Skip to main content
Download PDF

“Association of MTHFR and MS/MTR gene polymorphisms with congenital heart defects in North Indian population (Jammu and Kashmir): a case–control study encompassing meta-analysis and trial sequential analysis”

BMC Pediatrics volume 22, Article number: 223 (2022) Cite this article

Abstract

Background

The risk of Congenital Heart Defects (CHD) is greatly influenced by variants within the genes involved in folate-homocysteine metabolism. Polymorphism in MTHFR (C677T and G1793A) and MS/MTR (A2756G) genes increases the risk of developing CHD risk, but results are controversial. Therefore, we conducted a case–control association pilot study followed by an up-dated meta-analysis with trial sequential analysis (TSA) to obtain more precise estimate of the associations of these two gene variants with the CHD risk.

Methods

For case–control study, we enrolled 50 CHD patients and 100 unrelated healthy controls. Genotyping was done by PCR–RFLP method and meta-analysis was performed by MetaGenyo online Statistical Analysis System software. For meta-analysis total number of individuals was as follows: for MTHFR C677T 3450 CHD patients and 4447 controls whereas for MS A2756G 697 CHD patients and 777 controls.

Results

Results of the original pilot study suggested lack of association for MTHFR C677T and MS A2756G polymorphism with risk of CHD whereas MTHFR G1793A was significantly associated with the disease. On performing meta-analysis, a significant association was observed with MTHFR C677T polymorphism but not with MS A2756G. Trial sequential Analysis also confirmed the sufficient sample size requirement for findings of meta-analysis.

Conclusions

The results of the meta-analysis suggested a significant role of MTHFR in increased risk of CHD.

Peer Review reports

Introduction

Congenital heart diseases or defects (CHD) which share a significant proportion in CVD burden arises due to incomplete development of heart during the first 6-weeks of gestation [1]. The origin of CHD is diverse which can be associated with a syndrome or be isolated (non-syndromic). It is hypothesized that susceptibility of cardiac defects increases with dual interaction of key gene(s)/SNP-environmental factors which perturb normal cardiac developmental process during embryonic life. The risk of CHD is greatly influenced by variants within the genes involved in folate-homocysteine metabolism [2,3,4]. Many studies have revealed that the risk of CHD in new-borns of females carrying mutations in genes involved in folate metabolism can be reduced by maternal periconceptional use of multivitamins or folic acid [5], however, the mechanism underlying this effect is still under investigation. Folate and vitamin B12 are known to influence homocysteine concentration. Folates taken in diet are usually polyglutamates which are converted to simpler forms, particularly monoglutamates, dihydrofolate, tetrahydrofolate and finally to methylated form of folate i.e. 5, 10-methylenetetrahydrofolate (5,10-MTHF) and 5-methyltetrahydrofolate (5-MTHF) by a specialised enzyme of the pathway. Homocysteine and folate metabolism is dependent on a couple of genes performing their specific role but two genes namely MTHFR and MS are considered critical genes for development of diseased cardiovascular phenotypes. A common mutation, C677T (rs1801133), in exon 4 of the MTHFR gene results in decreased enzyme activity and contributes to increased plasma homocysteine, particularly in individuals with low folate status. Rady and co-workers reported a novel polymorphic site of the MTHFR gene at nucleotide position 1793 G to A transition in exon 11 (rs2274976) which results an arginine-to-glutamine change at codon 594 and modifies enzyme activity [6]. The A2756G mutation (rs1805087) in MS gene alters re-methylation process and is also associated with increased homocysteine levels and risk of CHD. Most of the research in relation to folate-homocysteine metabolising pathway with the risk of CHD is based on parent-of-origin effect. There are very few studies focussing on embryonic variation in candidate genes of folate-homocysteine metabolising pathway in association with the development of structural congenital heart malformations during early pregnancy. Consistent with this view, we attempted to perform a case–control pilot study involving evaluation of two important genes: MTHFR (C677T and G1793A) and MS (A2756G) gene variations with risk of CHD in Jammu region of UT of J&K, India. Further, we also performed an updated meta-analysis with trial sequential analysis to investigate the association between MTHFR (C677T and G1793A) and MS (A2756G) polymorphisms and risk of CHD with increased statistical power.

Methodology

Study population and area

The present study was ethically approved by Institutional Ethical Committee, University of Jammu. The present study was carried out on 150 children, out of whom 50 children (0–12 years) were confirmed cases of CHD and 100 children (below 18 years) were unrelated healthy controls belonging to Jammu region of Union Territory of Jammu and Kashmir. The CHD cases were enrolled from In-patient Department of Paediatrics whereas controls were recruited from Out-patient Department of Paediatrics, Shri Maharaja Gulab Singh (SMGS) hospital, Jammu. Data and blood collection was done after having an informed written consent from attendant or guardian of the children. The diagnosis and classification of CHD was based on the clinical and the echocardiography findings. The inclusion/exclusion criteria were followed wherein patients with any form of CHD were included whereas patients with syndromes and neural tube defects were excluded. Controls admitted to hospital for minor ailments with no history of CHD or other major abnormality and also children visiting for blood typing were recruited for the study under reference. Power of the study for sample size calculation was done by using online tool based on mean and standard deviation of two groups of study subjects, two tail test and with alpha value of 5% (https://www.sphanalytics.com/statistical-power-calculator-using-average-values/). The power of the study obtained was more than 80%.

