The Frequency of the 677C>T and 1298A>C Polymorphisms in the Methylenetetrahydrofolate Reductase (MTHFR) Gene in the Population

Background: The gene for 5,10-methylenetetrahydrofolate reductase (NAD(P)H) or MTHFR gene encodes protein methylenetetrahydrofolate reductase (MTHFR), an enzyme important in folate metabolism. Aim: The aim of this study was to determine the frequencies of 677C>T and 1298A>C polymorphisms in the MTHFR gene of healthy subjects from the population. Material and methods: The blood samples were collected from 164 unrelated and healthy donors from population consisted of 98 females and 66 males. Both the MTHFR 677C>T and 1298A>C single nucleotide polymorphisms (SNPs) were analyzed by Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Linkage disequilibrium (LD) between pair of SNPs was calculated through Haploview analysis. Results: The frequency of MTHFR 677T allele in the population (32.62%) was in agreement with the frequency of this allele in most other populations, however, the frequency of MTHFR 1298C allele (38.41%) was higher than that reported for most other populations in the world. Haploview analysis showed a relatively strong LD between 677C>T and 1298A>C SNPs with D′ values of 0.87. Conclusion: Regarding the two MTHFR polymorphisms, three of the nine combined genotypes were present in 87.2% of the population. 33.54% subjects were complex heterozygous (677CT/1298AC genotype), 34.15% subjects had 677CC/1298AC and 19.51% of 677CT/1298AA genotype. The subjects with 677TT genotype had a 1298AA or 1298AC genotype while subjects with 1298CC genotype had only 677CC genotype. The subjects with 677CC/1298AA genotype were only 3.05%. We were not found triple 677CT/1298CC and quadruple 677TT/1298CC mutation suggesting decreased viability of embryos with increased numbers of mutant alleles.

Although a number of mutations were described, 677C>T (rs1801133) and 1298A>C (rs1801131) SNPs (single nucleotide polymorphisms) are the two most common mutations in the MTHFR gene. Many of the MTHFR gene polymorphisms alter or decrease the activity of methylenetetrahydrofolate reductase, leading to an increase of homocysteine in the blood. Decreased folate and increased plasma Hcy levels are associated with a variety of common conditions such as cardiovascular disease, neural tube defects, cleft lip/palate, hypertension, preeclampsia, thrombosis, osteoporosis, dementia, Alzheimer's disease, Down syndrome, certain types of cancer, glaucoma, pregnancy complications, migraine, epilepsy, depression and schizophrenia. Polymorphism 677C>T (OMIM: 607093.0003) in the coding region of the MTHFR gene (position: 11,796,321) was described by Frosst (1). The replacement of cytosine with thymine is a point mutation of the transition type in exon 4 of this gene (c.788C>T) which alanine changes to valine (p.Ala222Val or A222V) in the N-terminal catalytic domain of MTHFR protein and is responsible for the synthesis of a thermolabile form of MTHFR with reduced enzymatic activity. 677C>T polymorphism is relatively common and has been studied for a long time.
Another mutation that often occurs in the MTHFR gene 1298A>C (OMIM: 607093.0004) is the first time described by van der Put (2). 1298AC transversion is located in exon 7 (position: 11,794,419) that leads to the replacement of adenine with cytosine (c.1286A>C) resulting in transfer glutamic acid to alanine (p.Glu429Ala or E429A) within the C-terminal regulatory domain of the protein. The 1298AC mutation is associated with decreased MTHFR activity that is more pronounced in the homozygous than heterozygous state.
The frequency of the MTHFR 677C>T and 1298A>C polymorphisms varies in different geographical regions of the world and among different ethnic groups. Although the frequency of MTHFR 677C>T polymorphism has been established (3,4), MTHFR 1298A>C polymorphism has not been investigated in Bosnia and Herzegovina so far. The aim of this study was to determine the frequency of 677C>T and 1298A>C polymorphisms in the methylenethetrahydrofolate reductase (MTHFR) gene in peripheral blood samples of healthy subjects from the population using the polymerase chain reaction (PCR) and the restriction fragment length polymorphism (RFLP) method. Subsequently, we compared the obtained frequencies of minor alleles (MAF) with the frequencies of these polymorphisms in some other populations of the world.

