BR Biomedical Reports 2049-9434 2049-9442 D.A. Spandidos 10.3892/br.2014.222 br-02-02-0172 Articles MTHFR rs1801133 C>T polymorphism is associated with an increased risk of tetralogy of Fallot HUANGJIANBING MEIJU JIANGLIANYONG JIANGZHAOLEI LIUHAO DINGFANGBAO Department of Cardiothoracic Surgery, Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200092, P.R. China Correspondence to: Professor Fangbao Ding, Department of Cardiothoracic Surgery, Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, 1665 Kongjiang Road, Yangpu District, Shanghai 200092, P.R. China, E-mail: 40821247@qq.com 3 2014 15 01 2014 2 2 172 176 02 12 2013 02 01 2014 Copyright © 2014, Spandidos Publications 2014

Abnormal folate metabolism and common variants of folate-metabolizing enzymes have been described as possible risk factors for congenital heart disease (CHD). Two important folate-metabolizing enzymes involved in the folate/homocysteine metabolic pathway are 5,10-methylenetetrahydrofolate reductase (MTHFR) and methylenetetrahydrofolate dehydrogenase 1 (MTHFD1). MTHFR and MTHFD1 polymorphisms may be associated with CHD susceptibility. To evaluate the impact of MTHFR and MTHFD1 single-nucleotide polymorphisms (SNPs) on CHD susceptibility, we genotyped functional MTHFR SNPs rs1801133 C>T, rs1801131 A>C and rs2274976 G>A, and MTHFD SNPs rs2236225 C>T, rs1950902 G>A and rs1076991 A>G in a hospital-based case-control study of 173 tetralogy of Fallot (TOF) cases and 207 non-CHD controls. When MTHFR rs1801133 CC homozygote genotype was used as the reference group, the TT genotype was associated with a significantly increased risk for TOF [TT vs. CC: odds ratio (OR)=1.67; 95% confidence interval (CI): 1.01–2.75; P=0.046]. In the recessive model, when MTHFR rs1801133 CC/CT genotype was used as the reference group, the TT homozygote genotype was associated with a significantly increased risk for TOF (OR=1.81, 95% CI: 1.15–2.84; P=0.010). In conclusion, our findings suggest that MTHFR rs1801133 C>T polymorphism may play a role in susceptibility for TOF. Large-scale studies with a more rigorous study design including diverse ethnic populations are required to confirm these findings.

 5,10-methylenetetrahydrofolate reductase congenital heart disease polymorphisms tetralogy of Fallot molecular epidemiology Introduction

Congenital heart disease (CHD) is currently the leading non-infectious cause of death in children and the most common type of structural malformation of the heart and large blood vessels, with a prevalence of 4–10 per 1,000 live births (1). In China, the prevalence of CHD is 73.2 per 10,000 births in high-prevalence areas.

Abnormal folate metabolism and common variants of folate-metabolizing enzymes have been previously described as possible risk factors for CHD. It has been hypothesized that the peri-conceptional supplementation of multiple vitamins (particularly folic acid) may significantly reduce the incidence of conotruncal heart defects (2). One important folate-metabolizing enzyme involved in the folate/homocysteine metabolic pathway is 5,10-methylenetetrahydrofolate reductase (MTHFR) (3). The MTHFR gene is located on chromosome 1p36.3 and encodes an enzyme that is essential for folate-mediated one-carbon metabolism. The common polymorphism C677T (rs1801133) in the gene that encodes MTHFR causes reduced enzymatic activity and elevated levels of homocysteine (4). Individuals who are homozygous for the 677T allele have only 30% of the MTHFR enzymatic activity measured in individuals homozygous for the wild-type C allele, whereas heterozygotes retain 65% of wild-type MTHFR enzymatic activity (5).

