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 ::  Abstract
 ::  Introduction
 ::  Materials and Me...
 ::  Results
 ::  Discussion
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Year : 2010  |  Volume : 56  |  Issue : 4  |  Page : 267-269

MTHFR A1298C polymorphism and idiopathic male infertility

1 Department of Molecular & Human Genetics, Banaras Hindu University, Varanasi - 221 005, India
2 Department of Endocrinology & Metabolism, Institute of Medical Science, Banaras Hindu University, Varanasi - 221 005, India
3 Department of Zoology, Cytogenetics Laboratory, Banaras Hindu University, Varanasi - 221 005, India

Date of Submission11-Feb-2010
Date of Decision24-Apr-2010
Date of Acceptance16-Jun-2010
Date of Web Publication7-Oct-2010

Correspondence Address:
K Singh
Department of Molecular & Human Genetics, Banaras Hindu University, Varanasi - 221 005
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0022-3859.70935

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 :: Abstract 

Background : DNA methylation is an important epigenetic feature of DNA that plays a pivotal role in gene expression regulation during spermatogenesis. The enzyme methylenetetrahydrofolate reductase (MTHFR) catalyses the formation of folate intermediates that are vital for DNA synthesis and methylation reactions. C677T and A1298C variants of MTHFR result in reduced plasma folate and increase the susceptibility to various multifactorial disorders. We have already shown that homozygosity for 677 (C ®T) mutation in the MTHFR gene, is a risk factor for idiopathic male infertility in an Indian population. Aim: Recently, we showed that homozygosity for the 677(C;T) mutation in the MTHFR gene is a risk factor for idiopathic male infertility and now we aim to assess whether the A1298C mutation in the same gene is an additional risk factor for idiopathic male infertility in an Indian population. Setting and Designs : In a case-control study 151 idiopathic male infertile patients and 140 healthy fertile control individuals were recruited from the University hospital and infertility clinics in Varanasi city, India. Materials and Methods: Genotyping for A1298C change of the MTHFR gene was done by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Statistical Analysis : Allele frequencies were calculated using Fisher's exact test. Odds ratio was calculated as the measure of the association between the MTHFR genotype and idiopathic male infertility. Results : The homozygous (C/C) A1298C polymorphism of the MTHFR gene was present at a statistically high significance in idiopathic azoospermic infertile men (OR=3.4494, CI: 1.0092 to 11.7899, P<0.05). Conclusion : The MTHFR 1298CC genotype is an additional genetic risk factor for idiopathic male infertility in an Indian population.

Keywords: Azoospermia, male infertility, methylene tetrahydrofolate reductase

How to cite this article:
Singh K, Singh S K, Raman R. MTHFR A1298C polymorphism and idiopathic male infertility. J Postgrad Med 2010;56:267-9

How to cite this URL:
Singh K, Singh S K, Raman R. MTHFR A1298C polymorphism and idiopathic male infertility. J Postgrad Med [serial online] 2010 [cited 2023 Jun 4];56:267-9. Available from:

 :: Introduction Top

Male infertility is a multifactorial disorder and the spectrum of factors affecting male infertility is diverse ranging from genetic to environmental. Spermatogenesis is governed by a tightly controlled series of gene expression events. Both Y-chromosomal as well as autosomal genes regulate the spermatogenesis at different steps and the aberrant expression of these genes may lead to infertility. [1] One of the mechanisms regulating their expression is DNA methylation. Folate is indispensable for DNA synthesis and methylation of DNA and histones. Methylenetetrahydrofolate reductase (MTHFR) is a candidate gene of the folate and homocysteine metabolic pathway and catalyses methylation of 5, 10 methylenetetrahydrofolate, which contributes to the methyl group in the conversion of homocysteine to methionine. Methionine gets converted to 5-adenosylmethionine, the lone donor of -CH 3 to cytosine and lysine residues, respectively in DNA and histones. [2]

DNA and histone methylation regulates the expression of candidate genes of spermatogenesis pathway. Hypomethylation leading to aberrant expression of these genes may result from MTHFR deficiency. Inactivation of MTHFR results in absence of germinal cells and spermatogenesis arrest. [3] The above reports suggest the role of MTHFR in spermatogenesis and indicate that its deregulation could be involved in male infertility.

