The polymorphism 5,10-methylenetetrahydrofolate reductase (MTHFR) c.1298A>C is associated with various diseases. 45 DNA samples homozygous for the A allele and 40 DNA probes homozygous for the C allele were taken from healthy German subjects of white Caucasian origin to analyze the haplotype of the two MTHFR c.1298A>C alleles. Samples were genotyped for the polymorphism MTHFR c.677C>T and for the silent polymorphisms MTHFR c.129C>T, IVS2 533 G>A, c.1068C>T and IVS10 262C>G.
Findings
Haplotype construction revealed that the C-allele of MTHFR c.1298A>C was more frequently observed in cis with c.129T, IVS2 533A, c.677C, c.1068T, and IVS10 262 G than expected from normal distribution. Estimation of the most recent common ancestor with the DMLE + 2.3 program resulted in an estimated age of approximately 36,660 years of the MTHFR c.1298C allele.
Conclusion
Given that the era from 30,000 to 40,000 years ago is characterised by the spread of modern humans in Europe and that the prevalence of the MTHFR c.1298C allele is significantly higher in Central Europe in comparison to African populations, a selective advantage of MTHFR c.1298C could be assumed, e. g. by adaption to changes in the nutritional environment. The known founder ancestry of the T allele of MTHFR c.677C>T allele, together with the present data suggests that the MTHFR mutant alleles c.677T and 1298C arose from two independent ancestral alleles, that both confer a selective advantage.
Background
The monomeric enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR, EC 607093, OMIM 236250) catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate (5-MTHF). 5-MTHF is a methyl group donor for the remethylation of homocysteine to methionine. The T-allele of the MTHFR polymorphism c.677C>T (A222V, rs1801133) is associated with reduced MTHFR activity and, thus with an increased total plasma homocysteine level [1]. The frequency of the T-allele of MTHFR c.677C>T differs between ethnic groups and ranges from 6-10% in African countries [2, 3] to more than 17% in Caucasians in North America [1, 4, 5] and more than 50% in Mexican populations [3]. In the literature, the presence of the T-allele has been associated with cardiovascular and cerebrovascular diseases, venous thrombosis, neural tube defects and various cancers [6]. It was hypothesized that this might be due to increased plasma homocysteine levels in carriers of the MTHFR c.677T allele [7]. In addition, the allele's function for DNA synthesis and methylation could be an important factor for the development of such diseases [8]. Previously, we demonstrated that the polymorphism MTHFR c.677C>T (A222V, rs1801133) is associated with a defined MTHFR haplotype in the German population suggesting a founder effect [9]. This result could be confirmed by other groups for other populations [10].
In the present study, we investigated the second functionally relevant MTHFR SNP, the c.1298A>C (E429A, rs1801131) [11]. The derived C-allele of MTHFR c.1298A>C has been found to be associated with decreased MTHFR activity in lymphocytes and cultured human fibroblasts, although this effect was smaller than the effect shown for the T-allele of MTHFR c.677C>T [11–13]. However, the C-allele of the MTHFR c.1298A>C polymorphism could be linked to later onset of neurodegenerative diseases and a lower risk for different types of cancer [12, 14]; [15–18]. Here, we report haplotype data on the c.1298A>C polymorphism in a healthy German population selected by the c.1298A>C genotype suggesting a founder allele in the late ancestry.
