ASD subjects, relative to non-ASD controls, revealed that amino acid alterations in neurexin 1 are not present at high frequency in ASD. However, a number of rare sequence variants in the coding region, including two missense changes in conserved residues of the α-neurexin 1 leader sequence and of an epidermal growth factor (EGF)-like domain, respectively, suggest that even subtle changes in NRXN1 might contribute to susceptibility to ASD. Autism and related developmental disabilities, clinically referred to as autism spectrum disorders (ASDs; MIM 209850), affect up to ∼1 in 150 children1Autism and Developmental Disabilities Monitoring Network Surveillance Year 2002 Principal InvestigatorsPrevalence of autism spectrum disorders–autism and developmental disabilities monitoring network, 14 sites, United States, 2002.MMWR Surveillance Summaries. 2007; 56: 12-28PubMed Google Scholar and include language impairment, difficulties in social interaction and communication, a restricted pattern of interests, unusual behaviors, and/or stereotyped and repetitive motor mannerisms. ASDs are likely to result from a complex interaction of genetic factors, environment, experience, and, possibly, chance. Most readily analyzed are the genes because the fully sequenced human genome presents a finite universe to explore for contributing factors. ASDs are among the most heritable behavioral disorders, on the basis of familial relative risk and twin studies.2Santangelo S.L. Tsatsanis K. What is known about autism: Genes, brain, and behavior.Am. J. Pharmacogenomics. 2005; 5: 71-92Crossref PubMed Scopus (128) Google Scholar Although several monogenic disorders might involve autism-like symptoms, their relevance to most cases of ASD is not clear. Genetic-linkage studies have suggested a few novel chromosomal locations for ASD genes, but the richest source of candidates might lie in chromosomal abnormalities that target particular genomic segments.3Grice D.E. Buxbaum J.D. The genetics of autism spectrum disorders.Neuromolecular Med. 2006; 8: 451-460Crossref PubMed Scopus (33) Google Scholar, 4Xu J. Zwaigenbaum L. Szatmari P. Scherer S.W. Molecular cytogenetics of autism.Curr. Genomics. 2004; 5: 347-364Crossref Scopus (64) Google Scholar, 5Vorstman J.A. Staal W.G. van Daalen E. van Engeland H. Hochstenbach P.F. Franke L. 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Senman L. et al.Mapping autism risk loci using genetic linkage and chromosomal rearrangements.Nat. Genet. 2007; 39: 319-328Crossref PubMed Scopus (1065) Google Scholar Cytogenetic abnormalities occur in >5% of ASD cases and, although some regions, such as 2q37, 15q11–q13, and 22q13.3, are seen recurrently, the Autism Chromosome Rearrangement Database4Xu J. Zwaigenbaum L. Szatmari P. Scherer S.W. Molecular cytogenetics of autism.Curr. Genomics. 2004; 5: 347-364Crossref Scopus (64) Google Scholar currently reports 391 visible chromosomal breakpoints spread throughout the genome. Recent application of higher-resolution techniques has revealed frequent submicroscopic genomic abnormalities and documented a significant increase in de novo copy-number changes, suggesting that many different genomic disruptions might predispose to ASD.6Sebat J. Lakshmi B. Malhotra D. Troge J. Lese-Martin C. Walsh T. Yamrom B. Yoon S. Krasnitz A. 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Genet. 2007; 39: 319-328Crossref PubMed Scopus (1065) Google Scholar These genetic analyses are consistent with the view that many different genes contribute, possibly leading by different means to a final common neurodevelopmental pathway that produces the autism phenotype. ASD genes are likely to involve both common variants that contribute broadly to susceptibility and rare variants that contribute in fewer individuals but might have stronger effect. Either could provide fundamental insights into the mechanism(s) leading to ASD. In the Developmental Genome Anatomy Project (DGAP), we have sought genes of developmental importance disrupted by apparently balanced chromosomal aberrations.9Alkuraya F.S. Saadi I. Lund J.J. Turbe-Doan A. Morton C.C. Maas R.L. SUMO1 haploinsufficiency leads to cleft lip and palate.Science. 