De Novo Variation in Autism by Sex and Diagnostic Status in 41,367 Parent-Child Trios

Autism shows a consistent sex bias, yet how sex shapes de novo variant (DNV) risk across coding and noncoding sequence remains unclear. We analyzed DNVs in 41,367 parent-child sequenced trios from three autism family-based cohorts and compared DNV characteristics and enrichment patterns in males and females. Importantly, these trios consisted of some trios with individuals with autism and some without autism. We developed a new sex-aware DNV caller and performed intensive, feature-based investigation of each candidate DNV to produce a high-confidence callset. We identified enrichment of missense and loss-of-function (LOF) DNVs both overall and within known autism-related genes (i.e., SFARI genes). Gene-specific enrichment analyses revealed twelve genes that were exome-wide significant and specific to males, for significance, including FOXP1, SMAD6, AUTS2, CCDC168, PIEZO1, EML6, ZNF84, IGSF23, OTOG, SLC6A1, GIGYF1, and FREM3 and four genes that were specific to females, for significance, including TAOK1, MECP2, DDX3X, and TBL1XR1 within a variant class. Direct comparisons of DNVs in males and females revealed GABBR2 as the only gene trending toward enrichment in the direct males with autism comparison to females with autism. Finally, we analyzed promoters and identified a single significant promoter region (p = 3.8x10-13), associated with the WDR74 gene, with the signal driven by DNVs observed in males with autism. Surprisingly, the noncoding RNA gene RNU2-2 lies within this significant WDR74 promoter and accounted for most of the DNVs in the region. RNU2-2 DNVs were present in 0.2% of males with autism, and several are predicted to potentially alter RNA folding. We also observed RNU2-2 DNVs in 0.2% of females with autism, including two DNVs that were recurrent (i.e., shared) with unrelated, affected males. Notably, RNU2-2 DNVs were detected in 0.1% of unaffected males and were not observed in unaffected females. Together, these results suggest that although RNU2-2 does not show a sex bias, it contributes to autism risk, which is intriguing due to a prior study implicating RNU2-2 in a severe neurodevelopmental disorder.