DACT1错义突变与先天性心脏病易感性的相关性研究

Association study between DACT1 missense mutations and susceptibility to congenital heart disease

目的:筛选与先天性心脏病(CHD)相关的DACT1基因的错义突变,并研究其生物学功能。方法:研究对象为中国山东地区汉族人群,病例组为2008年8月至2011年1月散发CHD的连续就诊患儿,排除综合征型和有心脏病家族史患者;对照组为同时期收集的排除CHD以及有心脏病家族史的健康儿童。①提取两组外周静脉血WBC的基因组DNA,靶向DACT1基因的编码区进行测序,将测序结果中的单核苷酸变异(SNV)与数据库dbSNP(version137)和千人基因组比对,病例组与对照组的SNV比对,筛选出病例特异性的新发现或稀有DACT1突变位点,通过Sanger法验证。②构建野生型和各突变型DACT1的表达质粒,在HEK293T细胞中通过蛋白质免疫印迹实验观察DACT1和DVL2蛋白表达水平。③通过双荧光素酶报告基因实验观察突变后DACT1对经典Wnt信号通路和非经典Wnt信号通路(PCP)调控作用的变化。结果:病例组404例,年龄(2.9±2.7)岁;对照组213名,年龄(7.1±3.7)岁。病例组和对照组的男女比例分别为1.2∶1和1∶1,均为汉族儿童。在3例CHD中筛选到4个DACT1基因编码区的新发现或稀有的错义突变c.1486C>T(p.S441L)、c.1598C>A(p.S478R)、c.1659G>C(p.E499Q)和c.1891T>A(p.M576K),经Sanger测序验证均为杂合突变。DACT1^S478R和DACT1^E499Q在不同物种中保守性很高。DACT1^S441L SIFT评价为有害突变;DACT1^E499Q PolyPhen-2评价为可能有害(0.978),DACT1^S478R和DACT1^M576K SIFT和PolyPhen-2预测为无有害性。4种突变型对DACT1蛋白的表达水平均无明显影响。与野生型DACT1相比较,突变型DACT1^E499Q对DVL2蛋白的下调作用减弱(P<0.05),对经典Wnt信号通路以及PCP信号通路的抑制作用也减弱(P<0.01)。结论:DACT1^E499Q错义突变可能通过减弱对Wnt信号通路活性的抑制来促进CHD的发生。

Objective To screen mutations in the coding region of DACT1 gene in patients with congenital heart disease (CHD) and to study their biological functions. Methods The subjects of study were from Chinese Han population in Shandong Province. CHD samples were collected from sporadic CHD patients diagnosed at the Cardiovascular Disease Institute in General Hospital of Jinan Military Region from Aug. 2008 to Jan. 2011. The samples collected from the patients with other syndromes or familial heart diseases were excluded. The control samples were collected from healthy children without CHD or familial heart diseases in the same hospital during the same time.①Genomic DNAs were extracted from the CHD and control samples. The second-generation sequencing technology was used for target sequencing of 404 samples of CHD and 213 healthy controls for the exons of DACT1 gene. Bioinformatics methods were used to screen case-specific new missense mutations or rare missense mutations, which were further verified by Sanger Sequencing.②The plasmids expressing wild type or mutant DACT1 genes were transfected into HEK293T cells and the effects of the mutants on the level of DACT1 protein were examined by Western blot.③The effects of the mutations on canonical Wnt signaling and PCP signaling were evaluated by luciferase reporter assay. Results There were 404 CHD cases at the age of (2.9±2.7) years and 213 controls at the age of (7.1±3.7) years. The ratios of male to female for CHD and control samples were 1.2∶1 and 1∶1 respectively. Four novel or rare missen mutations, c.1486C>T (p.S441L), c.1598C>A (p.S478R), c.1659G>C (p.E499Q) and c.1891T>A (p.M576K),(thereafter called DACT1^ S441L , DACT1 S478R , DACT1 E499Q and DACT1 M576K , respectively) were identified from three CHD samples. The four mutations were verified by Sanger sequencing. The mutations of DACT1^S478R and DACT1^E499Q occurred at highly conserved sites and the mutations of DACT1^S441L and DACT1^E499Q were predicted to be harmful. The four mutations have no effects on the protein levels of DACT1 mutant protein themselves, respectively. However, the mutation of DACT1^E499Q attenuated the degradation of DVL2 protein and reduced the inhibition of the canonical Wnt signaling pathway and PCP signaling pathway. Conclusion DACT1^E499Q mutation may contribute to the development of CHD by decreasing the inhibitation of Wnt signalings.