Uttar Pradesh Methionine Synthase Reductase A66g Polymorphism in Rural Population of Uttar Pradesh (India)
The present study was aimed to analyze methionine synthase reductase (MTRR) A66G polymorphism in Uttar Pradesh (UP) population and PCR-RFLP method was used for the mutation analysis. Total 104 samples were analyzed and AA genotype was found in 10 individuals, AG genotype in 67 individuals and GG in 27 individuals. The frequencies of AA, AG and GG genotypes in the present study were also assessed with the expected distribution (i.e. Hardy-Weinberg equilibrium) by using χ2 test. The genotype frequencies of AA, AG and GG were 0.096, 0.644 and 0.259, respectively. Allelic frequencies of A and G were 0.418 and 0.581. It was reflected from the results of the present study that the percentage of heterozygous genotype (AG) is highest in the target population.
Received: June 29, 2010;
Accepted: May 11, 2011;
Published: June 03, 2011
Methionine synthase reductase (MTRR) gene is located on chromosome 5p15.2-15.3
and enzyme catalyzes the conversion of the inactive form of methionine synthase
(MTR) into its active form, by regeneration of methyl (III) cobalamin, the cofactor
of MTR. Mutation analysis of homocystinuria patients with severe deficiency
of methionine synthase reductase led to the discovery of a mutation that is
common in the general population (Wilson et al.,
1999). Wilson et al. (1999) identified this
common variant of MTRR (A66G) in which methionine replaces isoleucine in the
enzyme with a reported population frequency of ~30% (Wilson
et al., 1999; Gaughan et al., 2001;
Rady et al., 2002; Brilakis
et al., 2003). The I22M variant is located in the putative FMN-binding
domain of the MTRR enzyme that is suggested to interact with methionine synthase
(Leclerc et al., 1998). MTRR A66G polymorphism
was associated with an increase in plasma homocysteine, with the GG genotype
having a greater effect than the AG genotype (Gaughan et
al., 2001; Rady et al., 2002). During
homocysteine/methionine cycle, methionine is synthesized from homocysteine by
Vitamin B12-dependent methionine synthase. The methyl groups released
upon the transformation of methionine to homocysteine (S-adenosylmethionine
being the most important methyl-group donor) facilitate the methylation of DNA,
lipids and proteins. Higher level of homocysteine is itself toxic and an independent
risk factor for several complex disorders like-cardiovascular disease (Robinson
et al., 1995), Alzheimers disease (McCaddon et
al., 1998), Schizophrenia (Applebaum et al.,
2004) end stage renal disease (Van Guldener and Stehouwer,
2003), type II diabetes (De Luis et al., 2005)
and Neural tube defects (Mills et al., 1995)
etc. After Methylenetetrahydrofolate reductase (MTHFR), Methionine synthase
reductase (MTRR) is the second enzyme providing an active state for the remethylation
of homocysteine to methionine and when MTRR enzyme variants is coupled with
MTHFR polymorphism the combined deleterious effect may be additive. Several
reports about MTHFR C677T mutation frequency are available for Indian population
(Bhat et al., 2008; Saraswathy
et al., 2008) but not a single report regarding frequency of MTRR
A66G polymorphism is available from India and the rural population of Uttar
Pradesh is genetically unexplored. Hence, the present study aimed to evaluate
the mutant allele (G) frequency in the rural area of Uttar Pradesh.
MATERIALS AND METHODS
Samples: Total 104 healthy individuals were included in the present
study which are unrelated and randomly selected from the rural area of the UP.
Out of which 70 were males and 34 were females. Three milliliter blood was collected
from each subject by veinipuncture in EDTA coated vials. Informed consent was
obtained from each subject. The present study was conducted in the Human Molecular
Genetics Laboratory, Department of Biotechnology, VBS Purvanchal University,
Jaunpur, India during the period 2008-2009.
