Objective: This study aims to investigate the association of 5,10-methylenetetrahydrofolate reductase (MTHFR C677T and A1298C) and methionine synthase reductase (MTRR A66G) gene polymorphisms with neural tube defects (NTDs) in a Tunisian population. Methods: Genotyping was performed by polymerase chain reaction with restriction fragment length polymorphisms (PCR-RFLPs) using the restriction enzymes. Allele and genotype frequencies were compared between mothers and fathers of fetuses with NTDs with matched controls based on an association analysis using SPSS software. Results: MTHFR (C677T, A1298C) and MTRR A66G polymorphisms were found to be protector factors for NTD fetuses in the mother group. In addition, a combination of the three wild-type alleles C677/A1298/A66 has increased four-fold the incidence of NTDs (p = 0.004, OR = 3.96, 95% CI: 1.53–10.23). In the father group, MTHFR C677T was a risk factor for NTDs. However, no association was found between MTHFR A1298C, MTRR A66G, and the occurrence of this anomaly. The analysis of MTHFR C677T and MTRR A66G polymorphisms has demonstrated a significant difference in vitamin B12 levels between recessive and dominant genotypes in case mothers (p < 0.05). Conclusion: Additional studies are required to better understand the roles of parental gene polymorphisms related to folate-homocysteine metabolism in the pathogenesis of NTD.

Congenital malformations are considered one of the major causes of mortality and morbidity in childhood. One of these principle congenital malformations is neural tube defects (NTDs) which constitute failures of neural tube closure [1]. Multifactorial causes have been associated with the occurrence of NTDs such as nutritional, environmental, and genetic factors [2-4], where folic acid deficiency is one of the major causes of 50–70% of NTD appearance.

During the period (1991–2011), a significant increase in the prevalence of NTD cases has been observed in Tunisia – from 0.57 to 3.47 NTD cases per 10,000 births. This increase may be due to the fact that no folic acid fortifications have been implemented in this country until now [5].

Recently, we have demonstrated that low folate and vitamin B12 as well as high homocysteine (Hcy) levels in mothers of fetuses with NTDs are risk factors for these malformations in Tunisia. In this study, case and control mothers were recruited similarly according to folate supplementation. So, no difference was found between the two groups, which indicated that the trouble may be in folate metabolism and not related to supplementation issue [6]. This finding helped us to develop a hypothesis regarding the origins of these malformations, mainly the mechanisms implicated in the metabolism of folic acid.

Methylenetetrahydrofolate reductase (MTHFR) plays a key role in folate-dependent homocysteine metabolism. It reduces 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, and engages in the vitamin B12-dependent remethylation of homocysteine to methionine [7]. In 1-carbon metabolism, this reaction is an imperative step. In fact, it is important for several processes, including DNA synthesis and gene transcription, which appear at a rapid rate in the embryo development [8].

The gene encoding the MTHFR enzyme is positioned on the short arm of chromosome 1 (1p36.3) [9], where a number of polymorphisms have been described. MTHFR C677T polymorphism has been considered a contributing factor to hyperhomocysteinemia [10]. The latter, associated with reduced folate levels, was implicated in the occurrence of NTDs in many studies [11]. Therefore, this polymorphism has been identified as a risk factor for NTDs in certain populations, and it has been suggested that the effects of the MTHFR polymorphism may be reduced with dietary supplementation of folate [12].

However, not all populations have found a comparable association between the MTHFR C677T polymorphism and NTDs. As a matter of fact, the association between the C677T polymorphism in the MTHFR gene and NTDs is controversial among numerous populations worldwide. This variant was associated with the appearance of NTDs in some studies [13-15]. However, no association was found in Italy or in Ireland, where a higher 677 T-allele frequency was observed [14-16].

The discovery of a second polymorphism within the MTHFR gene, MTHFR A1298C (E429A) [17, 18] and its association with decreased enzyme activity [18, 19] has led to many studies exploring the association between MTHFR A1298C polymorphism and NTDs. Results from these studies have been conflicting. No difference in enzyme activity of A1298C variants was shown by Yamada et al. [20] (2001). However, De Marco et al. [21] (2002) have found an association of the 1298C allele with NTDs.

