Original paper
Association of the rs1801133 variant in the MTHFR gene and sporadic Parkinson’s disease

Introduction

Parkinson’s disease (PD) is a multifactorial neurodegenerative disease that affects about 1-2% of people older than 65 years [23], sporadic cases (sPD) being more frequent than familial ones. Among genetic factors influencing PD, rare variants in PARK genes such as LRRK2 (PARK8) and SNCA (PARK1) [2,5] are known to play a major role in PD pathogenesis (rare variant common disease hypothesis) [3,14]. However, it is possible that common variants in other genes account for part of the unrevealed heritability of PD (the common variant common disease hypothesis) [16]. In this regard, the MTHFR gene has been recently proposed as a candidate risk gene for PD by two independent meta-analyses [24,25].
The enzyme MTHFR (EC 1.5.1.20) catalyzes the transformation of 5,10-methylenetetrahydrofolate to 5 methyltetrahydrofolate, a co-substrate for homocysteine remethylation to methionine. The T allele of the functional rs1801133 variant in this gene generates a thermolabile enzyme with reduced activity. The homozygous state of this variant (TT) has been linked to elevated plasma homocysteine (Hcy) levels [9], although optimal folate and vitamin B12 intake can counteract the effect of genotype [12]. Elevated Hcy may hasten dopaminergic cell death through oxidative stress and excitotoxicity [7,18].
Variants in the MTHFR gene may also influence the response to treatment, since impaired transmethylation potential has been detected in hyperhomocysteinemic L-dopa-treated PD patients [6].
Among different populations, Mexican-Mestizos (most present-day Mexicans) have the highest frequency of the T allele [20]; therefore we sought to determine whether rs1801133 is associated with SPD in our population.

Material and methods

Patients and controls

We conducted a case-control study that included 140 SPD patients and 216 neurologically healthy controls. Institutional Committees approved the study and informed written consent was obtained from participants. Patients were recruited from February 2009 to June 2010, from four tertiary-care level hospitals in Mexico (Neurology Departments from Centro Médico Nacional “20 de Noviembre” – ISSSTE, Centro Médico Nacional Siglo XXI-IMSS, Instituto de Ciencias Médicas y de la Nutrición “Salvador Zubirán”, Mexico City; and División de Genética, Centro de Investigación Biomédica de Occidente-IMSS, Jalisco, Mexico). Diagnosis was performed according to Queen Square Brain Bank criteria [15]. The threshold for early-onset Parkinson’s disease (EOPD) was considered as onset earlier than 40 years old. Cognitive impairment was assessed using the Folstein Mini Mental State Examination Test. We did not measure plasma Hcy levels because most PD patients could show elevated levels derived from pharmacological therapy with L-dopa. Controls were healthy blood bank donors or patients’ spouses who agreed to participate in an additional neurological evaluation; they were Mexican-Mestizos without family history of neurodegenerative disorders.

DNA isolation and genotyping

DNA was extracted from peripheral blood samples by the DTAB CTAB method [13]. Genotyping was performed by real-time PCR using TaqMan probes (Hydrolysis probes) using the C_1202883_20 assay (Applied Biosystems, Foster City, CA, USA). Real-time PCR was performed on a LightCycler 480 II (Roche Diagnostics GmbH, Switzerland); PCR reactions were conducted according to the manufacturer’s instructions. Random samples were confirmed by high resolution melting curves (Fig. 1). The samples were previously screened for common variants in six PARK genes, including A30P of SNCA and G2019S and G2385R of LRRK2; the prevalence of DNA changes was low [10].

Statistical analysis

Statistical analysis was performed using SPSS software v. 18.0 (SPSS Inc., Chicago, IL, USA) for the 2 test, logistic regression and ANOVA. Hardy-Weinberg equilibrium (HWE) was estimated in both groups using the 2 test (http://ihg.gsf.de/cgi-bin/hw/hwa1.pl [20/08/2013]). Statistical power was calculated a posteriori using Quanto Software Version 1.2.

