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ORIGINAL ARTICLE MTRR A66G POLYMORPHISM AMONG TWO CASTE GROUPS OF UTTAR PRADESH (INDIA) VANDANA RAI, UPENDRA YADAV, PRADEEP KUMAR
ABSTRACT OBJECTIVE: This study was aimed to evaluate the Methionine synthase reductase (MTRR) A66G mutation in Yadav and Scheduled Caste (SC) population of Uttar Pradesh. MATERIALS AND METHODS: Blood samples were collected from 100 subjects after taking informed written consent and PCR‑RFLP method was used for the analysis of A66G mutation. After NdeI digestion, 66‑bp amplicon of normal allele was cut into 22- and 44-bp long fragments, whereas mutant G allele was not digested. RESULTS: Frequencies of genotypes in Yadav population AA, AG, and GG were 12%, 60%, and 28%, respectively, and in SC population, genotypic frequencies were 12% (AA), 52% (AG), and 36% (GG). CONCLUSION: MTRR gene A66G mutation is found to be polymorphic in both the target populations with G allele frequencies being 0.58 for Yadav and 0.62 for Scheduled Caste. Key words: A66G polymorphism, homocysteine, methionine synthase reductase, methionine synthase reductase
INTRODUCTION Methionine synthase reductase (MTRR) enzyme plays a key role in folate‑dependent homocysteine metabolism and is a member of the electron transferase family. The enzyme has three characteristic sites which bind FMN, FAD, and NADH. MTRR is responsible for methionine synthase (MTR) regulation by reductive methylation and disturbances in its catalytic activity can lead Department of Biotechnology, Human Molecular Genetics Laboratory, VBS Purvanchal University, Jaunpur, Uttar Pradesh, India
to higher levels of homocysteine. MTRR is a housekeeping gene and localized at chromosome 5 (5p15.2‑p15.3).[1,2] The most common polymorphism in MTRR gene is A66G substitution, leading to a change of isoleucine to methionine at amino acid 22 (I22M). Although A66G polymorphism does not change the catalytic activity of the protein, the frequency of 66G genotype is reported to be higher in NTD cases and their mothers than in the control groups.[2,3] The I22M variant is Access this article online Quick Response Code:
Address for correspondence: Dr. Vandana Rai, Human Molecular Genetics Laboratory, Department of Biotechnology, VBS Purvanchal University, Jaunpur ‑ 222 001 Uttar Pradesh, India. E‑mail:
[email protected]
Indian Journal of Medical Sciences, Vol. 66, No. 5 and 6, May and June 2012
Website: www.indianjmedsci.org DOI: 10.4103/0019-5359.114200 PMID: *****************************
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located in the putative FMN‑binding domain of the MTRR enzyme that is suggested to interact with MTR. Substitution of an isoleucine by a methionine in this part of the enzyme might disrupt the binding of MTRR to the MTR‑cob (I) alanine‑complex, thereby decreasing the rate of homocysteine remethylation.[4] Malnutrition and malabsorption of folate and vitamin B 12 or an inherited enzymatic defect, such as MTHFR deficiency, may result in hyperhomocysteinemia. MTRR A66G polymorphism has also been associated with elevated plasma homocysteine levels, [5,6] although in some studies,[2,7] an effect of the MTRR A66G SNP on plasma homocysteine has not been observed but higher concentration of serum homocysteine is reported as risk factor for disorders like ‑ Neural tube defects,[8] cardiovascular disease,[9] Alzheimer disease,[10] psychiatric disorders as Schizophrenia,[11] etc., Hyperhomocysteinemia and the associated diseases are probably caused by an interaction between nutritional and genetic factors. Despite the susceptibility of the Indian population to elevated homocysteine levels due to their dietary habits, there are no reports to date in which MTRR polymorphism frequency in random population was studied. The present study is the first report of the frequency of MTRR A66G polymorphism from two caste groups of eastern UP.
MATERIALS AND METHODS Blood samples were collected from randomly selected 50 Yadav and 50 Scheduled Caste individuals of both sexes. Informed written consent was obtained from each subject. The study obtained prior ethical clearance from the
Institutional Ethics Committee for the collection of samples, following the guidelines of Indian Council of Medical Research. Genomic DNA was extracted according to method of Bartlett and White.[12] MTRR A66G polymorphism was identified by PCR amplification followed by NdeI digestion. Primers were as described by Wilson et al.[2] PCR was performed in MJ Mini Thermo Cycler (Bio‑Rad, USA), and program consisted of an initial melting step of 4 minutes at 940C, followed by 40 cycles of 1 minute denaturation at 940C, 1 minute annealing at 650C, 1.30 minutes extension at 720C, and final elongation step of 10 minutes at 720C. The amplified product was digested with NdeI restriction enzyme (Genei, India), which cleaves only normal allele into 44and 22-bp fragments [Figures 1 and 2]. Restricted products were analyzed in 4% agarose (Fermentas) gel electrophoresis. Allele frequencies were calculated by gene counting method. χ2 test was performed to test Hardy‑Weinberg equilibrium with respect to each population.
