Thymidylate synthase promoter tandem repeat and MTHFD1 R653Q polymorphisms modulate the risk for migraine conferred by the MTHFR T677 allele

Molecular Brain Research 139 (2005) 163 – 168 www.elsevier.com/locate/molbrainres

Research Report

Thymidylate synthase promoter tandem repeat and MTHFD1 R653Q polymorphisms modulate the risk for migraine conferred by the MTHFR T677 allele Agustı´n Oterinoa,*, Natalia Vallea, Julio Pascuala, Yolanda Bravoa, Pedro Mun˜ozc, Jesu´s Castillod, Carlos Ruiz-Alegrı´ab, Pablo Sa´nchez-Velascob, Francisco Leyva-Cobia´nb, Carmen Cida a

Services of Neurology, University Hospital Marque´s de Valdecilla (UC), Santander-39008, Spain b Immunology, University Hospital Marque´s de Valdecilla (UC), Spain c Primary Care Management, Cantabria, Spain d Health Center of Camargo, Cantabria, Spain Accepted 12 May 2005 Available online 13 June 2005

Abstract There is growing evidence that folate metabolism is involved in migraine pathophysiology, mainly in migraine with aura. Even though folate metabolism is regulated by a number of enzymes, only two functional polymorphisms have been tested in association studies with migraine. Here, we have explored the possible role in migraine of other folate-metabolizing enzymes which are in close interdependency with 5V,10V-methylenetetrahydrofolate reductase analyzing functional polymorphisms of these enzymes in a case-control study. Individually, thymidylate synthase (TS), methenyltetrahydrofolate cyclohydrolase formyltetrahydrofolate synthase (MTHFD1), or methionine synthase (MS) polymorphisms did not modify the general risk for suffering migraine. Nevertheless, we observed a strong interaction between TS and MTHFR mutated genotypes, which increased over 8-fold the risk for experiencing aura among migraineurs; MTHFD1 and MTHFR mutated genotypes also increased together the risk for migraine in general (OR = 3.08; 95% CI = 1.3 – 7.4). We conclude that the pathogenetic role of the MTHFR T677 allele in migraine is modulated by functional polymorphisms of TS and MTHFD1. D 2005 Elsevier B.V. All rights reserved. Theme: Migraine genetics Topic: Folate metabolism Keywords: Migraine; Genetic association study; Thymidylate synthase; Methenyltetrahydrofolate cyclohydrolase formyltetrahydrofolate synthase; Methylenetetrahydrofolate reductase

1. Introduction Homocysteine has been implicated in the pathophysiology of a variety of neurological disorders. Two polymorphisms of 5V,10V-methylenetetrahydrofolate reductase (MTHFR), C677T and A1298C, that yield higher homocysteine plasma

* Corresponding author. Fax: +34 942202655. E-mail addresses: [email protected], [email protected] (A. Oterino). 0169-328X/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.molbrainres.2005.05.015

levels, have been associated with migraine in three independent case-control studies [11,14,15]. We previously failed to demonstrate a significant genetic association between migraine in general and the MTHFR C677T polymorphism in a case-control study [21]. Nevertheless, we observed a significant genetic association of the mutant MTHFR T677 allele and the risk for experiencing migraine aura among migraineurs. Folate metabolism is under the influence of several enzymes and cofactors such as B12, B2, and folic acid; thus, we hypothesized that other folate-metabolizing enzymes could also influence the risk for suffering migraine,