Genomic DNA extraction: Genomic DNA was extracted according to the method
of Bartlett and White (2003) and extracted genomic DNA
was kept at -20°C until the genotype analysis.
Genotype analysis: PCR-RFLP method was used for the genotype analysis.
For amplification primers as described by Wilson et al.
(1999) were used. The primers 5-GCAAAGGCCATCGCAGAAGACAT-3 and
5-GTGAAGATCTGCAGAAAATCCATGTA-3 were used to amplify 66-bp fragment.
PCR was performed in Multi Gene II thermo cycler (Labnet, USA) and the profile
consisted of an initial melting step of 4 min at 94°C, followed by 40 cycles
of 1 min denaturation at 94°C, 1 min annealing at 65°C, 1.30 min extension
at 72°C and a final elongation step of 10 min at 72°C. Amplification
and restricted products were analyzed by electrophoresis in 2 and 4% agarose
(Fermentas) gels, respectively. Allele frequencies were calculated using the
gene counting method. χ2 test was performed to test Hardy-Weinberg
RESULTS AND DISCUSSION
Amplification with MTRR gene specific primer generated 66 bp amplicon (Fig. 1) and after Nde I digestion homozygous AA genotype produced two bands of 44 and 22 bp, heterozygous AG genotype produced three bands 66,44 and 22 bp and GG genotype remained uncut (Fig. 2).
The distribution of the MTRR genotypes (AA, AG and GG) within the study population
is shown in Table 1, total 104 samples were analyzed for the
||MTRR amplification product of 66-bp for A66G polymorphism
Out of which AA genotype were found in 10 (9.61%) individuals, AG genotype
in 67 (64.42%) individuals and GG in 27 (25.96%) individuals (Fig.
3). The frequencies of AA, AG and GG genotypes in the present study were
also assessed with the expected distribution (i.e., Hardy-Weinberg equilibrium)
by using χ2 test. The genotype frequencies of AA, AG and GG
were 0.096, 0.644 and 0.259, respectively. Allelic frequencies of A and G were
0.418 and 0.581. It was reflected from the results of the present study that
the percentage of heterozygous genotype (AG) is highest in the target population.
||Nde I digested A66G polymorphism analysis. Lane 1 and 5: AA
(normal homozygous); Lane 2 and 6: AG (hetrozygous), Lane 3 and 7: GG (mutant
homozygous), Lane 4: M (Marker) 100-bp ladder; Lane 8: Undigested amplicon
||Bar diagram showing AA, AG and GG genotypes in UP population
||MTRR genotype and allele frequency distribution among Uttar
The world-wide frequency of A66G polymorphism is ~30% (Wilson
et al., 1999; Gaughan et al., 2001;
Brilakis et al., 2003). However, its frequency varies in different
ethnic and geographical regions as reported by Rady et
al. (2002) 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 present study the frequency of G allele (0.581)
is also higher than the A allele and is comparable with the Caucasian population
Olteanu et al. (2002) have reported that the
I22M variant (A66G) MTRR enzyme exhibits four-fold lower activity than the wild-type
protein in the reactivation of MTR in vivo. Hence the level of active
MTR is reduced and so the availability of SAM, as methyl donor is also decreased,
thus leading to DNA hypomethylation and it was pointed out by several studies
that the DNA hypomethylation is the main causative factor in the chromosome
missegregation, micronucleus formation and defective gene expression etc (Zijno
et al., 2003). Missegregation of chromosomes and altered gene expression
are the main causative factors behind the role of A66G polymorphism and as risk
factor of hereditary disorders (Hobbs et al., 2000;
Lee et al., 2006). Several epidemiological and
case control studies have already reported that the GG genotype may be a risk
factor for several disease/disorders like Neural tube defects (Bailey
et al., 2001; Gos and Szpecht-Potocka, 2002;
Pietrzyk et al., 2003; Relton
et al., 2004; Van der Linden et al.,
2006), Down syndrome (Hobbs et al., 2000;
Scala et al., 2006; Coppede,
2010), Coronory artery disease (Brilakis et al.,
2003), male infertility (Lee et al., 2006;
Ravel et al., 2009), Cancer (Zhang
et al., 2007) etc. Screening of different Indian population for such
clinically important gene polymorphism is urgently needed for proper genetic
counselling and disease management strategies.