Other genetic variants, including genes of the folate and the homocysteine metabolic pathways, might be implicated in the pathophysiology of NTDs [22], and in the remethylation pathway of homocysteine, such as methionine synthase (MTR), methionine synthase reductase (MTRR), and glutamate carboxypeptidase II (GCP II). However, data on GCP II are rare [16, 23, 24].

In this current study, we investigated some polymorphisms of genes of the folate and homocysteine metabolic pathways including MTHFR C677T, MTHFR A1298C, MTRR A66C, and GCP II C1561T. The aim of our study was to determine the studied polymorphism genotype and haplotype distributions in parents of fetuses with NTDs and healthy controls, and their association with the appearance of this anomaly in Tunisia.

Such a study will support the comprehension of the etiopathogenesis of this malformation as well as the proving of strategies for prevention.

Study Population

The subjects of the present population-based case-control study (January 1, 2012, to December 31, 2013) are described in detail elsewhere [6]. In brief, cases and controls were paired (1:1) on the date of birth and the presence or the absence of folate supplementation.

A total of 148 pregnant women were recruited with 71 case women and 77 controls. In this study, fathers have been also included with 48 fathers of fetuses with NTDs and 50 healthy subjects as controls.

Genotyping

The extraction of genomic DNA from peripheral blood cells was made by a salting-out method, and genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Table 1 lists the polymorphisms examined and reaction conditions employed for amplification. The GCP II C1561T polymorphism was detected by PCR amplification and digested with AccI.

Table 1.

PCR-RFLP parameters

PCR-RFLP parameters
PCR-RFLP parameters

Statistical Analysis

Statistical analysis was performed with SPSS software (version 18.0; SPSS Inc., Chicago, IL, USA). The Hardy-Weinberg equilibrium was tested by a goodness-of-fit χ2 test to compare the observed genotype frequencies with the expected ones amongst parents of fetuses with NTDs and control groups.

Comparison of continuous variables was conducted using the independent samples t test, Mann-Whitney U test, or Kruskal-Wallis test as appropriate. Differences in the distribution of genotype and allele between groups were assessed by Pearson’s χ2 test (categorical variables). To test how the association of each polymorphism with NTD is independent of potential confounding factors, binary logistic regression models were used. Adjustment was applied for maternal age, fetal term, gravidity, and supplementation with folic acid (yes/no).

THESIAS (Testing Haplotype Effects In Association Studies) [25] was used to estimate haplotype frequencies and to perform a linkage disequilibrium test. A one-way analysis of variance was performed to evaluate the effect of the polymorphism on the variation of biochemical parameters.

Concentrations of folate, red blood cell (RBC) folate, vitamin B12, and plasma t-homocysteine were compared between mothers of cases and controls as a function of each polymorphism by medians of the Kruskal-Wallis nonparametric test. To further show the role of the mutations, we examined dominant (homozygous vs. others) effects on the genotype frequency as well as on concentrations of studied parameters.

Spearman’s correlation coefficients were performed to study the correlation between MTHFR and MTRR genotypes and the studied biochemical indicators. Concentration values of biochemical parameters used in this study were obtained from our previous study [6]. Results were considered statistically significant when the p value was <0.05.

Genotype distributions and allele frequencies for the MTHFR C677T, MTHFR A1298C, and MTRR A66C polymorphisms in parents of NTD fetuses and controls from the Tunisian population are presented in Table 2. Genotypes frequency distributions were, as expected, according to the Hardy-Weinberg equilibrium.

Table 2.

Genotypes distribution and allele frequencies of MTHFR and MTRR gene polymorphisms in parents of fetuses with NTDs and controls

Genotypes distribution and allele frequencies of MTHFR and MTRR gene polymorphisms in parents of fetuses with NTDs and controls
Genotypes distribution and allele frequencies of MTHFR and MTRR gene polymorphisms in parents of fetuses with NTDs and controls

Association analysis between parents of fetuses with NTDs versus the control group showed a significant difference for genotype and allele, suggesting that the C677T polymorphism is associated with NTD in both mothers and fathers.

For genotypes and alleles, the TT genotype and T allele in MTHFR significantly decreased the incidence of NTDs (p = 0.02, OR = 0.27, 95% CI: 0.09–0.81 with genotype, and p = 0.02, OR = 0.57, 95% CI: 0.35–0.94 with allele) in the mother group. Then, under the dominant model (CT + TT vs. CC), CC wild-type carriers were associated with lower risk of conceiving NTD infants compared with pregnant women with CT+TT mutants (OR = 0.48; 95% CI: 0.24–0.96; p = 0.03). On the contrary, in the father group, the TT genotype and T allele in MTHFR significantly increased the incidence of NTDs (p = 0.03, OR = 4.00, 95% CI: 1.11–14.35 with genotype, and p = 0.01, OR = 2.13, 95% CI: 1.14–4.00 with allele).