Methods
The MTHFR c.1298A>C polymorphism (rs 1801131) was studied in samples of genomic DNA prepared from peripheral leukocytes of 500 healthy controls recruited for previous studies [19, 20]. All individuals were Caucasians from Germany living in the area of Bonn. The distribution of AA/AC/CC was 260/200/40 or 0.52/0.40/0.08, respectively, which was in line with the Hardy-Weinberg-Equilibrium. Out of 500 DNA samples, we selected 40 samples that were homozygous for the C allele and 45 samples demonstrating homozygosity for the A-allele. All samples were genotyped for the missense polymorphism MTHFR c.677C>T (rs 1801133) and for the silent polymorphisms MTHFR c.129C>T (rs 2066462), IVS2 533 G>A (polymorphism at nucleotide number 533 in intron 2 [21]), c.1068C>T (rs 2066462) and IVS10 262C>G (polymorphism at nucleotide number 262 in intron 10 [21, 22]. Haplotypes were constructed from genotypes using the maximum likelihood method (Software HAPMAX, developed by M. Krawczak, http://www.uni-kiel.de/medinfo/mitarbeiter/krawczak/download/) and analysed with a χ2 test (one degree of freedom). Estimation of the most recent common ancestor of the derived allele of MTHFR c.1298C was performed by means of the DMLE + 2.3 program http://www.dmle.org[23]. The chromosome map distance of the polymorphisms was calculated from the pubmed SNP database http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?locusId=4524 (Table 1). The population growth rate in Germany was set to 0.0075 [24], the current German population to 82,200,000 (Population Reference Bureau. 2008 World Population Data Sheet) and the mean allele frequency of the MTHFR c.1298C allele to 28% according to the results from the present study derived from 500 samples which was in line with results from other German populations [14, 20, 25, 26]. The generation length was defined as 20 years.
Table 1
Chromosome map distances
SNP
Contig position NT_021937
Distance in bp to c.129C>T
c.129C>T
6.400.338
0
IVS2 533 G>A
6.398.006
2.332
c.677C>T
6.393.745
6.593
c.1068C>T
6.392.263
8.076
c.1298A>C
6.391.843
8.495
IVS10 262C>G
6.388.372
11.966
The study was approved by the local ethical committee of the University Bonn, Germany, and all individuals gave written informed consent.
Results
Selection of DNA samples was based on their MTHFR c.1298A>C genotype. Therefore, we only performed haplotype analysis stratified for this genotype and not for the other MTHFR SNPs since representative distribution of these SNPs in our sample could not be assumed. HAPMAX analysis revealed a linkage disequilibrium of MTHFR c.1298A>C to the other SNPs. The C-allele of MTHFR c.1298A>C was more often observed in cis with c.129T; IVS2 533A; c.677C; c.1068T; IVS10 262 G than expected from a normal distribution referring to the haplotype data obtained for the wildtype A-allele (Table 2). Accordingly, automated haplotype construction revealed different haplotypes of maximum likelihood for c.1298A and c.1298C (Table 3). Estimation of the most recent common ancestor of the MTHFR c.1298A>C alleles with the help of the DMLE + 2.3 software showed a major peak of the posterior distribution of 1833 generations (95% credible set, 1678-2176 generations) (Figure 1). Given a generation length of 20 years, this finding translates in an age of approximately 36,600 years (33560-435200).
Table 2
Relative frequency of polymorphisms of A- and C-alleles of MTHFR c.1298A>C, Pearson's Chi-Square Test.
c.129C>T
IVS2 533 G>A
c.677C>T
c.1068C>T
IVS10 262C>G
C
T
Chi2;
p
G
A
Chi2;
p
C
T
Chi2;
p
C
T
Chi2;
p
C
G
Chi2;
p
1298 A
0.99
0.01
15.92
0.84
0.16
19.81
0.49
0.51
52.63
0.99
0.01
15.92
0.70
0.30
30.53
1298 C
0.81
0.19
< 0.001
0.52
0.48
< 0,001
0.99
0.01
< 0.001
0.81
0.19
< 0.001
0.28
0.72
< 0,01
Table 3
Haplotypes of A- and C-alleles of MTHFR c.1298A>C.
