叶酸代谢通路关键酶基因非编码区多态的先天性心脏病易感性研究及功能分析

【作者】 赵健元；

【导师】 王红艳； 金力；

【作者基本信息】 复旦大学 ， 遗传学， 2011， 博士

【摘要】 先天性心脏病(Congenital heart disease, CHD)是我国排名第一的出生缺陷,是导致婴幼儿死亡和残疾的重要原因,严重影响了我国的人口素质,给社会和家庭带来了沉重的负担。近年来临床实验证明孕前补充叶酸可以显著减低CHD的发生,由此可知叶酸及其代谢通路基因与CHD的发生关系重大。尽管国内外在叶酸代谢基因和CHD易感性方面做了大量的研究工作,并鉴定出一些致病的多态位点,如MTHFR c.677 C>T、c.1298 A>C等,但不同研究中结果不一致,且缺乏相关的分子生物学功能验证,仍有许多问题尚待解决：(1)大多数研究将注意力集中在早期文献报道的、一直以来受到广泛关注的SNP上,而不是搜寻新的功能性致病多态,例如对MTHFR c.677 C>T、c.1298 A>C、MTRR c.66 A>G等多态的研究从十多年前持续至今,但却始终得不到一致结果；(2)研究的基因区域侧重基因编码区,关注因氨基酸变化导致的基因功能异常是否会提高先心病易感性,很少对基因非编码区进行系统研究来探讨基因剂量与CHD的关系；(3)随着dbSNP、HapMap数据库的日益完善,研究者们倾向于根据数据库信息挑选tagSNP开展自己的研究,然而不同种族、不同群体的SNP分布和频率存在差异,完全根据数据库信息挑选tagSNP难免会遗漏很多重要遗传信息；(4)先天性心脏病样本很难收集,因此国内外大多数研究样本量都有一定局限,很少有研究募集到500例以上病例进行遗传学方面研究,因此效力有限,且容易出现假阳性。为了探讨中国人群叶酸代谢途径核心基因和CHD易感性是否存在联系,并在研究方法上解决上述问题,我们在上海、江苏、山东三个省市收集了总计2,340例CHD病例和2,270例健康对照样本,挑选出32个样本对叶酸代谢途径中甲硫氨酸合成酶(MTR)、甲硫氨酸合成还原酶(MTRR)、胱硫醚p合酶(CB5)、胸苷酸合酶(TYMS)四个核心基因的启动子和3’UTR进行了测序,根据测序结果挑选最小等位基因频率大于10%的tagSNP,在三个人群中进行基因分型和关联分析。在找到与疾病易感性相关的阳性位点后,我们进一步对其进行了功能上的分析。因为叶酸代谢与癌症的发生息息相关,因此我们还分析了阳性位点与癌症患病风险的关联。通过关联分析,我们共找到4个非编码区多态位点与先天性心脏病易感性相关,并在三个人群中得到了一致的结果,其中MTR-186 T>G (rs28372871)、+905 G>A (rs1131450)、MTRR c.56 A>C (rs326119)是风险型SNP, CBS -551 C>G (rs2850144)是保护型SNP。MTR基因编码甲硫氨酸合成酶,在叶酸代谢中的主要功能是将5-甲基四氢叶酸(5m-THF)的甲基传递给同型半胱氨酸(HCY),生成四氢叶酸(THF)和甲硫氨酸(MET)。该基因中的两个风险因子分别位于启动子区和3’UTR区,二者均能显著增加先心病的患病风险,其中启动子区多态-186 T>G多态的G等位基因为风险因子,其GG基因型能增高1.56倍的患病风险(adjusted OR=1.56,95％CI=1.33.1.83,P=1.32×10-9)。in vivo的qRT-PCR和in vitro的Luciferase实验证明,-186 G等位基因启动转录的活性低于T等位基因,我们通过软件预测、EMSA、SPR、CHIP等手段证实了导致G等位基因转录活性低的原因是该多态T到G的变化使得启动子和转录激活因子USF的亲和力下降。另一方面从表观遗传学视角出发,我们发现-186 G等位基因的甲基化状况比T等位基因有显著升高,因此会进一步降低其转录活性。位于3’UTR的+905 G>A多态的A等位基因为风险因子,其GA、AA基因型分别能提高1.2倍和2.74倍的患病风险(GA: adjusted OR=1.2,95%CI=1.06-1.36;AA:adjusted OR=2.74,95% CI=2.09-3.60,P= 6.35×10-14)。功能研究发现,该多态由G到A的变化,会加大三种microRNA (miR-485、miR-608.miR-1293)对MTR基因的抑制力度,在翻译水平降低MTR的表达水平。我们还在522例健康志愿者血浆中检测了同型半胱氨酸的水平,结果发现-186 G和+905 A能显著增高体内血浆的HCY水平。关联分析以及HCY水平分析的结果表明这两个SNP可以协同作用,进一步增大CHD的患病风险。MTRR基因编码酶的主要功能是维持足够的活化形式的维生素B12,后者是MTR的辅酶。实验中发现的阳性位点c.56+781 A>C位于MTRR第一内含子,其C等位基因会显著提高CHD易感性,CA基因型和CC基因型分别会将CHD患病风险提高1.4倍和1.84倍(GA:ajust OR=1.4,95%CI=1.23-1.59;CC:ajust OR=1.84,95%CI=1.54-2.2;P=4.34×10-12)。在功能实验中我们通过qRT-PCR检测心组织中MTRR mRNA的量,通过Luciferase报告基因系统in vitro检测不同等位基因的转录活性,二方面的结论一致,表明c.56+781 C等位基因会显著降低转录活性,SPR实验证实C等位基因和核提取物物的亲和力确实低于野生型的A等位基因。我们也发现在健康人群中CC基因型携带者的血浆HCY浓度要高于AA基因型。CB5的功能是通过硫化作用直接催化HCY与丝氨酸缩合成胱硫醚,是HCY释放的途径之一。在关联分析中我们发现,位于启动子区的-551 C>G多态能降低CHD的患病风险,这种保护效应随着G等位基因数目的增加而增强,其CG基因型和GG基因型个体分别可降低15%、40%的CHD患病风险(CG:adjust OR=0.85,95%CI=0.75-0.96;GG:adjust OR=0.6,95%CI=0.49-0.73;P=6.62×10-7)。通过检测CBS的mRNA水平和Iuciferase报告基因验证,我们发现G等位基因的保护作用可能来源于提高的转录活性和表达量。在后续功能实验上我们发现-551C>G也受到了遗传学和表观遗传学两方面调控,一方面C到G的变化会降低转录抑制因子SP1与启动子的结合,从而激活转录；另一方面我们检测了-551 C>G附近区域的CpG位点甲基化状况,发现G等位基因对应的甲基化程度仅有C等位基因的1/7,无疑会进一步提高转录能力。以上4个多态主要围绕叶酸代谢通路的三个重要作用,即同型半胱氨酸的移除、甲基化和DNA合成原料的提供。MTR、MTRR中三个风险多态都能大幅度降低基因的表达量,从而使得HCY在体内堆积、THF和MET的供应不足,这种情况在胎儿在心脏发育期间更显严峻,因为其他代偿途径在心脏发育初期并未形成。而CBS的保护作用则强调了HCY在CHD发生中的风险作用,尽管有研究认为CBS在胚胎发育初期表达量仅有成人的20%,但本文中发现的多态能大幅提高CB5的表达量,对CHD的发生有着显著的保护作用。在实验中我们也对MTHFR c.677 C>T、c.1298A>C、MTR c.2756 A>G和MTRR c.66 A>G四个编码区多态位点的进行了分析,结果除了MTHFR c.1298 A>C在山东、江苏人群中会提高CHD患病风险之外,其他位点均与CHD易感性无关。将这4个编码区多态分型信息与我们发现的4个非编码区多态结果放在一起,进行多元Logistic回归分析后发现MTR-186 T>G、+905 G>A、MTRR c.56+781 A>C、CBS-551 C>G对表型的贡献是独立的。而在HCY队列中,通过多元回归分析发现,上述8个位点总共可以解释中国汉族人群HCY总变异的24.6%。其中MTR-186 T>G、+905 G>A和MTHFR c.677 C>T分别贡献了10.6%、5.7%、6.7%。叶酸代谢与癌症的发生关系密切,因此我们最后检测了文中所述各多态与前列腺癌、乳腺癌、肺癌、肝癌的易感性关联。结果在关联分析中发现MTR+905 G>A多态与上述四种肿瘤均存在关联,MTR-186 T>G只与前列腺癌和肺癌的发生相关,而CBS-551 C>G则能降低乳腺癌和肺癌的发生风险。更多还原

