摘要
背景：先天性心脏病(CHD)是人类最常见而严重的先天性缺陷之一,在活产婴儿中的发生率为6-8%o。CHD的发生主要受环境和遗传两个主要因素影响,90%属单或多基因遗传因素所致散发性、非综合性CHD (ns-CHD)。心脏间隔缺损(ASD、VSD)是ns-CHD最常见的类型,发病率分别占CHD的10%～20%和20%～30%。研究显示,以胰岛素抵抗为主要发病机制的妊娠期糖尿病(GDM)会增加新生儿心脏畸形发生率,而胰岛素信号转导通路异常与胰岛素抵抗关系密切,胰岛素相关信号通路与ns-CHD的发生是否相关的研究尚少。胰岛增强因子1(ISL1)是一种多效性转录因子,影响胚胎细胞的增值、分化和迁徙的作用,在胎儿心脏和胰腺的发育过程中均占据重要地位,探究ISL1及其相关的胰岛素信号通路与ns-CHD发病的关系,有助于寻找ns-CHD的病因。家族性CHD虽仅占总CHD的一小部分,却是研究基因异常与心脏间隔发育缺陷相关性的宝贵资源,因此CHD家系的致病基因筛查有助于在心胚发育遗传机制探索中取得突破。
目的：探讨胰岛素相关信号转导与ns-CHD的发生是否享有共同的信号通路；探讨ISL1基因与中国汉族人群ns-CHD的风险是否关联；建立ASD家族永生细胞系,寻找该家系发病的致病基因,进一步明确CHD的发生机制,为CHD的早预防、早诊断、早治疗提供科学依据。
方法：1.(1)数字基因表达谱(DGE)检测5例ASD病例组与2例风湿性心脏病单纯性二尖瓣狭窄(RHD,MS)对照组右房心肌组织的基因表达差异,将表达量拟合统计学模型,比较组间差异表达基因,筛选目的基因并进一步实时荧光定量PCR (RT-QPCR)验证基因表达量。(2)RT-QPCR检测37例病例组(ASD、VSD)和12例对照组(MS)的右房心肌组织中目的基因的相对表达量,比较二者的基因表达差异。用REST软件比较组间基因CT值,迭代法统计检验,当P<0.05时,认为差异有统计学意义。
2.(1)限制性片段长度多态性(RFLP)技术和SNaPshot分型技术检测233例病例(ASD、VSD)和288例对照(体检健康人)ISL1的9个SNP位点,比较9个SNP位点等位基因频率和基因型与CHD患病风险的关系,组间比较采用卡方检验,Bonferroni校正P<0.006为差异有统计学意义。(2)应用RT-QPCR检测37例病例组(ASD、VSD)与12例对照组(MS)中有房心肌组织中ISL1、GATA4、Mef2c基因相对表达量,比较两组间的基因表达差异,当P<0.05时认为差异有统计学意义。
3.(1)收集一个ASD家系中5代158人的外周血,调查CHD发病类型和例数。以EB病毒转化淋巴细胞,建立永生细胞系。对发病成员永生细胞株(第8代)和外周血提取的DNA进行GATA4基因和ISL1基因全部外显子测序,对比细胞株样本与外周血样本基因序列,分析所建永生细胞系的遗传稳定性。(2)对家系发病成员的GATA4基因和ISL1基因的全部外显子进行测序,与已报道和数据库的突变位点进行对比,寻找致病基因。
结果：1.(1)DGE实验结果：病例组与对照组之间存在26个基因表达差异有显著性,其中18个基因表达上调,其他8个基因的表达下调,其中多个基因归属于发育过程、生物调控以及免疫系统过程等生物过程中。病例组与对照组单个样本两两比较结果显示,胰岛素信号通路的Ras/Raf/MEK/MAPK通路上的基因表达普遍下调,在这信号通路中筛选出20个表达下调的基因为目的基因。(2)RT-QRCR结果显示：经病例与对照比较,胰岛素信号通路Ras/Raf/MEK/MAPK通路中20个目的基因有8个基因(PTPRF、SHC4、ARAF、MAP2K2、MKNK2、 MOB3C、MOB3B、ELK1)在病例组中的表达量均明显下调,差异均具有统计学意义(P值均<0.05)。
2.(1)1SL1基因的一个基因位点(rs1017)等位基因频率在病例组和对照组中分布差异有统计学的意义(P<0.000)。该位点在显性模式下,带有rs1017位点AT或者TT基因型的人的CHD发生明显高于带有AA基因型的人(OR=2.199,95%CI：1.411-3.427,P<0.000)；在显性模式下,VSD组和ASD组患者带有rs1017位点AT或者TT基因型的人的CHD发生也分别显著高于带有AA基因型的人(OR=2.157,95%CI：1.217-3.821,P=0.008；OR=2.188,95%CI：1.319～3.629,P=0.002)。(2)RT-QPCR检测右房心肌组织样本中ISL1、GATA4、Mef2c基因表达的结果：病例组中ISL1基因表达量较对照组明显下调,差异具有统计学意义(P=0.019), GATA4、Mef2c基因表达量在两组间无明显统计学差异。
3.(1)ASD家系5代158人中发现9例发病成员,其中6例ASD,3例房间隔膨出瘤(ASA)。建成发病成员的外周血永生细胞株9例,建株成功率100%(9/9),父母等家庭成员建系成功率为94.6%(141/149)。所有建成的细胞株冻存、复苏成功率为100%。传至8代细胞株样本与外周血样本中GATA4基因和ISL1基因的全部外显子序列一致。(2)家系中9例发病成员进行GATA4基因和ISL1基因全部外显子测序未发现文献报道和数据库的已知突变及新的突变。结论：1.胰岛素信号转导Ras/Raf/MEK/MAPK通路上基因表达异常可能与ns-CHD发生相关,其可能是ns-CHD的发生机制之一,提示ns-CHD的致病通路也许和糖尿病致病通路有重叠,进一步研究对ns-CHD的预防和早期干预可能有重要意义。
2.ISL1基因表达异常与ns-CHD的发生相关；ISL1基因的rs1017位点的T等位基因可能是中国汉族人群中ns-CHD的患病风险因素；ISL1基因与ns-CHD及糖尿病发生均存在关联,这可能暗示了ISL1基因是这两种疾病致病通路上的“交织点”。
3.采集ASD家系成员血样和建立永生细胞系有助于CHD易患基因筛查和人群防治,探究CHD的发病机制。GATA4基因和ISL1基因可能未参与所有ASD家系的发病,尚需筛查ASD家系其他致病基因。
Backgroud and Objective