In a recent meta-analysis it was suggested that genotypes with MTHFR C677T polymorphism may be associated with risk of CHD, particularly in Caucasians (6). This risk may be due to several factors, including etiologic heterogeneity among the many and varied CHD phenotypes. Thus, additional etiologic studies that focus on subgroups of CHDs which appear to share common embryologic, genetic and epidemiologic features (e.g., conotruncal and left-sided heart defects) are necessary.

Another non-synonymous single nucleotide polymorphism (SNP) of the MTHFR gene, Arg594Gln (1793G>A, rs2274976), has been identified; however, its functional significance remains unknown (7). In the methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) gene, the rs2236225 polymorphism at nucleotide 1958 (c.1958A>G) results in an arginine→glutamate substitution at amino-acid position 653 (p.Arg653Gln), which is localized in the synthetase domain of the enzyme (8). To evaluate the impact of MTHFR rs1801133 C>T, rs1801131 A>C and rs2274976 G>A, and MTHFD rs2236225 C>T, rs1950902 G>A and rs1076991 A>G SNPs on CHD susceptibility, we performed genotyping analyses for six SNPs in a hospital-based case-control study with 173 tetralogy of Fallot (TOF) cases and 207 controls in a Chinese population.

 Materials and methods Study population

This study included 173 TOF patients and 207 non-CHD controls. Patients were consecutively recruited from the Xinhua Hospital, Shanghai, China, between March 2009 and October 2011. The patients were ultrasonic diagnosed non-syndromic CHDs and confirmed during surgery. All the cases were identified as TOF. The controls were non-CHD patients, frequency-matched to the cases with regard to age (±3 years) and gender, recruited from the same hospital during the same time period and most of them were with trauma or infectious diseases.

All the subjects were genetically unrelated ethnic Han Chinese. After informed consent was obtained from their parents, each subject and her/his parents were personally questioned by trained interviewers using a structured questionnaire to obtain information concerning maternal diabetes mellitus (DM), teratogenic contact during pregnancy and family history of CHD in first-degree relatives (parents, siblings and children). Information regarding rubella, influenza and febrile illnesses during pregnancy was also collected. Cases with structural malformations involving another organ system or known chromosomal abnormalities were excluded. Exclusion criteria also included a positive family history of CHD in a first-degree relative (parents, siblings and children), maternal DM, phenyl ketonuria, maternal exposure to teratogens (e.g., pesticides and organic solvents), as well as rubella, influenza and febrile illnesses during pregnancy. Controls with congenital anomalies were excluded.

A 2-ml venous blood sample was collected from each subject. This study was conducted in accordance with the Declaration of Helsinki. This study was conducted with approval from the Institutional Review Board and Ethics Committee of the Xin Hua Hospital Affiliated to the Shanghai Jiao Tong University School of Medicine, Shanghai, China.

 Isolation of DNA and genotyping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-time-of-flight-MS)

Samples of venous blood (2 ml) were collected from patients using vacutainer tubes. The samples were transferred to tubes containing ethylenediaminetetraacetic acid. Genomic DNA was isolated from whole blood with the QIAamp DNA blood mini kit (Qiagen, Hilden, Germany). Genotyping was undertaken by MALDI-TOF-MS as described previously (9–11). SNP genotyping was performed using the MassArray system (Sequenom, San Diego, CA, USA) by the MALDI-TOF-MS method according to the manufacturer’s instructions (9–11). Completed genotyping reactions were spotted onto a 384-well SpectroChip system (Sequenom) using a MassArray Nanodispenser (Sequenom) and the genotype determined by MALDI-TOF-MS. Genotype calling was conducted in real time with MassArray RT software version 3.1 (Sequenom) and analyzed using MassArray Typer software version 4.0 (Sequenom).

 Statistical analysis

Differences in the distributions of demographic characteristics, selected variables, the variant alleles and genotypes of the six SNPs between the cases and controls were evaluated using the Student’s t-test (for continuous variables) and χ2 test (for categorical variables). The associations between the six SNPs and risk of CHD were estimated by computing the odds ratios (ORs) and their 95% confidence intervals (CIs) from logistic regression analyses. All of the statistical analyses were performed with Statistical Analysis System software (version 9.1.3; SAS Institute, Inc., Cary, NC, USA).

