Donna M. Martin

Multiple Recurrent De Novo CNVs, Including Duplications of the 7q11.23 Williams Syndrome Region, Are Strongly Associated with Autism

We have undertaken a genome-wide analysis of rare copy-number variation (CNV) in 1124 autism spectrum disorder (ASD) families, each comprised of a single proband, unaffected parents, and, in most kindreds, an unaffected sibling. We find significant association of ASD with de novo duplications of 7q11.23, where the reciprocal deletion causes Williams-Beuren syndrome, characterized by a highly social personality. We identify rare recurrent de novo CNVs at five additional regions, including 16p13.2 (encompassing genes USP7 and C16orf72) and Cadherin 13, and implement a rigorous approach to evaluating the statistical significance of these observations. Overall, large de novo CNVs, particularly those encompassing multiple genes, confer substantial risks (OR = 5.6; CI = 2.6-12.0, p = 2.4 × 10(-7)). We estimate there are 130-234 ASD-related CNV regions in the human genome and present compelling evidence, based on cumulative data, for association of rare de novo events at 7q11.23, 15q11.2-13.1, 16p11.2, and Neurexin 1.

Phenotypic Heterogeneity of Genomic Disorders and Rare Copy-Number Variants

BACKGROUND Some copy-number variants are associated with genomic disorders with extreme phenotypic heterogeneity. The cause of this variation is unknown, which presents challenges in genetic diagnosis, counseling, and management. METHODS We analyzed the genomes of 2312 children known to carry a copy-number variant associated with intellectual disability and congenital abnormalities, using array comparative genomic hybridization. RESULTS Among the affected children, 10.1% carried a second large copy-number variant in addition to the primary genetic lesion. We identified seven genomic disorders, each defined by a specific copy-number variant, in which the affected children were more likely to carry multiple copy-number variants than were controls. We found that syndromic disorders could be distinguished from those with extreme phenotypic heterogeneity on the basis of the total number of copy-number variants and whether the variants are inherited or de novo. Children who carried two large copy-number variants of unknown clinical significance were eight times as likely to have developmental delay as were controls (odds ratio, 8.16; 95% confidence interval, 5.33 to 13.07; P=2.11×10(-38)). Among affected children, inherited copy-number variants tended to co-occur with a second-site large copy-number variant (Spearman correlation coefficient, 0.66; P<0.001). Boys were more likely than girls to have disorders of phenotypic heterogeneity (P<0.001), and mothers were more likely than fathers to transmit second-site copy-number variants to their offspring (P=0.02). CONCLUSIONS Multiple, large copy-number variants, including those of unknown pathogenic significance, compound to result in a severe clinical presentation, and secondary copy-number variants are preferentially transmitted from maternal carriers. (Funded by the Simons Foundation Autism Research Initiative and the National Institutes of Health.).

Insights into Autism Spectrum Disorder Genomic Architecture and Biology from 71 Risk Loci

Analysis of de novo CNVs (dnCNVs) from the full Simons Simplex Collection (SSC) (N = 2,591 families) replicates prior findings of strong association with autism spectrum disorders (ASDs) and confirms six risk loci (1q21.1, 3q29, 7q11.23, 16p11.2, 15q11.2-13, and 22q11.2). The addition of published CNV data from the Autism Genome Project (AGP) and exome sequencing data from the SSC and the Autism Sequencing Consortium (ASC) shows that genes within small de novo deletions, but not within large dnCNVs, significantly overlap the high-effect risk genes identified by sequencing. Alternatively, large dnCNVs are found likely to contain multiple modest-effect risk genes. Overall, we find strong evidence that de novo mutations are associated with ASD apart from the risk for intellectual disability. Extending the transmission and de novo association test (TADA) to include small de novo deletions reveals 71 ASD risk loci, including 6 CNV regions (noted above) and 65 risk genes (FDR ≤ 0.1).

