De novo variants adjacent to the canonical splicing sites or in the well-defined splicing-related regions are more likely to impair splicing but remain under-investigated in autism spectrum disorder (ASD). By analyzing large, recent ASD genome sequencing cohorts, we find a significant burden of de novo potential splicing-disrupting variants (PSDVs) in 5048 probands compared to 4090 unaffected siblings. We identified 55 genes with recurrent de novo PSDVs that were highly intolerant to variation. Forty-six of these genes have not been strongly implicated in ASD or other neurodevelopmental disorders previously, including GSK3B. Through international, multicenter collaborations, we assembled genotype and phenotype data for 15 individuals with GSK3B variants and identified common phenotypes including developmental delay, ASD, sleeping disturbance, and aggressive behavior. Using available single-cell transcriptomic data, we show that GSK3B is enriched in dorsal progenitors and intermediate forms of excitatory neurons in the developing brain. We showed that Gsk3b knockdown in mouse excitatory neurons interferes with dendrite arborization and spine maturation which could not be rescued by de novo missense variants identified from affected individuals. In summary, our findings suggest that PSDVs may play an important role in the genetic etiology of ASD and allow for the prioritization of new ASD candidate genes. Importantly, we show that genetic variation resulting in GSK3B loss-of-function can lead to a neurodevelopmental disorder with core features of ASD and developmental delay.

与经典剪接位点相邻或位于定义明确的剪接相关区域的从头变异更有可能损害剪接，但在自闭症谱系障碍 （ASD） 中仍未得到充分研究。通过分析大型、最近的 ASD 基因组测序队列，我们发现与 4090 个未受影响的兄弟姐妹相比，5048 个先证者中存在明显的潜在剪接破坏变异 （PSDV）。我们确定了 55 个对变异高度不耐受的复发性新发 PSDVs 基因。这些基因中有 46 个以前与 ASD 或其他神经发育障碍（包括 GSK3B）没有强烈关系。通过国际、多中心合作，我们收集了 15 名具有 GSK3B 变异的个体的基因型和表型数据，并确定了常见的表型，包括发育迟缓、ASD、睡眠障碍和攻击性行为。使用可用的单细胞转录组数据，我们表明 GSK3B 在发育中的大脑中的背侧祖细胞和中间形式的兴奋性神经元中富集。我们发现小鼠兴奋性神经元中的 Gsk3b 敲除会干扰树突树枝化和脊柱成熟，而从受影响个体中鉴定的从头错义变异无法挽救。总之，我们的研究结果表明 PSDV 可能在 ASD 的遗传病因中发挥重要作用，并允许优先考虑新的 ASD 候选基因。重要的是，我们表明导致 GSK3B 功能丧失的遗传变异可导致具有 ASD 和发育迟缓核心特征的神经发育障碍。