【Abstract】 Congenital heart disease (CHD) is the first common type of birth defects in China and the leading cause of infant morbidity and disability, which seriously affects the population living quality and plays heavy burden to families as well as society. Clinical researches in recent decades have suggested that maternal preconception administration of folic acid can reduce the occurrence of CHD. Soit was indicated that the core genes of folate metabolism pathways might be the great candidate genes associated with increased risk of CHD. Association studies between core genes in folate metabolism pathways and susceptibility of CHD have pointed out that some pathogenic polymorphism, such as like MTHFR c.677 C>T and c.1298 A>C. However, the worldwide association results are not pretty consistent. The suspending problems are waiting for being solved:(1) Researches mostly focus on published variants ortag SNPs instead of searching for the potentially functional pathogenic variants; (2) Coding region variants have been extensively explored, while the noncoding variants, which can regulate the dosage of the CHD associated gene expression, have been largely ingored; (3) With increasingly improved database of dbSNP and HapMap, researchers tend to select tagSNP directly and pay less attention tothe diversity of distribution and frequency of SNPs in different ethnics and groups, which will veil some significant genetic information; (4) Sample size limitation and less studies with a sample of more than 500 CHD cases leads to low validity and possibilities of false positive.To investigate the association between key genes in the folate metabolism pathway and the susceptibility of CHD, we totally recruited 2,340 CHD cases and 2,270 healthy controls from Shanghai, Jiangsu and Shandong. The researched key genes include Methionine synthase (MTR), methionine synthase reductase (MTRR), cystathionineβ-synthase (CBS), and Thymidylate Synthetase (TYMS). We selected common tagSNPs with minor allele frequency >0.1 according to our own resequencing screening results, and carried out genotyping and association analysis among the three pairs of CHD-control groups. Then we conducted functional analysis of the CHD associated variants. Concerning the close relation between folate metabolism and cancer occurrence, we also analyzed the association between positive variants and several types of cancer incidence. The three independent case-control studies identified four CHD susceptible variants. Among the four polymorphisms, MTR-186T>G (rs28372871),+905 G>A (rs1131450), MTRR c.56+781 A>C (rs326119) are associated with increased risk of congenital heart disease, while CBS-551 C>G (rs2850114) is associated with reduced