Methionine synthase polymorphisms (MTR 2756 A>G and MTR 2758 C>G) frequencies and distribution in the Jordanian population and their correlation with neural tube defects in the population of the northern part of Jordan

BACKGROUND: The human methionine synthase gene (MTR) is located on chromosome 1q43; it is of 105.24 kb and is made up of 33 exons. Methionine synthase is a cytoplasmic enzyme that requires methylcobalamin for activity and catalyzes the remethylation of homocysteine to methionine. In this reaction, the methyl group of 5-methyltetrahydrofolate is transferred to the enzyme bond cob(I) alamin to generate methylcobalamin, followed by the transfer of the methyl group to homocysteine to reform methionine. MATERIALS AND METHODS: The frequencies of the polymorphisms of MTR 2756A>G and MTR 2758C>G have been determined in this study in a sample of 491 individuals collected from all regions of Jordan and representing the Jordanian population. The different alleles and genotypes at the two polymorphic sites were identified using the Polymerase Chain Reaction - Restriction Fragment Length Polymorphism (PCR-RFLP) analysis. RESULTS: Showed that the percentages of the polymorphic alleles at the MTR 2756 position in the north, middle and south regions were 90.38, 92.65 and 83.69%, respectively, for the MTR 2756A allele, and were 9.61, 7.34 and 16.30%, respectively, for the MTR 2756G allele, with overall percentages in the whole Jordanian population of 90.73 and 9.27% for the MTR 2756A and MTR 2756G alleles, respectively. The percentages of the genotype MTR 2756AA were 82.90% in the northern region, 86.72% in the middle region and 71.73% in the southern region, and an overall percentage of MTR 2756AA in the whole Jordanian population was 83.50%. The frequencies of MTR 2756AG genotype in the northern, middle and southern regions were 14.95, 11.84 and 23.91%, respectively, with an overall percentage of 14.46% in the whole Jordanian population. The percentages of the genotype MTR 2756GG in the northern, middle and southern regions were 2.13, 1.42 and 4.34%, respectively, with an overall percentage of 2.04% in the whole Jordanian population. Only the wild type allele (C) of the MTR 2758C>G polymorphism was detected in this study. In addition, the association of MTR 2756A>G and MTR 2758C>G polymorphisms with the development of neural tube defects (NTDs) was examined using 17 cases of mothers from the northern part of Jordan, who gave birth to NTD affected children during the period of this study. Results showed no association between these two examined polymorphisms and the increase in maternal risk for giving birth to NTD children. CONCLUSION: results of this study recommend that examination should be done on larger populations to arrive at better conclusions. Also, more studies on gene–gene interaction should be done to examine the associations with NTDs.


Introduction
The human methionine synthase gene (MTR) is located on chromosome 1q43; it is of 105.24 kb and is made up of 33 exons. [1,2] Also, it produces about 1265 amino acid residues and weighs 140.5 kDa. [3] Methionine synthase is a cytoplasmic enzyme that requires methylcobalamin for activity and catalyzes the remethylation of homocysteine to methionine. In this reaction, the methyl group of 5-methyltetrahydrofolate is transferred to the enzyme bond cob(I) alamin to generate methylcobalamin, followed by the transfer of the methyl group to homocysteine to reform methionine. In MTR, a polymorphism exists that is located at nucleotide position 2756 (MTR 2756A>G) and it changes an aspartic acid into a glycine. [4] MTR 2758 C>G is another polymorphism and it appears to have rare effects in the populations. [5] MTR 2756A>G polymorphism affects formyltetrahydropteroylglutamic acid (H4PteGlu) disposition of erythrocytes and the MTR 2756AG genotype is associated with more formyl-H4PteGlu, relative to 5-methyl-H4PteGlu, found in individuals with wild-type alleles. This relationship was not present in red blood cells of individuals with a neural tube defect (NTD). [6] The influence of MTR 2756A>G on total homocysteine plasma levels is still a matter of debate.
An association was observed between MTR 2756A>G and increased total homocysteine levels, [7] while such an association was not confirmed in other studies. [6,8] Furthermore, an association between MTR 2756A>G and low plasma levels of homocysteine was observed. [9] Humans lacking MTR activity have severe clinical consequences. [10] In mice, complete loss of MTR activity leads to early embryonic lethality. [11] The polymorphism MTR 2756A>G as well as the heterozygous genotype MTR 2756AG was reported to be associated with the severity of coronary artery disease [12] and was considered as high risk factor for their occurrence. [13] Different studies, however, showed no association between MTR 2756A>G polymorphism and birth defects, [6,8] cerebrovascular, cardiovascular diseases, [14] and early onset vascular thrombosis. [15] The homozygous MTR 2756GG was however linked to a higher susceptibility for malignant lymphoma. [16]  at 65 o C for 15 minutes. [5] The separation of the digested normal 2756A/A genotype on 3% agarose gels resulted in a single undigested fragment of 176 bp, while the mutant polymorphic homozygote genotype 2756G/G resulted in two fragments of 146 and 30 bp and the digestion of the heterozygous 2756A/G genotype produced three fragments of 176, 146 and 30 bp. [18] Restriction enzyme digestion was performed using the Sau96I restriction enzyme on the same fragment as in the previous section. Each restriction digestion reaction mixture contained 10 µl of the PCR product,  [5] The digestion of the normal 2758C/C genotype resulted in two fragments of 148 and 28 bp, while the mutant polymorphic 2758G/G resulted in one fragment of 176 bp.