HT
c.129C>T
IVS2 533 G>A
c.677C>T
c.1068C>T
IVS10 262C>G
1298A
1298C
1
C
G
C
C
C
0.3201
0.1043
2
C
G
T
C
C
0.2956
0
3
C
G
T
C
G
0.1113
0
4
C
G
C
C
G
0.1026
0.2287
5
C
A
T
C
C
0.0661
0
6
C
A
C
C
G
0.0432
0.3468
7
C
A
T
C
G
0.0384
0
8
T
A
C
C
C
0.0114
0.1279
9
C
G
C
T
C
0.0114
0.1128
10
T
G
C
T
C
0
0.0390
11
T
A
C
T
C
0
0.0160
12
T
G
C
T
C
0
0.0122
13
C
G
T
T
C
0
0.0122
Haplotypes with an estimated frequency below 0.01 are not shown. Numbering refers to haplotypes of c.1298A alleles (1-13; the additional c.1298 haplotypes not observed in c.1298A (C) alleles were numbered consecutively regarding their frequency). Frequencies are shown as the ratio of total amount of each 1298A and 1298C-alleles.
Figure 1
Estimation of the most recent common ancestor of MTHFR c.1298C with the DMLE + 2.3 program.
Discussion
In our German population the A- and the C-allele of MTHFR c.1298A>C demonstrated different haplotypes. The most recent common ancestor of these alleles was calculated with approximately 36,600 years. However, analysis of additional populations is required to definitely determine the age of the ancestor allele.
The selection of samples for homozygosity was performed to achieve an easier allocation of haplotypes to the A versus C allele which would have been more difficult in heterozygotes (AC) for MTHFR c.1298A>C. Due to this biased selection, the observed frequencies of the other MTHFR SNPs are not representative for a general population. Nevertheless, this approach was feasible to approximate haplotype frequencies in A versus C alleles of MTHFR c.1298A>C and to estimate the age of the derived allele, c.1298C. Although the DMLE program predicted the most likely age to be approximately 36,600 years according to the major peak, our analysis revealed at least one additional lower peak (Figure 1) that might be artificial or due to the presence of an undefined subpopulation in our study cohort. This as well as the large confidence interval of the estimated age requires replication of our findings in additional samples and other populations.
Our age estimation of the most recent common ancestor of the studied alleles falls into a time period (30,000 to 40,000 years ago, the Aurignacian) which is typically associated with the spread of modern humans (Homo sapiens) in Europe [27, 28]. The frequency of the MTHFR c.1298C allele is significantly higher in central Europe in comparison to African populations [3]. In addition, dietary folate intake can profoundly modify the effect of the C-allele on disease association, and the availability and intake of folate is different in Europe and Africa [29]. In summary, this could indicate an increased frequency of the C-allele due to changes in the (nutritional) environment leading to selective advantages of the C-allele.
Presence of the C-allele has been associated with a later onset of neurodegenerative diseases [12, 14], better fertility (however, only observed for in vitro fertilisation) [30], rarer occurrence of chromosomal aberrations (Down's Syndrome [31]), and lower cancer risk [15–18]. However, adverse effects like an increased risk of neural tube defects [11], hypertension [32] or acute myeloid leukemia in Brazilian children [33] were also described.
Besides historical genetic aspects, the haplotypes presented in this study might be helpful for perinatal diagnosis of MTHFR deficiency based on haplotype analysis in the case of an unknown MTHFR mutation (OMIM 236250). Furthermore, haplotypes might be used for loss of heterozygosity studies comprising chromosome 1p36, which is involved e.g. in glioblastoma formation [34]. In addition, the known linkage disequilibrium of the two MTHFR c.677T and c.1298C alleles [35, 36] was reconfirmed in our study.
Conclusion
The previously described founder effect for the c.677T allele [9] supported by the present data suggests that the MTHFR c.677T and c.1298C alleles arose from two independent ancestral alleles, that both confer a selective advantage.
Declarations
Acknowledgements
We are very grateful to Dr Jeff Reeve, Department of Medical Genetics, University Alberta, Canada, in guiding us through the DMLE + 2.3. program.