Blood collection and DNA isolation

500 μl-1 ml of blood was collected in EDTA coated vacutainers from each child by trained paramedical staff of the Hospital. Isolation of DNA from whole blood was carried out using commercially available kits (DNeasy Blood and Tissue Kit, QIAGEN). The quantitative and qualitative analysis of isolated DNA was performed by spectrophotometry and 1.5% agarose gel electrophoresis respectively.

Genotyping

Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) technique. Briefly, PCR was carried out in a reaction volume of 25 μl each in thin walled tubes, consisting of 5.0 μl of PCR buffer (10X), 2.5 μl of MgCl2 (25 mM), 0.5 μl of dNTPs (10 mM), 0.5 μl (100 pmol/µl) of each of the forward and reverse primers, 0.3 μl (5unit/μl) of Taq DNA polymerase enzyme and 2 μl (40 ng) of genomic DNA. PCR amplification was carried out using the Veriti, Applied Biosystems by life technology, Singapore and amplification and RFLP conditions for all the three polymorphisms are given in Table 1. The gel images of PCR–RFLP for MTHFR (C677T and G1793A) and MS (A2756G) polymorphisms with band sizes have been depicted in Fig. 1, 2 and 3 respectively.

Table 1 Details of Primer sequence, amplification conditions and restriction enzymes
Full size table
Fig. 1
figure 1

RFLP Gel Image of MTHFR C677T Polymorphism. L: 100 bp Ladder; Lane 1,2,3,5,6,8: 198 bp (CC: Wild); Lane 4,7: 198 + 175 + 23 bp (CT: Hetero). *23 bp band will not be visible on agarose gel

Full size image
Fig. 2
figure 2

RFLP Gel Image of MTHFR G1793A Polymorphism. L: 100 bp Ladder; Lane 3,4,6,7,8: 233 + 77 bp (GG: Wild); Lane 1,2,5: 310 + 233 + 77 bp (GA: Hetero)

Full size image
Fig. 3
figure 3

RFLP Gel Image of MS A2756G Polymorphism. L: 100 bp Ladder; Lane 1,2,6,7: 211 bp (AA: Wild); Lane 2,4,5: 131 + 80 + 211 bp (AG: Hetero)

Full size image

Statistical analyses

Genotypic frequency as well as allelic frequency was calculated by gene counting method. Hardy–Weinberg equilibrium (HWE) analysis and the differences in genotypic frequencies between two study groups were examined by using Pearson’s goodness of fit Chi-square test. To assess the association, odds ratios (OR) with 95% CI were calculated under different genetic models by using Statistical Package for Social Sciences (SPSS-version 20) software and also by another method provided by the Institute of Human Genetics accessed via the link: http://ihg.gsf.de/cgi-bin/hw/hwa1.pl. A p-value of < 0.05 was considered as statistically significant.

Meta-analysis

Literature search

Research papers (published up to February, 2021) examining the association between MTHFR C677T, MTHFR G1793A and MS A2756G polymorphisms and congenital heart defects were extracted from databases such as PubMed, Science direct, Proquest, Ovid and Google Scholar. Key words used for the database search were as follows: methylenetetrahydrofolate reductase; MTHFR gene polymorphisms; Methionine synthase; MS/MTR gene polymorphisms; Congenital heart defects; Congenital heart diseases; MTHFR C677T; MTHFR G1793A and MS/MTR A2756G. Reference records of studies included in our meta-analysis were manually searched for possible eligible articles.

Inclusion and exclusion criteria

The inclusion/exclusion criteria used for screening of eligible study are given in Table 2.

Table 2 Inclusion/Exclusion criteria for eligible studies
Full size table

Data extraction and quality assessment

From each eligible study, the following data were extracted by the two investigators independently using a standardized form: first author, publication year, country of origin, ethnicity, number of cases and controls, genotype frequency, source of controls, genotyping method, and Hardy–Weinberg equilibrium (HWE). We investigated the quality of each study based on the nine-point Newcastle–Ottawa Scale (NOS). The characteristics and results of NOS for all the included studies are shown in Table 3. The NOS scores for all eligible studies in this Meta analysis exceeded 6 points, indicating that our analysis is updated and is of good quality.