 Results Characteristics of the study population

The demographic and clinical characteristics of all the subjects are shown in Table I. Age for the cases and controls was 2.54 (±2.04) and 2.70 (±2.71 years), respectively, (P=0.521), with 56.1 of the cases and 57.5% of the controls being males and the difference was not statistically significant (P=0.781), indicating that the frequency-matching by age and gender was adequate. Of the 173 TOF patients, 114 (65.9%) were isolated TOF, 27 (15.6%) were TOF with atrial septal defect, 11 (6.4%) were TOF with patent ductus arteriosus, 3 (1.7%) were TOF with absent pulmonary valve, 6 (3.5%) were TOF with pulmonary atresia and 5 (2.9%) were TOF with muscular ventricular septal defect. The primary information for the six genotyped SNPs is shown in Table II. The genotyping success rate ranged from 97.89 to 99.21% for the six SNPs in all 380 samples. Minor allele frequency (MAF) in our controls was similar to that for Chinese individuals in the database for all six SNPs (Table II). The observed genotype frequencies for five polymorphisms in the controls were all in Hardy-Weinberg equilibrium (HWE) instead of MTHFR rs1801133 C>T (P<0.050) (Table II).

 MTHFR and MTHFD polymorphisms and TOF susceptibility

The MTHFR and MTHFD genotype distributions in the cases and controls were shown in Table III. In single-locus analyses, the genotype frequencies of MTHFR rs1801133 C>T were 37.50 (CC), 26.79 (CT) and 35.71 (TT) in TOF patients; and 41.18 (CC), 35.29 (CT) and 23.53% (TT) in the control subjects, with the difference being statistically significant (P=0.028). When the MTHFR rs1801133 CC homozygote genotype was used as the reference group, the CT genotype was not associated with the risk for TOF (CT vs. CC: OR=0.83, 95% CI: 0.51–1.37, P=0.471), the TT genotype was associated with a significantly increased risk for TOF (TT vs. CC: OR=1.67; 95% CI: 1.01–2.75; P=0.046). In the recessive model, when the MTHFR rs1801133 CC/CT genotypes were used as the reference group, the TT homozygote genotype was associated with a significantly increased risk for TOF (OR=1.81, 95% CI: 1.15–2.84, P=0.010). In the dominant model, the MTHFR rs1801133 CT/TT variants were not associated with the risk of TOF, compared with the MTHFR rs1801133 CC genotype (OR=1.17, 95% CI: 0.77–1.77, P=0.471) (Table III).

MTHFR rs1801131 A>C and rs2274976 G>A, as well as MTHFD rs2236225 C>T, rs1950902 G>A and rs1076991 A>G polymorphisms did not achieve a significant difference in the genotype distributions between the cases and controls. Logistic regression analyses revealed that the five SNPs were not associated with the risk of TOF (Table III).

 Discussion

In this case-control study, we investigated the association of MTHFR rs1801133 C>T, rs1801131 A>C and rs2274976 G>A, and MTHFD rs2236225 C>T, rs1950902 G>A and rs1076991 A>G SNPs and risk of TOF in a Chinese population. We found that the MTHFR rs1801133 C>T polymorphism was associated with risk for TOF. MTHFR rs1801131 A>C and rs2274976 G>A, and MTHFD rs2236225 C>T, rs1950902 G>A and rs1076991 A>G polymorphisms may not be associated with risk for TOF.

