TBX20错义突变和非编码区功能性遗传变异的鉴定及其致散发型先天性心脏病的机理研究

Congenital heart disease (CHD) is the most common category of birth defects worldwide nowadays, and its morbility has been increasing yearly. The previous reports focused mainly on finding mutations in coding regions of the core transcription factors such as TBX5, TBX20, GATA4 and NKX2-5 in pedigree studies. While in our previous case-control studies, a de novo mutation and a protective haplotype in promoter region of TBX20 were also demonstrated to be involved in sporadic CHD. Hereby, in the present study, we will apply this strategy further to the candidate genetic variants affecting CHD susceptibility, investigate the novel missense mutation, and determine the genotpe diaparitis of the non-coding regions of TBX20. Following identifying the positive variants, a collective operations, including luciferase reporters, EMSA, ChIP, SPR, methylation assessment,qRT-PCR, miRNAs screening and the zebrafish studies, would be applied to explore the genetic effects and the probable mechanism in spordic CHD from cellular, molecular and animal model levels. Therefore, our study will help to provide the individualized target for the susceptibility prediction in sporadic CHD.

先天性心脏病（congenital heart disease, CHD）已成为出生缺陷中最常见的先天畸形，发病率逐年增加。既往研究多集中于在家系中鉴定心脏发育相关转录因子编码区突变，包括TBX5、TBX20、GATA4、NKX2-5等。在前期研究中，我们选取临床上最常见的散发型CHD进行病例-对照研究，鉴定出TBX20编码区一处新的de novo错义突变及启动子区一处保护型单倍型。因此，我们拟在前期研究的基础上进一步鉴定TBX20新的错义突变，及其非编码区遗传变异的不同基因型在人群中的分布差异；在找到相关阳性位点后，我们将运用荧光素酶报告基因实验、EMSA、ChIP、SPR、甲基化检测、实时定量PCR、miRNAs检测及斑马鱼实验等方法，从细胞、分子和模式生物水平研究这些遗传变异对散发型CHD产生的遗传效应和作用机制，从个体化的角度为散发型CHD的疾病风险预测提供候选靶标。

先天性心脏病（CHD）的既往人群研究多集中于在家系中鉴定心脏发育相关转录因子编码区突变，而忽略了非编码区遗传变异的功能。在本研究题中，我们通过鉴定CHD病例-对照中分布差异的TBX20及TBX2非编码区变异，运用细胞转染、荧光素酶检测、EMSA和qRT-PCR等实验发现：1）TBX20启动子区位点rs6963934、rs6959887、rs10235849、rs6959846、rs6959920、rs10249005在病例（228例）和对照（292例）中的基因型分布频率存在显著差异，且该6个位点完全连锁。同时，rs10235849作为Tag（OR = 0.68，95% CI =0.51-0.90，P= 0.0069）参与的单倍型可显著降低CHD的发生风险（OR=0.61，P=0.0031，95% CI =0.44-0.85），提示其为保护型单倍型；CHD组织qRT-PCR及荧光素酶检测提示Minor单倍型（ACC）较Major单倍型（AAT）的转录活性显著下降，EMSA结果显示抑制性核蛋白与Minor单倍型的结合力更强，导致基因表达量下降；2）TBX20编码区一处新的错义突变c.G944A并未引起其编码区全序列转染后的荧光素酶活性，提示其并非真正的功能位点；3）TBX2 3’UTR位点rs59382073的GT-TT基因型使CHD（病例768例，对照660例）的患病风险增加2.13倍(95% CI=1.51-2.99, P = 1.44×10-5)。分层分析显示rs59382073主要与圆锥动脉干畸形的发病风险显著相关（OR = 2.75, 95% CI = 1.57-4.81，P = 3.99×10-4）；其中，携带GT/TT基因型的个体法洛四联症的发病风险将增加3.18倍（95% CI=1.53-6.61，P=1.90×10-3），室间隔缺损患病风险增加1.70 倍(95% CI=1.17-2.46, P=5.14×10-3)。功能研究发现，T等位基因可导致荧光素酶活性显著下降。本课题发现了TBX20及TBX2基因与CHD风险相关的非编码区遗传变异，，初步探讨了其致CHD的机制，为CHD的分子诊断提供新的思路。

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