Despite the known heritable nature of autism spectrum disorder (ASD), studies have primarily identified risk genes with de novo variants (DNVs). To capture the full spectrum of ASD genetic risk, we performed a two-stage analysis of rare de novo and inherited coding variants in 42,607 ASD cases, including 35,130 new cases recruited online by SPARK. In the first stage, we analyzed 19,843 cases with one or both biological parents and found that known ASD or neurodevelopmental disorder (NDD) risk genes explain nearly 70% of the genetic burden conferred by DNVs. In contrast, less than 20% of genetic risk conferred by rare inherited loss-of-function (LoF) variants are explained by known ASD/NDD genes. We selected 404 genes based on the first stage of analysis and performed a meta-analysis with an additional 22,764 cases and 236,000 population controls. We identified 60 genes with exome-wide significance (p < 2.5e-6), including five new risk genes (NAV3, ITSN1, MARK2, SCAF1, and HNRNPUL2). The association of NAV3 with ASD risk is entirely driven by rare inherited LoFs variants, with an average relative risk of 4, consistent with moderate effect. ASD individuals with LoF variants in the four moderate risk genes (NAV3, ITSN1, SCAF1, and HNRNPUL2, n = 95) have less cognitive impairment compared to 129 ASD individuals with LoF variants in well-established, highly penetrant ASD risk genes (CHD8, SCN2A, ADNP, FOXP1, SHANK3) (59% vs. 88%, p= 1.9e-06) . These findings will guide future gene discovery efforts and suggest that much larger numbers of ASD cases and controls are needed to identify additional genes that confer moderate risk of ASD through rare, inherited variants.

In the past decade, several studies have estimated the human per-generation germline mutation rate using large pedigrees. More recently, estimates for various non-human species have been published. However, methodological differences among studies in detecting germline mutations and estimating mutation rates make direct comparisons difficult. Here, we describe the many different steps involved in estimating pedigree-based mutation rates, including sampling, sequencing, mapping, variant calling, filtering, and how to appropriately account for false-positive and false-negative rates. For each step, we review the different methods and parameter choices that have been used in the recent literature. Additionally, we present the results from a 'Mutationathon', a competition organized among five research labs to compare germline mutation rate estimates for a single pedigree of rhesus macaques. We report almost a two-fold variation in the final estimated rate among groups using different post-alignment processing, calling, and filtering criteria and provide details into the sources of variation across studies. Though the difference among estimates is not statistically significant, this discrepancy emphasizes the need for standardized methods in mutation rate estimations and the difficulty in comparing rates from different studies. Finally, this work aims to provide guidelines for computational and statistical benchmarks for future studies interested in identifying germline mutations from pedigrees.

While 9p deletion and duplication syndromes have been studied for several years, small sample sizes and minimal high-resolution data have limited a comprehensive delineation of genotypic and phenotypic characteristics. In this study, we examined genetic data from 719 individuals in the worldwide 9p Network Cohort: a cohort seven to nine times larger than any previous study of 9p. Most breakpoints occur in bands 9p22 and 9p24, accounting for 35% and 38% of all breakpoints, respectively. Bands 9p11 and 9p12 have the fewest breakpoints, with each accounting for 0.6% of all breakpoints. The most common phenotype in 9p deletion and duplication syndromes is developmental delay, and we identified eight known neurodevelopmental disorder genes in 9p22 and 9p24. Since it has been previously reported that some individuals have a secondary structural variant related to the 9p variant, we examined our cohort for these variants and found 97 events. The top secondary variant involved 9q in 14 individuals (1.9%), including ring chromosomes and inversions. We identified a gender bias with significant enrichment for females (p = 0.0006) that may arise from a sex reversal in some individuals with 9p deletions. Genes on 9p were characterized regarding function, constraint metrics, and protein-protein interactions, resulting in a prioritized set of genes for further study. Finally, we achieved precision genomics in one child with a complex 9p structural variation using modern genomic technologies, demonstrating that long-read sequencing will be integral for some cases. Our study is the largest ever on 9p-related syndromes and provides key insights into genetic factors involved in these syndromes.

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Autism spectrum disorder (ASD) is a genetically heterogeneous condition, caused by a combination of rare de novo and inherited variants as well as common variants in at least several hundred genes. However, significantly larger sample sizes are needed to identify the complete set of genetic risk factors. We conducted a pilot study for SPARK (SPARKForAutism.org) of 457 families with ASD, all consented online. Whole exome sequencing (WES) and genotyping data were generated for each family using DNA from saliva. We identified variants in genes and loci that are clinically recognized causes or significant contributors to ASD in 10.4% of families without previous genetic findings. Additionally, we identified variants that are possibly associated with autism in an additional 3.4% of families. A meta-analysis using the TADA framework at a false discovery rate (FDR) of 0.2 provides statistical support for 34 ASD risk genes with at least one damaging variant identified in SPARK. Nine of these genes (BRSK2, DPP6, EGR3, FEZF2, ITSN1, KDM1B, NR4A2, PAX5 and RALGAPB) are newly emerging genes in autism, of which BRSK2 has the strongest statistical support as a risk gene for autism (TADA q-value = 0.0015). Future studies leveraging the thousands of individuals with ASD that have enrolled in SPARK are likely to further clarify the genetic risk factors associated with ASD as well as allow accelerate autism research that incorporates genetic etiology. Rolen=465 Average age of ASD dx (years) Average age at registration (years) Has Intellectual disability(%) Non-verbal (%) Has Epilepsy (%) Has ADHD (%) Affected male offspring 376 4.8 12 22% (78/356) 13% (46/356) 7% (25/356) 30%(106/356) Affected female offspring 89 5.6 12 33% (28/84) 10% (8/84) 13% (11/84) 23% (19/84)Table 1: Phenotypic description of the 457 families with at least 1 offspring affected with ASD in the SPARK pilot study. 1379 individuals in 39 multiplex and 418 simplex families were genomically characterized, including 472 individuals (465 offspring and 7 parents affected with ASD) were sequenced. Phenotypic variables are not available for each person, so they are reported for whom they are available. All offspring with ASD

Autism is a highly heritable, complex disorder where de novo mutation (DNM) variation contributes significantly to disease risk. Using whole-genome sequencing data from 3,474 families, we investigate another source of large-effect risk variation, ultra-rare mutations. We report and replicate a transmission disequilibrium of private likely-gene disruptive (LGD) 5 mutations in probands but find that 95% of this burden resides outside of known DNM-enriched genes. This variant class more strongly affects multiplex family probands and supports a multihit model for autism. Candidate genes with private LGD variants preferentially transmitted to probands converge on the E3 ubiquitin-protein ligase complex, intracellular transport, and Erb signaling protein networks. We estimate these mutations are ~2.5 generations old and 10 significantly younger than other mutations of similar type and frequency in siblings. Overall, private LGD variants are under strong purifying selection and act on a distinct set of genes not yet associated with autism. 15One sentence summary: Ultra-rare autism variants preferentially transmitted to probands are younger and identify distinct gene candidates and functional networks.

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Three variants were misnamed in the text and mislabeled in Figure 2. The relevant sentence has been changed to read, ''The three de novo CHD8 mutations observed in CM1 include two stop-gain mutations (GenBank: NM_001170629.2; c.4414C>T [p.Arg1472*] and c.4514G>A [p.Trp1505*]) and a splice donor site mutation (c.2907þ1G>T [p.?]).