新的先天性心脏病相关基因的鉴定

Congenital heart disease (CHD) is the most common birth defects. Genetic factors play important roles in pathogenesis and development of CHD. However, disease gene mutations identified over the past few decades only explain a small fraction of all CHD cases，many more CHD associated genes are yet to be discovered. In our previous study, we found 18 de novo copy number variations (CNVs) from 150 well clinically characterized patients with a common type of CHD, conotruncal defects (CTD), by using genome-wide high resolution microarray analysis.Fifty-nine CHD candidate genes were identified from the genes within the CNVs.In this study,we plan to develop a next generation sequencing (NGS) based mutation screening system to interrogate sequence variants in 59 CHD candidate genes. We intend to capture and sequence all the coding and exon-intron boundary sequences of 59 CHD candidate genes in samples from 500 clinically evaluated cases with CTD as well as 500 normal controls with Agilent SureSelect Target enrichment system and Miseq sequencing platform. Sequencing data will be assembled and aligned to the reference human genome (hg19). Variants calling, annotation, and evaluation on functional impacts of variants will be performed with a variety of bioinformatic tools. Sanger sequencing will be used to validate the resultant variants.The primary functions of the identified novel CHD gene(s) will be studied by using zebrafish model. The findings of this project will lead to the discovery of new CHD genetic biomarker(s) and help elucidate novel mechanism of CHD pathogenesis as well as facilitate early molecular diagnosis and even prevention of CHD.

先天性心脏病(CHD)是一种最为常见的出生缺陷，遗传因素在CHD的发生、发展中具有重要作用，但目前已知的遗传变异只能解释一小部分CHD病例，许多新的CHD相关基因亟待发现。我们在前期研究中，对150例复杂CHD的代表类型心脏圆锥动脉干畸形（CTD）患者样本通过全基因组高分辨芯片分析，发现18个新生拷贝数变异（CNV），并从中遴选出59个新的CHD候选相关基因。本项目拟以这59个候选基因为基础，设计靶向外显子组捕获系统，通过下一代测序技术对500例CTD患者和500例正常对照样本进行CHD新候选相关基因外显子组高通量测序，并利用生物信息学软件进行分析，经Sanger测序法验证，遴选新的CHD相关基因；进一步建立相应的斑马鱼模型，对新鉴定的CHD相关基因进行初步功能研究。本项目的研究，将有利于阐明CHD的遗传发病机制，发现新的CHD遗传生物标志物，为CHD的早期分子诊断和干预治疗提供理论依据。

先天性心脏病（CHD）是一种最为常见的出生缺陷，遗传因素在CHD的发生、发展中具有重要作用，但目前已知的遗传变异只能解释一小部分CHD病例，新的CHD致病基因亟待发现。本项目借助前期研究基础，通过对502例CHD患者全基因组高分辨芯片数据进行分析，遴选出23个新的CHD候选相关基因。同时，通过数据库检索及文献资料，选取80个CHD已知致病基因，以这103个CHD候选及已知致病基因为基础，基于Illumina HiSeq技术平台，设计了靶向外显子组捕获系统。本项目主要研究成果如下：（1）自主研发了两种22q11.2拷贝数变异检测系统，为本项目靶向测序进行样本筛选提供可行的方法。（2）应用新建立的CHD候选及已知致病基因外显子组捕获系统，对300例22q11.2微缺失筛查结果阴性的心脏圆锥动脉干（CTD）患者进行靶向高通量测序，经生物信息学分析及Sanger测序验证，并通过与1447例中国正常人全外显子测序数据进行比对，最终确定SORBS2和PTCH1为新的CHD候选致病基因。（3）通过斑马鱼Sorbs2b基因敲减模型，发现Sorbs2b敲减后的斑马鱼均出现新生血管生长受阻、心包水肿等表型；进一步构建Sorbs2基因敲除小鼠，发现约42％（8/19）纯合Sorbs2基因敲除小鼠表现房间隔缺损表型，证实了SORBS2基因在心脏发育中的关键作用，是CHD新的致病基因。. 本项目共发表SCI论文4篇，申请发明专利1项，并培养了4名博士、1名硕士及1名博士后。本项目的研究，进一步阐明了CHD的遗传致病机制，发现新的CHD遗传生物标志物，为CHD的早期分子诊断和干预治疗提供理论依据。