Autism spectrum disorder (ASD) encompasses a range of neurodevelopmental conditions. Different mutations on a single ASD gene contribute to heterogeneity of disease phenotypes, possibly due to functional diversity of generated isoforms. SHANK2, a causative gene in ASD, demonstrates this phenomenon, but there is a scarcity of tools for studying endogenous SHANK2 proteins in an isoform-specific manner. Here, we report a point mutation on SHANK2, which is found in a patient with autism, located on exon of the SHANK2B transcript variant (NM_133266.5), hereby SHANK2B^Y29X. This mutation results in an early stop codon and an aberrant splicing event that impacts SHANK2 transcript variants distinctly. Induced pluripotent stem cells (iPSCs) carrying this mutation, from the patient or isogenic editing, fail to differentiate into functional dopamine (DA) neurons, which can be rescued by genetic correction. Available SMART-Seq single-cell data from human midbrain reveals the abundance of SHANK2B transcript in the ALDH1A1 negative DA neurons. We then show that SHANK2B^Y29X mutation primarily affects SHANK2B expression and ALDH1A1 negative DA neurons in vitro during early neuronal developmental stage. Mice knocked in with the identical mutation exhibit autistic-like behavior, decreased occupancy of ALDH1A1 negative DA neurons and decreased dopamine release in ventral tegmental area (VTA). Our study provides novel insights on a SHANK2 mutation derived from autism patient and highlights SHANK2B significance in ALDH1A1 negative DA neuron.

自闭症谱系障碍 (ASD) 涵盖一系列神经发育疾病。单个 ASD 基因的不同突变会导致疾病表型的异质性，这可能是由于所产生的亚型的功能多样性所致。 SHANK2 是 ASD 的致病基因，它证明了这种现象，但缺乏以异构体特异性方式研究内源 SHANK2 蛋白的工具。在这里，我们报告了 SHANK2 上的点突变，该突变在自闭症患者中发现，位于 SHANK2B 转录变体 (NM_133266.5) 的外显子上，特此为 SHANK2B ^Y29X。这种突变会导致早期终止密码子和异常剪接事件，从而明显影响 SHANK2 转录本变体。来自患者或同基因编辑的携带这种突变的诱导多能干细胞（iPSC）无法分化为功能性多巴胺（DA）神经元，而这可以通过基因校正来挽救。来自人类中脑的可用 SMART-Seq 单细胞数据揭示了 ALDH1A1 阴性 DA 神经元中 SHANK2B 转录本的丰度。然后我们表明，SHANK2B ^Y29X突变主要影响早期神经元发育阶段体外的 SHANK2B 表达和 ALDH1A1 阴性 DA 神经元。携带相同突变的小鼠表现出类似自闭症的行为，ALDH1A1 阴性 DA 神经元的占用减少，腹侧被盖区 (VTA) 的多巴胺释放减少。我们的研究提供了关于源自自闭症患者的 SHANK2 突变的新见解，并强调了 SHANK2B 在 ALDH1A1 阴性 DA 神经元中的重要性。