Authors’ Affiliations
(1)
Department of Neurology, University Zurich
(2)
Department of Neurology, University Bonn
(3)
Institute of Molecular Medicine
References
Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RG, Boers GJ, den Heijer M, Kluijtmans LA, van den Heuvel LP: A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. NatGenet. 1995, 10 (1): 111-113.Google Scholar
Schneider JA, Rees DC, Liu YT, Clegg JB: Worldwide distribution of a common methylenetetrahydrofolate reductase mutation. AmJHumGenet. 1998, 62 (5): 1258-1260.Google Scholar
Gueant-Rodriguez RM, Gueant JL, Debard R, Thirion S, Hong LX, Bronowicki JP, Namour F, Chabi NW, Sanni A, Anello G, et al: Prevalence of methylenetetrahydrofolate reductase 677T and 1298C alleles and folate status: a comparative study in Mexican, West African, and European populations. Am J Clin Nutr. 2006, 83 (3): 701-707.PubMedGoogle Scholar
Ma J, Stampfer MJ, Christensen B, Giovannucci E, Hunter DJ, Chen J, Willett WC, Selhub J, Hennekens CH, Gravel R, et al: A polymorphism of the methionine synthase gene: association with plasma folate, vitamin B12, homocyst(e)ine, and colorectal cancer risk. Cancer EpidemiolBiomarkers Prev. 1999, 8 (9): 825-829.Google Scholar
Jacques PF, Bostom AG, Williams RR, Ellison RC, Eckfeldt JH, Rosenberg IH, Selhub J, Rozen R: Relation between folate status, a common mutation in methylenetetrahydrofolate reductase, and plasma homocysteine concentrations. Circulation. 1996, 93 (1): 7-9.PubMedView ArticleGoogle Scholar
Fodinger M, Horl WH, Sunder-Plassmann G: Molecular biology of 5,10-methylenetetrahydrofolate reductase. JNephrol. 2000, 13 (1): 20-33.Google Scholar
Klerk M, Verhoef P, Clarke R, Blom HJ, Kok FJ, Schouten EG: MTHFR 677C-->T polymorphism and risk of coronary heart disease: a meta-analysis. Jama. 2002, 288 (16): 2023-2031. 10.1001/jama.288.16.2023.PubMedView ArticleGoogle Scholar
Sohn KJ, Jang H, Campan M, Weisenberger DJ, Dickhout J, Wang YC, Cho RC, Yates Z, Lucock M, Chiang EP, et al: The methylenetetrahydrofolate reductase C677T mutation induces cell-specific changes in genomic DNA methylation and uracil misincorporation: a possible molecular basis for the site-specific cancer risk modification. Int J Cancer. 2009, 124 (9): 1999-2005. 10.1002/ijc.24003.PubMedPubMed CentralView ArticleGoogle Scholar
Linnebank M, Homberger A, Nowak-Gottl U, Koch HG: A common haplotype for the 677T thermolabile variant of the 5,10-methylenetetrahydrofolate reductase gene in thrombophilic patients and controls. HumMutat. 2002, 20 (6): 478-Google Scholar
Ogino S, Wilson RB: Genotype and haplotype distributions of MTHFR677C>T and 1298A>C single nucleotide polymorphisms: a meta-analysis. J Hum Genet. 2003, 48 (1): 1-7. 10.1007/s100380300000.PubMedView ArticleGoogle Scholar
van der Put NM, Gabreels F, Stevens EM, Smeitink JA, Trijbels FJ, Eskes TK, van den Heuvel LP, Blom HJ: A second common mutation in the methylenetetrahydrofolate reductase gene: an additional risk factor for neural-tube defects?. AmJHumGenet. 1998, 62 (5): 1044-1051.Google Scholar
