A complementary study pipeline unravels novel players in the pathoetiology of Hirschsprung's disease

Purpose: Hirschsprung’s disease (HSCR, OMIM 142623) represents one of the main causes of neonatal intestinal obstruction. It is caused by dysfunction of neural crest cells (NCCs) and their progeny during development of the enteric nervous system (ENS). HSCR is considered a multifactorial disorder, however, associated risk genes only account for a minority of cases. Consequently, defining disease-relevant variants is still a demanding task. Methods: To reduce the number of candidate genes identified by Whole Exome Sequencing (WES) and to examine their disease-causing relevance, we established a complementary study pipeline including transcriptome data of murine embryonic ENS-relevant tissues, literature and database searches, in silico network analyses as well as functional assays using genome-edited candidate-specific cell clones. Results: Applying this strategy on a pilot set of two HSCR patients and their non-affected parents led to the identification of four novel HSCR candidate genes: ATP7A, SREBF1, ABCD1 and PIAS2. This candidate gene selection was corroborated by the discovery of further rare variants in additional HSCR cases. Moreover, expression analyses revealed that all four genes are expressed in embryonic murine gastrointestinal tissues. Functional analyses using candidate gene-specific, neuronal-like CRISPR/Cas9-edited knockout cell clones demonstrated impaired differentiation, proliferation and/or cell survival capacity. Conclusions: Taken together, the presented study pipeline was proven to be suitable for the selection and validation of candidate genes as well as to gain insight into underlying pathomechanisms of HSCR. For mRNA expression analyses by microarray profiling, murine embryonic regions of interest (pre-migratory vagal NCCs at stages E8.75 and E9.5; embryonic gut at E13.5, without stomach) were prepared. Embryonic tissues from each litter were pooled and three litters were analyzed per stage.

研究背景（Purpose）：先天性巨结肠（Hirschsprung’s disease, HSCR, OMIM 142623）是新生儿肠梗阻的主要致病原因之一。该病由肠神经系统（enteric nervous system, ENS）发育过程中神经嵴细胞（neural crest cells, NCCs）及其子代细胞的功能异常所引发。HSCR被归类为多因子疾病，但目前已明确的相关风险基因仅能解释少数病例，因此鉴定疾病相关遗传变异仍是一项极具挑战性的工作。
研究方法（Methods）：为减少全外显子组测序（Whole Exome Sequencing, WES）筛选得到的候选基因数量，并验证其致病相关性，本研究建立了一套互补性研究流程，涵盖小鼠胚胎肠神经系统相关组织的转录组数据、文献与数据库检索、计算机模拟网络分析，以及采用基因组编辑候选基因特异性细胞克隆的功能实验。
研究结果（Results）：在包含2例HSCR患者及其未患病父母的先导队列中应用该策略后，成功鉴定出4个新型HSCR候选基因：ATP7A、SREBF1、ABCD1及PIAS2。在额外HSCR病例中发现的更多罕见变异进一步佐证了该候选基因筛选结果。此外，表达分析显示，上述4个基因均在小鼠胚胎胃肠道组织中存在表达。通过针对候选基因的类神经元CRISPR/Cas9编辑敲除细胞克隆开展的功能实验证实，这些基因的缺失会导致细胞分化、增殖及/或细胞存活能力受损。
研究结论（Conclusions）：综上，本研究提出的分析流程可有效用于候选基因的筛选与验证，并为解析HSCR的潜在发病机制提供新的研究视角。在通过微阵列芯片分析开展mRNA表达研究时，我们制备了目标小鼠胚胎组织：E8.75和E9.5时期的迁移前迷走神经嵴细胞，以及E13.5时期的胚胎肠道（不含胃）。每窝来源的胚胎组织进行混合，每个发育阶段分析3窝胚胎组织。