2006; 313: 1751Crossref PubMed Scopus (137) Google Scholar, 10Kim H.G. Herrick S.R. Lemyre E. Kishikawa S. Salisz J.A. Seminara S. MacDonald M.E. Bruns G.A. Morton C.C. 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Genet. 2007; 80: 616-632Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar We have focused where possible on “double-hit” cases, in which independent breakpoints in two individuals with related phenotypes occur in the same chromosomal region, maximizing the likelihood that the chromosomal disruption is causative. The independent occurrence among our first 200 DGAP cases, encompassing a wide variety of clinical phenotypes, of two subjects displaying ASD in association with a chromosomal breakpoint in 2p16.3 has revealed NRXN1 (MIM 600565) as an autism-associated gene, consistent with recent reports of both de novo heterozygous deletion of NRXN1 and rare sequence variants in the β-NRXN1 leader sequence in ASD.8Szatmari P. Paterson A.D. Zwaigenbaum L. Roberts W. Brian J. Liu X.Q. Vincent J.B. Skaug J.L. Thompson A.P. Senman L. et al.Mapping autism risk loci using genetic linkage and chromosomal rearrangements.Nat. Genet. 2007; 39: 319-328Crossref PubMed Scopus (1065) Google Scholar, 13Feng J. Schroer R. Yan J. Song W. Yang C. Bockholt A. Cook Jr., E.H. Skinner C. Schwartz C.E. Sommer S.S. High frequency of neurexin 1beta signal peptide structural variants in patients with autism.Neurosci. Lett. 2006; 409: 10-13Crossref PubMed Scopus (248) Google Scholar Two DGAP subjects with an ASD phenotype, DGAP123 and DGAP200, display chromosomal breakpoints in 2p16.3: DGAP123 is a female subject with a familial rearrangement, 46,XX,ins(16;2)(q22.1;p16.1p16.3)pat, and DGAP200 is a male with a de novo apparently balanced translocation, 46,XY,t(1;2)(q31.3;p16.3)dn (Figure 1). Blood samples were obtained from both and from the parents of DGAP123 for preparation of DNA and EBV-transformed lymphoblastoid cell lines.14Anderson M.A. Gusella J.F. Use of cyclosporin A in establishing Epstein-Barr virus-transformed human lymphoblastoid cell lines.In Vitro. 1984; 20: 856-858Crossref PubMed Scopus (290) Google Scholar All human studies were performed under informed consent protocols approved by the Partners HealthCare System Human Research Committee (Boston, MA) or the Yale University School of Medicine (New Haven, CT). The detailed phenotypes of DGAP123, her mother (DGAP123-2), and her father (DGAP124) were determined by diagnostic instruments, behavioral questionnaires, and neuropsychological assessment in the Psychiatric and Neurodevelopmental Genetics Unit (PNGU) of the Center for Human Genetic Research, Massachusetts General Hospital. This was not possible for DGAP200, whose phenotypic information and diagnoses were provided by communication with the clinician. DGAP123 meets criteria for autism on both the ADOS and ADI-R, with manifestations that include ritualized behaviors, vocal and motor mannerisms, limited eye contact, minimal verbal output, little change in affect or facial expression, and minimal seeking of interaction.15Lord C. Rutter M. Goode S. Heemsbergen J. Jordan H. Mawhood L. Schopler E. Autism diagnostic observation schedule: A standardized observation of communicative and social behavior.J. Autism Dev. Disord. 1989; 19: 185-212Crossref PubMed Scopus (1385) Google Scholar, 16Lord C. Rutter M. Le Couteur A. Autism diagnostic interview-revised: A revised version of a diagnostic interview for caregivers of individuals with possible pervasive developmental disorders.J. Autism Dev. Disord. 