Some individuals with autism spectrum disorder (ASD) carry functional mutations rarely observed in the general population. We explored the genes disrupted by these variants from joint analysis of protein-truncating (PTV), missense, and copy number variants (CNVs) in a cohort of 63,237 individuals. We discovered 72 ASD risk genes at false discovery rate (FDR)≤0.001 (185 at FDR≤0.05). De novo PTVs, damaging missense variants, and CNVs represented 57.5%, 21.1%, and 8.44% of association evidence, while CNVs conferred greatest relative risk. Meta-analysis with cohorts ascertained for developmental delay (DD, N=91,605) yielded 373 ASD/DD risk genes at FDR≤0.001 (664 at FDR≤0.05), some of which differed in relative frequency of mutation between ASD and DD. The DD-associated genes were enriched in transcriptomes of progenitor and immature neuronal cells whereas genes displaying stronger evidence in ASD were more enriched in maturing neurons and overlapped with schizophreniaassociated genes, emphasizing that these neuropsychiatric disorders share common pathways to risk.

Individuals with autism spectrum disorder (ASD) or related neurodevelopmental disorders (NDDs) often carry disruptive mutations in genes that are depleted of functional variation in the broader population. We build upon this observation and exome sequencing from 154,842 individuals to explore the allelic diversity of rare protein-coding variation contributing risk for ASD and related NDDs. Using an integrative statistical model, we jointly analyzed rare protein-truncating variants (PTVs), damaging missense variants, and copy number variants (CNVs) derived from exome sequencing of 63,237 individuals from ASD cohorts. We discovered 71 genes associated with ASD at a false discovery rate (FDR) ≤ 0.001, a threshold approximately equivalent to exome-wide significance, and 183 genes at FDR ≤ 0.05. Associations were predominantly driven by de novo PTVs, damaging missense variants, and CNVs: 57.4%, 21.2%, and 8.32% of evidence, respectively. Though fewer in number, CNVs conferred greater relative risk than PTVs, and repeat-mediated de novo CNVs exhibited strong maternal bias in parent-of-origin (e.g., 92.3% of 16p11.2 CNVs), whereas all other CNVs showed a paternal bias. To explore how genes associated with ASD and NDD overlap or differ, we analyzed our ASD cohort alongside a developmental delay (DD) cohort from the deciphering developmental disorders study (DDD; n=91,605 samples). We first reanalyzed the DDD dataset using the same models as the ASD cohorts, then performed joint analyses of both cohorts and identified 373 genes contributing to NDD risk at FDR ≤ 0.001 and 662 NDD risk genes at FDR ≤ 0.05. Of these NDD risk genes, 54 genes (125 genes at FDR ≤ 0.05) were unique to the joint analyses and not significant in either cohort alone. Our results confirm overlap of most ASD and DD risk genes, although many differ significantly in frequency of mutation. Analyses of single-cell transcriptome datasets showed that genes associated predominantly with DD were strongly enriched for earlier neurodevelopmental cell types, whereas genes displaying stronger evidence for association in ASD cohorts were more enriched for maturing neurons. The ASD risk genes were also enriched for genes associated with schizophrenia from a separate rare coding variant analysis of 121,570 individuals, emphasizing that these neuropsychiatric disorders share common pathways to risk.

Large genomic databases of neurodevelopmental disorders (NDD) are helpful resources of genomic variations in complex and heterogeneous conditions, as Autism Spectrum Disorder (ASD). We evaluated the role of rare copy number variations (CNVs) and exonic de novo variants, in a molecularly unexplored Brazilian cohort of 30 ASD trios (n = 90), by performing a meta‐analysis of our findings in more than 20,000 patients from NDD cohorts. We identified three pathogenic CNVs: two duplications on 1q21 and 17p13, and one deletion on 4q35. CNVs meta‐analysis (n = 8,688 cases and n = 3,591 controls) confirmed 1q21 relevance by identifying duplications in other 16 ASD patients. Exome analysis led the identification of seven de novo variants in ASD genes (SFARI list): three loss‐of‐function pathogenic variants in CUL3, CACNA1H, and SHANK3; one missense pathogenic variant in KCNB1; and three deleterious missense variants in ATP10A, ANKS1B, and DOCK1. From the remaining 12 de novo variants in non‐previous ASD genes, we prioritized PRPF8 and RBM14. Meta‐analysis (n = 13,754 probands; n = 2,299 controls) identified six and two additional patients with validated de novo variants in PRPF8 and RBM14, respectively. By comparing the de novo variants with a previously established mutational rate model, PRPF8 showed nominal significance before multiple test correction (P = 0.039, P‐value adjusted = 0.079, binomial test), suggesting its relevance to ASD. Approximately 60% of our patients presented comorbidities, and the diagnostic yield was estimated in 23% (7/30: three pathogenic CNVs and four pathogenic de novo variants). Our uncharacterized Brazilian cohort with tetra‐hybrid ethnic composition was a valuable resource to validate and identify possible novel candidate loci. Autism Res 2020, 13: 199–206. © 2019 International Society for Autism Research, Wiley Periodicals, Inc. Lay Summary We believed that to study an unexplored autistic population, such as the Brazilian, could help to find novel genes for autism. In order to test this idea, with our limited budget, we compared candidate genes obtained from genomic analyses of 30 children and their parents, with those of more than 20,000 individuals from international studies. Happily, we identified a genetic cause in 23% of our patients and suggest a possible novel candidate gene for autism (PRPF8).

Autism Spectrum Disorder (ASD) is characterized by impaired social communication, restricted interests, and repetitive and stereotyped behaviors. The TRPC6 (transient receptor potential channel 6) represents an ASD candidate gene under an oligogenic/multifactorial model based on the initial description and cellular characterization of an individual with ASD bearing a de novo heterozygous mutation disrupting TRPC6, together with the enrichment of disruptive TRPC6 variants in ASD cases as compared to controls. Here, we perform a clinical re-evaluation of the initial non-verbal patient, and also present eight newly reported individuals ascertained for ASD and bearing predicted loss-of-function mutations in TRPC6. In order to understand the consequences of mutations in TRPC6 on nervous system function, we used the fruit fly, Drosophila melanogaster, to show that null mutations in transient receptor gamma (trpγ; the fly gene most similar to TRPC6), cause a number of behavioral defects that mirror features seen in ASD patients, including deficits in social interactions (based on courtship behavior), impaired sleep homeostasis (without affecting the circadian control of sleep), hyperactivity in both young and old flies, and defects in learning and memory. Some defects, most notably in sleep, differed in severity between males and females and became normal with age. Interestingly, hyperforin, a TRPC6 agonist and the primary active component of the St. John’s wort antidepressant, attenuated many of the deficits expressed by trpγ mutant flies. In summary, our results provide further evidence that the TRPC6 gene is a risk factor for ASD. In addition, they show that the behavioral defects caused by mutations in TRPC6 can be modeled in Drosophila, thereby establishing a paradigm to examine the impact of mutations in other candidate genes.