Analysis of the MTHFR A1298C polymorphism has shown that the AC genotype was associated with lower risk of being pregnant with NTD infants (p = 0.03, OR = 0.43, 95% CI: 0.19–0.94). For the MTRR A66G polymorphism, the AG mutant was also associated with a protective effect for NTDs (p = 0.03, OR = 0.33, 95% CI: 0.12–0.91). In addition, a significant difference was found in the mother group when comparing the effect of the presence of the G allele (AG+GG) with its absence (GG) (p = 0.03, OR = 0.35, 95% CI: 0.13–0.93) (Table 2).

The association of MTHFR and the MTRR polymorphisms with NTDs was only significant with the MTRR A66G genotype when adjusting for potential confounding factors (maternal age, fetal term, gravidity, and supplementation with folic acid) (p = 0.04, OR = 0.22, 95% CI: 0.05–0.97) (Table 2).

Seven major haplotypes of MTHFR and MTRR were present in the study population (Table 3). C677/A1298/A66 and C677/A1298/66G were the most common haplotypes in case mothers (0.24 and 0.21, respectively). In control mothers, the most common haplotypes were 677T/A1298/66G and C677/A1298/66G (0.26 and 0.21, respectively). The C677/A1298/A66 haplotypes occurred at greater frequencies in case mothers versus controls, suggesting that it is a risk factor for NTDs (p = 0.004, OR = 3.96, 95% CI: 1.53– 10.23). On the other hand, there was no significant association between clinical subtypes of NTDs and the studied genotypes (p > 0.05) (Fig. 1).

Table 3.

Comparison of haplotype frequencies in the MTHFR(C677T and A1289C) and MTRR A66G polymorphisms between mothers of fetuses with NTDs and controls

Comparison of haplotype frequencies in the MTHFR(C677T and A1289C) and MTRR A66G polymorphisms between mothers of fetuses with NTDs and controls
Comparison of haplotype frequencies in the MTHFR(C677T and A1289C) and MTRR A66G polymorphisms between mothers of fetuses with NTDs and controls
Fig. 1.

Distribution of clinical subtypes of NTDs by MTHFR (C677T, A1298C) and MTRR (A66G) genotypes in case mothers.

Fig. 1.

Distribution of clinical subtypes of NTDs by MTHFR (C677T, A1298C) and MTRR (A66G) genotypes in case mothers.

Close modal

In addition, we evaluated whether these polymorphisms are associated with some of the feto-maternal characteristics in control and case mothers (maternal age, fetal term, gravidity, parity, consanguinity, folate supplementation). Surprisingly, in the control group, significant differences in gravidity and parity levels were shown between the 3 genotypes. No differences were observed for any other variables between the genotypes (Table 4).

Table 4.

Distribution of MTHFR and MTRR genotypes according to some feto-maternal characteristics in control and case mothers

Distribution of MTHFR and MTRR genotypes according to some feto-maternal characteristics in control and case mothers
Distribution of MTHFR and MTRR genotypes according to some feto-maternal characteristics in control and case mothers

Table 5 summarizes the biochemical characteristics of the study mother groups according to the MTHFR and MTRR genotypes. Significant differences were found in folate and vitamin B12 levels according to MTHFR A1298C and MTRR A66G, respectively (p = 0.016 and p = 0.004) in case mothers.

Table 5.

Biochemical parameters of control and case mothers according to MTHFR and MTRR genotypes

Biochemical parameters of control and case mothers according to MTHFR and MTRR genotypes
Biochemical parameters of control and case mothers according to MTHFR and MTRR genotypes

Because of the reduced number of studied subjects, we also decided to analyze the data on the basis of two models – dominant, in which effects are expected from both heterozygotes and mutant type, or recessive, in which effects are expected only from homozygotes. A significant difference in vitamin B12 levels was found between the two groups (recessive and dominant) in mothers of NTD fetuses in MTHFR C677T and MTRR A66G polymorphisms (p = 0.03 and p = 0.002, respectively). Figure 2 indicates that mothers with CT+TT genotypes had higher levels of vitamin B12. On the other hand, mothers with AG+GG genotypes had lower levels of vitamin B12.