Samples Collection
The blood samples were collected from 164 unrelated and healthy donors from population consisted of 98 females and 66 males between 17 and 79 years of age (Table  1). Blood samples (3 ml) were taken and collected into tubes with EDTA. All individuals who participated in this study belonged to Caucasians from different regions of Bosnia and Herzegovina between 2011 and 2015. All the subjects included in this analysis gave written informed consent to participate in the study. The study was approved by the Ethics Committee of the Faculty of Science on University of Sarajevo (01/01-556/2-2018).

DNA isolation and detection of MTHFR gene mutations
After genomic DNA was extracted from whole peripheral blood according to a previously described method proposed by Miller et al. (5) with several small modifications, the MTHFR 677C>T and 1298A>C polymorphisms were analyzed using the PCR-RFLP method. A fragment of 198 bp containing the polymorphic site in the exon 4 of MTHFR gene was amplified by PCR on a thermal cycler (Eppendorf Mastercycler gradient, Hamburg, Germany) in 0.2-ml thin-walled tubes using previ ously described couples of primers for 677C>T: 5'-TGAAGGAGAAGGTGTCTGCGGGA -3' as the forward and 5'-AGGACGGTGCGGTGAGAGTG -3' as the reverse primer (1) while a fragment of 163 bp containing the polymorphic site in the exon 7 of MTHFR gene was amplified using 5'-CTTTGGGGAGCTGAAGGAC-TACTAC -3' as the forward and 5'-CACTTTGTGAC-CATTCCGGTTTG -3' as the reverse primer (2).
After gel electrophoresis, each remaining PCR product was digested with the restriction endonucleases Hinf I and MBoII (New England BioLabs, USA), separately. The digestion reactions contained: 10 µl of PCR product (0.1-0.5μg of DNA), 2.5 µl of buffer (10x NEBuffer Cut Smart, supplied with enzyme) 0.5 µl of restriction enzyme (concentration: 10U/µl) and 12 μl of nuclease-free water. Restriction analysis was performed for 4 hours at 37 °C. The digestion products were separated on ethidium bromide-stained 3% agarose gels at 70 V and visualized under ultraviolet light.
Regarding the 677C>T SNP, since mutation creates a Hinf I restriction site (5'-G|ANTC -3'), the amplicon of the wild-type allele (198 bp) is not cut, while the mutant one is cut into two frag ments of 175 and 23 bp respectively. In agarose gel electrophoresis it is possible to see the 198 and 175 bp fragments, the 23-bp fragment is not retained in the gel. Nevertheless, concerning the 1298A>C SNP, since mutation abolishes a restriction site for MboII enzyme (5'-GAAGAN8 -3'), the amplicon (163 bp) of the mutated allele will be cut into four (84, 31, 30 and 18 pb) instead of five fragments (56, 31, 30, 28 and 18 bp) as a wild type allele. In agarose gel electrophoresis it is possible to see the 84 and 56 bp fragments and fragments of 30, 31 and 28 bp as one band.