1994; 24: 659-685Crossref PubMed Scopus (6298) Google Scholar Her overall level of functioning falls within the range of mental retardation. Neither parent meets formal criteria for autism, although each has minor functional abnormalities. The parents' overall intellectual abilities are in the normal range, although the mother exhibits a relative deficit in working memory. The father, who shares the chromosome rearrangement with his daughter, displayed stuttering as a child, has received speech services in the past, and has some persistent difficulty in articulation. He also exhibits some features of obsessive compulsive disorder (OCD; MIM 143465) and attention deficit disorder (ADHD; MIM 143465), although he has never received a formal diagnosis of either. DGAP200 has diagnoses of PDD-NOS (MIM 209850), along with ADHD, conduct disorder with early onset, and intermittent explosive disorder. The chromosomal abnormality in DGAP123 involves the excision of ∼8.9 Mb of DNA between 2p16.1 and 2p16.3 and its insertion into 16q22.1 (Figure 1). By array comparative genomic hybridization (array CGH; 244K Human Oligo chip, Agilent Technologies, Palo Alto CA), this anomaly is balanced, showing no significant loss or gain of copy number at either site or elsewhere in the genome. FISH with whole BAC clones, chosen from NCBI and UCSC websites and obtained from CITB-D and RP11 libraries (Invitrogen, San Diego, CA, and the Children's Hospital of Oakland Research Institute, CA), mapped the 2p16 breakpoints within BAC clones RP11-391D19 (Figure 2) and RP11-579K4. Southern-blot analysis in DGAP123 narrowed the breakpoint to a 1368 bp region (Figure 3) in intron 5 of the large NRXN1 gene, which spans 1.1 Mb and encodes α-neurexin 1 from 24 exons and β-neurexin 1 from seven exons (Figure 4). Because each isoform is produced from a different promotor and has a different first exon,17Rowen L. Young J. Birditt B. Kaur A. Madan A. Philipps D.L. Qin S. Minx P. Wilson R.K. Hood L. Graveley B.R. Analysis of the human neurexin genes: Alternative splicing and the generation of protein diversity.Genomics. 2002; 79: 587-597Crossref PubMed Scopus (140) Google Scholar, 18Tabuchi K. Sudhof T.C. Structure and evolution of neurexin genes: Insight into the mechanism of alternative splicing.Genomics. 2002; 79: 849-859Crossref PubMed Scopus (201) Google Scholar the DGAP123 breakpoint directly disrupts the α-NRXN1 sequence but leaves β-NRXN1 coding sequences intact.Figure 3Mapping of the 2p16.3 and 2p16.1 Breakpoints of DGAP123Show full caption(A) The mapping of the two chromosome 2 breakpoints (dashed red vertical lines) at the edges of the 8.9 Mb of DNA (shown in red) excised and inserted into chromosome 16. Below the map schematic are BAC clones that in FISH experiments detect both der(2) and der(16) (blue lines) or only one of the derivative chromosomes (black lines). Below the BACs are shown restriction fragments used in DNA-blotting experiments (see [B]) to confine the breakpoints to small segments of 2p16.3 and 2p16.1, respectively.(B) Genomic DNA blots hybridized with probes from the 2p16.3 breakpoint region (left) and the 2p16.1 breakpoint region (right), respectively. Each lane contains genomic DNA digested with the designated restriction enzyme from either DGAP123 (P) or a normal control (C). Additional bands in the P lanes indicate novel restriction fragments generated by the interchromosomal exchange. The hybridization probes P258C and P328, which detected aberrant bands containing breakpoints at 2p16.3 and 2p16.1, respectively, were amplified using the following primer pairs:P258C: 5′-ATGTCTGATATTATAAGGTGAAACTCCGGTCTTCC-3′ and5′-CAAGTCCTGTGTTGCTATATAGCGAATTTGTCTG-3′;P328: 5′-CTGTTTTCTTCTCTCACTATATGAGTTGAACATATACAAATAGGC-3′ and5′-GGAAGTGGAAAGCTGCTGTTTCTCAGCCATTGCTCA-3′.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4NRXN1 Region of 2p16.3 and DGAP Subject BreakpointsShow full captionThis schematic diagram, reworked from tracks provided by the UCSC Genome Browser, shows exon locations and transcript orientation for α-NRXN1 and the overlapping β-NRXN1, along with the upstream region devoid of known genes, below a black bar indicating 2p16.3 and base-pair locations from the telomeric side (left) toward the centromere (right). The blue graph between these shows the estimated regulatory potential (0–0.4) calculated by comparing frequencies of short alignment patterns between known regulatory elements and neutral DNA across seven species (human, chimpanzee, macaque, mouse, rat, dog, and cow).19Taylor J. Tyekucheva S. King D.C. Hardison R.C. Miller W. Chiaromonte F. ESPERR: Learning strong and weak signals in genomic sequence alignments to identify functional elements.Genome Res. 2006; 16: 1596-1604Crossref PubMed Scopus (98) Google Scholar