Linnebank M, Linnebank A, Jeub M, Klockgether T, Wullner U, Kolsch H, Heun R, Koch HG, Suormala T, Fowler B: Lack of genetic dispositions to hyperhomocysteinemia in Alzheimer disease. AmJMedGenetA. 2004, 131 (1): 101-102.Google Scholar
Weisberg I, Tran P, Christensen B, Sibani S, Rozen R: A second genetic polymorphism in methylenetetrahydrofolate reductase (MTHFR) associated with decreased enzyme activity. MolGenetMetab. 1998, 64 (3): 169-172.Google Scholar
Wullner U, Kolsch H, Linnebank M: Methylenetetrahydrofolate reductase in Parkinson's disease. AnnNeurol. 2005, 58 (6): 972-973.Google Scholar
Skibola CF, Smith MT, Kane E, Roman E, Rollinson S, Cartwright RA, Morgan G: Polymorphisms in the methylenetetrahydrofolate reductase gene are associated with susceptibility to acute leukemia in adults. ProcNatlAcadSciUSA. 1999, 96 (22): 12810-12815.View ArticleGoogle Scholar
Matsuo K, Hamajima N, Suzuki R, Ogura M, Kagami Y, Taji H, Yasue T, Mueller NE, Nakamura S, Seto M, et al: Methylenetetrahydrofolate reductase gene (MTHFR) polymorphisms and reduced risk of malignant lymphoma. AmJHematol. 2004, 77 (4): 351-357.Google Scholar
Keku T, Millikan R, Worley K, Winkel S, Eaton A, Biscocho L, Martin C, Sandler R: 5,10-Methylenetetrahydrofolate reductase codon 677 and 1298 polymorphisms and colon cancer in African Americans and whites. Cancer EpidemiolBiomarkers Prev. 2002, 11 (12): 1611-1621.Google Scholar
Wang J, Gajalakshmi V, Jiang J, Kuriki K, Suzuki S, Nagaya T, Nakamura S, Akasaka S, Ishikawa H, Tokudome S: Associations between 5,10-methylenetetrahydrofolate reductase codon 677 and 1298 genetic polymorphisms and environmental factors with reference to susceptibility to colorectal cancer: a case-control study in an Indian population. IntJCancer. 2006, 118 (4): 991-997.Google Scholar
Moskau S, Golla A, Grothe C, Boes M, Pohl C, Klockgether T: Heritability of carotid artery atherosclerotic lesions: an ultrasound study in 154 families. Stroke. 2005, 36 (1): 5-8. 10.1161/01.STR.0000149936.33498.83.PubMedView ArticleGoogle Scholar
Linnebank M, Fliessbach K, Kolsch H, Rietschel M, Wullner U: The methionine synthase polymorphism c.2756Aright curved arrow G (D919G) is relevant for disease-free longevity. IntJMolMed. 2005, 16 (4): 759-761.Google Scholar
Rosenberg N, Murata M, Ikeda Y, Opare-Sem O, Zivelin A, Geffen E, Seligsohn U: The frequent 5,10-methylenetetrahydrofolate reductase C677T polymorphism is associated with a common haplotype in whites, Japanese, and Africans. AmJHumGenet. 2002, 70 (3): 758-762.Google Scholar
Linnebank M, Homberger A, Koch HG, Bova I, Sylantiev C, Bornstein NM, Chapman J, Korczyn AD: Frequent polymorphism of the human methylenetetrahydrofolate reductase. Stroke. 2000, 31 (4): 990-PubMedView ArticleGoogle Scholar
Rannala B, Reeve JP: Joint Bayesian estimation of mutation location and age using linkage disequilibrium. Pac Symp Biocomput. 2003, 526-534.Google Scholar
Pritchard JK, Seielstad MT, Perez-Lezaun A, Feldman MW: Population growth of human Y chromosomes: a study of Y chromosome microsatellites. Mol Biol Evol. 1999, 16 (12): 1791-1798.PubMedView ArticleGoogle Scholar