Statistical Analysis
Allelic and genotypic frequencies were calculated by the gene counting method. The chi-square (χ 2 ) test was used to evaluate the Hardy-Weinberg equilibrium for the distribution of genotypes of subjects and to determine differences in the distribution of genotypes and allele frequencies between males and females. Linkage disequilibrium (LD) analysis was performed using the Haploview software version 4.2 (Daly Lab at the Broad Institute, Cambridge, USA). Comparisons of SNP fre-quencies between population and other ethnic populations were done using a chi-square test with significance level of 0.05 and 95% confidence intervals (95% CI) using the MedCalc statistical software package version 12.5.0.0 (Ostend, Belgium). Table 1 gives frequencies of genotypes and alleles of the MTHFR 677C>T and 1298A>C polymorphisms. The frequencies of 677CC, CT and TT genotypes among subjects from the population were 40.85%, 53.05% and 6.1%, respectively resulting in a T allele frequency of 32.62%. The genotype distribution for males and females was in the Hardy-Weinberg equilibrium. The frequencies of 1298AA homozygotes, AC heterozygotes and CC homozygote were 26.83%, 69.51% and 3.66%, respectively. The 1298A>C genotype distribution deviated from the expected Hardy-Weinberg distribution. The overall C allele frequency was 38.41%. It was found that there was no statistically significant difference in the distribution of genotypes for 677C>T polymorphism between males and females (P = 0.4030). The frequency of 677T allele was higher in females (34.18%) than in males (30.3%) but this difference was not statistically significant according to chi-square test (P = 0.5385). Similarly, there were no significant differences in the distribution of genotypes (P = 0.3325) and allele frequencies (P = 0.9617) for 1298A>C polymorphism between males and females (Table 1).
Haploview analysis showed a relatively strong LD between MTHFR 677C>T and 1298A>C SNPs with D' values of 0.87 and the correlation r 2 of 0.23.
The frequency of MTHFR 677T allele in the population (32.62%) was in agreement with the frequency of this allele in most other populations in the world (Brazil, Turkey, Germany, UK, France, Italy, Denmark, Spain, Ireland, China, Bosnia and Herzegovina, Serbia, Croatia, Slovenia, USA and Australia) but significantly different from the frequency of 677T allele in the population of Turkey for two samples (P = 0.0028 and P = 0.0001), the samples from Egypt (P = 0.0126) and India (P <0.0001) ( Table 2). However, the frequency of MTHFR 1298C allele in the population (38.41%) was much higher (except for one population from Turkey: 43.96%) than that reported for other populations in the world (Brazil, Turkey, France, Italy, Spain, Egypt, India, Sweden, South Korea, Germany, Russia, USA, Australia and UK). These differences were statistically significant (P < 0.05) except for population from Turkey (one sample), Italy (one sample), Egypt, Russia and Australia (Table 3).

DISCUSSION
MTHFR 677T and 1298C allele frequencies differ between populations. The frequency of MTHFR 677T allele in the population (32.62%) observed in this study is consistent with results of previous studies (3,4) as well as with the frequency of allele in most other populations, however, the frequency of MTHFR 1298C allele (38.41%) was higher than frequency of this allele in the most other populations in the world. These differences were statistically significant (P < 0.05). We found that there were no statistically significant differences in the distribution of genotypes and allele frequencies for MTHFR 677C>T and 1298A>C polymorphisms between males and females.
After analysis of combined genotypes for these two polymorphisms we observed that the most frequent subjects (34.15%) were homozygous for 677C allele and heterozygous of the 1298 locus (677CC/1298AC genotype). But, those who were complex heterozygous for 677 and 1298 locus (677CT/1298AC genotype) were 33.54%. Subjects who were heterozygous for the 677 locus and homozygous for 1298C allele (677CT/1298AA genotype) were 19 jects with 1298CC genotype had only 677CC genotype (3.66%). These genotypes represent 12.8%. Just three subjects had a triple 677TT/1298AC genotype, probably as result of recombination between the two ancestor genotypes (most likely 677CT/1298AC genotype). These results suggest that 677T allele is linked with the A allele of 1298 locus and 1298C allele is associated with C allele of locus 677. Only 3.05% subjects were heterozygous for both MTHFR wild-type alleles (677CC/1298AA genotype). The triple 677CT/1298CC and quadruple 677TT/1298CC mutation has not been found suggestion decreased viability of embryos with increased numbers of mutant alleles. Combined genotypes which contain three or four mutant alleles were not detected or detected several implying linkage disequilibrium between two polymorphisms. The pattern of LD in the MTHFR gene showed a relatively strong LD between 677C>T and 1298A>C SNPs with D' values of 0.87 and the correlation r 2 of 0.23. The examples of LD observed in natural populations are the result of a complex interaction between genetic factors and the demographic history of the population. Particularly, recombination shows a significant role in determining the patterns of LD in a population.

CONCLUSIONS
The frequency of MTHFR 677T allele in the population (32.62%) was in agreement with the frequency of this allele in most other populations, however, the frequency of MTHFR 1298C allele (38.41%) was higher than frequency of this allele in the most other populations of the world.