Regulatory potential is highest at the recognized α-NRXN1 and β-NRXN1 promotors and especially at an anonymous site more than 1 Mb upstream of NRXN1. Red lines indicate the position in α-NRXN1 intron 5 and approximate position upstream of NRXN1 for the DGAP123 and DGAP200 breakpoints, respectively. FISH-mapped BAC clones crossing the former are shown in red, with selected clones mapping to der(2) and der(16) shown in dark blue and teal, respectively. For the latter, FISH-mapped BAC clones mapping to der(1) and der(2) are shown in dark brown and light brown, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) The mapping of the two chromosome 2 breakpoints (dashed red vertical lines) at the edges of the 8.9 Mb of DNA (shown in red) excised and inserted into chromosome 16. Below the map schematic are BAC clones that in FISH experiments detect both der(2) and der(16) (blue lines) or only one of the derivative chromosomes (black lines). Below the BACs are shown restriction fragments used in DNA-blotting experiments (see [B]) to confine the breakpoints to small segments of 2p16.3 and 2p16.1, respectively. (B) Genomic DNA blots hybridized with probes from the 2p16.3 breakpoint region (left) and the 2p16.1 breakpoint region (right), respectively. Each lane contains genomic DNA digested with the designated restriction enzyme from either DGAP123 (P) or a normal control (C). Additional bands in the P lanes indicate novel restriction fragments generated by the interchromosomal exchange. The hybridization probes P258C and P328, which detected aberrant bands containing breakpoints at 2p16.3 and 2p16.1, respectively, were amplified using the following primer pairs: P258C: 5′-ATGTCTGATATTATAAGGTGAAACTCCGGTCTTCC-3′ and 5′-CAAGTCCTGTGTTGCTATATAGCGAATTTGTCTG-3′; P328: 5′-CTGTTTTCTTCTCTCACTATATGAGTTGAACATATACAAATAGGC-3′ and 5′-GGAAGTGGAAAGCTGCTGTTTCTCAGCCATTGCTCA-3′. This schematic diagram, reworked from tracks provided by the UCSC Genome Browser, shows exon locations and transcript orientation for α-NRXN1 and the overlapping β-NRXN1, along with the upstream region devoid of known genes, below a black bar indicating 2p16.3 and base-pair locations from the telomeric side (left) toward the centromere (right). The blue graph between these shows the estimated regulatory potential (0–0.4) calculated by comparing frequencies of short alignment patterns between known regulatory elements and neutral DNA across seven species (human, chimpanzee, macaque, mouse, rat, dog, and cow).19Taylor J. Tyekucheva S. King D.C. Hardison R.C. Miller W. Chiaromonte F. ESPERR: Learning strong and weak signals in genomic sequence alignments to identify functional elements.Genome Res. 2006; 16: 1596-1604Crossref PubMed Scopus (98) Google Scholar Regulatory potential is highest at the recognized α-NRXN1 and β-NRXN1 promotors and especially at an anonymous site more than 1 Mb upstream of NRXN1. Red lines indicate the position in α-NRXN1 intron 5 and approximate position upstream of NRXN1 for the DGAP123 and DGAP200 breakpoints, respectively. FISH-mapped BAC clones crossing the former are shown in red, with selected clones mapping to der(2) and der(16) shown in dark blue and teal, respectively. For the latter, FISH-mapped BAC clones mapping to der(1) and der(2) are shown in dark brown and light brown, respectively. The 2p16.1 breakpoint in DGAP123 was mapped to a 584 bp segment within BAC clones RP11-303F17, RP11-66A9, and CTD-3245F16 (Figure 3), in a gene desert flanked by BCL11A (MIM 606557) and FANCL (MIM 608111), located 589 kb proximal and 1.6 Mb distal, respectively. The chromosome 16 breakpoint was flanked, by RP11-52E21 and RP11-311C24, but not further delineated in a 325 kb segment of 16q22.1 that contains the genes SNTB2 (basic beta 2 syntrophin; MIM 600027), VPS4A (vacuolar-protein-sorting factor 4A; MIM 609982), PDF (peptide deformylase, mitochondrial precursor), COG8 (oligomeric golgi complex component 8; MIM 606979), NIP7 (60S ribosome subunit biogenesis protein NIP7 homolog), TMED6 (transmembrane emp24 domain-containing protein 6 precursor), TERF2 (telomeric repeat binding factor 2; MIM 602027), CYB5B (cytochrome b5 outer mitochondrial membrane