Folate is known to play a crucial role during the development of the cardiovascular system in the embryo. Variation in the genes involved in the folate pathway plays an important part in the etiology of birth defects, including CHD (12). It has been hypothesized that genetic polymorphisms in folate-metabolizing enzymes affect global DNA methylation as well as changes in the availability of nucleotides for the synthesis and repair of DNA. Animal experiments have shown that disruption of the MTHFR gene results in decreased methylation capacity (13). Studies of MTHFR polymorphisms in humans provide evidence of an association between genotype and global DNA methylation, particularly in the presence of a low-folate diet (14). MTHFR C677T also causes reduced enzymatic activity and elevated concentrations of homocysteine. The results from previous meta-analyses investigating MTHFR C677T polymorphism and CHD risk suggested that genotypes for the MTHFR C677T polymorphism may be associated with CHD risk (6). The importance of the MTHFR C677T polymorphism has been confirmed in a large-scale, methodologically independent genome-wide association study (15).

Genetic polymorphisms often vary between ethnic groups. In this study with 207 non-CHD controls, we reported that the allele frequency of MTHFR rs1801133 C>T (0.412) was similar to that reported in Chinese (0.439) and Japanese (0.367) populations. However, the mutant homozygote among controls was higher compared to that reported in European (0.237) and Sub-Saharan African (0.110) controls.

Taking into consideration MTHFR rs1801133 C>T mutant alleles in the control group, OR, TOF samples and control samples, the power of our analysis (α=0.05) was 0.818 in 173 TOF cases and 207 controls with OR=1.81 for MTHFR rs1801133 C>T.

Previously reported data concerning the association between MTHFR C677T polymorphism and CHD risk together with the designation of the MTHFR C677T risk allele are contradictory and inconclusive. Junker et al (16) were the first to identify a significant association between the incidence of CHD and the MTHFR C677T polymorphism. Marinho et al (17) suggested that the mechanisms underlying the MTHFR C677T mutation may be partially responsible for TOF cardiac defects. Their results are in concordance with some of our data; the present study provides additional evidence concerning the importance of the MTHFR 677TT genotype for conotruncal heart defects in a Han Chinese population.

Several limitations of the study need to be addressed. First, we did not obtain blood samples from the mothers to evaluate the etiological role of MTHFR rs1801133 C>T SNPs in CHDs. Second, since our study was a hospital-based case-control study and selection bias could not be fully excluded, large population-based studies are required to elucidate the role of MTHFR rs1801133 C>T SNPs and susceptibility of TOF. Third, the sample size of this study was not large enough to evaluate the low penetrance effect of the SNPs. Fourth, there was a lack of information on the maternal folate status and maternal genotype. Investigations are required to explore the gene-environment and maternal-fetal genotype interactions. Transient elevations in serum concentrations of folate from the mother due to supplementation or dietary intake may prevent birth defects by overcoming metabolic inefficiencies or transport-related issues. Absence of information on low folate status increases the difficulty of identification of putative genotypes.

In conclusion, our findings suggest that MTHFR rs1801133 C>T polymorphism may play a role in susceptibility for TOF. To confirm the current findings, larger studies with more rigorous study designs that include other ethnic populations are necessary.

 Acknowledgements

The study was supported by the Outstanding Young Talent Fund of Xinhua Hospital.

