Vitamin D receptor and binding protein genes variants in patients with migraine

Abstract Background/Objectives Several studies have shown a relationship between vitamin D and migraine, including the association between decreased serum 25‐hydroxyvitamin D in patients with migraine and the positive effects of vitamin D supplementations in the therapy of this disease. Two single‐nucleotide variants (SNVs) vitamin D receptor (VDR) gene, VDR rs2228570, and VDR rs731236 have shown an association with migraine risk in a previous case–control association study, while an exome sequencing study identified a rare variant in GC vitamin D binding protein gene. This study aims to look for the association between several common variants in these two genes and the risk for migraine. Methods We genotyped 290 patients diagnosed with migraine and 300 age‐matched controls using specific TaqMan assays for VDR rs2228570, VDR rs731236, VDR rs7975232, VDR rs739837, VDR rs78783628, GC rs7041, and GC rs4588 SNVs. Results We did not find an association between these SNVs and the risk for migraine. None of these SNVs were related to the positivity of a family history of migraine or with the presence of aura. The VDR rs731236A allele showed a significant association with the triggering of migraine attacks by ethanol (Pc = 0.007). Conclusions In summary, the results of the current study suggest a lack of association between common SNVs in the VDR and GC gene and the risk of developing migraine. The possible relationship between VDR rs731236 and the triggering of migraine episodes with ethanol deserves future studies.


Introduction
Migraine affects 10%-18% of the population, approximately 2-3 women for each man, therefore being one of the most frequent neurological disorders.Both patients diagnosed with migraine with aura (MWA) and migraine without aura (MWoA) refer very frequently (50%-70%) positive family history of migraine and, on the other hand, individuals with first-degree parents affected with MWA or MWoA have increased risk for both MWA and MWoA have shown an increased risk for these conditions.Despite these data suggesting an important role of genetic factors in the risk for migraine, the genetics of this disease is not completely established, except for the identification of CACNA1A, ATP1A2, and SCN1A genes as causative for familial hemiplegic migraine. 1A recent meta-analysis of previous hypothesis-free genome-wide association studies (GWAS), which involved 102,084 migraine cases and 771,257 controls identified 123 susceptibility loci for migraine 86 of them previously unknown. 2 A lot of hypothesis-driven case-control association studies involving many candidate genes have been reported during the last 30 years, but the results of these studies regarding association with migraine have been inconsistent.It is out of the scope of the present article a detailed revision of these studies.
There are many studies describing a relationship between serum 25-hydroxyvitamin D3 levels and migraine.Mottaghi et al. 3 described a weak correlation of serum vitamin D with the frequency of migraine attacks but not with migraine severity.Several authors found decreased levels of serum vitamin D levels in patients with migraine compared with controls, [4][5][6][7] or with reference values, 8 that was more marked 5 or similar 6 in patients with chronic migraine than in those with episodic migraine, and a correlation between vitamin D status and headache frequency 7,8 or severity 7 has been described in several studies.In contrast, other authors found similar serum 25-hydroxyvitamin D3 levels in migraine patients and controls, 9,10 and a lack of correlation of serum vitamin D status with the severity of headache. 9A recent meta-analysis of previous publications confirmed decreased serum 25-hydroxyvitamin D levels in patients with migraine compared with controls. 11inally, a Mendelian randomization study involving three migraine datasets (48,975, 28,852, and 10,536 cases each) described an association between increased serum vitamin D levels and decreased risk for migraine. 12uettner et al. 13 described decreased risk for a headache of migraine type (although not with the severity of headache) in subjects with higher serum levels of vitamin D and statin use, in a series of 5938 participants.Serum vitamin D receptor levels have been found to be decreased, and serum vitamin D-binding protein levels are similar in migraine patients compared with controls in a single study. 4everal randomized clinical trials have shown a beneficial effect of vitamin D supplementation in the therapy of migraine in adults [14][15][16][17][18] and children, 19 one of them using vitamin D in combination with simvastatin, 14 and another showing a synergistic effect with topiramate.Two meta-analyses of randomized clinical trials showed improvement in migraine based on a reduced number of headache days, 20,21 the frequency of headache attacks. 20,21eadache severity, 20 and Migraine Disability Assessment Score, 20,21 but no effect in the duration of the migraine attack. 21In one study, patients under vitamin D therapy showed a reduction in serum levels of inducible nitric oxide synthase (iNOS) and a trend towards lower serum interleukin 6 (IL-6) levels compared with the placebo group, while serum levels of IL-10 and cyclooxygenase-2 (Cox-2) were similar in both groups. 17Another study showed a significant reduction in the serum calcitonin gene-related peptide (CGRP, the most important mediator of migraine pain pathogenesis) in patients treated with vitamin D compared with those under placebo. 18he beneficial effect of vitamin D in migraine could be related to their effects on neuronal and immune homeostasis, and their anti-inflammatory and antioxidant effects 22 as inflammatory factors seem to play an important role in the pathogenesis of migraine. 23he nuclear hormone receptor for vitamin D3 is encoded by the VDR gene (link http://www.ncbi.nlm.nih.gov/gene/7421; chromosome 12q13.11;Gene ID 7421; MIM 601769), which, according to the gnomAD database, has five common single-nucleotide variants (SNV) with frequencies higher than 0.0010 and known functional impact in Caucasians: (a) rs2228570 (Fok1) causes an amino acid substitution (Met 1, start loss), (b) rs731236 (Taq1) is a synonymous SNV not causing amino acid substitution (Ile352Ile), (c and d) rs739837, and rs78783628 are two common variants located in the 3 0 untranslated area, and (e) rs7975232 (Apa1) is a common intronic variant.
Vitamin D binding protein, which binds to vitamin D and their plasma metabolites and acts as a transporter of vitamin D to target tissues, is encoded by the GC vitamin D binding protein gene (link http://www.ncbi.nlm.nih.gov/gene; chromosome 4q13.3;Gene ID 2638; MIM 139200).Two main SNVs in the GC gene with frequencies higher than 0.0025 and known functional impact affecting this gene have been described in Caucasians, according to the gnomAD database: (a) rs7041, which is a missense SNV causing the amino acid substitution Asp 451 Glu, and (b) and rs4588, which is another missense SNV causing the amino acid substitution Thr 455 Lys.][26] Two previous studies addressed the possible relationship between VDR or GC variants and the risk for migraine.Motaghi et al., 27 in a case-control association study involving 103 patients diagnosed with migraine with aura and 100 healthy controls from Iran, described an association of VDR rs2228570 and VDR rs731236 SNVs and the risk of migraine with aura.Nagata et al. 28 identified a variant (R21L) in the exon 2 of the GC vitamin D binding protein gene through linkage analysis and exome sequencing in a family with four affected individuals.
The main aim of our study is to replicate the finding of Motaghi et al. 27 In addition, we studied other common SNVs in the VDR and GC genes in Caucasian Spanish patients diagnosed with migraine and in healthy controls.

