Hirschsprung disease (HSCR, OMIM 142623) involves congenital intestinal obstruction caused by dysfunction of neural crest cells and their progeny during enteric nervous system (ENS) development. HSCR is a multifactorial disorder; pathogenetic variants accounting for disease phenotype are identified only in a minority of cases, and the identification of novel disease-relevant genes remains challenging. In order to identify and to validate a potential disease-causing relevance of novel HSCR candidate genes, we established a complementary study approach, combining whole exome sequencing (WES) with transcriptome analysis of murine embryonic ENS-related tissues, literature and database searches, in silico network analyses, and functional readouts using candidate gene-specific genome-edited cell clones. WES datasets of two patients with HSCR and their non-affected parents were analysed, and four novel HSCR candidate genes could be identified: ATP7A, SREBF1, ABCD1 and PIAS2. Further rare variants in these genes were identified in additional HSCR patients, suggesting disease relevance. Transcriptomics revealed that these genes are expressed in embryonic and fetal gastrointestinal tissues. Knockout of these genes in neuronal cells demonstrated impaired cell differentiation, proliferation and/or survival. Our approach identified and validated candidate HSCR genes and provided further insight into the underlying pathomechanisms of HSCR.

先天性巨结肠症（HSCR，OMIM 142623）涉及肠神经系统（ENS）发育过程中神经嵴细胞及其后代功能障碍引起的先天性肠梗阻。HSCR 是一种多因素疾病；仅在少数病例中鉴定出导致疾病表型的致病变异，并且鉴定新的疾病相关基因仍然具有挑战性。为了识别和验证新型 HSCR 候选基因的潜在致病相关性，我们建立了一种补充研究方法，将全外显子组测序 (WES) 与小鼠胚胎 ENS 相关组织的转录组分析、文献和数据库搜索相结合，计算机网络分析，以及使用候选基因特异性基因组编辑细胞克隆的功能读数。分析了两名 HSCR 患者及其未受影响父母的 WES 数据集，可以识别出四个新的 HSCR 候选基因：ATP7A、SREBF1、ABCD1和PIAS2。在其他 HSCR 患者中发现了这些基因的更多罕见变异，表明与疾病相关。转录组学显示这些基因在胚胎和胎儿胃肠组织中表达。神经元细胞中这些基因的敲除表明细胞分化、增殖和/或存活受损。我们的方法识别并验证了候选 HSCR 基因，并进一步深入了解 HSCR 的潜在病理机制。