Congenital heart disease (CHD) is one of the commonest congenital defects of the human beings, with occurrence of6-8%o in live-born babies. As research showed, CHD were mainly affected by both genetics and environment factors, about90%of the which were sporadicand non syndrome congenital heart disease (ns-CHD) in which affected by single of multiple genetics. Septal defect including VSD and ASD are the most common types of ns-CHD with incidence of10%-20%and20%-30%respectively. Researches have shown that gestational diabetes mellitus (GDM) increases the incidence of infant cardiac malformation and the abnormal of insulin signal transduction pathway can directly cause insulin resistance, which induced GDM. Therefore, influence of insulin on the development of cardiac is one of the most active investigate direction. On the other side, as a multiple-effect transcript factor, Insulin enhancement factor1(ISL1) does not only influences the heart development, but also the development of pancreas system and nerve system. In Han Chinese population, ISL1gene is relevant to the existence of ns-CHD, which is another direction worth exploring. It is worth mention that, CHD pedigree provides valuable resources for the research on the relevance between the genetic defect and the heart interval development. As such, the disease-causing gene of CHD family is also one of the purposes of our research object.
Our investigations are aimed to discuss whether a common signal pathway between the insulin-related signal pathway and the occurrence of ns-CHD, explore the relevance between IS L1and the occurrence of ns-CHD among the Han Chinese people, establish the ASD family immortal cell system and find out the disease-causing gene of the family, so as to understand the pathogenic mechanism of ns-CHD.
Methods:
1. Digital gene expression profiling (DGE) was conducted to the right atrium cardiac muscle tissue of5CHD cases and2rheumatic heart disease,mitral stenosis(RHD,MS) controls, gene expression difference test between groups was implemented to a reported statistic model, to find out the possible common pathway gene of CHD and insulin signal, we also executed pathway and GO term enrichment analysis on the difference expression genes, real time quantity PCR (RT-QPCR) was conducted to the right atrium tissue of37CHD cases and12MS controls to verify the DGE result and other genes which have been reported to be associated with CHD, REST toolset were used to compare CT value of genes between groups, then we got different expression genes with p-value less than0.05in iteration statistic test.