RESULTS AND DISCUSSION Frequencies of genotypes in Yadav population AA, AG, and GG were 12%, 60%, and 28%, respectively, and in SC population, genotypic frequencies were 12% (AA), 52% (AG), and 36% (GG). MTRR gene A66G mutation is found to be polymorphic in both the target population with G allele frequencies being 0.58 for Yadav and 0.62 for SCs [Table 1]. Both the populations are in Hardy‑Weinberg equilibrium with χ2 value 2.6801 and 0.5361 for Yadav and SC populations. The G allele among SC is found to be more frequent (0.62) than Yadav (0.58).
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Figure 1: Amplified product of 66-bp for A66G polymorphism Table 1: Distribution of MTRR genotype and allele frequencies among the yadav and scheduled caste population Population
Observed genotype
Allelic frequency
Figure 2: RFLP analysis for the A66G mutation on 66-bp MTRR PCR products with NdeI. Mutant-type homozygous remains uncut after NdeI digestion gives one band, and heterozygous gives three bands (66-bp,44-bp,22-bp). The figure shows mutant GG (lane: 2-4, 6 and 8), heterozygous CT (lane:5), and homozygous wild AA (lane:7) genotypes
χ2 value
AA
AG
GG
A
G
Yadav
6
30
14
0.42
0.58
2.6801
SC
6
26
18
0.38
0.62
0.5361
MTRR=Methionine synthase reductase
The world‑wide frequency of A66G polymorphism is ~30%. [2,5,13] However, its frequency varies in different ethnic and geographical regions as reported by Rady et al.,[14] the lowest frequency in the Hispanic population (28.65%) compared to 34% among African‑Americans, 43.1% among Ashkenazi Jews, and 54.45 among Caucasians (54.4%). In our study, the frequency of G allele is higher in both the population, 58% for Yadav and 62% for SC populations. Wilson et al.[2] have found the frequency of both the alleles
of other species A allele has been assigned as wild allele, it has been suggested that the ancestral human MTRR sequence contains the isoleucine codon and that the G allele represents the mutation.[2] In several studies, either case control or epidemiological survey, the frequency of G allele was found to be higher than A allele (>50%).[15‑17] In addition, Wilson et al. [2] reported that homozygous mutant genotype (GG), when combined with low cobalamin levels, greatly increases the risk for giving birth to child with Down syndrome and congenital defects especially neural tube defects.[2] Since then, several studies reported A66G polymorphism as risk factor for Down syndrome, [18,19] neural tube defects, [2,4] and coronary artery diseases.[13,20]
A and G same, so it was not possible for them to designate the wild type and mutated allele; however, on the basis of sequence homology with related flavin nucleotide binding proteins
The allelic frequency of the MTRR A66G allele in our sample (0.58 and 0.62) was slightly higher than those reported in
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other studies, where frequencies ranging from 0.51 (Brazilian Caucasian population) to 0.55 (European population) of the 66G allele were reported, [2,15,16] suggesting that this population might be susceptible to some type of factor contributing to the modification of these allelic frequencies. In addition, a majority of Indian population are known to be deficient in vitamin B12, presumably due to their adherence of a strict vegetarian diet.[21] Thus, this polymorphism of folate pathway gene may be perceived to have a great impact in relation to hyperhomocysteinemia in Indian population. Population frequency of this clinically important SNP is not reported from any Indian population, few reports are available which are based on the case‑control studies. So, it is urgently needed to screen the ethnic and geographical populations for this clinically important polymorphism. However, the sample size in the present study is small, so it is critical to carry out the MTRR mutation analysis in larger sample size to confirm the finding of the present study.
Genetic variation in genes of folate metabolism and neural‑tube defect risk. Proceeding of Nutrition Society 2006;65:204‑15. 5. Gaughan D, Kluijtmans LA, Barbaux S, McMaster D, Young IS, Yarnell JW, et al. The methionine synthase reductase (MTRR) A66G polymorphism is a novel genetic determinant of plasma homocysteine concentrations. Atherosclerosis 2001;157:451‑6. 6. Vaughan JD, Bailey LB, Kristina M, Dunwoody C, Mavenal DR, Davis SR, et al. Methionine synthase reductase 66A‑>G polymorphism is associated increased plasma homocysteine concentration when combined with the homozygous methylenetetrahydrofolate reductase 677>T variant. J Nutr 2004;134:2985‑90. 7. Jacques PF, Bostom AG, Selhub J, Rich S, Ellison RC, Eckfeldt JH, et al. Effects of polymorphisms of methionine synthase and methionine synthase reductase on total plasma homocysteine in the NHLBI Family Heart Study. Atherosclerosis 2003;166:49‑55. 8. Mills JL, Mcpartlin JM, Kirke PN, Lee YJ, Conley MR, Weir DG, et al. Homocysteine metabolism in pregnancies complicated by neural tube defects. Lancet 1995;345:149‑52.
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Indian Journal of Medical Sciences, Vol. 66, No. 5 and 6, May and June 2012