We are grateful to the subjects who participated in this study without their
cooperation, this study could not be completed. The present study was supported
by grant No. BT/PR98887/SPD/11/1028/2007 to Vandana Rai and Sanjay Gupta from
Department of Biotechnology, Government of India, as Major Research Project.
1: Applebaum, J., H. Shimon, B.A. Sela, R.H. Belmaker and J. Levine, 2004. Homocysteine levels in newly admitted schizophrenia patients. J. Psychiatry Res., 38: 413-416.
2: Bailey, L.B., S. Moyers and J.F. Gregory, 2001. Folate. In: Present Knowledge in Nutrition, Bowman, B.A. and R.M. Russell (Eds.). International Life Sciences Institute, Washington, DC., pp: 214-229
3: Bartlett, J.M. and A. White, 2003. Extraction of DNA from Blood. In: Methods in Molecular Biology, PCR Protocls, Bartlett, J.M.S. and D. Stirling (Eds.). 2nd Edn., Humana Press Inc., Totowa, New Jersey
4: Bhat, T.A., M.R. Mir, I. Qasim, S.S. Misra, and M.A. Kirmani, 2008. Genetic polymorphism of 5,10-methylenetetrahydrofolate reductase C677T in Kashmiri population. Biotechnology, 7: 822-825.
5: Brilakis, E.S., P.B. Berger, K.V. Ballman and R. Rozen, 2003. Methylenetetrahydrofolate reductase (MTHFR) 677C>T and methionine synthase reductase (MTRR) 66A>G polymorphism association with serum homocysteine and angiographic coronary artery disease in the era of flour product fortified with folic acid. Atherosclerosis, 168: 315-322.
6: Coppede, F., 2010. Reflections on the possible role of the methionine synthase reductase (MTRR) A66G polymorphism as a maternal risk factor for Down syndrome in Italy. Am. J. Obstet. Gynecol., 202: e7-e8.
7: De Luis, D.A., N. Fernandez, M.L. Arranz, R. Aller, O. Izaola and E.J. Romeo, 2005. Total homocysteine levels relation with other cardiovascular risk factors in a population of patients with diabetes mellitus type 2. Diabetes Complications, 19: 42-46.
8: Gaughan, D., L.A.J. Kluijtmans, S. Barbaux, D. McMaster and I.S. Young et al., 2001. The methionine synthase reductase (MTRR) A66G polymorphism is a novel genetic determinant of plasma homocysteine concentrations. Atherosclerosis, 157: 451-456.
9: Gos, M. and A. Szpecht-Potocka, 2002. Genetic basis of neural tube defects. II. Genes correlated with folate and methionine metabolism. J. Applied Genet., 43: 511-524.
10: Hobbs, C.A., S.I. Sherman, P. Yi, C.P. Torfs and R.J. Hine et al., 2000. Polymorphism in genes involved in folate metabolism as maternal risk factors for Down syndrome. Am. J. Hum. Genet., 67: 623-630.
11: Lee, H.C., Y.M. Jeong, S.H. Lee, K.Y. Cha, S.H. Song, N.K. Kim, K.W. Lee and S. Lee, 2006. Association study of four polymorphisms in three folate-related enzyme genes with non-obstructive male infertility. Hum. Reprod., 21: 3162-3170.
12: Leclerc, D., A. Wilson and R. Dumas, C. Gafuik and D. Song et al., 1998. Cloning and mapping of a cDNA for methionine synthase reductase, a flavoprotein defective in patients with homocystinuria. Proc. Natl. Acad. Sci. USA., 95: 3059-3064.
