基于小家系鉴定遗传性肝内胆汁淤积症新致病基因及其功能初步分析

Hereditary intrahepatic cholestasis accounts for significant proportion of childhood liver morbidity and mortality. Its prompt diagnosis either significantly improves the prognosis of treatable diseases, or could prevent the birth of new victims of untreatable disorders. It also acts as a natural model in exploring liver physiology and pathophysiology of cholestasis. However, even after extensive explorations, the etiology still could not be identified in many cholestatic children with a family history or sibling victim. Most of the victims die in their early age, so traditional method to identify causing gene is difficult for the unavailability of big kindreds. Next generation sequencing is a powerful method for identification of novel genes from small kindreds. In our proposal, 33 patients from small kindreds with suspicious hereditary intrahepatic cholestasis have been collected, all of whom had undergone extensive investigations already, including Sanger sequencing and CNV analysis of suspicious genes, but no etiology detected. In this project, Next Generation Sequencing-based targeted panel, whole exome sequencing will be performed to find new pathogenic genes in 50 core kindreds, and functional experiments to explore how it happens. The significance of our proposal is to find new pathogenic genes of inherited cholestasis, and then explore its mechanism and try to reveal novel therapeutic targets of cholestasis.

遗传性肝内胆汁淤积症是儿童期肝病的重要病因，亦是了解肝脏生理及胆汁淤积症疾病机制的天然模型。课题组在国内率先认识十余种遗传性胆汁淤积症，针对性措施显著改善其预后。但仍有许多高度可疑遗传性的患儿无法用已知致病基因解释，推测存在新的致病基因；且多数患儿早期致死，缺乏大家系，应用经典遗传学方法定位克隆新致病基因非常困难。二代测序(NGS)技术是通过小家系鉴定遗传病新致病基因的高效手段。课题组前期已基于NGS开发能同时检测69个已知及可疑胆汁淤积致病基因的Panel，并结合全外显子组测序(WES)在两个亚组胆汁淤积患者中分别筛选到一个可疑致病基因。本项目拟收集50个高度怀疑遗传性肝内胆汁淤积症、但除外已知致病基因的患儿家系，进行Panel筛查及WES检测，并通过病例-对照验证、功能学实验进一步明确候选基因与该疾病关系及机制。本研究有望鉴定遗传性胆汁淤积新致病基因，并为其他胆汁淤积症治疗提供新靶标。

遗传性肝内胆汁淤积症是儿童期肝病的重要病因。课题组前期在国内率先认识十余种遗传性胆汁淤积症，针对性措施显著改善其预后。但仍有许多高度可疑遗传性的患儿无法用已知致病基因解释，推测存在新的致病基因，而传统遗传学研究方法对鉴定这些新基因存在限制。本项目中，课题组基于能够高效鉴定新致病基因的二代测序(NGS)技术，首先通过前期自主开发、能同时检测69个已知胆汁淤积症致病基因的靶向测序panel筛选不明原因遗传性胆汁淤积症核心家系，而后应用全外显子组测序 (WES)结合全基因组测序 (WGS)、肝组织RNA测序筛选可疑新致病基因。我们总共检测了109个胆汁淤积核心家系，其中低GGT胆汁淤积组67个，高GGT胆汁淤积组42个，其它肝病组50个；包括228人次WES测序，22人次WGS测序和4人次肝组织RNA测序。经过分析，一共62个家系病例得到确诊，包括33种基因缺陷病。胆汁淤积组病例中，我们鉴定了3个新致病基因（低GGT胆汁淤积组MYO5B、USP53，高GGT组ZFYVE19），并对MYO5B的表型谱、基因型-表型关系、胆汁酸代谢谱进行阐述；针对鉴定的3个胆汁淤积症新致病基因，我们分别构建了细胞模型、转基因敲除鼠模型进行表型模拟和机制初探。我们还发现TJP2缺陷可致低GGT胆汁淤积症谱系、VPS33B缺陷可致仅有低GGT胆汁淤积症的不全ARC综合征表型，以及脑腱黄瘤病（CYP27A1缺陷）可致婴儿严重胆汁淤积症。在对照的其它肝病组中，我们参与了反复发热相关肝衰竭/肝功能异常病例新致病基因RINT1的鉴定和报道，针对该表型的另外两个致病基因NBAS、SCYL1，我们报道了国内首例病例。依托本项目我们一共发表SCI论文20篇（单篇最高影响因子14.979），中文论著2篇，获奖2项; 培养硕士研究生3名，博士研究生3名，博后1名。

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