Materials and Methods
The digestion of the heterozygous 2758C/G genotypes produced three fragments of 176, 148 and 28 bp. [18] Enumeration data including the number of individuals with various genotypes and the comparisons between regions were evaluated using the Chi-Square and Fisher's-exact tests. P value equal to 0.05 [19] was statistically significant and all the analysis had been done using the SPSS software version 15.5.

Results
The   in the middle region and 4.34% in the southern region.
A two-way contingency table analysis showed that  This analysis showed that there were no correlations (P = 0. 107) between NTD cases and the MTR 2756A>G polymorphism.
The MTR 2758C>G polymorphism was not found either in the NTD case mothers or in the controls.

Discussion
This is the first study that has examined the  [20] Besides, this study has also tried to evaluate the correlation between the polymorphisms MTR 2756A>G and MTR 2758C>G and maternal risk of delivering NTD affected babies in the northern part of Jordan.
These polymorphisms were examined due to their role in the folate metabolic pathway, which is essential for the methylation and regulation of developmental processes in embryos. [21] The etiology of NTD is poorly understood, but it is now suggested that there is a complex interplay between environmental and genetic factors. [22] Knowledge of the association between NTD The results also showed that the overall percentages of MTR 2756 genotype in Jordan were 83.5% for AA, 14.46% for AG and 2.04% for GG. This was relatively different from the genotype percentages reported in the small sample of the Moroccan population, which were 73.4, 21.9 and 4.7% for AA, AG and GG, respectively, [20] but more close to those genotype percentages reported which was reported only in one Caucasian male who had failure to severe eczema, megaloblastic anemia, and methylmalonic aciduria [4] and is similar to the results reported in Texas, USA (Zhu et al 2003).
The fact that the results of this study showed no association between MTR 2756A>G and MTR 2758C>G polymorphisms and maternal risk for NTDs, in the northern part of Jordan, is in agreement with the absence of association between MTR 2756A>G polymorphism and NTDs in Alabama, USA, [18] the Irish [24] and the Netherland populations. [25] Concerning our attempt to investigate the association b e t w e e n M T R 2 7 5 6 A > G a n d M T R 2 7 5 8 C > G polymorphisms with the maternal risk for NTDs, in the northern part of Jordan, during 1 year period study (as part of the requirements for the MSc degree), we ended up with a limited small sample of case mothers due to the incidence of 3.8 affected with NTDs in 1000 live births in Jordan and 1.5 per 1000 live births in the northern region in Jordan, [26][27][28] which may have possibly affected our conclusion. Further tests of more mothers of NTD affected children in the future would be essential to confirm our results. Finally, the results of this study recommend that examination should be done on larger populations to arrive at better conclusions. Also, more studies on gene-gene interaction should be done to examine the associations with NTDs.