Fig. 1. Analysis workflow.

In the discovery stage, we identified DNVs in 16,877 ASD trios and rare LoF variants in 20,491 parents without ASD diagnoses and intellectual disability. We compared properties of de novo and rare variants to identify rare LoFs that contribute to genetic risk in individuals with ASD. We also evaluated their associations with cognitive impairment and enriched gene sets. We performed an initial exome-wide scan of genes enriched by DNVs or showing transmission disequilibrium of rare LoFs to affected offspring and selected a total of 404 genes for further replication, including 159 de novo enriched genes and 260 prioritized transmission disequilibrium genes from enriched gene sets (15 genes were in both). In the meta-analysis stage, we first evaluated evidence from de novo enrichment and transmission disequilibrium of rare inherited LoFs in an expanded set of family-based samples including over 6,000 additional ASD trios and around 2,000 additional duos. The DNVs in ASD were combined with those from an additional 31,565 NDD trios to refine the filters of high-confidence LoF variants in de novo LoF enriched genes. We also constructed an independent dataset of LoF variants of unknown inheritance from 15,780 cases that were not used in de novo or transmission analysis. We compared LoF rates in cases with two population-based sets of controls (n = ~104,000 and ~132,000, respectively). For 367 LoF-intolerant genes on autosomes, the final gene-level evidence was obtained by meta-analyzing P values of de novo enrichment, transmission disequilibrium of high-confidence rare inherited LoFs, and comparison of high-confidence LoFs from cases and controls not used in the de novo or transmission analysis. We also performed a mega-analysis that analyzed high-confidence LoFs identified in all 31,976 unrelated ASD cases and compared their rates with population-based controls. HC, high-confidence.