Methylenetetrahydrofolate Reductase Gene Polymorphisms in Egyptian Women with Unexplained Recurrent Pregnancy Loss

Introduction

Recurrent pregnancy loss (RPL), defined as the spontaneous loss of two or more consecutive intra-uterine pregnancies, is known to affect 0.5%-2% of pregnant women (Wilcox et al., 1988; Daya, 1996). In Egypt, early and late pregnancy wastage was demonstrated to be a great problem, especially among low socioeconomic class and high-parity groups (Serour et al., 1981). Of the factors that might contribute to RPL are the thrombophilias which are hemostatic disorders predisposing to thrombotic phenomena. They affect about 15% of the Caucasian population and are classified as inherited and acquired forms. A common acquired thrombophilia is due to antiphospholipid antibodies, whereas the thermolabile variant of the methylenetetrahydrofolate reductase (MTHFR) gene represents one example of diverse hereditary thrombophilic elements (Walker et al., 2001).

The enzyme MTHFR plays a critical role in homocysteine metabolism by catalyzing the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, the methyl-group donor in the B12-dependent remethylation of homocysteine to methionine (Boers et al., 1985; Clarke et al., 1991). A common polymorphism of the MTHFR gene is the 677 C→T (Ala-to-Val) transition, which produces thermolability and somewhat reduced enzyme activity in vitro (Kang et al., 1991). Homozygosity for the C677T polymorphism has been implicated as a risk factor for venous thrombosis (Frosst et al., 1995; Gemmati et al., 1999). More recently, a second prevalent polymorphism, which is also associated with decreased enzyme activity in vitro, is due to A→C transversion at nucleotide 1298, which produces a Glu-to-Ala substitution. Studies have shown that ∼10% of individuals are homozygous for the 1298C allele, and roughly 20% of individuals are heterozygous carriers of both the C677T and A1298C polymorphisms (van der Put et al., 1998; Weisberg et al., 1998).

A strong association between MTHFR genetic polymorphisms with RPL was confirmed by some researchers, whereas others denied this association (Resch et al., 2004; Wang et al., 2004; Mtiraoui et al., 2006). Since data pertinent to the contribution of genetically based thrombophilia due to MTHFR gene mutations to unexplained RPL among Egyptian women are relatively lacking, we planned this study to check for this association.

Subjects and Methods

This is a case-controlled study including 206 women with an age ranging from 19 to 38 years selected from those admitted and followed up in the Obstetrics/Gynecology and Genetics Departments of Mansoura University Hospitals, Egypt. Of them, 70 women had a history of two or more events of fetal loss in the form of abortion, miscarriage, or still birth. The other 136 women were clinically healthy and had a good obstetric history. They were assigned as a control group for comparison. Exclusion criteria included women who were documented to have anatomic or endocrine abnormalities or to be carriers of genetic or chromosomal aberrations.

After obtaining approval of local scientific and ethical committees as well as an informed consent from all cases and controls, blood sampling and DNA extraction (Gentra Systems) were carried out. Detection of MTHFR C677T and A1298C mutations was done using a polymerase chain reaction with restriction fragment length polymorphisms (PCR-RFLP) assay.

Each PCR reaction was performed using 300 ng of DNA, 200 mM of each dNTP, 500 nM of each primer, and 2.5 units of Taq DNA polymerase (Amplitaq Gold; Perkin- Elmer Cetus). The DNA was initially denatured for 10 min at 95°C; followed by 35 cycles each in the form of 94°C for 45 s, 60°C for 45 s, and 72°C for 1 min; completed with a final extension cycle at 72°C for 7 min. For identification of the 677C/T variant, the following primer pair was used: F: 5′-TGA AGG AGA AGG TGT CTG CGG GA-3′; R: 5′-AGG ACG GTG CGG TGA GAG TG-3′. The amplified segment (198 bps) was digested using the restriction enzyme HinfI, which yields 2 fragments of 175 and 23 bps in the presence of the T allele (Frosst et al., 1995; Skibola et al., 1999). On the other hand, the 1298A/C variant was identified using the primer pair: F1: 5′-CTT TGC GGA GCT GAA GGA CTA CTA C-3′ and R1: 5′-CAC TTT GTG ACC ATT CCG GTT TG-3′. The amplified product (163 bp) was digested using the restriction enzyme MboII, which yields 84, 31, 30, and 18 bp fragments in the presence of the mutant allele C; whereas the wild-type variant A gives rise to 56, 31, 30, 28, and 18 bp fragments (Fig. 1) (van der Put et al., 1998; Weisberg et al., 1998; Skibola et al., 1999).

OPEN IN VIEWER

FIG. 1.

Top panel shows MTHFR 1298 PCR digestion products; lane 1, 4, and 7: AC genotype; lane 2, 3, and 6: CC genotype; and lane 5 AA genotype. The bottom panel shows MTHFR 677 PCR digestion products; lane 1, 5, and 6: CC genotype; lane 2, 4, and 7: CT genotype and lane 3: TT genotype; lane M: DNA size marker (50-1000 bp); Note that the small fragments have run off the gel. MTHFR, methylenetetrahydrofolate reductase; PCR, polymerase chain reaction.

