Folate Metabolism Pathway Key Enzyme Gene Polymorphic Non-coding Regions Of Congenital Heart Disease Susceptibility And Functional Analysis

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.