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either as independent factors or by modifying the risk conferred by the MTHFR C677T polymorphism. Regarding enzymes, thymidylate synthase (TS) encodes an enzyme that utilizes 5V,10V-methylenetetrahydrofolate (MTHF) as a cofactor to maintain the dTMP pool critical for DNA replication and repair. TS competes with MTHFR for the availability of MTHF, the substrate of MTHFR reduced into methylTHF [25]. Methionine synthase (MS) catalyzes the transfer of methyl base from 5-methylTHF to homocysteine, producing methionine and THF [2]. MS gene polymorphism D919G (aspartic to glycine) results from A to G transition at nucleotide 2756, and is thought to result in homocysteine elevation and DNA hypomethylation, and has been moderately associated with colorectal cancer [17] and vascular diseases [3,31]. Methylenetetrahydrofolate dehydrogenase, methenyltetrahydrofolate cyclohydrolase formyltetrahydrofolate synthetase (MTHFD1, [MIM 172460]) is a trifunctional nicotinamide adenine dinucleotide phosphatedependent cytoplasmic enzyme that has been associated with neural tube defects [1]. Also, one MTHFD1 function utilizes MTHF and competes for this substrate with MTHFR. Those MTHFR and MS alleles that reduce their respective enzymatic activity, and those TS or MTHFD1 alleles associated with higher gene expression or enzymatic activity will theoretically yield higher homocysteine plasma levels, and therefore might be associated with a higher risk for suffering migraine or at least migraine aura. In this rationale, here we have explored the potential implication in migraine of these three additional folate enzymes which are each other in close metabolic dependence with MTHFR.

2. Patients and methods 2.1. Subjects We recruited 329 migraine consecutive unrelated patients attending our general Neurological clinic (aged 38.9 T 14.9 years) and 237 healthy controls (aged 38.9 T 13.7 years) (Table 1). All of them were Caucasians and living in the same region. Patients with a history of stroke and controls with any type of migraine were excluded. All cases and healthy controls were interviewed by an experienced neurologist. The migraine patients were diagnosed as having IHS migraine without aura (MO) (191 cases; aged 42.5 T 13.7 years) and migraine with aura (MA) (138 cases; aged 35.0 T 12.6 years). Forty-three MA patients had also experienced MO attacks. Informed consent was obtained from all patients and controls. The protocol had been approved by our local ethics committee. 2.2. Genetic analysis Genotyping of each polymorphism was performed on leukocyte genomic DNA samples by polymerase chain reaction (PCR).

2.2.1. MTHFR polymorphism Identification of the HinfI restriction fragment-length polymorphism (RFLP) was performed using primers, PCR, and digestion conditions described elsewhere [14]. 2.2.2. TS tandem repeat polymorphism Presence of the tandem repeat polymorphism in the 5Vterminal of the regulatory region of the TS gene was detected basically using the protocol described by Skibola et al. [26], except for the amount of each primer used (10 pM) and by the addition of 10% of DMSO (vol:vol; SIGMAAldrich\) for each reaction. Amplified products were electrophoresed in 3% agarose gels. Wild-type allele containing two repeats (2R) is 220 bp long; 3R allele produces a 250-bp band, and 4R allele a 300-bp band. Table 1 Frequency distribution of genotypes and alleles of MTHFR C677T, TS 2R/3R, MS D919G, and MTHFD1 R653Q polymorphisms

N Females (%)

Migraine in general

MA

MO

Controls

329 77.5

138 76.8

191 78.0

237 61.6

Genotype (MTHFR)* CC 0.432 CT 0.447 TT 0.122 Alleles C 0.655 T 0.345 Genotype (TS) 2R2R 2R3R 3R3R Alleles 2R 3R Genotype MS DD DG GG Alleles D G