Table 3 Characteristics of the included studies in the meta-analysis
Full size table

Statistical analysis for meta-analysis

The association between the selected polymorphisms and congenital heart defects was evaluated for each study by the crude odds ratios (ORs) with 95% confidence intervals (CIs). For each study, HWE was assessed by the chi-square goodness of fit test. For all studies, we estimated the association under three different genetic models [Allele contrast, dominant model and recessive model]. Statistical heterogeneity between studies was assessed by Cochran’s Q test and I-square (I2) > 50% indicated the significance [31]. When I2 > 50%, a random-effect model should be taken otherwise fixed model is used. To calculate the OR and draw inference for each study, we used both random effects model and fixed effect model. Sensitivity analyses were conducted by omitting any single study, which predisposed the observed heterogeneity excessively and there should be no change in OR’s. Egger’s test and Begg’s funnel plot is used to solve the problem of Publication bias. All statistical analyses were performed in the MetaGenyo online Statistical Analysis System software [32].

Trial sequential analysis (TSA)

Meta-analysis may result in Type I error owing to an increased risk of random errors (play of chance) which can be due to dispersed data and repeated significance testing. Bias from low trial with low methodological qualities, publication bias and small trial bias may result in false p-value. Trial Sequential analysis is a methodology that can be used in meta-analysis to control random errors, and to assess whether the studies included in the meta-analysis have surpassed the requisite sample size. TSA was performed to calculate the required information size on the basis of overall 5% risk of Type-I error and a power of 80% for checking the reliability of meta analysis [33].

Results

Case–control study

Based on echocardiography reports, the different CHD phenotypes were categorised (Table 4). The observed prevalence of different CHD phenotypes in present study was highest for ventricular septal defect (VSD: 34%) and atrial septal defect (ASD: 26%) followed by tetralogy of fallot (TOF: 14%) and patent ductus arteriosus (PDA: 8%) and least for endocardial cushion defect (6%). The frequency of complex CHD forms (more than one CHD condition) were as follows: 4% for ASD with PDA, 2% for VSD with AV-canal defect, 4% for VSD with pulmonary arterial hypertension (VSD-PAH) and 2% for endocardial cushion defect along with dextrocardia.

Table 4 Prevalence of CHD phenotypes in present study
Full size table

The genotypic and allelic frequencies along with Chi square values for Hardy–Weinberg calculations for the all the three polymorphisms in study participants are depicted in Table 5. There observed frequencies of genotypes were in concordance with HWE in both the groups for all the polymorphisms except for MTHFR C677T in patient group. The genotypic frequency of CC, CT and TT (MTHFR C677T) in CHD patients was 88%, 8% and 4% whereas in controls it was 90%, 9% and 1% respectively. The frequency of variant allele T (0.08) was higher in CHD patients than controls (0.05) whereas wild allele C was reported to be in slightly higher frequency in controls (0.95) as compared to patients (0.92). The genotypic frequencies for MTHFR G1793A in CHD patients were 58%, 38% and 4% for GG, GA and AA respectively. The frequencies in control group were 90% for GG and 10% for GA genotypes; however we did not observe any AA genotype in controls. In general there was higher frequency of risk allele ‘A’ in CHD patients (0.23) in comparison to controls (0.05). The distribution of observed MS genotypes in CHD patients were 60%, 36% & 4% for AA, AG and GG genotypes respectively. In control group the distribution was as follows: 73% for AA, 26% for AG and 1% for GG genotype. The CHD patients were showing higher frequency of risk allele ‘G’ (0.22) than controls (0.14).

Table 5 Showing genotypic and allelic distribution of selected gene polymorphisms among cases and controls
Full size table

In order to investigate the possible association of these three polymorphisms with susceptibility of CHD, ORs with 95% confidentiality intervals was calculated for different genetic models which are presented in Table 6.

Table 6 Association between selected gene polymorphisms and CHD
Full size table

For both MTHFR C677T and MS A2756G polymorphisms, we observed that even though the values calculated for ORs under different models were above 1, but none of the values reached statistical significance level (p > 0.05). The present study proclaimed lack of association of MTHFR C677T and MS A2756G gene polymorphism with the risk of CHD in our population. Furthermore, the GA vs GG genotype depicted a strong significant association of MTHFR G1793A gene polymorphism. The G vs A frequency showed that the allele ‘A’ is adding a significant risk of approximately 5.7 folds in the development of CHD in the studied population. Distribution of MTHFR haplotypes in cases & controls and their association towards CHD susceptibility is depicted in Table 7.

Table 7 Association of MTHFR haplotypes with risk of CHD
Full size table

The frequency of C-G haplotype was higher in both cases and controls (0.690 & 0.895 respectively). There was complete absence of T-A haplotype in both study groups. The haplotype combination C-A was significantly associated with CHD risk (OR = 5.67 [2.58–12.48], p = 2.71e‐006) and C-G was significantly involved in protection against CHD development (OR = 0.26 [0.14–0.48], p = 1.00e‐005) in the population under reference. By analysing LD scores in two study groups it was observed that the MTHFR variants were in complete LD in both patients (D' = 0.999, r2 = 0.026) and controls (D' = 1, r2 = 0.003).