Fig. 2.

Distribution of biochemical parameter levels by dominant model genotype in case and control mothers. * p < 0.05.

Fig. 2.

Distribution of biochemical parameter levels by dominant model genotype in case and control mothers. * p < 0.05.

Close modal

The correlates of MTHFR and MTRR genotypes are shown in Tables 5 and 6. The MTRR A66G polymorphism was negatively correlated with vitamin B12 (p = 0.000). No other correlation was found between the different polymorphisms and the studied parameters. As for the GCP II C1561T polymorphism, all the studied subjects (cases and controls) in both the mother and father groups had the wild-type genotype (CC).

Table 6.

Correlations between MTHFR and MTRR genotypes and plasma RBC folate, homocysteine, and vitamin B12 levels in case mothers

Correlations between MTHFR and MTRR genotypes and plasma RBC folate, homocysteine, and vitamin B12 levels in case mothers
Correlations between MTHFR and MTRR genotypes and plasma RBC folate, homocysteine, and vitamin B12 levels in case mothers

To our knowledge, no investigation exploring the relation between genetic polymorphisms and the occurrence of congenital malformations in Tunisian people has been carried out until now. In this current research, our objective was to study whether MTHFR (C677T and A1298C) and MTRR (A66G) genetic polymorphisms are associated with NTDs in the Tunisian population.

Results have shown that the TT genotype and T allele in MTHFR C677T significantly decreased the incidence of NTDs in the mother group, but significantly increased this incidence in the father group. The association between MTHFR C677T polymorphism and NTDs was controversial, and the direct association was not demonstrated in several populations. These conflicts amongst diverse populations show that the thermolabile variant of MTHFR in mothers is not always related to a high risk for NTDs [26].

No association of NTDs with the C677T polymorphism has been revealed in a number of studies. In Korea, the prevalence of the C677T polymorphism in the MTHFRgene was not high in spina bifida mothers when compared to healthy subjects [27]. Also, no significant difference in genotypic frequencies between the spina bifida mothers and the controls was observed among American Caucasians in the USA [28].

CT and TT genotypes were found to be protective factors against NTD fetuses in our study. In the same way, a study carried out in Tunisia (2016) on the association between the MTHFR C677T polymorphism and depression disorder has shown that the CT genotype may be protective in this case [29].

In addition, the present study indicated that genetic variants of MTHFR A1298C and MTRR A66G conferred protective factors for NTDs in the mother group. Similar findings were obtained in several studies where these studied polymorphisms significantly decreased the incidence of NTDs [30-32]. These results are of interest because they are different from most of the other findings where MTHRF and MTRR polymorphisms were associated with increasing risk of NTDs.

Genetic variants of rs1801133 MTHFR C677T were risk factors for NTD fetus in the finding of Wang et al. [30] (2015) and Nauman et al. [33] (2018). In addition, the A1298C polymorphism was a contributing risk factor for NTDs in several studies [21, 34-36]. Zhu et al. [37] (2003) reported an association between the G allele and NTD risk in a US population.

There are probably a number of factors that may contribute to the conflicting conclusions between studies, such as sample size, different selection criteria, ethnicity, demographic differences, etc.… [30]. Such effects are complicated to explain but may go some way to clarify the changeable results of different polymorphism studies. Evidently, a broader haplotype should be considered in relation to NTD-related polymorphisms to permit a precise evaluation of disease risk.

In our population, haplotype analysis has confirmed our previous findings of the protective effects of mutant alleles and genotypes in mothers. In fact, a combination of the three wild-type alleles C677/A1298/A66 has increased four-fold the incidence of NTDs (p = 0.004, OR = 3.96, 95% CI: 1.53–10.23). In the father group, MTHFR C677T was a risk factor for NTDs. However, no association was found between MTHFR A1298C, MTRR A66G, and the occurrence of this anomaly.

The C677T polymorphism may have a greater association with NTDs than A1298C because of the locality of these two variants. The C677T polymorphism is in exon 4, which is within the N-terminal catalytic domain of the enzyme, while the A1298C polymorphism is in exon 7, which is within the C-terminal regulatory domain. The more dynamic effect of C677T is attributable to its position within the catalytic region.

