Association between the A1298C Polymorphism of the Methylenetetrahydrofolate Reductase Gene and Recurrent Spontaneous Abortion

Introduction

In medical terminology, spontaneous abortion or miscarriage is defined as the involuntary end of pregnancy before 20 weeks of gestation. Recurrent pregnancy loss (RPL), also known as recurrent miscarriages, is defined by the consecutive loss of two or more pregnancies with the same partner, and having no more than one living child (1).

Miscarriages may occur for many reasons, not all of which can be identified. Thrombophilia disorders are among the few recognized risk factors and potential causes of recurrent mis-carriages. During pregnancy, the maternal body adapts to the needs of the fetus, since maternal blood provides all the nutrients and oxygen the baby needs throughout the pregnancy (Figure 1). In patients with thrombophilia, placental damage

happens mostly due to blood clot formation in the maternal as well as the fetal vessels (2, 3).

        Single-nucleotide polymorphisms (SNPs) in metabolic pathways, which regulate enzymes such as MTHFR, are considered to be risk factors for thrombo-philia. MTHFR is the key enzyme in folate, methionine, and homocysteine metabolism (4). The disturbances in MTHFR activity could be the cause of increased serum level of homocysteine (a potentially toxic amino acid).

       Hyperhomocysteinemia is a risk factor for changes in coagulation cascade through direct cytotoxic influence on endothelium, atheroge-nesis, activation of coagulation factor V and VII, increased level of thrombin, platelet aggregation,

and a tendency toward venous thrombosis (4, 5).

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Figure 1. Diagram of  the circulation of blood within the human placenta

MTHFR irreversibly reduces 5,10-methyl-enetetrahydrofolate (substrate) to 5-methyltetra-hydrofolate (product). 5-Methyltetrahydrofolate is the dominant folate form in the circulating human plasma, and is used to convert homocysteine to methionine via methionine synthase (5-7). Decreased MTHFR activity hamstrings the methylation cycle, resulting in increasing levels of homocysteine (7). The enzyme is coded by the gene with the symbol MTHFR on chromosome 1, location p36.3 in humans, and is composed of 11 exons (8).A common polymorphism in the gene for the enzyme MTHFR is known as the A1298C MTHFR polymorphism, which leads to an altered amino acid sequence; this polymorphism has been associated with a decreased enzyme activity. The adenine (A) to cytosine (C) substitution at nucleotide 1298 in exon 7 of MTHFR gene leads to the substitution of glutamic acid by alanine residue at position 429 in the regulatory domain of the MTHFR enzyme, and therefore, reduces its enzymatic activity, which is more pronounced in the homozygous than the heterozygous state.

Reduced activity can lead to elevated levels of homocysteine (also called hyperhomocys-teinemia), especially when folate levels are low. A1298C polymorphism is known to have a less significant effect on reducing enzyme activity, compared to the 677 mutation (9, 10).

According to previous research, although individuals who are homozygous for the 1298C allele have about a 40% reduced enzyme activity in vitro, they do not appear to have higher plasma homocysteine levels compared to the controls. However, individuals who are compound heterozygous for the 677T and 1298C alleles (which produce a 677CT/1298AC genotype) have a 40-50% reduced MTHFR activity in vitro and a biochemical profile similar to that seen among 677T homozygotes with increased homocysteine levels and decreased folate levels.

The A1298C polymorphism by itself may have important clinical effects on folate metabolism under conditions such as low folate intake or elevated folate requirement during pregnancy and embryogenesis (11). Considering the effects of MTHFR A1298C polymorphism on enzyme activity and the increased risk of venous thrombosis, as well as the association between inherited thrombophilia and RPL, this study aimed to investigate the association between A1298C polymorphism of the MTHFR gene as a genetic risk factor for idiopathic recurrent miscarriage.

Materials and Methods

Study population

The present case-control prevalence study included 30 women with a previous history of two or more unexplained RPLs, who attended Avicenna Infertility Clinic (AIC), Tehran, Iran. In addition, 10 fertile women with at least two live births, attending Baqiyatallah Hospital, Tehran, were selected as the control group between August 2009 and March 2009.