Patients and controls
We studied the genotype and allelic variants VDR rs2228570, VDR rs731236, VDR rs7975232, VDR rs739837, VDR rs78783628, GC rs7041, and GC rs4588 in 290 patients fulfilling standardized diagnostic criteria for migraine 29 and 300 age-and sex-matched controls.We recruited patients diagnosed with migraine, not suffering from other headache types or other neurological diseases, from the general neurologic clinics of several University Hospitals during 2 periods (197 between September 2006-September 2007 and 93 between June 2017 and February 2019).The healthy controls were mainly students of staff from the University of Extremadura who had neither personal nor family history and migraine and did not suffer from other types of headache), and were recruited during the same periods (215 in the first and 85 in the second ones).1][32][33][34][35][36][37][38][39][40] Table 1 summarizes the demographic data of both migraine patients and control groups.

Ethical aspects
The study was approved by the Ethics Committees of the University Hospital of Badajoz, University Hospital "Pr ıncipe de Asturias" (Alcal a de Henares, Madrid, Spain), Hospital La Mancha-Centro (Alc azar de San Juan, Ciudad Real, Spain), and the Ethics Committee of the province of C aceres, and was performed according to the principles of the Declaration of Helsinki.After fully explaining the objectives and the procedure, participants signed informed consent to be included in the study.

Statistical analysis
The statistical analysis was performed by using the SPSS 27.0 version for Windows (SPSS Inc., Chicago, Illinois, USA), and the confirmation of Hardy-Weinberg equilibrium in both groups with the online program https://ihg.gsf.de/cgi-bin/hw/hwa1.pl.The chi-squared test, or Fisher's exact test where appropriate, were used to calculate the intergroup comparison values.We also calculated the 95% confidence intervals and the negative predictive values 42 and used the False Discovery Rate (FDR) to perform the correction for multiple comparison adjustments. 43he sample size was calculated according to a genetic model that analyzed the frequency of the lower allele with an odds ratio (OR) value = 1.5 (a = 0.05) from the allelic frequencies found in healthy subjects.According to this sample, the statistical power (two-tailed association) for variant alleles was, respectively, 92.83% for VDR rs2228570, 93.5% for VDR rs731236, 92.5% for VDR rs7975232, 92.5% for VDR rs739837, 93.5% for VDR rs78783628, 93.3% for GC rs7041, and 92.0% for rs4588.The Student's t-test was used for the comparison of the mean AE SD age at the onset of migraine across the different genotypes of the SNVs studied.
We also analyzed the possible influence in the frequency of the genotype or allelic variants of migraine patients according to several variables which included (a) positive or negative family history of migraine, (b) presence or absence of aura, (c) triggering or not of migraine attacks with ethanol.The chi-squared test, Fisher's exact test, or the student t-tests were used where appropriate.
The report of this study has been performed according to broad EQUATOR guidelines, 44 specifically STROBE and STREGA checklists, which are summarized in Tables S1 and S2.