0.377 0.435 0.188**

0.471 0.455 0.073

0.397 0.481 0.122

0.594 0.406

0.699 0.301

0.637 0.363

0.229 0.479 0.293

0.225 0.449 0.326

0.232 0.500 0.268

0.162 0.560 0.278

0.475 0.525

0.449 0.551

0.482 0.518

0.442 0.558

0.769 0.201 0.030

0.790 0.196 0.014

0.754 0.204 0.042

0.791 0.183 0.026

0.878 0.122

0.883 0.117

0.874 0.126

0.893 0.107

0.225 0.486 0.290

0.215 0.508 0.277

0.220 0.472 0.306

0.485 0.515

0.469 0.531

0.457 0.543

Genotype (MTHD1) RR 0.219 RQ 0.498 QQ 0.283 Alleles R 0.475 Q 0.525

Hardy – Weinberg equilibrium was fitted for all the genotypes and clinical groups except for MS in MO group (2P = 0.02), and for TS in control group (2P = 0.04). * v 2 for the distribution of genotypes among MA, MO, and controls = 11.12 (4 df); P = 0.02. ** OR = 3.21 (1.6 – 6.6; P = 0.001), MA vs. MO; CC genotype as reference.

A. Oterino et al. / Molecular Brain Research 139 (2005) 163 – 168

2.2.3. Methionine synthase D919G polymorphism (MS) Genotyping for MS was carried out using a modification of the method of Leclerc et al. [16] implemented by Chen et al. [3]. 2.2.4. MTHFD1 R659Q polymorphism (MTHFD1) MTHFD1 R653Q polymorphism was analyzed essentially as described by Hol et al. [9], except for the restriction enzyme MspI was used for the RFLP identification. Reverse PCR primer for the MTHFD1 R653Q polymorphism flanks a 98-bp intron that allows the identification of the pseudogene located on X-chromosome on the basis of size difference (i.e., 330 bp for the functional MTHFD1 gene and 232 bp for the pseudogene). In addition, two internal mismatches in the reverse primer avoided the amplification of the pseudogene. 2.3. Statistical analysis Analyses were carried out using a logistic regression model to obtain maximum likelihood estimates, ORs and 95% CIs. Even though the aim of this study was exploratory, here we considered significant association when P values were lower or equal than 0.01 when multiple comparisons were made. Initially, we examined the possible association of each polymorphism and migraine univariately. For the categorical analyses, we report both the individual ORs and CIs of the non-wild-type (wild type is baseline), and overall v 2 test of association. In addition, we also examined possible gene – gene interaction. For statistical analysis, MA and MO patients were grouped together and termed ‘‘migraine in general’’. To gain statistical power, MTHFR 677CT/TT, TS 2R3R/3R3R, MS 919DG/GG, and MTHFD1 653RQ/QQ genotypes were further grouped. Genotypic and allelic frequencies, and calculations were computed using the SPSS program, version 11.5. Hardy –Weinberg equilibrium adjustment and differences in the frequency of MTHFR C677T, TS 2R/3R, MS D919G, and MTHFD1 R653Q alleles and genotypes were calculated by v 2 test. t test was used to compare continuous variables (a = 0.05).

3. Results 3.1. Univariate analysis 3.1.1. MTHFR C677T polymorphism After entering new subjects (plus 30 controls and 99 cases, 60 having MA) into this study, our observations are very similar to those previously published by us with regard to the role of MTHFR C677T polymorphism in migraine [21]. Again, we observed significant differences in the TT genotype distribution in MA patients versus MO (Table 1). TT genotype confers an over 3-fold risk for suffering from migraine aura among migraineurs. Except for an excess of T

