Sage Journals: Discover world-class research

Abstract

The purpose of this study is to investigate the association of methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms with the risk of congenital heart diseases (CHD). The genotypes of the MTHFR genetic variant were determined by the polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing methods. Our data suggested that the allelic and genotypic frequencies of CHD patients were significantly different from non-CHD controls. The MTHFR c.1625A>C genetic variant was significantly associated with the increased risk of CHD (CC vs. AA: odds ratio [OR]=2.29, 95% confidence interval [CI] 1.15-4.53, p=0.016; C vs. A: OR=1.47, 95% CI 1.11-1.96, p=0.008). Results from this study indicate that the MTHFR c.1625A>C genetic variant influences the risk of CHD in the studied population.

Introduction

Congenital heart disease (CHD) is one of the most common birth defects, occurring in ∼4 to 10 per 1000 live births. It is a major cause of childhood morbidity and mortality worldwide (Hoffman and Kaplan, 2002; Marelli et al., 2007; Gong et al., 2012; Mamasoula et al., 2013; Wang et al., 2013; Wang et al., 2014; Zhang et al., 2014). It is generally accepted that genetic factors are considered to play important influences on the treatment effectiveness of CHD and the prognosis of patients. However, the exact etiology of CHD still remains poorly understood. Recently, several studies have indicated that the methylenetetrahydrofolate reductase (MTHFR) gene is an important candidate gene for influencing the prognosis of patients and the risk of CHD (Chambers et al., 2000; Junker et al., 2001; Klerk et al., 2002; Lee et al., 2005; Pereira et al., 2005; Hobbs et al., 2006; Zhu et al., 2006; van Beynum et al., 2007; Brandalize et al., 2009; Garcia-Fragoso et al., 2010; Nie et al., 2011; Gong et al., 2012; Sanchez-Urbina et al., 2012; Yin et al., 2012; Balderrabano-Saucedo et al., 2013; Mamasoula et al., 2013; Mehlig et al., 2013; Wang et al., 2013; Zidan et al., 2013; Wang et al., 2014; Zhang et al., 2014). MTHFR genetic variants, such as C677T and A1298C, have been reported and identified with respect to their genetic influences on the risk of CHD in different populations (Chambers et al., 2000; Junker et al., 2001; Klerk et al., 2002; Nie et al., 2011; Wang et al., 2013; Zidan et al., 2013). However, to date, no similar studies were reported about the association of the MTHFR c.1625A>C genetic variant with the risk of CHD. Thus, this study aimed to evaluate the influence of MTHFR c.1625A>C genetic variation on the risk of CHD.

Materials and Methods

Subjects

In the present study, we enrolled 507 subjects from the Shengjing Hospital of China Medical University between June 2009 and November 2013. All subjects were genetically unrelated Han Chinese and lived in Shenyang, China. CHD patients were diagnosed and confirmed by doctors. The non-CHD controls were free from CHD and present or past history of diseases. The non-CHD controls were frequency-matched to CHD patients by sex and age. The protocol of this study was approved by the Ethics Committee of the Shengjing Hospital of China Medical University (Shenyang, China). Informed written consent forms were obtained from all participants.

Genotyping of the MTHFR gene

Genomic DNA was isolated from peripheral venous blood using the Axygen DNA Isolation Kit (Axygen, CA). According to the human MTHFR gene reference sequences (DNA sequences, GenBank ID: NG_013351.1; mRNA sequences, GenBank ID: NM_005957.4), the polymerase chain reaction (PCR) primers were designed by the Primer Premier 5.0 software (Premier Biosoft International, Palo Alto, CA). Table 1 shows the detailed information of the primer sequences, amplification region, annealing temperature, and fragment size. The PCR amplification reaction was carried out in a total volume of 20 μL PCR solution, including 50 ng template DNA, 1× buffer (100 mmol Tris-HCl, pH 8.3; 500 mmol KCl), 0.25 μmol primers, 2.0 mmol MgCl₂, 0.25 mmol dNTPs (Bioteke Corporation, Beijing, China), and 0.5 U Taq DNA polymerase (Promega, Madison, WI). PCR cycling was performed using an initial denaturation at 94°C for 5 min, followed by 32 cycles at 94°C for 30 s, at 61.9°C for 30 s, at 72°C for 30 s, and a final extension at 72°C for 5 min. The created restriction site-PCR (CRS-PCR) method with one of the primers containing a nucleotide mismatch, which enables the use of restriction enzymes for discriminating sequence variations and DNA sequencing methods were utilized to investigate the genotypes of the MTHFR c.1625A>C genetic variant. Following the manufacturer's instructions, the PCR-amplified products (10 μL) were digested with 5 U TaqI restriction enzyme (MBI Fermentas, St. Leon-Rot, Germany) (Table 2) at 37°C for 10 h. The digested products were electrophoresed and separated on a 2.5% agarose gel and observed for different genotypes under ultraviolet light.