Meta-analysis

We found 26 eligible studies having 3450 cases and 4447 controls with reference to MTHFR C677T polymorphism and 6 studies with 697 cases and 777 controls concerning MS A2756G polymorphism. The main study characteristics are summarized in Table 3. The study selection process has been depicted in PRISMA diagram (Fig. 4). By pooling all the studies, it was found that there is statistically significant association between MTHFR C677T polymorphism and congenital heart defects under all applied genetic models (Dominant model: OR = 1.38, 95% CI: 1.14- 1.69; recessive model: OR = 1.49, 95% CI: 1.83–1.87; allele model: OR = 1.33, 95% CI: 1.14–1.55) as shown in Table 8 and Fig. 5, 6, and 7. When we stratified the studies according to ethnicity, a significant association was observed between this locus and CHD only in Asian populations (Dominant model: OR = 1.50, 95% CI: 1.12- 2.01; recessive model: OR = 1.67, 95% CI: 1.21–2.31; allele model: OR = 1.42, 95% CI: 1.15- 1.76), but not in Caucasian populations (dominant model: OR = 1.24, 95% CI: 0.95- 1.62; Recessive model: OR = 1.27, 95% CI: 0.99–1.63; allele model: OR = 1.21, 95% CI: 0.97–1.50) as given in Table 8.

Fig. 4
figure 4

PRISMA flow diagram showing the selection of various studies for the meta –analysis

Full size image
Table 8 Overall meta-analysis and subgroup analysis by ethnicity for MTHFR C677T polymorphism
Full size table
Fig. 5
figure 5

Pooled OR (Dominant model) and 95% CI for individual studies and pooled data for the association between the polymorphism C677T and congenital heart disease (CHD) in the overall population

Full size image
Fig. 6
figure 6

Pooled OR (Recessive model) and 95% CI for individual studies and pooled data for the association between the polymorphism C677T and congenital heart disease (CHD) in the overall population

Full size image
Fig. 7
figure 7

Pooled OR (Allele model) and 95% CI for individual studies and pooled data for the association between the polymorphism C677T and congenital heart disease (CHD) in the overall population

Full size image

However, it was observed that Caucasian population was also showing association but it did not reach statistical significance. For MS polymorphism, none of the applied genetic models found association with CHD in overall population or even after subgrouping (Table 9 and Fig. 8). Sensitivity analysis for both MTHFR and MS revealed that there is no change in the pooled ORs by omitting individual studies (Fig. 9 and 10). The publication bias was also estimated by using funnel plot for log-odds ratio for dominant model against the reciprocal of its standard error (Fig. 11 and 12). Further Egger regression asymmetry test was also used to evaluate publication bias (Table 9). No publication bias was observed in the present meta-analysis. Meta- analysis could not be performed for MTHFR G1793A gene polymorphism as we were able to find only one study other than the study under reference. Meta-analysis could not be performed for MTHFR G1793A gene polymorphism as we were able to find only one study other than the study under reference.

Table 9 Overall meta-analysis and subgroup analysis by ethnicity for MS A2756G polymorphism
Full size table
Fig. 8
figure 8

Pooled OR (Dominant model) and 95% CI for individual studies and pooled data for the association between the polymorphism MS/MTR A2756G and congenital heart disease (CHD) in the overall population

Full size image
Fig. 9
figure 9

Sensitivity analysis of association between MTHFR C677T polymorphism and CHD

Full size image
Fig. 10
figure 10

Sensitivity analysis of association between MS/MTR A2756G polymorphism and CHD

Full size image
Fig. 11
figure 11

Funnel plot of the MTHFR C677T polymorphism and susceptibility to CHD (Dominant model) in the overall population

Full size image
Fig. 12
figure 12

Funnel plot of the MS/MTR A2756G polymorphism and susceptibility to CHD (Dominant model) in the overall population

Full size image

Trial Sequential Analysis (TSA)

Trial sequential analysis was performed to calculate the requisite sample size for the meta-analysis of MTHFR C677T gene polymorphism. It revealed that sufficient number of studies have been included in the meta-analysis of this polymorphism. The results of TSA were in accordance with the findings of the conventional meta-analysis and revealed that C677T polymorphism was significantly associated with the risk of CHD (Fig. 13). For MS A2756G polymorphism, TSA could not be performed owing to very little information of sample size which revealed that there is need of more replicas of case control studies to reach the conclusive remarks on role of said polymorphism in conferring risk of CHD. Similarly for MTHFR G1793A gene polymorphism, TSA could not be performed as only two studies were available for meta-analysis.

Fig. 13
figure 13

Trial Sequential Analysis (TSA) of the studies included in the meta analysis of MTHFR C677T gene polymorphism with CHD