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alleles in MA versus controls, comparisons among migraine clinical groups and controls failed to show an overall genetic association. 3.1.2. TS tandem repeat polymorphism The TS triple tandem repeat (3R) allele frequency was 55% in controls (slightly above the range of 38% to 54% previously reported in Caucasians) [19]. We did not observe significant differences on the 3R allele frequency distribution among clinical groups (Table 1). We also identified two subjects having both a 4R allele in heterozygous state who were ruled out for further genetic association (4R and higher expanded alleles are common in Africans an other populations) [18]. 3.1.3. MS D919G MS G919 allele has a low frequency in general population and also in other case-control studies [3]. This issue yields very low number of G homozygotes that lowered the statistical power of tests. In our sample, G allele was more frequent in migraine patients; but among migraineurs, G allele was even more frequent among those having MO than MA, contrary to that observed with TMTHFR allele. There was no statistically significant genetic association of migraine with this polymorphism (Table 1). 3.1.4. MTHFD1 R653Q Genotypic and allelic distributions of MTHFD1 R653Q polymorphism displayed minor, not significant differences among the distinct clinical groups (Table 1). 3.2. Bivariate analysis Following this univariate analysis, we next investigated for possible gene – gene interactions using a logistic regression model constructed with mutant genotypes and the effect of interaction of TS, MTHFD1, and MS each with MTHFR. Here, we observed potential statistical interaction between the mutated genotypes of TS and MTHFR (Table 2). While TS alone did not seem to modify the risk for migraine in general or migraine aura, the relative risk for suffering from migraine aura among migraineurs was over 8-fold. Furthermore, we observed that the relative risk for suffering from migraine aura among migraineurs in MTHFR CT/TT carriers varied over levels of TS tandem repeat (likelihood ratio for homogeneity of ORs = 14.86 with 2 df; P = 0.001). Those patients’ TS 3R3R homozygotes (OR = 2.63; 95% CI = 1.2 –6.4) carrying at least one T allele of the MTHFR had over 9-fold increased risk for suffering from migraine aura than those TS 2R2R homozygotes (OR = 0.29; 95% CI = 0.1– 0.7), and those MTHFR 677CT/TT migraineurs carrying one 3R allele had an intermediate risk for having aura (OR = 2.1; 95% CI = 1.2– 4.3). Using a model of logistic regression analysis, in which genotypes MTHFR 677CT/TT, TS 2R3R/3R3R, and interaction between these genotypes were entered, we observed

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Table 2 Logistic regression analysis Variables in the equationa

MGb vs. controls

MA vs. MO

MA vs. controls

MO vs. controls

T * 3R

0.72 (0.3 – 1.8); ns

8.67 (2.8 – 26.6); P = 0.00016

2.4 (0.8 – 7.2); ns

0.28 (0.1 – 0.8); P = 0.02

OR and 95% CI for MTHFR and TS mutated genotypes on migraine. a The effect of T-MTHFR and 3R-TS, as individual parameters, was not significant for each comparison in this model. b Migraine in general.

that the main effect is supported by the interaction between the two genes (OR = 8.67; 95% CI = 2.8 – 26.6; P < 0.001). The isolated effect of MTHFR 677CT/TT genotypes is shadowed when MTHFR * TS interaction enters the equation (Table 2). Using the same logistic regression model, comparing MA, migraine in general, or MO with controls, ORs obtained were not significant. Therefore, it seems that the main effect conferred by MTHFR 677TC/TT genotypes depends on their interaction with TS 3R alleles. We did not observe any significant interaction of MTHFR C677T and MS D919G polymorphisms. Those patients having MTHFR 677CT/TT and MS D919 have no additional risk (OR = 1.6; 95% CI = 0.9– 2.69) for suffering from migraine aura, whereas among MS 919DG/GG patients, the same relative risk decreases (OR = 1.0; 95% CI = 0.4 –2.7; ns). Here, logistic regression model failed to demonstrate genetic interaction; although the reduction in the estimated ORs among strata could suggest possible interaction, our study did not have the power to statistically detect this magnitude of interaction. Using the same model to detect gene – gene interaction between MTHFD1 and MTHFR, we observed (Table 3) that the MTHFR 677TC/TT interacts with MTHFD1 653RQ/QQ genotypes increasing the risk for having migraine in general (OR = 3.08; 95% CI = 1.3 –7.4, P = 0.01), mainly MO but also for MA. In this model, the effect of MTHFR 677TC/TT genotypes is not modified. Other types of interaction were not significant (i.e., TS * MTHFD1, TS * MS, MS * MTHFD1). 3.2.1. Influence of MTHFR, TS, MTHFD1, and MS polymorphisms on clinical aspects Age, frequency, and severity of attacks did not significantly influence the distribution of genotypes of these polymorphisms. There was a tendency to recognize worse performance at work for those patients having TS * 3R allele (36% vs. 55%; P = 0.002). As recalled by our patients, age at onset was later in life for patients having MTHFR * T allele (17.5 T 9.3 years of age) or TS * 3R (17.0 T 8.9 years of age) or for T * 3R interaction (18.6 T 14.65 years of age) than those having wild-type genotypes for these enzymes (14.8 T 6.9 years, P = 0.03; 13.6 T 5.2; P = 0.005,