 :: Materials and Methods Top

This case-control study included a total of 151 patients classified as idiopathic azoospermic visiting the Out Patient Door facility of the Department of Endocrinology and Metabolism, Institute of Medical Sciences, Banaras Hindu University, Varanasi and infertility clinics in Varanasi city. 140 fertile control individuals of a comparable age group (30, SD+ 3), belonging to the same geographical region were included in the study. At least three seminal fluid examinations, carried out after three to four days of sexual abstinence, were performed to ascertain their infertility status. Each patient's family history, habits (smoking, alcoholism etc.), and disease were recorded through a questionnaire. Those who suffered from varicocele, diabetes, mumps and orchitis etc. were excluded from the study. Informed consent was obtained from all the subjects to carry out molecular analyses. Institutional ethical committee approval was obtained.

Genotyping for A1298C change in the MTHFR gene was done by PCR-RFLP method. An A;C change at base pair 1298 abolishes an MboII restriction site. PCR product of 163bp was digested with MboII following which wild type 1298AA and mutant 1298CC alleles would yield, respectively 5 (56, 31, 30, 28, 18bp) and 4 fragments (84, 31, 30, 18bp). The identification bands for each were of 56 and 84bp, [4] shown in [Figure 1].
Figure 1 :RFLP analysis for the 1298(A→C) mutation on a 163-bp PCR fragment with MboII. The 1298(A→C) abolishes an MboII restriction site. Digestion of the 163-bp fragment of the 1298AA genotype gives five fragments, of 56, 31, 30, 28 and 18 bp, whereas the 1298CC genotype results in four fragments, namely, 84, 31, 30, and 18 bp. The 18 bp fragment has been run off the gel

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Statistical analysis

Allele frequencies were calculated for each genotype and differences between the case and control were determined using Fisher's exact test crude. Odds ratio for the heterozygous and homozygous mutant between the MTHFR genotypes were calculated as the measure of the association between the MTHFR genotype and idiopathic male infertility, and used as an estimate of relative risk. Throughout, a two-tailed P value of < 0.05 was interpreted as statistically significant.

 :: Results Top

PCR-RFLP analysis was done for DNA extracted from both infertile patients and healthy fertile controls. PCR amplified product was digested with MboII and resolved in 4% agarose gel. [Figure 1] illustrates the three genotypes (AA, AC and CC). 151 idiopathic infertile subjects and 140 fertile control individuals were genotyped for A1298C single nucleotide polymorphism in the MTHFR gene. The genotype frequency for the MTHFR A1298C polymorphism is shown in [Table 1]. The population was in the Hardy-Weinberg Equilibrium (HWE) for C677T genotypes; [5] however, for A1298C genotypes only patient subpopulation was in HWE, the control subpopulation was not. The 1298CC genotype was higher in patients compared to controls and was associated with increased risk for male infertility (OR=3.4494, CI: 1.0092 to 11.7899, P<0.05). The cross-tabulation of genotype combinations of MTHFR C677T and A1298C revealed none of the subjects to be homozygous for both the mutations. Combined heterozygosity AC/TT frequency was quite comparable in both the groups (10.6% in cases and 9.3% in controls).
Table 1 :Genotype frequency of A1298C, MTHFR in case as well as controls

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 :: Discussion Top

Our study on MTHFR SNP A1298C arises out of the consideration that DNA methylation plays an important role in the regulation of spermatogenesis and that SAM, the lone donor of methyl group is the product of homocysteine (Hcy) metabolism. [6] It is also known that certain SNPs in one of the genes in this pathway, MTHFR , lead to impaired enzyme activity, elevation of Hcy levels and serve as risk factor for various multifactorial disorders. Animal model studies suggest that MTHFR plays a critical role in spermatogenesis. [3] Previously, we have reported that MTHFR, C677T, is a risk factor for infertility in an Indian population. [5] Since A1298C is associated with the lowering of MTHFR enzyme activity, and elevated Hcy level in Indian populations, [7] it is logical to see its association with infertility, particularly in the individuals already analyzed for C677T polymorphism. 1298CC genotype showed significant difference in its frequency between the cases and controls.