Results

Compared with controls, cases with unexplained pregnancy loss showed higher frequency of the homozygous mutant MTHFR 677 TT genotype (5.7% vs. 0.7%, p=0.05), 1298 CC genotype (8.6% vs. 2.2%, p=0.06), and the combined mutant haplotype 677T/1298C (10% vs. 9.6%, p=0.73), although not reaching a statistical significance (Table 1). It was also noted that the frequency of 677 mutant genotypes (TT or TC) combined with either the 1298 mutant (CC or AC) or normal (AA) genotypes was significantly increased among cases with late-stage pregnancy loss versus. those with an early-stage pregnancy loss (p=0.001). The frequency of the 677 mutant genotypes (TT or TC) was also increased—although not statistically significant—among cases with secondary infertility compared with those with primary infertility and among cases with a higher rate of pregnancy loss >4 times compared with those with pregnancy loss ≤4 times (Table 2).

Analyzing potential risk factors, it was noted that 24 cases were exposed to passive smoking, 22 cases had positive enzyme-linked immunosorbent assay test for perinatal infection (TORCH; toxoplasmoais, rubella, cytomegalovirus, and herpes), 28 had Rh negative blood group, 3 had hypertension, 2 had diabetes, 20 had blood-group O type, and 10 were positive for antiphospholipid and anticardiolipin antibodies. Testing the frequency of mutations among cases with no or just one risk factor versus those having two or more risk factors showed a nonsignificant difference (p=0.98). The same was observed when testing each factor alone comparing with cases who were positive versus those who were negative for that risk factor (Table 3).

Discussion

Unexplained pregnancy loss is speculated to be associated with inherited thrombophilias that encompass diverse conditions including the thermolabile variant of the MTHFR gene (Jilma et al., 2003).

This study showed an increased frequency of the mutant forms of MTHFR 677 and 1298 polymorphisms among Egyptian women with unexplained pregnancy loss, although not reaching a statistical significance probably due to the relatively small sample size of cases. In this regard, we recommend a wider scale study including other forms of thrombophilic genes as factor V Leiden, prothrombin, proteins C and S, and plasminogen activator inhibitor gene. This positive interaction was actually documented by previous studies among Italian women (D'Uva et al., 2008; Tranquilli et al., 2010).

Both mutations of MTHFR 677 and 1298 polymorphisms were also found to have significantly higher frequency among women with RPL compared with control fertile women in other studies as those done among Tunisian women (Mtiraoui et al., 2006), among American women in Chicago (Goodman et al., 2006), and among Iranian women (Jeddi-Tehrani et al., 2011). However, many studies have focused on the 677 polymorphism to be contributing to RPL rather than the 1298 polymorphism as those done among Chinese women (Wang et al., 2004; Ren and Wang, 2006; Xu et al., 2007), Iranian women (Behjati et al., 2006), Middle-European women (Unfried et al., 2002), Italian women (D'Uva et al., 2007), American women (Govindaiah et al., 2009), and Bulgarian women (Ivanov et al., 2007). In contrast, Sotiriadis et al. (2007) found that there was no difference in the distribution of MTHFR C677T polymorphism among Greek women with two and three or more miscarriages, whereas the MTHFR A1298C mutant variant was more common among those with two miscarriages.

Other studies that reported no association between MTHFR polymorphisms and RPL include those done among Jewish women (Brenner et al., 1999; Carp et al., 2002), Austrian women (Resch et al., 2004), German women (Pauer et al., 2003), Lebanese women (Zahed et al., 2006), Spanish women (Cardona et al., 2008), and Indian women (Biswas et al., 2008; Vettriselvi et al., 2008).

Regarding the phase of pregnancy loss, this study showed that the frequency of MTHFR C677T mutation was high among cases with late-phase loss. We may speculate that the effect of thrombosis if it occurs affects mainly the placental blood supply after being developed in the later phase of pregnancy. These results coincide with those of Mtiraoui et al. (2006), who found a higher prevalence of MTHFR 677 TT in late and mixed early/late RPL among Tunisian women. On the other hand, other researchers reported an increased MTHFR C677T gene mutation rate with early fetal loss as those done among Bulgarian women (Ivanov et al., 2007; Kovacheva et al., 2007) and among Indian women (Mukhopadhyay et al., 2009). We might speculate that—depending on other contributory and interactive factors—the effect of these mutations can be high enough to end the pregnancy at an early stage or have their effect later on in the course of pregnancy.

This study showed a relatively non-contributory role of the association of MTHFR mutant polymorphic variants with other risk factors that might lead to the occurrence of pregnancy loss such as diabetes, hypertension, smoking, Rh negative and O blood-group types, and positive antiphospholipid antibodies. This may be due to the relatively small sample size and the relative small percentage of women exposed to these risk factors.

So, we recommend another survey study with a large sample size to accurately analyze the interactive effect of such factors with potentially deleterious genetic mutations.

In conclusion, this study showed an increased frequency of mutations MTHFR gene—although not statistically significant—in Egyptian women with unexplained pregnancy loss. Interactions with other genetic variants might be speculated and need to be investigated.