TGFBR3错义突变与先天性心脏病遗传关联性研究

doi:10.3969/j.issn.1673-5501.2019.04.013

摘要/Abstract

摘要： 目的在先天性心脏病(CHD)人群中检测TGFBR3基因编码区的疾病特异性错义突变,并研究突变的生物学功能。方法 ①研究对象为中国山东地区的汉族人群。病例组为2008年8月至2011年1月连续就诊的404例散发CHD患儿;对照组为同时期收集的排除CHD以及有心脏病家族史的213例体检健康儿童。②提取样本外周血基因组DNA,进行TGFBR3基因外显子靶向测序。测序获得的病例组特异的单核苷酸变异(SNV)与数据库dbSNP(version137)和千人基因组比对,筛选出疾病特异性稀有或新发突变,行Sanger测序验证。③构建野生型和突变型TGFBR3表达载体,在HEK293T细胞中表达后用免疫印迹检测外源TGFBR3蛋白水平,并用荧光素酶报告基因检测突变对TGF-β信号通路的影响。结果分别于3个CHD患儿中筛选得到3个CHD特异性的TGFBR3新发/稀有杂合突变,其中TGFBR3^K685R为新发现的突变;TGFBR3^A791V和TGFBR3^A804S为稀有突变,并被SIFT和PolyPhen-2软件预测为有害突变。3个被突变的氨基酸都位于TGFBR3蛋白的高度保守区。功能分析实验显示,TGFBR3^K685R和TGFBR3^A804S突变导致TGFBR3蛋白表达水平显著降低,而且3个突变均导致TGFBR3蛋白对参与心脏发育重要信号通路TGF-β信号通路的抑制作用显著降低。结论突变TGFBR3^K685R、TGFBR3^A791V和TGFBR3^A804S可能通过降低TGFBR3对TGF-β信号通路的抑制作用,参与先天性心脏病的发生。

关键词: 先天性心脏病, TGFBR3, 错义突变, TGF-β信号通路

Abstract: Objective To screen disease-specific mutations in the coding region of TGFBR3 gene in congenital heart disease (CHD) and to study the biological functions of the mutations.Methods The study subjects were from Chinese Han population of Shandong province, composed of 404 sporadic CHD samples collected from CHD patients diagnosed during the period from Aug.2008 to Jan.2011. A total of 213 control samples were collected from healthy children without CHD or familial heart diseases in the same hospital during the same time period. The genomic DNAs were extracted from the peripheral blood of CHD and control samples and the target sequencing for the exons of TGFBR3 of the patients and controls was performed. Bioinformatics methods were used to screen case-specific new missense mutations or rare missense mutations, which were further verified by Sanger Sequencing. Wild-type and mutant TGFBR3 expression vectors were constructed and were then transfected into HEK293T cells for evaluating TGFBR3 protein levels by Western Blot and the effects of the mutants on TGF-β signaling pathway by luciferase reporter assay.Results Three disease-specific heterozygous mutations were identified from three different CHD patients separately, TGFBR3^K685R is a newly discovered mutation, while TGFBR3^A791V and TGFBR3^A804S were rare mutations and were predicted to be deleterious mutations by SIFT and PolyPhen-2 software. The protein levels of TGFBR3^K685R and TGFBR3^A804S mutants were significant lower than that of wild type TGFBR3 and all three mutations significantly impaired their function to inhibit TGF-β signaling.Conclusion Mutations TGFBR3^K685R, TGFBR3^A791V and TGFBR3^A804S may participate in the development of congenital heart disease by reducing their inhibition of TGF-β signaling.

Key words: Congenital heart disease, TGFBR3, Missense mutation, TGF-β pathway

吕昕, 王红艳, 张斌. TGFBR3错义突变与先天性心脏病遗传关联性研究[J]. 中国循证儿科杂志, 2019, 14(4): 308-312.

LV Xin, WANG Hong-yan, ZHANG Bin. Association study between missense mutations in TGFBR3 and susceptibility to congenital heart disease[J]. Chinese Journal of Evidence-Based Pediatrics, 2019, 14(4): 308-312.

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