Full size image

Discussion

The folate-homocysteine metabolic pathway performs a paramount role in neural tube formation and cardiac development during embryogenesis. Low folate and high homocysteine levels are a closely related with the manifestation of congenital heart defects, which indicates that single nucleotide polymorphisms (SNPs) in the genes controlling this pathway may be the genetic risk factors for these disorders [34]. Therefore, we performed a case–control association study and an updated meta-analysis along with TSA to investigate the association of MTHFR and MS gene polymorphisms with risk of CHD. We did not find a significant association of MTHFR C677T and MS A2756G polymorphism with risk of CHD in our studied population. The results were consistent with studies done by various workers [5, 7, 35,36,37,38]. Regarding MTHFR G1793A polymorphism in link with CHD risk we found significant association under co-dominant, dominant and allelic model in present study. The genotypic frequencies reported in the present study were almost compatible with frequencies as reported by Toganel and co-workers and the investigators also observed a strong significant association this SNP with susceptibility of CHD [AA + GA vs GG: OR = 4.18; 95% CI (1.25- 13.98), p = 0.02] in a Romanian population whereas antithetical findings were reported in Chinese population [39, 40]. Xu and co-workers found that the variant genotypes of MTHFR G1793A polymorphism were significantly associated with a decreased risk of CHD, especially in patients with isolated peri-membranous VSD [40]. The correlation between the MTHFR G1793A gene polymorphism and the CHD risk has not been extensively studied so far. To the best of our knowledge there is no previous report from India and we are the first to analyse G1793A variation of MTHFR gene from North India. The present study is first of its kind concentrating on the effect of MTHFR (C677T and G1793A) haplotypes with vulnerability of CHD. The haplotype C-A was conferring nearly 5.7-fold disease risk and C-G haplotype was giving a shielding outcome of approximately 3.8-fold (1/0.26). Based on measure of LD, the two MTHFR SNPs were in complete LD in both CHD cases and controls. The possible limitations of the present study may be the enrolment of study samples from single region of UT J&K and lack of homocysteine measurements in the study subjects. Besides these limitations and to the best of our knowledge, the study under reference here is the first attempt that evaluates the association of MTHFR and MS gene polymorphisms in CHD.

Genetic association studies have been a powerful approach for identifying susceptibility genes for common diseases but it has been experienced that most of the initial positive associations were not reproduced in the subsequent replication studies because of small sample size or false-positive reports [41, 42]. Meta-analysis solves this problem as it increases the statistical power to detect gene–disease associations by combining results from the original and subsequent replication studies [42]. Similarly, when we conducted case–control association, we did not observe significant association of MTHFR C677T with risk of CHD, as it was a pilot study and carried on limited number of samples. But after performing meta-analysis, the results suggested a positive association of MTHFR C677T with the risk of CHD. The results of the overall analysis depicted an increased risk of CHD with the presence of MTHFR 677 T- allele in fetus. The putative risk allele-677 T had a 1.33 folds increased risk of CHD against the C-allele. From the subgroup analysis, the increased risk of the T-allele was widely detected in Asians but not in Caucasians. Our results are compatible with the previous Meta analyses that investigated the association of the MTHFR C677T polymorphism in CHD [34, 43]. Further, this association revealed through conventional meta-analysis has also been confirmed by performing Trial Sequential Analysis. Lack of association was reported for MS A2756G both in pooled and in sub-grouped meta-analysis and the findings are consistent with study done by Cai and co-workers [44]. The findings of MS polymorphism needs to be further investigated as there are not enough studies on association of this polymorphism with risk of CHD and during our search we also found only six eligible studies and TSA has not been performed in lieu of lack of sufficient number of studies. Further, we were not able to perform meta- analysis for MTHFR G1793A polymorphism as to best of our efforts; we found only a few case–control studies which were not sufficient for performing meta-analysis.

Conclusion

In conclusion, the results of meta-analysis and TSA support the role of MTHFR C677T gene polymorphism as susceptibility factor for Congenital Heart Defects. For MTHFR G1793A and MS A2756G gene polymorphisms, there is need to perform large number of homogenous studies to evaluate these crude results further.

Availability of data and materials

The data and the material used in the research work under reference can be made available upon reasonable request from corresponding author.

Abbreviations

CHD:

Congenital Heart Defects

MTHFR:

Methylenetetrahydrofolate reductase

MTR:

5-Methyltetrahydrofolate-Homocysteine Methytransferase

TSA:

Trial sequential analysis

PCR:

Polymerase Chain Reaction

RFLP:

Restriction Fragment Length Polymorphism

PCR–RFLP:

Polymerase Chain Reaction- Restriction Fragment Length Polymorphism

CVD:

Cardiovascular Disease

5, 10-MTHF:

5, 10-Methylenetetrahydrofolate

5-MTHF:

5-Methyltetrafolate

MS:

Methionine synthase

HWE:

Hardy–Weinberg equilibrium

OR:

Odd Ratio

CI:

Confidence Interval

SPSS:

Statistical Package for Social Sciences

NOS:

Newcastle–Ottawa Scale

I2 :

I-square

p-value:

Probability value

VSD:

Ventricular septal defect

ASD:

Atrial septal defect

TOF:

Tetralogy of fallot

PDA:

Patent ductus arteriosus

VSD-PAH:

Ventricular septal defect with pulmonary arterial hypertension

LD:

Linkage Disequilibrium

PRISMA:

Preferred Reporting Items for Systematic Reviews and Meta-Analyses

SNPs:

Single nucleotide polymorphisms

AV canal defect:

Atrioventricular canal defect

References

  1. Marinho C, Alho I, Guerra A, Rego C, Areias J, Bicho M. The methylenetetrahydrofolate reductase gene variant (C677T) as a susceptibility gene for tetralogy of fallot. Rev Port Cardiol. 2009;28(7–8):809–12.