Table 1.

The Created Restriction Site-Polymerase Chain Reaction Analysis for the MTHFR c.1625A>C Genetic Variant

Primer sequences	Annealing temperature (°C)	Amplification fragment (bp)	Region	Restriction enzyme	Genotype (bp)
Forward primer: 5′-CGGGTTAATTACCACCTTGTCG-3′	61.9	217	Exon10	TaqI	AA: 187, 20 AC: 217, 187, 20
Reverse primer: 5′-CACTTACACTGGCTGGTGGTCAC-3′					CC: 217

Underlined nucleotides mark nucleotide mismatches enabling the use of the selected restriction enzymes for discriminating sequence variations.

MTHFR, methylenetetrahydrofolate reductase.

Table 2.

The Frequencies of Allelic and Genotypic MTHFR c.1625A>C Genetic Variants

	Genotypic frequencies (%)			Allelic frequencies (%)
Groups	AA	AC	CC	A	C	χ²	p
Cases (n=252)	135 (53.57)	90 (35.72)	27 (10.71)	360 (71.43)	144(28.57)	3.9375	0.1396
Controls (n=255)	160 (62.75)	81 (31.76)	14 (5.49)	401 (78.63)	109 (21.37)	0.7682	0.6811
Total (n=507)	295 (58.19)	171 (33.73)	41 (8.09)	761 (75.05)	253 (24.95)	5.0100	0.0817
	χ²=6.6968, p=0.0351			χ²=7.0156, p=0.0081

Statistical analyses

The chi-squared (χ²) test was utilized to estimate the Hardy-Weinberg equilibrium (HWE) and the differences in the genotype and allele distributions between CHD patients and non-CHD controls. The association of the MTHFR c.1625A>C genetic variant with the risk of CHD was evaluated by computing the odds ratios (ORs) with their 95% confidence intervals (CIs) using unconditional logistic regression analysis. The statistically significant level was settled at p-value <0.05. All statistical analyses were analyzed by the Statistical Package for Social Sciences software (SPSS, Windows version release 15.0; SPSS, Inc., Chicago, IL).

Results

General characteristics of subjects

A total of 507 unrelated individuals, including 252 CHD patients (Male: 154, Female: 98; mean±standard deviation [SD] age: 2.59±1.43) and 255 sex- and age-matched non-CHD controls (Male: 165, Female: 90; mean±SD age: 2.77±1.35), were recruited in this case-control study. The CHD patients and non-CHD controls were frequency-matched by sex and age, as indicated by χ²-tests (for sex, χ²=0.7020, p=0.4021; for age, χ²=0.3192, p=0.5721, respectively).

Frequencies and genotyping of the MTHFR genetic variant

Through the CRS-PCR and DNA sequencing methods, a genetic variant (c.1625A>C) of the MTHFR gene was detected in this study. Our sequence analyses indicate that this genetic polymorphism is a nonsynonymous mutation that causes an A to C mutations in exon10 of the MTHFR gene. This mutation alters asparagine (Asn) to threonine (Thr) (p.Asn542Thr, the reference sequences GenBank IDs: NG_013351.1, NM_005957.4, and NP_005948.3). The TaqI restriction enzyme has been utilized to digest the PCR-amplified products, which were divided into three genotypes, AA (187 and 20 bp), AC (217,187, and 20 bp), and CC (217 bp, Table 1). The allelic and genotypic frequencies in the studied populations are summarized in Table 2. The allelic frequencies of CHD patients (A, 71.43%; C, 28.57%) were not consistent with those of non-CHD controls (A, 78.63%; C, 21.37%; χ²=7.0156, p=0.0081, Table 2). The genotypic frequencies of CHD patients (AA, 53.57%; AC, 35.72%; CC, 10.71%) were significantly different from those in non-CHD controls (AA, 62.75%; AC, 31.76%; CC, 5.49%; χ²=6.6968, p=0.0351, Table 2). The genotype and allele distributions of this genetic variant between CHD patients and non-CHD controls were in accordance with the HWE (all p-values >0.05, Table 2).