Results

In total, 356 individuals were genotyped, 140 patients with SPD (95 males and 45 females, aged: 65.46 ± 11.5 [mean ± standard deviation] years old), age at onset 58 ± 13.66), whereas 216 healthy individuals constituted the control group (140 males and 76 females, aged: 63.68 ± 8.8 years old). Hardy-Weinberg equilibrium test showed that alleles were distributed according to expected frequencies in both groups (corrected p-values, controls p = 0.13, cases p = 1.00). Distribution of genotypes between groups is shown in Table I.
The association test showed that the C allele was associated with PD only under the recessive model (OR = 2.02, CI: 1.08-3.77, p = 0.02); after logistic regression for known confounding factors, the association remained significant (exp() = 2.06, CI: 1.101-3.873, p = 0.024). There was no association of any allele and cognitive impairment (p = 0.33). One-way ANOVA showed no differences between genotypes and age at onset (p > 0.05). Additionally, when age at onset was categorized (EOPD), the 2 test did not show an association of any allele or genotype and EOPD (p > 0.05) (TT group n = 31).

Discussion

In contrast to the hypothesis that the TT genotype of rs1801133 in the MTHFR gene leads to elevated levels of Hcy, cell death and therefore a higher risk of neurodegeneration [25], our data suggest that the CC genotype is associated with PD. This may be explained by the fact that the TT genotype has the greatest influence on Hcy levels in populations with low folate and high B12 vitamin plasma concentration such as Africans but not Mexican-Mestizos, in whom folate and B12 levels were high and moderate respectively [11]. Thus the TT genotype does not always imply high Hcy levels; in fact, a protective effect of the TT genotype against preeclampsia was reported in Maya-Mestizo women [1].
Others have also suggested that homozygosis of the T allele may confer a survival advantage in populations with sufficient dietary folate consumption [11,17,19]. Therefore, it is expected that in some populations such as Mexican-Mestizos, the T allele even when linked to an impaired biochemical function (elevated Hcy) may represent the wild-type allele, since an advantage may fix an allele within a population under particular environmental conditions. A presumptive advantage may exist, as described previously in an intervention where folate sources and dosages where controlled [4]. In contrast to its counterpart, the TT genotype showed only a slight decrease in global DNA methylation after folate depletion; conversely, under low folate basal levels, the TT genotype has shown significantly diminished global DNA methylation [8]. Thus, the finding that the derived CC genotype has the greatest decrease in DNA methylation after folate depletion may represent a different mechanism linked to neurodegeneration, besides the known effect of hyperhomocysteinemia on neuronal cytotoxicity [4].
The ancestral C allele may be considered the risk variant for PD in the Mexican-Mestizo population. Although it is speculative, hypomethylation as observed with the CC genotype under folate depletion may potentially impact epigenetic regulation of other genes such as LRRK2 and SNCA [21]. SNCA protein is also involved in the arrest of DNMT1 (a major element in epigenetic regulation) in postmortem brains of PD patients, worsening in this way the hypomethylation phenomenon [21]. Interestingly, another study found this same genotype (CC of rs1801133) related to earlier age at onset of PD [22]. Our data do not replicate the observation, probably because in our group patients with EOPD were uncommon. Larger studies documenting age at onset and MTHFR genotype may confirm this finding. To our knowledge this is the first report on association of the CC genotype of MTHFR and SPD. Other studies in different populations with larger samples may add support to our hypothesis in which convergent pathways between common and rare variants may potentially affect complex neurodegenerative disorders such as PD.

Limitations of the study

Some of the limitations of the present study were that since a considerable proportion of patients were treated with L-dopa among other anti-parkinsonian drugs, homocysteine levels or global methylation were not measured and therefore the genotype-phenotype correlation could not be explored to support our hypothesis. The presence of essential hypertension could not be assessed accurately, since the study was not intended to do so. The statistical power reached was 72% (lower than the expected 80%).

Acknowledgments

This project was financed by the E-015 institutional program (ISSSTE).
We are also grateful to Luis Javier Cano Martínez and Benjamín Gómez Díaz for useful suggestions and comments.

Disclosure

Authors report no conflict of interest.