In this study, the paternal MTHFR C677T polymorphism increased the incidence of NTDs. This suggests that this malformation may be transmitted to fetuses via paternal genes [38]. Noiri et al. [39] (2000) were the first to examine the sex dependence of the MTHFR genotype distribution. Stangler et al. [40] (2013) found that only male probands contributed to the association with fertility problems, suggesting that the MTHFRpolymorphism may be a gender-specific factor that disturbs the fertility of grown adults.

In human sperm, MTHFR polymorphic variants were connected with decreased sperm counts, leading to male infertility in some populations [41] and recurrent spontaneous abortion [42]. Besides, MTHFR promoter hypermethylation is correlated with impaired spermatogenesis in infertile men and men with idiopathic infertility [43]. Altered DNA methylation samples of the MTHFRpromoter in sperm cells from males imply a possible mechanism that may explain the observedassociation. Further studies are needed to clarify and confirm this hypothesis.

In case mothers of our study, the C677T polymorphism did not reduce the concentrations of folate, RBC folate, and vitamin B12. Among the CC/CT/TT genotypes, mothers with the 677CT genotype showed increased folate, RBC folate, vitamin B12, and homocysteine, but this was not significant.

However, when analyzing the data on the basis of two models, dominant versus recessive (CT+TT vs. CC), a significant difference in vitamin B12 levels was found between the two groups in case mothers (p = 0.03). This confirms the protector effect of the TT genotype and T allele in the MTHFR gene and their role in decreasing the incidence of NTDs in case mothers. This result was not expected and was in contrast with several studies which indicated that the MTHFR C677T polymorphism induced decreased levels of folate and vitamin B12 and increased frequency of offspring with NTDs [14]. The prevalence of TT homozygosity and the plasma homocysteine levels in spina bifida mothers were similar to those of the control mothers in Brazil [44]. In Ireland, the homozygosity for the C677T MTHFRvariant was not associated with the reduced red cell folate or plasma folate concentrations in mothers of NTD fetuses [45].

When analyzing the MTHFR A1298C polymorphism by the codominant model, a significant difference in folate concentrations between the three genotypes has been shown in cases mothers (p = 0.016) (Table5). This significance was not found when analyzing this polymorphism by the dominant model (Fig. 2).

Cunha et al. [26] (2002) have suggested that the MTHFR A1298C mutation affects homocysteine metabolism. They have revealed that the MTHFR 1298AA genotype showed increased total homocysteine and slightly reduced RBC folate and vitamin B12 versus the 1298AC/CC genotype in NTD children.

As for the MTRR A66G polymorphism, a significant difference in vitamin B12 levels between genotypes has been demonstrated in case mothers in the codominant (p = 0.004) (Table 5) and dominant models (p < 0.05) (Fig. 2). No association was found between MTRR A66G and folate concentrations in the finding of Yu et al. [31] (2014).

In our study, no correlations between MTHFR genotypes and the studied biochemical parameters were noticed. However, the polymorphism MTRR A66G was negatively correlated with vitamin B12 (p = 0.000). Golbahar et al. [46] (2004) observed that MTHFR genotypes were negatively and significantly correlated with plasma and RBC MTHF. But, RBC 5-MTHF was the strongest correlate of the MTHFR polymorphism.

To conclude, such results are essential for planning a program in order to prevent the occurrence of NTDs. However, additional studies are required to better understand the roles of parental gene polymorphisms related to folate and homocysteine metabolism in the pathogenesis of NTD.

This study is limited by the small number of participants because this anomaly is considered rare in our country. Therefore, larger samples may be suggested with the intention of resolving this limitation. Despite the limitation, our study focused on the relationship between the MTHFRC677T/A1298C, MTRR A66G genotypes/haplotypes and NTDs in a Tunisian population.

There is a necessity for further case-control studies to investigate the role of MTHFR and other genetic and nutritional issues that are expected to affect folate-homocysteine metabolism in the pathophysiology of NTDs in the Tunisian population.

The present work was supported by the Ministry of Higher Education, Scientific Research and Technology of Tunisia.

Ethical approval for the study was obtained from the Ethics Committee of the Maternity and Neonatology La Rabta Center in Tunis. All subjects gave written informed consent

The authors have no conflicts of interest to declare.

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