    PubMed  Google Scholar 

  2. Lupo PJ, Goldmuntz E, Mitchell LE. Gene-gene interactions in the folate metabolic pathway and the risk of conotruncal heart defects. J Biomed Biotechnol. 2010;2010:1–7.

    Article  CAS  Google Scholar 

  3. Czeizel AE, Dudás I, Vereczkey A, Bánhidy F. Folate deficiency and folic acid supplementation: the prevention of neural-tube defects and congenital heart defects. Nutrients. 2013;5:4760–75. https://doi.org/10.3390/nu5114760.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. van Beynum IM, Kapusta L, den Heijer M, VermeulenSita HHM, Kouwenberg M, Daniels O, Blom HJ. Maternal MTHFR 677C>T is a risk factor for congenital heart defects: effect modification by periconceptional folate supplementation. Eur Heart J. 2006;27:981–7. https://doi.org/10.1093/eurheartj/ehi815.

    Article  CAS  PubMed  Google Scholar 

  5. Shi H, Yang S, Liu Y, Huang P, Lin N, Sun X, et al. Study on environmental causes and SNPs of MTHFR, MS and CBS genes related to congenital heart disease. PLoS ONE. 2015;10(6):1–10.

    Google Scholar 

  6. Rady PL, Szucs S, Grady J, Hudnall SD, Kellner LH, Nitowsky H, et al. Genetic polymorphisms of methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) in ethnic populations in Texas; a report of a novel MTHFR polymorphic site, 1793G>A. Am J Med Genet. 2002;107(2):162–8.

    Article  PubMed  Google Scholar 

  7. Pereira AC, Xavier-Netro J, Mesquita SM, Mota GF, Lopes AA, Krieger JE. Lack of evidence of association between MTHFR C677T polymorphism and congenital heart disease in a TDT study design. Int J Cardiol. 2005;105(1):15–8.

    Article  PubMed  Google Scholar 

  8. Mohamad NA, Vasudevan R, Ismail P, Jafar NI, Etemad A, Aziz AFA, et al. Analysis of homocysteine metabolism enzyme gene polymorphisms in non-syndromic congenital heart disease patients among Malaysians. Life Science Journal. 2014;11(8):318–25.

    Google Scholar 

  9. Junker R, Kotthoff S, Vielhaber H, Halimeh S, Kosch A, Koch HG, et al. Infant methylenetetrahydrofolate reductase 677TT genotype is a risk factor for congenital heart disease. Cardiovascular Res. 2001;51(2):251–4.

    Article  CAS  Google Scholar 

  10. Lee CN, Su YN, Cheng WF, Lin MT, Wang JK, Wu MH, Hseih FJ. Association of the C677T methylenetetrahydrofolate reductase mutation with congenital heart diseases. Acta Obstet Gynecol Scand. 2005;84(12):1134–40.

    Article  PubMed  Google Scholar 

  11. Li Y, Cheng J, Zhu WL, Dao JJ, Yan LY, Li MY, et al. Study of serum Hcy and polymorphisms of Hcy metabolic enzymes in 192 families affected by congenital heart disease. Beijing Xue Xue Bao Yi Xue Ban. 2005;37(1):75–80.

    Google Scholar 

  12. Shaw GM, Iovannisci DM, Yang W, Finnell RH, Carmichael SL, Cheng S, et al. Risks of human conotruncal heart defects associated with 32 single nucleotide polymorphisms of selected cardiovascular disease-related genes. Am J Med Genet A. 2005;138(1):21–6.

    Article  PubMed  Google Scholar 

  13. Zhu WL, Li Y, Yan L, Dao J, Li S. Maternal and offspring MTHFR gene C677T polymorphism as predictors of congenital atrial septal defect and patent ductus arteriosus. MHR: Basic Sci Reprod Med. 2006;12(1):51–4.

    Google Scholar 

  14. Galdieri LC, Arrieta SR, Silva CM, Pedra CA, D’Almeida V. Homocysteine concentrations and molecular analysis in patients with congenital heart defects. Arch Med Res. 2007;38(2):212–8.

    Article  CAS  PubMed  Google Scholar 

  15. Van-Driel LM, de Jonge R, Helbing WA, van Zelst BD, Ottenkamp J, Steegers EA, et al. Maternal global methylation status and risk of congenital heart diseases. Obstet Gynecol. 2008;112(2, Part 1):277–83.

    Article  PubMed  Google Scholar 

  16. Xu J, Xu X, Xue L, Liu X, Gu H, Cao H, et al. MTHFR c.1793G>a polymorphism is associated with congenital cardiac disease in a Chinese population. Cardiol Young. 2010;20(3):318–26.

    Article  PubMed  Google Scholar 

  17. Kuehl K, Loffredo C, Lammer EJ, Iovannisci DM, Shaw GM. Association of congenital cardiovascular malformations with 33 single nucleotide polymorphisms of selected cardiovascular disease-related genes. Birth Defects Res A Clin Mol Teratol. 2010;88(2):101–10.