A Multisite Study of the Clinical Diagnosis of Different Autism Spectrum Disorders

CONTEXT Best-estimate clinical diagnoses of specific autism spectrum disorders (autistic disorder, pervasive developmental disorder-not otherwise specified, and Asperger syndrome) have been used as the diagnostic gold standard, even when information from standardized instruments is available. OBJECTIVE To determine whether the relationships between behavioral phenotypes and clinical diagnoses of different autism spectrum disorders vary across 12 university-based sites. DESIGN Multisite observational study collecting clinical phenotype data (diagnostic, developmental, and demographic) for genetic research. Classification trees were used to identify characteristics that predicted diagnosis across and within sites. SETTING Participants were recruited through 12 university-based autism service providers into a genetic study of autism. PARTICIPANTS A total of 2102 probands (1814 male probands) between 4 and 18 years of age (mean [SD] age, 8.93 [3.5] years) who met autism spectrum criteria on the Autism Diagnostic Interview-Revised and the Autism Diagnostic Observation Schedule and who had a clinical diagnosis of an autism spectrum disorder. MAIN OUTCOME MEASURE Best-estimate clinical diagnoses predicted by standardized scores from diagnostic, cognitive, and behavioral measures. RESULTS Although distributions of scores on standardized measures were similar across sites, significant site differences emerged in best-estimate clinical diagnoses of specific autism spectrum disorders. Relationships between clinical diagnoses and standardized scores, particularly verbal IQ, language level, and core diagnostic features, varied across sites in weighting of information and cutoffs. CONCLUSIONS Clinical distinctions among categorical diagnostic subtypes of autism spectrum disorders were not reliable even across sites with well-documented fidelity using standardized diagnostic instruments. Results support the move from existing subgroupings of autism spectrum disorders to dimensional descriptions of core features of social affect and fixated, repetitive behaviors, together with characteristics such as language level and cognitive function.

Common genetic variants, acting additively, are a major source of risk for autism.

BackgroundAutism spectrum disorders (ASD) are early onset neurodevelopmental syndromes typified by impairments in reciprocal social interaction and communication, accompanied by restricted and repetitive behaviors. While rare and especially de novo genetic variation are known to affect liability, whether common genetic polymorphism plays a substantial role is an open question and the relative contribution of genes and environment is contentious. It is probable that the relative contributions of rare and common variation, as well as environment, differs between ASD families having only a single affected individual (simplex) versus multiplex families who have two or more affected individuals.MethodsBy using quantitative genetics techniques and the contrast of ASD subjects to controls, we estimate what portion of liability can be explained by additive genetic effects, known as narrow-sense heritability. We evaluate relatives of ASD subjects using the same methods to evaluate the assumptions of the additive model and partition families by simplex/multiplex status to determine how heritability changes with status.ResultsBy analyzing common variation throughout the genome, we show that common genetic polymorphism exerts substantial additive genetic effects on ASD liability and that simplex/multiplex family status has an impact on the identified composition of that risk. As a fraction of the total variation in liability, the estimated narrow-sense heritability exceeds 60% for ASD individuals from multiplex families and is approximately 40% for simplex families. By analyzing parents, unaffected siblings and alleles not transmitted from parents to their affected children, we conclude that the data for simplex ASD families follow the expectation for additive models closely. The data from multiplex families deviate somewhat from an additive model, possibly due to parental assortative mating.ConclusionsOur results, when viewed in the context of results from genome-wide association studies, demonstrate that a myriad of common variants of very small effect impacts ASD liability.