isoform; MIM 602027), and NFAT5 (nuclear factor of activated T cells 5 isoform a; MIM 604708). For DGAP200, FISH mapping placed the 2p16.3 breakpoint between BAC clones RP11-1141D22 and RP11-18O19, ∼750 kb from exon 1 of α-NRXN1, within a 2.6 Mb upstream segment that is devoid of annotated genes (Figure 4). FISH studies localized the chromosome 1 breakpoint between RP11-25C18 and RP11-173E24 in a gene desert with no genes within 1 Mb on either side. Array CGH analysis of DGAP200 genomic DNA revealed a cryptic deletion of ∼534 kb within 2p16.2, 3 Mb proximal to NRXN1, removing the genes ACYP2 (muscle-type acylphosphatase 2; MIM 102595) and TSPYL6 (Testis-specific protein Y-linked-like 6), FLJ40298 (a hypothetical protein), and SPTBN1 (spectrin, beta, nonerythrocytic 1 isoform 1; MIM 182790). It is conceivable that the deletion of one or more of these loci could contribute to the severe behavioral phenotype of DGAP200. The gene desert upstream of NRXN1 contains sites predicted by ESPERR to have strong regulatory potential on the basis of comparative genomics, in seven species, that used a combination of conservation, composition and short-pattern structure information (Figure 4).19Taylor J. Tyekucheva S. King D.C. Hardison R.C. Miller W. Chiaromonte F. ESPERR: Learning strong and weak signals in genomic sequence alignments to identify functional elements.Genome Res. 2006; 16: 1596-1604Crossref PubMed Scopus (98) Google Scholar This suggested that the DGAP200 translocation, although not directly truncating NRXN1, might still disrupt its expression, perhaps by separating a long-range regulatory element from the coding portions of the gene. Because NRXN1 expression has been studied previously mainly in neuronal tissue, we first determined whether the gene is expressed in lymphoblastoid cells, the only tissue readily available from DGAP subjects. We used RT-PCR of RNA from control lymphoblasts to amplify portions of the α-NRXN1 mRNA, as follows: exons 1–2, exons 2–7, exons 7–9, exons 12–14, exons 20–22 (exons 3–5 of β-NRXN1) and exons 22–24 (exons 5–7 of β-NRXN1), as well as exons 1–5 of β-NRXN1. A typical result is shown in Figure 5. All primer pairs produced an appropriately spliced product (albeit at much lower abundance) of expected size and correct DNA sequence compared to brain mRNA, except α-NRXN1 exons 1–2, exons 2–7 and β-NRXN1 exons 1–5, suggesting that while neurexin 1 is expressed, the precise equivalent of neither neuronal α- nor β-neurexin 1 is present. Rather, any neurexin 1 isoform expressed in lymphoblasts is apparently produced from an mRNA that remains to be fully delineated but that does not share a 5′ end with either the brain α or β isoforms. NRXN1 mRNA was detected in all control lymphoblasts tested, as well as in cell lines from DGAP123 and family members and from DGAP200, but variation in the levels of individual RT-PCR products from different exons and mRNA preparations precluded reproducible quantitation. To test for an effect of the DGAP200 translocation, we capitalized on a polymorphism in the NRXN1 3′ untranslated region in which alleles possess either one or two consecutive copies of a 4 bp TTAC stretch. Control heterozygotes showed expression of both allelic variants in lymphoblastoid cell RNA. By contrast, whereas DGAP200 genomic DNA showed heterozygosity, only one allelic variant was present in the corresponding RNA (Figure 6). We also performed western-blot analyses of protein lysates from cultured lymphoblastoid cell lines of DGAP123, her mother (DGAP123-2), her father (DGAP124), DGAP200, and normal controls with a neurexin-1-specific antibody directed against the carboxyl-terminal region expected to be expressed from our mRNA studies. Figure 7 shows typical results. A specific neurexin 1 band at ∼82 kDa was detected reproducibly in all samples. This band is not seen in brain-tissue extracts, in which α-NRXN1 and β-NRXN11 products migrate at 160–200 kDa and 90–100 kDa, respectively, depending on alternative splicing and glycosylation. The ∼82 kDa band was consistently and significantly reduced in intensity in lysates from DGAP123 and from her father, DGAP124, relative to the proband's