 Abbreviations CI

confidence interval

 CHD

congenital heart disease

 LD

linkage disequilibrium

 OR

odds ratio

 SNP

single-nucleotide polymorphism

 References BottoLDCorreaAEricksonJDRacial and temporal variations in the prevalence of heart defectsPediatrics107E322001 LewisDPVan DykeDCStumboPJBergMJDrug and environmental factors associated with adverse pregnancy outcomes. Part II: Improvement with folic acidAnn Pharmacother329479611998 GoyettePSumnerJSMilosRHuman methylenetetrahydrofolate reductase: isolation of cDNA mapping and mutation identificationNat Genet75511994 FrosstPBlomHJMilosRA candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductaseNat Genet101111131995 RozenRMolecular genetics of methylenetetrahydrofolate reductase deficiencyJ Inherit Metab Dis195895941996 NieYGuHGongJMethylenetetrahydrofolate reductase C677T polymorphism and congenital heart disease: a meta-analysisClin Chem Lab Med49210121082011 BufalinoARibeiro ParanaibaLMNascimento de AquinoSMaternal polymorphisms in folic acid metabolic genes are associated with nonsyndromic cleft lip and/or palate in the Brazilian populationBirth Defects Res A Clin Mol Teratol889809862010 EtheredgeAJFinnellRHCarmichaelSLMaternal and infant gene-folate interactions and the risk of neural tube defectsAm J Med Genet A158A243924462012 GongDGuHZhangYMethylenetetrahydrofolate reductase C677T and reduced folate carrier 80 G>A polymorphisms are associated with an increased risk of conotruncal heart defectsClin Chem Lab Med50145514612012 GuHGongDDingGA variant allele of ADH1B and ALDH2, is associated with the risk of esophageal cancerExp Ther Med41351402012 GuHDingGZhangWReplication study of PLCE1 and C20orf54 polymorphism and risk of esophageal cancer in a Chinese populationMol Biol Rep39910591112012 BaileyLBBerryRJFolic acid supplementation and the occurrence of congenital heart defects, orofacial clefts, multiple births, and miscarriageAm J Clin Nutr81S1213S12172005 ChenZKaraplisACAckermanSLMice deficient in methylenetetrahydrofolate reductase exhibit hyperhomocysteinemia and decreased methylation capacity, with neuropathology and aortic lipid depositionHum Mol Genet104334432001 CurtinKSlatteryMLUlrichCMGenetic polymorphisms in one-carbon metabolism: associations with CpG island methylator phenotype (CIMP) in colon cancer and the modifying effects of dietCarcinogenesis28167216792007 TanakaTScheetPGiustiBGenome-wide association study of vitamin B6, vitamin B12, folate, and homocysteine blood concentrationsAm J Hum Genet844774822009 JunkerRKotthoffSVielhaberHInfant methylenetetrahydrofolate reductase 677TT genotype is a risk factor for congenital heart diseaseCardiovasc Res512512542001 MarinhoCAlhoIGuerraAThe methylenetetrahydrofolate reductase gene variant (C677T) as a susceptibility gene for tetralogy of FallotRev Port Cardiol288098122009

Comparison of TOF patients and controls by selective characteristics.

Variable TOF (n=173) Controls (n=207) P-value
Age, years (mean ± SD) 2.54±2.04 2.70±2.71 0.521
Male/female (n) 97/76 119/88 0.781
Isolated TOF, n (%) 114 (65.9) - -
TOF with ASD, n (%) 27 (15.6) - -
TOF with PDA, n (%) 11 (6.4) - -
TOF with ASD and PDA, n (%) 7 (4.0) - -
TOF with absent pulmonary valve, n (%) 3 (1.7) - -
TOF with pulmonary atresia, n (%) 6 (3.5) - -
TOF with muscular VSD, n (%) 5 (2.9) - -

TOF, tetralogy of Fallot; ASD, atrial septal defect; PDA, patent ductus arteriosus; VSD, ventricular septal defect.

Primary information for six genotyped SNPs.

Genotyped SNPs Chr Regulo-meDB scorea TFBSb nsSNP MAF for Chinese in database MAF in our controls (n=207) P-value for HWE test in our controls % Genotyping value
MTHFR: rs1801133 C>T 1 4 - Y 0.439 0.412 <0.050 97.89
MTHFR: rs1801131 A>C 1 1f - Y 0.198 0.160 0.712 98.95
MTHFR: rs2274976 G>A 1 4 - Y 0.093 0.066 0.896 99.21
MTHFD: rs2236225 C>T 14 5 - Y 0.221 0.243 0.418 98.95
MTHFD: rs1950902 G>A 14 No data - Y 0.268 0.274 0.599 99.21
MTHFD: rs1076991 A>G 14 4 Y - 0.276 0.238 0.328 98.42

http://www.regulomedb.org/;

http://snpinfo.niehs.nih.gov/snpinfo/snpfunc.htm.