13: McCaddon, A., G. Davies, P. Hudson, S. Tandy and H. Cattell, 1998. Total serum homocysteine in senile dementia of Alzheimer type. Int. J. Geriatr. Psychiatry, 13: 235-239.
14: Mills, J.L., J.M. Mcpartlin, P.N. Kirke, Y.J. Lee, M.R. Conley, D.G. Weir and J.M. Scott, 1995. Homocysteine metabolism in pregnancies complicated by neural tube defects. Lancet, 345: 149-152.
15: Olteanu, H., T. Munson and R. Banerjee, 2002. Differences in the efficiency of reductive activation of methionine synthase and exogenous electron acceptors between the common polymorphic variants of human methionine synthase reductase. Biochemistry, 41: 13378-13385.
16: Pietrzyk, J.J., M. Bik-Multanowski, M. Sanak and M. Twardowska, 2003. Polymorphism of 5,10-methylenetetrahydrofolate and methionine synthase reductase genes as independent risk factors for spina bifida. J. Applied Genet., 44: 111-113.
17: Rady, P.L., S. Szues, J. Grady, S.D. Hudnall and L.H. Kellner et al., 2002. Genetic polymorphisms of methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) in ethnic populations in Texas; a report of a novel MTHFR polymorphic site, G1793A. Am. J. Hum. Genet., 107: 162-168.
18: Ravel, C., S. Chantot-Bastaraud, C. Chalmey, L. Barreiro and I. Aknin et al., 2009. Lack of association between genetic polymorphisms in enzymes associated with folate metabolism and unexplained reduced sperm counts. Plos, 4: 6540-6540.
19: Relton, C.L., C.S. Wilding, M.S. Pearce, A.J. Laffling and P.A. Jonas et al., 2004. Gene-gene interaction in folate-related genes and risk of neural tube defects in a UK. population. J. Med. Gent., 41: 256-260.
20: Robinson, K., E.I. Mayer, P.D. Miller, R. Green and F. van Lente et al., 1995. Hyperhomocysteinemia and low pyridoxal phosphate common and independent reversible risk factors for coronary artery disease. Circulation, 92: 2825-2830.
Direct Link |
21: Saraswathy, K.N., R. Mukhopadhyay, E. Sinha, S. Aggarwal, M.P. Sachdeva and A.K. Kalla, 2008. MTHFR C677T polymorphisms among the Ahirs and Jats of Haryana (India). Am. J. Hum. Biol., 20: 116-117.
22: Scala, I., B. Granese, M. Sellitto, S. Salome and A. Sammartino et al., 2006. Analysis of seven maternal polymorphisms of genes involved in homocysteine/folate metabolism and risk of Down syndrome offspring. Genet. Med., 8: 409-416.
23: Van Guldener, C. and C.D. Stehouwer, 2003. Homocysteine metabolism in renal disease. Clin. Chem. Lab. Med., 41: 1412-1417.
24: Van der Linden, I.J.M., L.A. Afman, S.G. Heil and H.J. Blom, 2006. Genetic variation in genes of folate metabolism and neural-tube defect risk. Proc. Nutr. Soc., 65: 204-215.
25: Wilson, A., R. Platt, R.K. Wu, D. Leclerc, B. Christensen, H. Yang, R.A. Gravel and R. Rozen, 1999. A common variant in methionine synthase reductase combined with low cobalmin (vitamin B12) increased risk for spina bifida. Mol. Genet. Metabol., 67: 317-323.
26: Zhang, F.F., M.B.Terry, L. Hou, J. Chen and J. Lissowska et al., 2007. Genetic polymorphism in folate metabolism and the risk of stomach cancer. Cancer Epid. Mark., 16: 115-121.
27: Zijno, A., C. Andreli, F. Leopardi, F. Marcon and S. Rossi et al., 2003. Folate status, metabolic genotype,and biomarkers of genotoxicity in healthy subjects. Carcinogenesis, 24: 1097-1103.
Direct Link |