Molecular and Phenotypic Aspects of CHD7 Mutation in CHARGE Syndrome

CHARGE syndrome [coloboma of the eye, heart defects, atresia of the choanae, retardation of growth and/or development, genital and/or urinary abnormalities, and ear abnormalities (including deafness)] is a genetic disorder characterized by a specific and a recognizable pattern of anomalies. De novo mutations in the gene encoding chromodomain helicase DNA binding protein 7 (CHD7) are the major cause of CHARGE syndrome. Here, we review the clinical features of 379 CHARGE patients who tested positive or negative for mutations in CHD7. We found that CHARGE individuals with CHD7 mutations more commonly have ocular colobomas, temporal bone anomalies (semicircular canal hypoplasia/dysplasia), and facial nerve paralysis compared with mutation negative individuals. We also highlight recent genetic and genomic studies that have provided functional insights into CHD7 and the pathogenesis of CHARGE syndrome.

Loss of Chd7 function in gene-trapped reporter mice is embryonic lethal and associated with severe defects in multiple developing tissues.

CHD7 is a novel chromodomain gene mutated in 60%–80% of humans with CHARGE syndrome, a multiple congenital anomaly condition characterized by ocular coloboma, heart defects, atresia of the choanae, retarded growth and development, genital hypoplasia, and characteristic ear abnormalities including deafness. Phenotypic features of CHARGE are highly variable and incompletely penetrant. To explore developmental roles of CHD7, we generated mice carrying the Chd7Gt allele from a Chd7-deficient, gene-trapped lacZ reporter ES cell line. RT-PCR of embryo RNA demonstrated significantly reduced levels of wild-type transcript in Chd7Gt/Gt embryos. Chd7Gt/Gt embryos survive only up to embryonic day 10.5 (E10.5). Chd7Gt/+ male and female mice are viable, small, and exhibit variable degrees of head-bobbing and circling, consistent with vestibular dysfunction. Paint-filling of E16.5 heterozygous inner ears revealed defects of the semicircular canals. The pattern of β-galactosidase activity in Chd7Gt/+ embryos mimics Chd7 mRNA expression in wild-type embryos, confirming the fidelity of the lacZ reporter. We observed tissue-specific β-galactosidase in the E12.5 and E14.5 Chd7Gt/+ brain, pituitary, ear, heart, and craniofacial structures, indicating survival of Chd7Gt/+ cells in CHARGE-relevant organs. These studies demonstrate the utility of Chd7Gt as a reporter-tagged loss-of-function allele for future studies exploring developmental mechanisms of Chd7 deficiency.

Characterization of Progenitor Domains in the Developing Mouse Thalamus

To understand the molecular basis of the specification of thalamic nuclei, we analyzed the expression patterns of various transcription factors and defined progenitor cell populations in the embryonic mouse thalamus. We show that the basic helix‐loop‐helix (bHLH) transcription factor Olig3 is expressed in the entire thalamic ventricular zone and the zona limitans intrathalamica (ZLI). Next, we define two distinct progenitor domains within the thalamus, which we name pTH‐R and pTH‐C, located caudal to the ZLI. pTH‐R is immediately caudal to the ZLI and expresses Nkx2.2, Mash1, and Olig3. pTH‐C is caudal to pTH‐R and expresses Ngn1, Ngn2, and Olig3. Short‐term lineage analysis of Olig3‐, Mash1‐, Ngn1‐, and Ngn2‐expressing progenitor cells as well as tracing the Pitx2 cell lineage suggests that pTH‐C is the only major source of thalamic nuclei containing neurons that project to the cerebral cortex, whereas pTH‐R and ZLI are likely to produce distinct postmitotic populations outside of the cortex‐projecting part of the thalamus. To determine if pTH‐C is composed of subdomains, we characterized expression of the homeodomain protein Dbx1 and the bHLH protein Olig2. We show that Dbx1 is expressed in caudodorsal‐high to rostroventral‐low gradient within pTH‐C. Analysis of heterozygous Dbx1nlslacZ knockin mice demonstrated that Dbx1‐expressing progenitors preferentially give rise to caudodorsal thalamic nuclei. Olig2 is expressed in an opposite gradient within pTH‐C to that of Dbx1. These results establish the molecular heterogeneity within the progenitor cells of the thalamus, and suggest that such heterogeneity contributes to the specification of thalamic nuclei. J. Comp. Neurol. 505:73–91, 2007.

