从基因、组织水平的相互作用探究先天性巨结肠患者症状差异的分子机制

先天性巨结肠（HSCR）属肠神经系统（ENS）发育异常性疾病，遗传方式复杂，表型异质性强。既往研究多只发现在患者群体层面呈显著富集的基因（座），而对每个先证者个体的疾病发生机制解释不清。我们的前期研究发现RET基因复合杂合致病机制、RET-EDNRB协同累积作用参与HSCR发生等多种复杂、独特的致病模式，提示基因水平和组织水平的相互作用研究势在必行。为了确立同一基因内或不同基因间的相互作用机制及其影响HSCR患者临床表型的分子路径，本项目拟利用细胞水平“滴定”实验、临床遗传学证据收集及遗传规律分析、成对肠组织样本RNA-Seq与全基因组测序相结合的方法确定ENS特异性表达定量性状遗传位点及其基因调控网络，阐明不同变异（基因）组合的致病作用强度，观察散发HSCR家系的风险累积模式及症状外显阈值，辅以肠组织类器官快速功能筛选及基因编辑小鼠模型验证分析，为更好地解释患者的临床症状差异提供新线索。

Hirschsprung disease (HSCR) is a congenital malformation of the enteric nervous system (ENS) with complicated patterns of inheritance and strong phenotypic heterogeneity. Previous studies have found only genes (loci) that are significantly enriched at the patient-population level, but the pathogenesis of each proband is often unclear. Our previous study detected a variety of complex and unique pathogenic patterns such as RET compound-inheritance and RET-EDNRB synergistic cumulative effect, suggesting that it is imperative to study the interaction between gene level and tissue level. With the aim of establishing the interaction mechanism within the same gene or between different genes, and delineating the molecular pathway that affects the clinical phenotype of HSCR patients, we here plan to use cell level "titration" experiment, clinical genetic evidence collection and genetic analysis, paired-colon tissue transcriptome sequencing and whole-genome sequencing to identify ENS-specific expression Quantitative Trait Locus and its regulatory gene network, clarify the pathogenicity intensity of different combinations of variants (genes), observe the risk accumulation pattern and symptom explicit threshold of sporadic HSCR families. Finally, intestinal organoid and transgenic mouse model will be utilized to further demonstrate their role in inducing ENS defects.