mother, DGAP123-2, and to normal controls (Figure 7). Interestingly, DGAP200 also displayed significantly reduced expression of this neurexin 1 band, consistent with a position effect of the upstream breakpoint. The precise structure of this ∼82 kDa neurexin 1 isoform relative to the neuronal isoforms and its extent of glycosylation remain to be determined by more detailed studies. Because the “double-hit” nature of the DGAP cases provided a strong argument for these breakpoints contributing to the phenotypes in the respective subjects, we sought to determine whether NRXN1 coding-sequence alterations, such as nonsense mutations, missense alterations, or frame shifts, might be a frequent predisposing factor in ASD. We performed exon scanning of all NRXN1 coding exons by direct sequencing to identify variants, initially in 57 subjects with ASD (autism, Asperger syndrome, and PDD-NOS; MIM 209850) compared with 57 individuals with OCD (MIM 164230) or Tourette syndrome (TS; MIM 137580) (27 and 30, respectively). In addition to common polymorphisms or previously reported SNPs, we identified seven novel variants that are within the coding sequence (Table 1) and that occurred as heterozygous differences in single individuals, all in the ASD cohort. None was found on the 114 chromosomes of the TS or OCD cohorts or on 354 control chromosomes from 177 unrelated members of the CEPH collection. Two were missense changes, L18Q in the signal peptide of α-neurexin 1 and L748I in an epidermal growth factor (EGF)-like domain, but the other five variants (three in exons specific to α-NRXN1) did not change amino acids. Subsequently, we sequenced the coding exons of 192 individuals (87 affected) from 53 families of the Autism Genetic Resource Exchange (AGRE) cohort but did not identify any rare coding-sequence variants cosegregating with ASD. Of the seven novel variants reported above, only one, L748I, was seen, in two independent ASD families in a total of three of four ASD affected individuals and one of two unaffected individuals, consistent with the possibility that it is an ASD susceptibility allele with incomplete penetrance.Table 1NRXN1 Coding-Sequence Variants in ASD Screening CohortExonAlterationCodonSNPASDnon-ASDaOCD, TS, and unaffected controls.rs NumberVariant Alleles/Total ChromosomesVariant Alleles/Total Chromosomes2c.53T > Ap.L18Q–1/1140/4682c.105C > Ap.G35–1/1120/4662c.511C > Tp.L171rs104587444/114N.D.5c.912C > Tp.G304–1/1140/4647c.999C > Tp.P333rs23032983/1143/45811c.2242C > Ap.L748I–1/1140/47616c.3165C > Tp.A1055– –1/1140/46222c.3975C > Tp.G1325–1/1140/46224c.4374A > Gp.P1458–1/1140/464a OCD, TS, and unaffected controls. Open table in a new tab The observation of ASD in independent DGAP subjects establishes clearly that disruption of NRXN1 can contribute to autism but further indicates that such disruption, and by extension the consequences of heterozygous deletion, might show incomplete penetrance. DGAP123 and DGAP200 reinforce the recent report from the Autism Genome Project of a family in which two sisters with ASD share a de novo heterozygous deletion of 2p16.3 that directly affects the NRXN1 gene.8Szatmari P. Paterson A.D. Zwaigenbaum L. Roberts W. Brian J. Liu X.Q. Vincent J.B. Skaug J.L. Thompson A.P. Senman L. et al.Mapping autism risk loci using genetic linkage and chromosomal rearrangements.Nat. Genet. 2007; 39: 319-328Crossref PubMed Scopus (1065) Google Scholar However, the lack of ASD phenotype in DGAP124, the father of DGAP123 who shares her chromosomal abnormality, demonstrates that heterozygous inactivation of NRXN1 is not by itself sufficient to cause the symptoms of autism. This is consistent with the report of a deletion of exons 6–9 of α-NRXN1 in a Japanese participant in the International HapMap Project, an individual expected to be without obvious abnormal phenotype.20Redon R. Ishikawa S. Fitch K.R. Feuk L. Perry G.H. Andrews T.D. Fiegler H. Shapero M.H. Carson A.R. Chen W. et al.Global variation in copy number in the human genome.Nature. 2006; 444: 444-454Crossref PubMed Scopus (3066) Google ScholarNRXN1 mutation probably contributes by predisposing or sensitizing the individual, but addition