Klotz L, Farkas M, Bain N, Keskitalo S, Semmler A, Ineichen B, Jelcic J, Klockgether T, Kolsch H, Weller M, et al: The variant methylenetetrahydrofolate reductase c.1298A>C (p.E429A) is associated with multiple sclerosis in a German case-control study. Neurosci Lett. 2009Google Scholar
Semmler A, Linnebank M, Krex D, Gotz A, Moskau S, Ziegler A, Simon M: Polymorphisms of Homocysteine Metabolism Are Associated with Intracranial Aneurysms. Cerebrovasc Dis. 2008, 26 (4): 425-429. 10.1159/000155638.PubMedView ArticleGoogle Scholar
Bailey SE, Weaver TD, Hublin JJ: Who made the Aurignacian and other early Upper Paleolithic industries?. J Hum Evol. 2009, 57 (1): 11-26. 10.1016/j.jhevol.2009.02.003.PubMedView ArticleGoogle Scholar
Mellars PA: Archaeology and the population-dispersal hypothesis of modern human origins in Europe. Philos Trans R Soc Lond B Biol Sci. 1992, 337 (1280): 225-234. 10.1098/rstb.1992.0100.PubMedView ArticleGoogle Scholar
Xu WH, Shrubsole MJ, Xiang YB, Cai Q, Zhao GM, Ruan ZX, Cheng JR, Zheng W, Shu XO: Dietary folate intake, MTHFR genetic polymorphisms, and the risk of endometrial cancer among Chinese women. Cancer Epidemiol Biomarkers Prev. 2007, 16 (2): 281-287. 10.1158/1055-9965.EPI-06-0798.PubMedView ArticleGoogle Scholar
Haggarty P, McCallum H, McBain H, Andrews K, Duthie S, McNeill G, Templeton A, Haites N, Campbell D, Bhattacharya S: Effect of B vitamins and genetics on success of in-vitro fertilisation: prospective cohort study. Lancet. 2006, 367 (9521): 1513-1519. 10.1016/S0140-6736(06)68651-0.PubMedView ArticleGoogle Scholar
Rai AK, Singh S, Mehta S, Kumar A, Pandey LK, Raman R: MTHFR C677T and A1298C polymorphisms are risk factors for Down's syndrome in Indian mothers. JHumGenet. 2006, 51 (4): 278-283.Google Scholar
Markan S, Sachdeva M, Sehrawat BS, Kumari S, Jain S, Khullar M: MTHFR 677 CT/MTHFR 1298 CC genotypes are associated with increased risk of hypertension in Indians. Mol Cell Biochem. 2007Google Scholar
da Costa Ramos FJ, Cartaxo Muniz MT, Silva VC, Araujo M, Leite EP, Freitas EM, Zanrosso CW, Hatagima A, de Mello MP, Yunes JA, et al: Association between the MTHFR A1298C polymorphism and increased risk of acute myeloid leukemia in Brazilian children. Leuk Lymphoma. 2006, 47 (10): 2070-2075. 10.1080/10428190600800132.PubMedView ArticleGoogle Scholar
Houillier C, Lejeune J, Benouaich-Amiel A, Laigle-Donadey F, Criniere E, Mokhtari K, Thillet J, Delattre JY, Hoang-Xuan K, Sanson M: Prognostic impact of molecular markers in a series of 220 primary glioblastomas. Cancer. 2006, 106 (10): 2218-2223. 10.1002/cncr.21819.PubMedView ArticleGoogle Scholar
Linnebank M, Homberger A, Nowak-Gottl U, Marquardt T, Harms E, Koch HG: Linkage disequilibrium of the common mutations 677C>T and 1298A>C of the human methylenetetrahydrofolate reductase gene as proven by the novel polymorphisms 129C>T, 1068C>T. EurJPediatr. 2000, 159 (6): 472-473.Google Scholar
Stegmann K, Ziegler A, Ngo ET, Kohlschmidt N, Schroter B, Ermert A, Koch MC: Linkage disequilibrium of MTHFR genotypes 677C/T-1298A/C in the German population and association studies in probands with neural tube defects(NTD). AmJMedGenet. 1999, 87 (1): 23-29.Google Scholar
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