References

1. Canto P, Canto-Cetina T, Juarez-Velazquez R, Rosas-Vargas H, Rangel-Villalobos H, Canizales-Quinteros S, Velazquez-Wong AC, Villarreal-Molina MT, Fernandez GCoral-Vazquez R. Methylenetetrahydrofolate reductase C677T and glutathione S-transferase P1 A313G are associated with a reduced risk of preeclampsia in Maya-Mestizo women. Hypertens Res 2008; 31: 1015-1019.
2. Cardo LF, Coto E, de Mena L, Ribacoba R, Lorenzo-Betancor O, Pastor P, Samaranch L, Mata IF, Diaz M, Moris G, Menendez M, Corao AIAlvarez V. A search for SNCA 3’ UTR variants identified SNP rs356165 as a determinant of disease risk and onset age in Parkinson’s disease. J Mol Neurosci 2012; 47: 425-430.
3. Clark LN, Wang Y, Karlins E, Saito L, Mejia-Santana H, Harris J, Louis ED, Cote LJ, Andrews H, Fahn S, Waters C, Ford B, Frucht S, Ottman RMarder K. Frequency of LRRK2 mutations in early- and late-onset Parkinson disease. Neurology 2006; 67: 1786-1791.
4. Crider KS, Quinlivan EP, Berry RJ, Hao L, Li Z, Maneval D, Yang TP, Rasmussen SA, Yang Q, Zhu JH, Hu DJ, Bailey LB. Genomic DNA methylation changes in response to folic acid supplementation in a population-based intervention study among women of reproductive age. PLoS One 2011; 6: e28144.
5. Chung SJ, Armasu SM, Biernacka JM, Lesnick TG, Rider DN, Lincoln SJ, Ortolaza AI, Farrer MJ, Cunningham JM, Rocca WA, Maraganore DM. Common variants in PARK loci and related genes and Parkinson’s disease. Mov Disord 2011; 26: 280-288.
6. De Bonis ML, Tessitore A, Pellecchia MT, Longo K, Salvatore A, Russo A, Ingrosso D, Zappia V, Barone P, Galletti P, Tedeschi G. Impaired transmethylation potential in Parkinson’s disease patients treated with L-Dopa. Neurosci Lett 2010; 468: 287-291.
7. Duan W, Ladenheim B, Cutler RG, Kruman, II, Cadet JL, Mattson MP. Dietary folate deficiency and elevated homocysteine levels endanger dopaminergic neurons in models of Parkinson’s disease. J Neurochem 2002; 80: 101-110.
8. Friso S, Choi SW, Girelli D, Mason JB, Dolnikowski GG, Bagley PJ, Olivieri O, Jacques PF, Rosenberg IH, Corrocher R, Selhub J. 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: 5606-5611.
9. Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RG, Boers GJ, den Heijer M, Kluijtmans LA, van den Heuvel LP et al. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat Genet 1995; 10: 111-113.
10. García S, López-Hernández LB, Suarez-Cuenca JA, Solano-Rojas M, Gallegos-Arreola MP, Gama-Moreno O, Valdez-Anguiano P, Canto P, Dávila-Maldonado L, Cuevas-García CF, Coral-Vázquez RM. Low prevalence of most frequent pathogenic variants of six PARK genes in sporadic Parkinson’s disease. Folia Neuropathol 2014; 52: 22-29.
11. Gueant-Rodriguez RM, Gueant JL, Debard R, Thirion S, Hong LX, Bronowicki JP, Namour F, Chabi NW, Sanni A, Anello G, Bosco P, Romano C, Amouzou E, Arrieta HR, Sanchez BE, Romano A, Herbeth B, Guilland JC, Mutchinick OM. 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: 701-707.
12. Gueant JL, Chabi NW, Gueant-Rodriguez RM, Mutchinick OM, Debard R, Payet C, Lu X, Villaume C, Bronowicki JP, Quadros EV, Sanni A, Amouzou E, Xia B, Chen M, Anello G, Bosco P, Romano C, Arrieta HR, Sanchez BE, Romano A, Herbeth B, Anwar W, Namour F. Environmental influence on the worldwide prevalence of a 776C->G variant in the transcobalamin gene (TCN2). J Med Genet 2007; 44: 363-367.