Association of the MTHFR genetic variant with the risk of CHD

The association of the MTHFR c.1625A>C genetic variant with the risk of CHD is shown in Table 3. Our data suggested that the MTHFR c.1625A>C genetic variant was statistically associated with the increased risk of CHD in a homozygote comparison (CC vs. AA: OR=2.29, 95% CI 1.15-4.53, χ²=5.80, p=0.016), dominant model (CC/AC vs. AA: OR=1.46, 95% CI 1.02-2.08, χ²=4.38, p=0.036), recessive model (CC vs. AC/AA: OR=2.07, 95% CI 1.06-4.04, χ²=4.64, p=0.031), and allele contrast (C vs. A: OR=1.47, 95% CI 1.11-1.96, χ²=7.01, p=0.008, Table 3).

Table 3.

The Association of the MTHFR c.1625A>C Genetic Variant with the Risk of Congenital Heart Diseases

	Test of association
Comparisons	OR (95% CI)	χ²-value	p-Value
Homozygote comparison (CC vs. AA)	2.29 (1.15-4.53)	5.80	0.016
Heterozygote comparison (AC vs. AA)	1.32 (0.90-1.92)	2.04	0.153
Dominant model (CC/AC vs. AA)	1.46 (1.02-2.08)	4.38	0.036
Recessive model (CC vs. AC/AA)	2.07 (1.06-4.04)	4.64	0.031
Allele contrast (C vs. A)	1.47 (1.11-1.96)	7.01	0.008

The χ² tests were used for statistical analyses.

CI, confidence interval; OR, odds ratio.

Discussion

In recent years, a large number of epidemiologic studies have suggested that CHD is a huge medical problem and presents a significant economic burden to the families and society around the world (Junker et al., 2001; Hoffman and Kaplan, 2002; Nie et al., 2011; Gong et al., 2012; Sanchez-Urbina et al., 2012; Mamasoula et al., 2013; Wang et al., 2013). It is caused by complex interactions between environmental and genetic factors; furthermore, growing evidence indicated that genetic factors play key roles in the development of CHD. Previous studies reported that the MTHFR gene is one of the most important candidate genes for influencing the susceptibility to CHD. In the present study, we firstly investigated the distribution of the MTHFR c.1625A>C genetic variant by CRS-PCR, and evaluated the influence of the MTHFR c.1625A>C genetic variant on the risk of CHD in a Chinese Han population through association analyses on the basis of analysis of 252 CHD patients and 255 sex- and age-matched non-CHD controls. Our findings suggested that there were significant differences for the allelic and genotypic frequencies between the CHD patients and non-CHD controls (Table 2). Compared with allele-A and genotype-AA, the allele-C and genotype-CC were significantly associated with increased susceptibility to CHD (All p-values <0.05, Table 3). Allele-C and genotype-CC may be significantly associated with the increased risk of CHD. The MTHFR c.1625A>C genetic variant lead to the Asn to Thr amino acid replacement and might impact the expression and function of the MTHFR protein, thus influencing the risk of CHD. It also might be linked to other nonsynonymous polymorphisms, such as C677T and A1298C in the MTHFR gene, which have been proven to associate with the risk of CHD (Chambers et al., 2000; Junker et al., 2001; Klerk et al., 2002; Nie et al., 2011; Wang et al., 2013; Zidan et al., 2013). The findings from this study could provide more evidence to explain the biological function and role of the MTHFR gene in the development of CHD.

In conclusion, to our knowledge, we are the first to investigate the potential association of the MTHFR c.1625A>C genetic variant with the risk of CHD. Results from this case-control study suggest that the MTHFR c.1625A>C genetic variant is significantly associated with the increased risk of CHD in the Chinese Han population, and could be a useful molecular biomarker for evaluating the susceptibility to CHD. Further studies with larger, different ethnic populations are needed to confirm these conclusions and to reveal the underline molecular mechanisms.

Footnotes

Author Disclosure Statement

The authors have no conflicts of interest.

References

Balderrabano-Saucedo

, Sanchez-Urbina

, Sierra-Ramirez

, et al. (2013) Polymorphism 677C->T MTHFR gene in Mexican mothers of children with complex congenital heart disease. Pediatr Cardiol, 34:46-51.

Brandalize

, Bandinelli

, dos Santos

, et al. (2009) Evaluation of C677T and A1298C polymorphisms of the MTHFR gene as maternal risk factors for Down syndrome and congenital heart defects. Am J Med Genet A, 149A:2080-2087.