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Obermann-Bors SA, van Driel LM, Helbing WA, de Jonge R, Wildhagen MF, Steegers EA, et al. Congenital heart defects and biomarkers of methylation in children: a case-control study. Eur J Clin Invest. 2011;41(2):143–50.

    Article  CAS  Google Scholar 

  19. Kotby A, Anwar M, El-Masry OAEA, Awady M, El-Nashar A, Meguid NA. Genetic variants in the methylenetetrahydrofolate reductase gene in Egyptian children with conotruncal heart defects and their mothers. Maced J Med Sci. 2012;5(1):78–84.

    Article  Google Scholar 

  20. Gong D, Gu H, Zhang Y, Gong J, Nie Y, Wang J, et al. Methylenetetrahydrofolate reductase C677T and reduced folate carrier 80 G>a polymorphisms are associated with an increased risk of conotruncal heart defects. Clin Chem Lab Med. 2012;50(8):1455–61.

    Article  CAS  PubMed  Google Scholar 

  21. El-Abd DM, Said RN, Hanna BM, El-Naggar NF. Maternal and offspring methylenetetrahydrofolate reductase gene C677T polymorphism: does it influence the prevalence of congenital heart defects in Egyptian neonates? Comp Clin Pathol. 2012;23:317–22.

    Article  CAS  Google Scholar 

  22. Wang W, Wang Y, Gong F, Zhu W, Fu S. MTHFR C677T polymorphism and risk of congenital heart defects: evidence from 29 Case-control and TDT studies. PLoS ONE. 2013;8(3):e58041.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Kocakap BDS, Sanli C, Cabuk F, Koc M, Kutsal A. Association of MTHFR A1298C polymorphism with conotruncal heart disease. Cardiol Young. 2015;25(7):1326–31.

    Article  Google Scholar 

  24. Chao CS, Wei J, Huang HW, Yang SC. Correlation between methyltetrahydrofolate reductase (MTHFR) polymorphisms and isolated patent ductus arteriosus in Taiwan. Heart Lung Circ. 2014;23(7):655–60.

    Article  PubMed  Google Scholar 

  25. Sahiner UM, Alanay Y, Alehan D, Tuncbilek E, Alikasifoglu M. Methylene tetrahydrofolate reductase polymorphisms and homocysteine level in heart defects. Pediatr Int. 2014;56(2):167–72.

    Article  CAS  PubMed  Google Scholar 

  26. Li WX, Dai SX, Zheng JJ, Liu JQ, Huang JF. Homocysteine metabolism gene polymorphisms (MTHFR C677T, MTHFR A1298C, MTR A2756G and MTRR A66G) jointly elevate the risk of folate deficiency. Nutrients. 2015;7(8):6670–87.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Shi H, Yang S, Liu Y, Hang P, Lin N, Sun X, et al. Study on environmental causes and SNPs of MTHFR, MS and CBS genes related to congenital heart disease. PLoS ONE. 2015;10(6):e0128646.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Wang Y, Zhang H, Yue S, Zhang K, Wang H, Dong R, et al. Evaluation of high resolution melting for MTHFR C677T genotyping in congenital heart disease. PLoS ONE. 2016;11(3):e0151140.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. Noori N, Miri-Moghaddam E, Dejkam A, Garmie Y, Bazi A. Are polymorphisms in MTRR A66G and MTHFR C677T genes associated with congenital heart diseases in Iranian population? Caspian J Intern Med. 2017;8(2):83–90.

    PubMed  PubMed Central  Google Scholar 

  30. Wang X, Wei H, Tian Y, Wu Y, Luo L. Genetic variation in folate metabolism is associated with the risk of conotruncal heart defects in a Chinese population. BMC Pediatr. 2018;18(1):287.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Higgins JP, Thompson SG, Deeks JJ, Altman DG. Measuring inconsistency in meta-analyses. BMJ. 2003;327(7414):557–60.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Martorell-Marugan J, Toro-Dominguez D, Alarcon-Riquelme ME, Carmona-Saez P. MetaGenyo: a web tool for meta-analysis of genetic association studies. BMC Bioinformatics. 2017;18:1990–4.

    Article  CAS  Google Scholar 

  33. Thorlund K, Engstrom J, Wetterslev J, Brok J, Imberger G, Gluud C. User manual for trial sequential analysis (TSA). Copenhagen: Copenhagen Trial Unit, Centre for Clinical Intervention Research; 2011. p. 1–119.

    Google Scholar 

  34. Zhang R, Huo C, Wang X, Dang B, Mu Y, Wang Y. Two common MTHFR gene polymorphisms (C677T and A1298C) and fetal congenital heart disease risk: an updated meta-analysis with trial sequential analysis. Cell Physiol Biochem. 2018;45(6):2483–96.