Mutation analysis

Genomic DNA was extracted from an 80 μl aliquot of whole blood, collected in EDTA, using the standard salting-out method; the extracted DNA was stored at −20°C until analysis. To detect the presence or absence of the C677T polymorphism,

Table 1. Primer sequence, amplified product, and restriction enzyme for the detection of A1298C polymorphism in MTHFR gene

Note:    * Normal allele         ** Mutant allele

Figure 2. The electrophoresis image of the PCR product for a 256-bp fragment of the MTHFR gene on agarose gel; the size of PCR product (256 bp) was determined in comparison with 50 bp molecular weight marker (in bp) on lines 20 and 11 of the image

we selectively amplified a 256-bp fragment of the MTHFR gene by polymerase chain reaction (PCR) method (Table 1). PCRs were performed with 40 ng of genomic DNA, 1 U/µl of Taq DNA polymerase, 3.5 µL of 10X PCR buffer [500 mM KCl and Tris-HCl (pH 8.4)], 2.0 mM MgCl₂, 0.25 mM of all four deoxynucleoside triphosphates, and 0.4 mM of each primer, in a volume of 20 µL.

 After denaturation at 95°C for 5 min, the temperature was cycled 30 times (95°C for 1 min, 63°C for 1 min, and 72°C for 2 min), followed by extension at 72 °C for 7 min to amplify the target DNA.

MTHFR A1298C polymorphism was assayed by polymerase PCR amplification, using primers and under conditions previously described (Figure 2). Ten μl of PCR product was digested overnight at 37°C with 1 unit of MboII restriction enzyme in a final volume of 15 μl, according to the manufacturers' instructions (New England Biolabs). This reaction yielded fragments of 176, 30, 28, and 22  base pairs in the presence of the A allele, and 204, 30, and 22 base pairs in the presence of the C allele.

A heterozygous individual gives five fragments of lengths 204, 176, 30, 28, and 22 bp. Digestion results were submitted to electrophoresis in a 3% agarose gel, and the bands were visualized using ethidium bromide staining on a ultraviolet trans illuminator. Control on each gel included a known homozygous.

Statistical analysis

The genotypes and allele frequencies of MTHFR A1298C variations in both groups of patients were compared with the controls using the χ² test. P-value less than 0.05 was considered statistically significant. SPSS version 16 was used for the statistical analysis.

Results

According to the results demonstrated in the electrophoresis image, the genotype RFLP product per patient was established for each poly-morphism (Figure 3). Thirteen women (43.3 %) with RPL and 6 women (20 %) in the control group were heterozygous for MTHFR A1298C poly-morphism. No homozygous individual was found among patients with RPL or the control group. C allele frequency in the patient group was higher than the control group (41.7% and 40% for the patient and control groups, respectively, P<0.05) (Table 2).

Discussion

Studies on different human populations indicate different results regarding the association between MTHFR A1298C polymorphism and spontaneous abortion. Some studies show that the scarcity of folic acid during pregnancy, hyperhomocysteinemia, and homozygosity for MTHFR polymorphisms are associated with an increased risk for spontaneous abortion and the destruction of placenta, whereas others reported no such association (12-14).

Wang et al from China reported that the prevalence of MTHFR A1298C, associated with 3 genotypes and A/C alleles, in the group with unexplained recurrent spontaneous abortion (URSA) did not differ significantly from the controls; however, the frequency of 677CC/1298AA

Figure 3.  Agarose gel electrophoretic analysis of the MTHFR A1298C polymorphism; lines 10 and 20 represent 50 bp molecular weight marker; line 1 represents the 256 bp fragment of PCR product before RFLP; lines 7, 9, and 11 represent the 204 bp fragment corresponding to the genotype CC after digestion with MboII as a positive control; lines 2, 4, 5, 12, 15, 17, and 19 represent the 176 bp fragment corresponding to the genotype AA after digestion with MboII; lines 2, 4, 5, 12, 15, 17, and 19 represent fragments corresponding to the genotype AC after digestion with MboII (204, 176 bp)

Table 2. The prevalence and allelic frequencies of MTHFR A1298C polymorphism among RPL patients and the control group

Note: No significant difference was observed in terms of genotypes between the two groups