Chambers

, Ireland

, Thompson

, et al. (2000) Methylenetetrahydrofolate reductase 677C- >T mutation and coronary heart disease risk in UK Indian Asians. Arterioscler Thromb Vasc Biol, 20:2448-2452.

Garcia-Fragoso

, Garcia-Garcia

, Leavitt

, et al. (2010) MTHFR polymorphisms in Puerto Rican children with isolated congenital heart disease and their mothers. Int J Genet Mol Biol, 2:43-47.

Gong

, Gu

, Zhang

, et al. (2012) 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, 50:1455-1461.

Hobbs

, James

, Jernigan

, et al. (2006) Congenital heart defects, maternal homocysteine, smoking, and the 677 C >T polymorphism in the methylenetetrahydrofolate reductase gene: evaluating gene-environment interactions. Am J Obstet Gynecol, 194:218-224.

Hoffman

, Kaplan

(2002) The incidence of congenital heart disease. J Am Coll Cardiol, 39:1890-1900.

Junker

, Kotthoff

, Vielhaber

, et al. (2001) Infant methylenetetrahydrofolate reductase 677TT genotype is a risk factor for congenital heart disease. Cardiovasc Res, 51:251-254.

Klerk

, Verhoef

, Clarke

, et al. (2002) MTHFR 677C- >T polymorphism and risk of coronary heart disease: a meta-analysis. JAMA, 288:2023-2031.

10.

Lee

, Su

, Cheng

, et al. (2005) Association of the C677T methylenetetrahydrofolate reductase mutation with congenital heart diseases. Acta Obstet Gynecol Scand, 84:1134-1140.

11.

Mamasoula

, Prentice

, Pierscionek

, et al. (2013) Association between C677T polymorphism of methylene tetrahydrofolate reductase and congenital heart disease: meta-analysis of 7697 cases and 13,125 controls. Circ Cardiovasc Genet, 6:347-353.

12.

Marelli

, Mackie

, Ionescu-Ittu

, et al. (2007) Congenital heart disease in the general population: changing prevalence and age distribution. Circulation, 115:163-172.

13.

Mehlig

, Leander

, de Faire

, et al. (2013) The association between plasma homocysteine and coronary heart disease is modified by the MTHFR 677C>T polymorphism. Heart, 99:1761-1765.

14.

Nie

, Gu

, Gong

, et al. (2011) Methylenetetrahydrofolate reductase C677T polymorphism and congenital heart disease: a meta-analysis. Clin Chem Lab Med, 49:2101-2108.

15.

Pereira

, Xavier-Neto

, Mesquita

, et al. (2005) Lack of evidence of association between MTHFR C677T polymorphism and congenital heart disease in a TDT study design. Int J Cardiol, 105:15-18.

16.

Sanchez-Urbina

, Galaviz-Hernandez

, Sierra-Ramirez

, et al. (2012) Methylenetetrahydrofolate reductase gene 677CT polymorphism and isolated congenital heart disease in a Mexican population. Rev Esp Cardiol (Engl Ed), 65:158-163.

17.

van Beynum

, den Heijer

, Blom

, et al. (2007) The MTHFR 677C- >T polymorphism and the risk of congenital heart defects: a literature review and meta-analysis. QJM, 100:743-753.

18.

Wang

, Liu

, Yan

, et al. (2013) Association of SNPs in genes involved in folate metabolism with the risk of congenital heart disease. J Matern Fetal Neonatal Med, 26:1768-1777.

19.

Wang

, Zhu

, Lv

, et al. (2014) The association between c.1333C>T genetic polymorphism of MTHFR gene and the risk of congenital heart diseases. Biomarkers, 19:77-80.

20.

Yin

, Dong

, Zheng

, et al. (2012) Meta analysis of the association between MTHFR C677T polymorphism and the risk of congenital heart defects. Ann Hum Genet, 76:9-16.

21.

Zhang

, Zha

, Dong

, et al. (2014) Association analysis between MTHFR genetic polymorphisms and the risk of congenital heart diseases in Chinese Han population. J Pharm Pharmacol, 66:1259-1264.

22.

Zhu

, Li

, Yan

, et al. (2006) Maternal and offspring MTHFR gene C677T polymorphism as predictors of congenital atrial septal defect and patent ductus arteriosus. Mol Hum Reprod, 12:51-54.

23.

Zidan

, Rezk

, Mohammed

(2013) MTHFR C677T and A1298C gene polymorphisms and their relation to homocysteine level in Egyptian children with congenital heart diseases. Gene, 529:119-124.