risk of congenital heart disease.MTR encodes Methionine synthase, which catalyzes the transfer of a methyl group from 5-methyltetrahydrofolate to homocysteine (HCY). The promoter polymorphism-186 G allele in MTR is the risky factor, and the homozygous GG genotype increases a 1.56-fold CHD risk compared with wildtype (adjusted OR=1.56,95% CI=1.33-1.83, P =1.32×10-9). Both in vivo quantitative real-time PCR analysis of MTR hnRNA and mRNA in cardiac tissue samples of CHD and in vitro luciferase assay in transfected cells showed that the -186 G allele remarkably deceases MTR transcription. We confirmed that the reason why G allele leads to lower promoter activity is that the transversion T to G destroys the binding site of the promoter to the transcriptional activator USF. In the cis-regulation, the MTR -186G allele is always associated with elevated methylation ratio allover the promoter CpG islands, which leads to inhibition of MTR expression. It appears that genetic variation could be the primary determinant for the regulation of MTR expression because the methylation status also changed based on the polymorphism. The A allele of 3’UTR polymorphism +905 G>Ain MTR gene is also a CHD risk factor, and the GA and AA genotype can raise 1.2 and 2.74 times of CHD risk respectively (GA:adjusted OR=1.2,95% CI=1.06-1.36; AA:adjusted OR=2.74,95% CI=2.09-3.60, P=6.35×10-14). Functional study showed that the transversion G to A would corroborate the inhibition of three microRNA (miR-485, miR-608, miR-1293) to MTR, thus reduces the expression of MTR at the translating stage. We also measured the HCY level in 522 healthy volunteered controls. The examination result suggested that both-186 T>G and +905 G>A correlates with the elevated plasma HCY level. The further analysis indicated that these two SNPs increase the CHD risk synergistically.Enzyme coded by MTRR gene acts mainly on maintaining adequate amount of activated vitamin B12, a coenzyme of MTR. Experiments showed that variant c.56+781 A>C in the first intron of MTRR is associated with CHD. The C allele of variant c.56+781 A>C will increases CHD susceptibility significantly, while the CA and CC genotype increase CHD risk by 1.4 and 1.84-fold respectively (GA:adjust OR=1.4, 95%CI=1.23-1.59; CC:ajust OR=1.84,95%CI=1.54-2.2; P=4.34×10-12). Both in vivo quantitative real-time PCR analysis of MTRR mRNA in cardiac tissue samples of CHD and in vitro luciferase assay in transfected cells showed that the c.56+781 C allele remarkably deceases MTRR transcription. Additionally, healthy individuals with homozygous CC genotype have significantly elevated homocysteine level compared with wild-type AA carriers.CBS catalyzes the condensation of HCY and serine to cystathionine via the sulfidation, which is one of the HCY-releasing pathways. Correlated analysis showed that-551 C>G polymorphisms in promoter region of CBS can reduce the CHD risk. This protective effect is correlated with the copy number of G alleles, among which the CG and GG genotype can reduce 15% and 40% CHD risk respectively (CG:adjust OR=0.85,95%CI=0.75-0.96; GG:adjust OR=0.6,95%CI=0.49-0.73; P=6.62×10-7). We