中国人群非综合征法洛四联症可遗传致病突变的分子流行病学研究

Congenital heart disease (CHD) is one of the most common birth defects, of which tetralogy of Fallot (TOF) is the most common cyanotic CHD. Previously, our group conducted a genome-wide association study (GWAS) in China and found that there are several susceptible regions of CHD. However, the genetic model and genetic pathology model of TOF is still not clear enough due to the heterogeneous subtypes and genetic heterogeneity of CHD. And the real pathogenic mutation of TOF and its biological function have yet to be identified. The applicant had already performed whole exome sequencing of 150 non-syndromic TOF cases and completed preliminary data processing. This study will systematically discover new pathogenicity-related genes and rare mutations of TOF by establishing a database of known causative genes in human CHD and in combination with gene-based association analysis. Then validate the mutations by using target sequencing in large TOF samples and other different subtypes CHD cases. Further study of gene-phenotype function in mouse model will be conducted to investigate the biological mechanism of pathogenic genes and mutations in the develop of TOF. This study is helpful to determine the frequency of specific pathogenic mutations and the frequency of TOF in Chinese Han population and will provide references and evidences for prenatal diagnosis and genetic counseling of tetralogy of Fallot.

先天性心脏病是一种最为常见的出生缺陷，其中法洛四联症是最常见的青紫型先心病。本课题组前期开展的中国人群全基因组关联研究（GWAS）发现了多个先心易感区域，然而先心病众多的亚型以及遗传异质性导致法洛四联症的遗传模型及遗传致病模式仍不够清晰，真正的致病变异及其生物学功能也有待鉴定。申请人前期已经利用全外显子测序，完成了150例非综合征型法洛四联症的测序和数据处理。本研究将通过建立人类先心已知致病基因数据库并结合以基因为单位的关联分析，系统性发现新的法洛四联症致病相关基因和罕见突变，继而在大样本人群中进行捕获测序验证和亚型特异性验证，进一步在小鼠模型中展开基因-表型功能研究，探讨致病基因及突变在法洛四联症发生过程中的生物学机制。本研究有助于确定中国汉族人群法洛四联症特异性致病突变及其频率，为法洛四联症的产前诊断和遗传咨询提供参考和依据。

本研究通过对150例单纯TOF患者进行全外显子组测序，结合课题组建立的人群和小鼠相关的先心已知致病基因，对单纯TOF患者携带的突变进行致病性判断，并从TOF的遗传模式、非编码致病变异、表观遗传学致病机制和未知致病基因鉴定层面进行了综合分析。. 首先，鉴定了TOF家系中复合杂合遗传模式，我们发现患者同时携带NOTCH1编码区和调控区突变。该编码区突变位于NOTCH1基因EGF样重复序列，导致蛋白第685位天冬酰胺变成异亮氨酸，该区域错义突变会引起蛋白错误折叠。并通过体外实验证实非编码突变所在的调控区域发挥沉默子的功能，同时证实该调控区突变增加了沉默子的活性。因此，NOTCH1编码区突变可能导致蛋白错误折叠，而调控区突变进一步导致其表达降低，最终导致心脏发育异常。. 其次，对功能丧失突变进行富集分析，发现功能丧失突变富集于纤毛相关通路。通过构建基因敲除小鼠模型，发现纤毛相关基因敲除小鼠表现出心脏畸形，纯合敲除小鼠胚胎心脏流出道宽度变窄，长度缩短。流出道细胞的纤毛数量减少，长度缩短，结构异常。功能实验发现纤毛基因参与Hedgehog信号通路，纯合敲除小鼠心脏中GATA4，NKX2-5等基因表达下降，心脏发育异常。. 第三，我们通过人群研究发现NSUN5潜在功能性突变在法四患者中显著富集，构建的NSUN5纯合敲除小鼠也表现出室间隔缺损和主动脉骑跨畸形，其室间隔和心室壁厚度显著低于野生型；细胞增殖实验证实纯合敲除小鼠心脏不同部位细胞增殖减弱。进一步甲基化测序和蛋白质谱实验发现，NSUN5基因失活导致细胞增殖相关基因Tpm1 mRNA甲基化水平下降，进而导致其蛋白水平下降，心肌细胞增殖减弱，最终导致流出道发育异常。. 本研究利用150例单纯TOF测序数据进行突变分析，从多个不同的角度阐明了TOF可能的发病机制，法洛四联症遗传学病因探索提供了新的证据，同时为今后TOF的产前筛查和分子诊断提供了理论依据。