2.(1) Using Restricted fragment length polymorphisms (RFLP) technology and SNaPshot sequencing,9loci in ISL1gene of233ns-CHD patients (ASD, VSD) and288control patients were genotyped, in order to find out the relationship between different genetic types and ns-CHD disease risks, we used test as our statistic method, and p-value after Bonferroni correction less than0.006were considered as significance.(2) To evaluate the expression differences of gene ISL1, GATA4and Mef2c between37cases (VSD, ASD) and12(MS) controls, we executeda RT-QPCR experiment and test the differences using a t-test with p-value<0.05as the significance cutoff.
3. Peripheral blood of158samples in an ASD family was collected,9persons in this five-generation family were suffering CHD. Among these9cases,6were diagnosed as ASD and3were as atrial septal aneurysm (ASA). Immortal cell lines were established with EB virus transformation lymphocyte. To estimated the genetic stability of our immortal cell lines, we applied a whole exon sequencing to gene GATA4and ISL1, gene sequence of these two genes were compared between the8th generation cell lines and the original peripheral blood samples.(2) Exonsof GATA4and ISL1genes were sequenced in all CHD members in ASD family, to search for disease-causing mutations, we compare our data to disease related locus have been reported.
Results:
1.DGE experiment found26genes were significantly different expressed between the cases and controls, in which18genes were upregulated expressed, and8were downregulated. Most of these genes were attributed to the development process, biological control, the immune system process and other important biological processes. During the one by one comparison of differential expression genes between cases and controls samples, we found that in most of the comparisons, genes in Ras/Raf/MEK/MAPK signal pathway ofthe insulin signal pathway were downregulated. our researches were then focused on the Ras/Raf/MEK/MAPK signal pathway that belong to the insulin signal pathway. RT-QPCR was used to evaluate20genes includingin Ras/Raf/MEK/MAPK signal pathway of the insulin signal pathway.Through the comparison of relative expre-ssion quantity, statistical verification was conducted to the result of RT-QPCR with iteration method. The result showed that in the comparison between cases and controls,8genes (PTPRF,SHC4,ARAF,MAP2K2,MKNK2,MOB3C,MOB3B,ELK1)inRas/Raf/MEK/MAPK signal pathway were significant difference expres-sed(P-value<0.05).
2.(1)Allele frequency of one locus ingene1SL1was statistically significantly difference distributedin case group and the control group(.P<0.000). Under the dominance model, samples with AT or TT genotypes belong to CHD were significantly higher than those with AA genotype (OR=2.199,95%CI:1.411-3.427, P<0.000);ventricular septal defect (VSD) and atrial septal defect (ASD) patients with AT or TT genetic types diagnosed as CHD were obviously higher than those with AA genetic types (OR=2.157,95%CI:1.217-3.821,P=0.008;OR=2.188,95%CI:1.319～3.629, P=0.002).(2) The RT-QPCR result of ISL1within cardiac muscle tissues indicated that it was statistically significant downregulated in CHD (P=0.019).
3.9cases of peripheral blood immoral cell strains in the ASD family were establishedwith100%(9/9)success rate, the rate thatof family members like parents was94.6%(141/149). All established cell lines were frozen for storage, and the recovery rate was100%. We consider our immoral cell lines were constructed successfully since the8lhgeneration cell lines carried a gene sequence of GATA4and ISL1with the ancestor cell lines from blood samples.Whole exon sequencing of GATA4and ISL1gene was conducted to9members who were diagnosed as CHD in the family (6cases of ASD and3cases of ASA), no reported or novel disease related mutation were founded.
Conclusions:
1.Our experiment demonstrated that, the occurrence of ns-CHD were correlated to the abnormal expression of genes in the Ras/Raf/MEK/MAPK pathway of the insulin pathway, this may be one of the mechanisms that driving the ns-CHD, indicated that the pathway influence CHD may have overlaps with diabetes, both of which are very important to the prevention and early diagnosis of ns-CHD.
2. We believe that ISL1gene is related to the occurrences of ns-CHD, and allele T of SNP rs1017located in gene ISL1may be one of the risk factor of ns-CHD in Han Chinese population. Polymorphisms of ISL1gene associated with Diabetes and ns-CHD at the same time. It maybe implies that this gene could be a'crossing point' of molecular mechanism of these two kinds of diseases.
3.Peripheral blood collection and the construction of immoral cell lines of ASD pedigree are useful in screening disease related genes, disease control and pathogenesis investigation. Further investigation should be applied in searching disease related genes besides GATA4and ISL1gene in ASD pedigree.
引文
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