PITX2, β-catenin and LEF-1 interact to synergistically regulate the LEF-1 promoter

PITX2, β-catenin and lymphoid enhancer factor (LEF-1) are required for the inductive formation of several epithelial-derived organs, including teeth. Lef-1 is expressed in the dental epithelium after Pitx2, and both factors have overlapping expression patterns in the tooth bud and cap stages. Our analysis of Pitx2–/– mutant mice showed reduced Lef-1 expression in facial tissues by RT-PCR and quantitative RT-PCR. Consistent with these results we show that the human 2.5 kb LEF-1 promoter is activated by PITX2. Furthermore, the LEF-1 promoter is differentially activated by PITX2 isoforms, which are co-expressed in dental epithelium. The 2.5 kb LEF-1 promoter contains two regions that act to inhibit its transcription in concert with PITX2. The proximal region contains a Wnt-responsive element (WRE) that attenuates PITX2 activation. LEF-1 cannot autoregulate LEF-1 expression; however co-transfection of PITX2 and LEF-1 result in a synergistic activation of the 2.5 kb LEF-1 promoter. LEF-1 specifically interacts with the PITX2 C-terminal tail. Deletion of a distal 800 bp segment of the LEF-1 promoter resulted in enhanced PITX2 activation, and increased synergistic activation in the presence of LEF-1. Furthermore, β-catenin in combination with PITX2 synergistically activates the LEF-1 promoter and this activation is independent of the Wnt-responsive element. β-catenin directly interacts with PITX2 to synergistically regulate LEF-1 expression. We show a new mechanism where LEF-1 expression is regulated through PITX2, LEF-1 and β-catenin direct physical interactions. LEF-1 and β-catenin interactions with PITX2 provide new mechanisms for the regulation of PITX2 transcriptional activity.

SEMA3E mutation in a patient with CHARGE syndrome

CHARGE association (MIM 214800) is a sporadic disorder, characterised by coloboma of the eye, choanal atresia, cranial nerve dysfunction, characteristic external and inner ear abnormalities, cardiac anomalies, genitourinary abnormalities, and growth retardation. Various chromosomal rearrangements have been reported in rare patients with a CHARGE-like phenotype. These include balanced translocation between chromosomes 6 and 8,1 unbalanced translocations involving chromosomes 2 and 18, 3 and 22,2 partial trisomy of 19q with partial monosomy 21q,3 inverted duplication of chromosome 14 (14q22→q24.3),4 and partial trisomy of 2q.5 Based in part on the inconsistent chromosomal aberrations in rare patients with CHARGE association, it is most likely that this condition is genetically heterogeneous. Within the group of children with CHARGE association, there is clearly a subgroup with distinctive clinical characteristics that appears to have a recognisable syndrome.6 Previously, a systematic scan for loss of heterozygosity using microsatellite markers in 10 such patients failed to identify a discernible submicroscopic deletion.7 Although several candidate genes such as PITX2 8 and PAX2 9 have been investigated, no mutations have been identified in patients with CHARGE syndrome. Here, we demonstrate a de novo mutation in SEMA3E in an affected patient, identified upon mapping the translocation breakpoints in an unrelated individual with a de novo balanced translocation involving chromosomes 2 and 7: karyotype 46,XY,t(2;7)(p14;q21.11). The study sample includes 72 patients, of whom 43 have either four major criteria for CHARGE (coloboma, choanal atresia, characteristic ear abnormality, and cranial nerve dysfunction) or three major and three minor criteria (genital hypoplasia, developmental delay, cardiovascular malformations, growth deficiency, orofacial cleft, tracheo-oesopageal fistula, and characteristic face) as described by Blake et al .10 This set of patients was used for all FISH analyses, DHPLC, and candidate gene sequencing. An additional 29 patients with clinical diagnosis of CHARGE, …