Heterogeneous distribution of C677T and A1298C is recorded globally and may be attributable to different ethnic, environmental and genetic backgrounds. The present study has certain limitations. For A1298C polymorphism only patient group was in HWE not the control group. Similar results has previously been reported from North India where patient subpopulation was only in HWE for A1298C polymorphism. [8],[9] The present study was done in the Indian context thus generalizability to the other ethnic groups remains uncertain.

The identification of genetic risk is an important step for the management of idiopathic male infertility. The findings from the present study support the hypothesis that 1298CC homozygosity is an additional risk for idiopathic male infertility in an Indian population.

 :: References Top

1.Saunders PT, Turner JM, Ruggiu M, Taggart M, Burgoyne PS, Elliot D, et al. Absence of mDazl produces a final block on germ cell development at meiosis. Reproduction 2003;126:589-97.  Back to cited text no. 1      
2.Friso S, Choi SW, Girelli D, Mason JB, Dolnikowski GG, Bagley PJ, et al. A common mutation in the 5, 10-methylenetetrahydrofolate reductase gene affects genomic DNA methylation through an interaction with folate status. Proc Natl Acad Sci U S A 2002;99:5606-11.  Back to cited text no. 2  [PUBMED]  [FULLTEXT]  
3.Kelly TL, Neaga OR, Schwahn BC, Rozen R, Trazler JM. Infertility in 5, 10 methylenetetrahydrofolate reductase (MTHFR)-deficient male mice is partially alleviated by lifetime dietary betaine supplementation. Biol Reprod 2005;72:667-77.  Back to cited text no. 3      
4.van der Put NM, Gabreels F, Stevens EM, Smeitink JA, Trijbels FJ, Eskes TK, et al. A second common mutation in the Methylenetetrahydrfolate reductase gene: An additional risk factor for Neural -Tube defects? Am J Hum Genet 1998;62:1044-51.  Back to cited text no. 4      
5.Singh K, Singh SK, Sah R, Singh I, Raman R. Mutation C677T in the methylenetetrahydrofolate reductase gene is associated with male infertility in an Indian population. Int J Androl 2005;28:115-9.  Back to cited text no. 5  [PUBMED]  [FULLTEXT]  
6.Kelly TL, Li E, Trasler JM. 5-Aza-2'-Deoxycytidine induces alterations in murine spermatogenesis and pregnancy outcome. J Androl 2003;24:822-30.  Back to cited text no. 6  [PUBMED]  [FULLTEXT]  
7.Kumar J, Das SK, Sharma P, Karthikeyan G, Ramakrishnan L, Sengupta S. Homocysteine levels are associated with MTHFR A1298C polymorphism in Indian population. J Hum Genet 2005;50:655-63.  Back to cited text no. 7  [PUBMED]  [FULLTEXT]  
8.Poduri A, Mukherji D, Sud K, Kohli HS, Sakhuja V, Khullar M. MTHFR A1298C polymorphism is associated with cardiovascular risk in end stage renal disease in North Indians. Mol Cell Biochem 2008;308:43-50.  Back to cited text no. 8      
9.Freitas AI, Mendonηa I, Guerra G, Briσn M, Reis RP, Carracedo A, et al. Methylenetetrahydrofolate reductase gene, homocysteine and coronary artery disease: The A1298C polymorphism does matter: Inferences from a case study (Maderira, Portugal). Thromb Res 2008;122:648-56.  Back to cited text no. 9      


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  [Table 1]

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