respectively) or lacking T * 3R interaction (14.6 T 6.5, P = 0.004). The presence or absence of MO attacks in patients having MA (33%) did not alter genotypic or allelic distribution for any of the polymorphisms or their interactions (data not shown).

4. Discussion To our knowledge, this is the first study investigating the relevance of TS promoter tandem repeat polymorphism and MTHFD1 and migraine, and its interaction with MTHFR C677T alleles. Here, we failed to demonstrate genetic association of TS 2R/3R, MS D919G, MTHFD R653Q polymorphisms and migraine in general, MA or MO, neither for migraine aura among migraineurs. Nevertheless, our results indicate that the risk conferred by MTHFR 677TC/ TT genotypes is modulated well by the number of TS 3R promoter repeats and MTHFD1 653Q allele. Previously, we have reported that MTHFR C677T polymorphism could modify the risk for suffering migraine aura among migraineurs, but failed to discriminate from controls [21]. Other reports had found a significant association of MTHFR C677T polymorphism and migraine, mainly MA; but these studies have two main drawbacks: the relative small sample sizes and that both studies corresponded to specific populations (Japanese [14] and Turkish [11], respectively). In our study, again, with a higher sample size, we failed to demonstrate any genetic association of MTHFR C677T mutation and migraine in general, but we observed that migraine aura is over threefold more frequent among migraineurs carrying the TT homozygosis. Lea et al. [15] have very recently published similar results in other Caucasian population. They observed significant T allele excess only in MA (40%) versus controls (33%), and even T homozygosis was more frequent in controls (9%) than in MO patients (7%). Therefore, our results agree with those of Lea et al. and support the hypothesis that migraine aura and migraine do not necessarily share the same genetic background.

Table 3 OR and 95% CI for MTHFR and MTHFD1 mutated genotypes on migraine Variables in the equation