found that the protective function of G allele might result from increased transcriptional activity and expression of CBS, which was verified through both in vivo mRNA level measurement and luciferase assay. Followed function experiments showed that variant -551 C>G tales play genetically and epigenetically on the CBS expression. The conversion C to G reduces the binding affinity of transcriptional repressor SP1 and promoter, and thus activates the transcription as a result. Moreover, the methylation level of CpG sites in CBS promoter region with G allele was only 1/7 compared with that of C allele, which will improve the transcription of G allelewith no doubt.The above four variants come from the folate metabolism pathways core genes of MTR, MTRR and CBS, which are coordinately responsible for in the removal of HCY, the resource of methyl group for DNA methylation, as well as the supplement of materials for DNA synthesis. Due tothe three risk variants in MTR and MTRR can reduce gene expression, the substrat HCY would accumulate in the body, while,THF as well as MET would be out of supply. This situation can become severer in the developing period of infant heart, for the reason that other alternative pathways haven’t developed well, such as BHMT which is expressed mainly in liver and kidney. And the protection of CBS emphasizes the risky function of HCY in the CHD occurrence. Although some studies consider that the expression of CBS in the early period of infant development is only 20% compared with that of an adult, the polymorphism we identified by uscan improve the express of CBS significantly, thus has a significant protection against the occurrence of CHD.In the experiments we also analyzed four extensively reported folate SNPs, MTHFR c.677 C>T, c.1298 A>C, MTR c.2756 A>G and MTRR c.66 A>G. Except for MTHFR c.1298 A>C raising the CHD risk, all the rest sites proved irrelative with CHD susceptibility. Using multiple Logistic regression analysis on all of the 4 noncoding variants and 4 extensively explored coding SNPs, considering both gender and age, we discovered that MTR-186 T>G,+905 G>A, MTRR c.56+781 A>C and CBS-551 C>G contributed to CHD in Chinese Han population independently. Additionally, all the 8 tested SNPs could explain 24.6% variation of HCY which was evaluated by partial correlation coefficient. The main contribution to the HCY concentration are from MTR-186 T>G (10.6%),+905 G>A (5.7%), MTHFR c.677 C>T (6.7%) respectively.Since folate metabolism is closely related to the occurrence of cancer, we finally examined the correlation between the above polymorphisms and susceptibilities of several kinds of cancer, including prostatic cancer, breast cancer, liver cancer and lung cancer. In the correlation analysis, we found that MTR +905 G>A variant was strongly correlated to all types of the cancer occurrences, MTR-186 T>G variant was associated with increased risk only in prostatic cancer and liver cancer, while the CBS-551 C>G showed protective effect on breast cancer and lung cancer. These results would provide a new thought for the prognosis of CHD patients.更多还原