SNPs, single nucleotide polymorphisms; Chr, chromosome; TFBS, transcription factor binding site; nsSNP, non-synonymous single-nucleotide polymorphism; MAF, minor allele frequency; HWE, Hardy-Weinberg equilibrium; MTHFR, 5,10-methylenetetrahydrofolate reductase; Y, yes; MTHFD, methylenetetrahydrofolate dehydrogenase.

Logistic regression analyses of associations between six genotyped polymorphisms and risk of TOF.

Cases (n=173) Controls (n=207)
Genotype n % n % OR (95% CI) χ2 P-value
MTHFR rs1801133 C>T
 CC 63 37.50 84 41.18 1.00
 CT 45 26.79 72 35.29 0.83 (0.51–1.37) 0.520 0.471
 TT 60 35.71 48 23.53 1.67 (1.01–2.75) 3.997 0.046
 CT+TT 105 62.50 120 58.82 1.17 (0.77–1.77) 0.521 0.471
 CC+CT 108 64.29 156 76.47 1.00
 TT 60 35.71 48 23.53 1.81 (1.15–2.84) 6.566 0.010
MTHFR rs1801131 A>C
 AA 111 65.29 146 70.87 1.00
 AC 56 32.94 54 26.21 1.36 (0.87–2.14) 1.845 0.174
 CC 3 1.76 6 2.91 0.66 (0.16–2.69) 0.340 0.560
 AC+CC 59 34.71 60 29.13 1.29 (0.84–2.00) 1.338 0.247
 AA+AC 167 98.24 200 97.09 1.00
 CC 3 1.76 6 2.91 0.60 (0.15–2.43) 0.515 0.473
MTHFR rs2274976 G>A
 GG 147 85.96 180 87.38 1.00
 GA 24 14.04 25 12.14 1.18 (0.64–2.14) 0.278 0.598
 AA 0 0.00 1 0.49 - 0.0003 0.986
 GA+AA 24 14.04 26 12.62 1.13 (0.62–2.05) 0.163 0.686
 GG+GA 171 100.00 205 99.51 1.00
 AA 0 0.00 1 0.49 - 0.0003 0.986
MTHFD rs2236225 C>T
 CC 107 62.94 116 56.31 1.00
 CT 60 35.29 80 38.83 0.81 (0.53–1.24) 0.908 0.341
 TT 3 1.76 10 4.85 0.33 (0.09–1.21) 2.796 0.095
 CT+TT 63 37.06 90 43.69 0.76 (0.50–1.15) 1.694 0.193
 CC+CT 167 98.24 196 95.15 1.00
 TT 3 1.76 10 4.85 0.35 (0.10–1.30) 2.452 0.117
MTHFD rs1950902 G>A
 GG 84 49.12 110 53.40 1.00
 GA 71 41.52 79 38.35 1.18 (0.77–1.81) 0.556 0.456
 AA 16 9.36 17 8.25 1.23 (0.59–2.58) 0.307 0.580
 GA+AA 87 50.88 96 46.60 1.19 (0.79–1.78) 0.683 0.409
 GG+GA 155 90.64 189 91.75 1.00
 AA 16 9.36 17 8.25 1.15 (0.56–2.35) 0.143 0.706
MTHFD rs1076991 A>G
 AA 97 57.06 116 56.86 1.00
 AG 63 37.06 79 38.73 0.95 (0.62–1.46) 0.047 0.828
 GG 10 5.88 9 4.41 1.33 (0.52–3.40) 0.351 0.553
 AG+GG 73 42.94 88 43.14 0.99 (0.66–1.50) 0.0015 0.970
 AA+AG 160 94.12 195 95.59 1.00
 GG 10 5.88 9 4.41 1.35 (0.54–3.41) 0.413 0.520

Bold values indicate statistically significant differences (P<0.05). TOF, tetralogy of Fallot; OR, odds ratio; CI, confidence interval; MTHFR, 5,10-methylenetetrahydrofolate reductase; MTHFD, methylenetetrahydrofolate dehydrogenase.