    Article  CAS  PubMed  Google Scholar 

  35. Mohamad NA, Vasudevan R, Ismail P, Jafar NI, Etemad A, Aziz AFA, et al. Analysis of homocysteine metabolism enzyme gene polymorphisms in non-syndromic congenital heart disease patients among Malaysians. Life Sci J. 2014;11(8):318–26.

    Google Scholar 

  36. McBride KL, Fernbach S, Menesses A, Molinari L, Quay E, Pignatelli R, et al. A family-based association study of congenital left-sided heart malformations and 5, 10-methylenetetrahydrofolate reductase. Birth Defects Res A Clin Mol Teratol. 2004;70(10):825–30.

    Article  CAS  PubMed  Google Scholar 

  37. Shaw GM, Iovannisci DM, Yang W, Finnell RH, Carmichael SL, Cheng S, et al. Risks of human conotruncal heart defects associated with 32 single nucleotide polymorphisms of selected cardiovascular disease-related genes. Am J Med Genet A. 2005;138(1):21–6.

    Article  PubMed  Google Scholar 

  38. Pishva SR, Vasudevan R, Etemad A, Heidari F, Komara M, Ismail P, et al. Analysis of MTHFR and MTRR gene polymorphisms in Iranian ventricular septal defect subjects. Int J Mol Sci. 2013;14(2):2739–52.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Toganel R, et al. Correlations between three variants of MTHFR gene polymorphisms and congenital heart defects risk: a Romanian case-control study. Exp Clin Cardiol. 2014;20:6336–44.

    Google Scholar 

  40. Xu J, Xu X, Xue L, Liu X, Gu H, Cao H, et al. MTHFR c.1793G>a polymorphism is associated with congenital cardiac disease in a Chinese population. Cardiol Young. 2010;20(3):318–26.

    Article  PubMed  Google Scholar 

  41. Nakaoka H, Inoue I. Meta-analysis of genetic association studies: methodologies, between-study heterogeneity and winner’s curse. J Hum Genet. 2009;54:615–23.

  42. Lee YH. Meta-analysis of Genetic Studies. Ann Lab Med. 2015;35(3):283–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Liu PF, Ding B, Zhang JY, Mei XF, Li F, Wu P, et al. Association between MTHFR C677T polymorphism and congenital heart disease. Int Heart J. 2020;61(3):553–61.

    Article  CAS  PubMed  Google Scholar 

  44. Cai B, Zhang T, Zhong R, Zou L, Zhu B, Chen W, et al. Genetic variant in MTRR, but not MTR, Is associated with risk of congenital heart disease: an integrated meta-analysis. PLoS ONE. 2014;9(3):e89609.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

Download references

Acknowledgements

The authors humbly acknowledge the support rendered by the patients and families for providing their consent for participation in the study.

Funding

The study under reference forms the part of the doctoral thesis of first author and thus the authors are thankful to the overall support (financial and infrastructural) by the Institute of Human Genetics, University of Jammu. The laboratory used for the purpose operates under the administrative and academic control of University of Jammu.

Author information

Authors and Affiliations

  1. Institute of Human Genetics, University of Jammu Jammu and Kashmir, 180006, Jammu, India

    Jyotdeep Kour Raina,  Anupriya & Parvinder Kumar

  2. Department of Zoology, University of Jammu, Jammu and Kashmir, 180006, Jammu, India

    Rakesh Kumar Panjaliya & Parvinder Kumar

  3. Department of Zoology Govt. Degree College, Samba, J&K, Jammu, India

    Vikas Dogra

  4. Department of Neonatology, University Hospital Southampton, Hampshire, UK

    Sushil Sharma

Authors
  1. Jyotdeep Kour Raina
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rakesh Kumar Panjaliya
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Vikas Dogra
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sushil Sharma
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Anupriya
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Parvinder Kumar
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

JKR and A: carried out the sampling and lab work for the conduct of research under reference, VD: carried out data interpretation and manuscript writing, RKP and PK: participated in the study design and conceptualization, SS carried out the clinical diagnosis and recruitment of patients. All the authors undertake to declare that they have read the complete manuscript before submission to the journal. The author(s) read and approved the final manuscript. 

Corresponding authors

Correspondence to Rakesh Kumar Panjaliya or Parvinder Kumar.

Ethics declarations

Ethics approval and consent to participate

Ethical approval for the conduct of present research work was taken from the Institutional Ethical Committee, University of Jammu. All the methods were carried out in accordance with relevant guidelines and regulations. Data collection and blood sampling were done after getting prior informed consent from mother/guardian of the subject(s).

Consent for publication

Not applicable.

Competing interests

The authors declare that they do not have any conflict of interest.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Raina, J.K., Panjaliya, R.K., Dogra, V. et al. “Association of MTHFR and MS/MTR gene polymorphisms with congenital heart defects in North Indian population (Jammu and Kashmir): a case–control study encompassing meta-analysis and trial sequential analysis”. BMC Pediatr 22, 223 (2022). https://doi.org/10.1186/s12887-022-03227-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12887-022-03227-z

Keywords

BMC Pediatrics

ISSN: 1471-2431

Contact us