MG vs. controls

MA vs. MO

MA vs. controls

MO vs. controls

T-MTHFR Q635-MTHFD1 T*Q

0.62 (0.4 – 0.9); P = 0.03 0.87 (0.6 – 1.4); ns 3.08 (1.3 – 7.4); P = 0.01

1.52 (0.9 – 2.6); ns 0.92 (0.5 – 1.6); ns 0.9 (0.3 – 2.6); ns

0.79 (0.4 – 1.3); ns 0.83 (0.5 – 1.4); ns 2.93 (1.0 – 8.5); P = 0.049

0.52 (0.3 – 0.8); P = 0.009 0.9 (0.6 – 1.5); ns 3.25 (1.2 – 8.7); P = 0.018

A. Oterino et al. / Molecular Brain Research 139 (2005) 163 – 168

Folate metabolic pathway is controlled by several enzymes and cofactors like B12, B2, and folate, that are subject to dietary modifications which may alter the plasma levels of homocysteine; this metabolite plays a pivotal role in the pathophysiology related to the imbalance of folate route and methylation pathway. Allelic variants of MTHFR C677T [7] and TS 2R/3R [28] have been associated with higher homocysteine plasma levels, but not MTHFR A1298C [32]. MTHFR T677 is associated with reduced enzymatic activity. The resultant inhibition of the MTHF pathway leads to increased levels of methyleneTHF, and this elevation accelerates the methylation of uridylate to thymidylate. On the other hand, increased translational activity of TS competes with MTHFR for their substrate [12]. TS gene expression could likely affect the homocysteine and folate levels [28]. In addition to the highly significant association of interaction of TS and MTHFR mutant alleles to migraine aura, we have found a delay in the age at onset for those carriers having either of the mutant genotypes, such interaction between them being also significant. This observation may be related to some environmental factor acting differentially for both subpopulations. Horie et al. [10] have characterized the human TS promoter region, identifying several potential mechanisms for gene regulation. These studies found that the TS 28-bp triple repeat found in the 5VUTR near the ATG start codon led to gene expression that was 2.6 times greater than that found in the double repeat. At least one set of repeated sequence is necessary for efficient expression of the human TS gene [12]. Theoretically, TS and MTHFR compete for their common substrate 5V-10V-MTHF, but this effect is only afforded, in terms of the ability to increase plasma homocysteine, when MTHFR T677 allele, that decreases the MTHFR enzymatic activity to 30%, interacts with higher TS expression. Some association studies regard this possible interaction to explain the modification of risk conferred by MTHFR T677 allele, mainly in solid cancer and leukemias [20,29], and also in neural tube defects [22]. The effects of these polymorphisms varied with levels of dietary folate intake. Furthermore, interaction among genes that influence the flux of onecarbon moieties has been suggested and a common translational autoregulatory process that could couple the control of TS and SHMT1 gene expression has been proposed to control DNA and thymidylate synthesis [27]. Our observation of significant interaction between the TS and MTHFR genes, suggesting a gene-dose effect related to both the number of MTHF T677 and TS 3R alleles, may relate to some factor that varies between MTHFR C and T alleles that affects their biologic interaction with TS. However, whether TS tandem repeats really affect the interaction with MTHFR cannot be concluded from this study, and further biochemical studies are needed. MS D919G has been analyzed in casecontrol surveys for association with malignant lymphoma [20] and spina bifida [5], showing a higher risk for these diseases for carriers, maternally transmitted in the latter disease, of mutant G allele.

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In our sample, MS D919G does not modify the risk for migraine or migraine aura individually. Our results suggest that this polymorphism could modulate the risk conferred by MTHFR C677T for suffering migraine aura among migraineurs, but the observed low frequencies of the mutated MS G919 allele decreased our statistical power to detect interaction with MTHFR T677 allele. The genetic risk conferred by mutant folate-metabolizing allelic variants may be reduced with higher dietary folate intake. Such an environment– genetic interaction has been well assessed (vide supra) for cancer and neural tube defects. To date, surveys regarding genetic association of folate-metabolizing enzyme polymorphisms with migraine have not taken into account environmental factors such as folate and vitamin B complex (B2, B6, and B12) intake. A combination of diet, vitamin B, and folate status, and unfavorable genotypes may influence the risk for suffering migraine, especially the presence of aura among migraineurs. Evers et al. [6] found moderately elevated homocysteine plasma levels only in those migraineurs having aura, which was not corroborated by others [8]. Some previous studies have pointed out the possible role of folic acid, B2, and B12 in migraine patients [4,13,23,24,30]. An outstanding finding was that B2 had a more preventive effect on migraineurs having MA than those having MO [24]. As demonstrated in our study, many of the folatemetabolizing enzymes are polymorphic and may interact synergistically or even antagonistically. Therefore, the isolated analysis of one enzymatic variant may not disclose clear conclusions. These factors and their interactions should be further analyzed in a selected population. Finally, the accumulated evidence for the role, although moderate, of folate route in migraine, suggested in at least three different populations, is a consistent stimulus to undertake clinical trials with folate or vitamin B complex under the stratification of polymorphisms that we have studied here.

Acknowledgments This work was supported by Grants A28/02 of ‘‘Fundacio´n, Marque´s de Valdecilla’’ and by the ‘‘Centro Investigacio´n de Enfermedades, Neurolo´gicas’’, Nodo HUMV/ UC, ISCIII, Spain.

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