13. Gustincich S, Manfioletti G, Del Sal G, Schneider C, Carninci P. A fast method for high-quality genomic DNA extraction from whole human blood. Biotechniques 1991; 11: 298-300, 302.
14. Healy DG, Falchi M, O’Sullivan SS, Bonifati V, Durr A, Bressman S, Brice A, Aasly J, Zabetian CP, Goldwurm S, Ferreira JJ, Tolosa E, Kay DM, Klein C, Williams DR, Marras C, Lang AE, Wszolek ZK, Berciano J, Schapira AH, Lynch T, Bhatia KP, Gasser T, Lees AJ, Wood NW. Phenotype, genotype, and worldwide genetic penetrance of LRRK2-associated Parkinson’s disease: a case-control study. Lancet Neurol 2008; 7: 583-590.
15. Hughes AJ, Daniel SE, Ben-Shlomo Y, Lees AJ. The accuracy of diagnosis of parkinsonian syndromes in a specialist movement disorder service. Brain 2002; 125: 861-870.
16. Manolio TA, Collins FS, Cox NJ, Goldstein DB, Hindorff LA, Hunter DJ, McCarthy MI, Ramos EM, Cardon LR, Chakravarti A, Cho JH, Guttmacher AE, Kong A, Kruglyak L, Mardis E, Rotimi CN, Slatkin M, Valle D, Whittemore AS, Boehnke M, Clark AG, Eichler EE, Gibson G, Haines JL, Mackay TF, McCarroll SA, Visscher PM. Finding the missing heritability of complex diseases. Nature 2009; 461: 747-753.
17. Mayor-Olea A, Callejon G, Palomares AR, Jimenez AJ, Gaitan MJ, Rodriguez A, Ruiz M, Reyes-Engel A. Human genetic selection on the MTHFR 677C>T polymorphism. BMC Med Genet 2008; 9: 104.
18. Miller JW. Homocysteine, folate deficiency, and Parkinson’s disease. Nutr Rev 2002; 60: 410-413.
19. Munoz-Moran E, Dieguez-Lucena JL, Fernandez-Arcas N, Peran- Mesa S, Reyes-Engel A. Genetic selection and folate intake during pregnancy. Lancet 1998; 352: 1120-1121.
20. Mutchinick OM, Lopez MA, Luna L, Waxman J, Babinsky VE. High prevalence of the thermolabile methylenetetrahydrofolate reductase variant in Mexico: a country with a very high prevalence of neural tube defects. Mol Genet Metab 1999; 68: 461-467.
21. Tan YY, Wu L, Zhao ZB, Wang Y, Xiao Q, Liu J, Wang G, Ma JF, Chen SD. Methylation of alpha-synuclein and leucine-rich repeat kinase 2 in leukocyte DNA of Parkinson’s disease patients. Parkinsonism Relat Disord 2014; 20: 308-313.
22. Vallelunga A, Pegoraro V, Pilleri M, Biundo R, De Iuliis A, Marchetti M, Facchini S, Formento Dojot P, Antonini A. The MTHFR C677T polymorphism modifies age at onset in Parkinson’s disease. Neurol Sci 2014; 35: 73-77.
23. Wickremaratchi MM, Perera D, O’Loghlen C, Sastry D, Morgan E, Jones A, Edwards P, Robertson NP, Butler C, Morris HR, Ben-Shlomo Y. Prevalence and age of onset of Parkinson’s disease in Cardiff: a community based cross sectional study and meta-analysis. J Neurol Neurosurg Psychiatry 2009; 80: 805-807.
24. Wu YL, Ding XX, Sun YH, Yang HY, Sun L. Methylenetetrahydrofolate reductase (MTHFR) C677T/A1298C polymorphisms and susceptibility to Parkinson’s disease: a meta-analysis. J Neurol Sci 2013; 335: 14-21.
25. Zhu ZG, Ai QL, Wang WM, Xiao ZC. Meta-analysis supports association of a functional SNP (rs1801133) in the MTHFR gene with Parkinson’s disease. Gene 2013; 531: 78-83.