Results
The Hardy-Weinberg equilibrium was fulfilled by the frequencies of the genotypes and allelic variants of VDR rs2228570, VDR rs731236, VDR rs7975232, VDR rs739837, VDR rs78783628, GC rs7041, and GC rs4588 SNVs, both in migraine patients and in controls.The frequencies of most of the genotypes and allelic variants did not differ significantly between the two groups, with the exception of a lower frequency of the VDR rs731236 (A/ G) and GC rs4588 (G/T) variants, and a higher frequency of GC rs7041 (C/C) and GC rs4588 (G/G) variants in migraine patients, that disappeared after correction for multiple comparisons (Table 2).When analyzing each sex separately migraine women showed a lower frequency of the VDR rs731236 (A/G) genotype, which disappeared after correction for multiple comparisons as well (Table S3).
Mean AE SD age at onset of migraine attacks was significantly lower in patients carrying the VDR rs731236 (G/ G) genotype compared with those carrying the VDR rs731236 (A/G) genotype and in those carrying the GC rs7041 (A/C) compared with GC rs7041 (A/A) genotype (Table 3).The genotype and allele frequencies in patients with migraine were similar in patients with versus without positive family history of migraine (Table S4), and in patients with migraine with aura compared with those with migraine without aura (Table S4).The frequencies of VDR rs731236 (A/A) and VDR rs731236 (G/G) genotypes were, respectively, significantly higher and significantly lower in patients in which alcohol was a triggering factor for migraine attacks (Table S4).Despite these genotype differences disappearing after correction for multiple comparisons, the allele frequencies were different even after correction for multiple comparisons (Pc = 0.007; Table S4), and the test for trend for variant alleles showed a significant association of the VDR rs731236 SNV with the ethanol effect, with an OR = 0.58 (p = 0.0016).

Discussion
The decreased serum 25-hydroxyvitamin levels found in patients with migraine, together with the described beneficial effects of vitamin D in this disease make it reasonable to investigate the possible association between SNVs in vitamin D-related genes and the risk for migraine.
While Motaghi et al. 27 described the association of VDR rs2228570 and VDR rs731236 SNVs with the risk of migraine with aura, data from the current study, did not confirm an association of the five analyzed SNVs in the VDR gene with the risk for migraine, with or without aura, and we did not find association either in patients with or those without a family history of migraine.In addition, we did not find an association between the two most common SNVs in the GC vitamin D binding protein gene and migraine risk.On the other hand, subjects carrying the minor allele of VDR rs731236 SNV showed a lower frequency of induction of migraine attacks by ethanol, being this finding difficult to explain.
Association between SNVs in the VDR and GC vitamin D-binding protein genes and the risk for other neurological diseases has been a matter of several studies.VDR rs2228570 45,46 and VDR rs7975232 45 have been associated with the risk for Parkinson's disease, at least in the Asian population, 46 VDR rs731236 with the risk for Alzheimer's disease, 45,46 at least in Caucasians, 47 and VDR rs1544410 and VDR rs7975232, respectively, with increased and decreased risk for mild cognitive impairment. 47Pooled data of two studies 48,49 showed an association between VDR rs2228570 and essential tremor, while VDR rs731236, 49,50 VDR rs7975232, 49 VDR rs739837, 49 and VDR rs78783628 49 showed lack of association with this disease.Other described associations of SNVs in the VDR gene include an association of VDR rs7975232 and the risk for multiple sclerosis 51 and amyotrophic lateral sclerosis, 52 VDR rs2228570 with ischemic stroke, 53 VDR rs2228570 and VDR rs7975232 with childhood temporal lobe epilepsy, 54 and VDR rs731236 with adult nonthymoma myasthenia gravis with the negativity of acetylcholine receptors antibodies. 55An association of VDR rs2228570 and VDR rs731236 with the risk for restless legs syndrome reported in one study 41 was not confirmed in another. 56C rs7041 SNV has been associated with the risk for Parkinson's disease, 57 while GC rs7041, GC rs4588, and GC rs2282679 were not associated with the risk for multiple sclerosis, 58 and GC rs7041and GC rs4588 were not associated to restless legs syndrome.56 Taking into account the relatively low sample size as the main limitation of the current study, our results suggest the lack of association of the most common SNVs in the two genes analyzed with the risk for migraine in the Caucasian Spanish population.The finding on the possible influence of the VDR rs731236 SNV in triggering migraine attacks by ethanol deserves further studies.

ª
2023 The Authors.Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.

Table 1 .
Demographic and clinical data of the series studied.
ª 2023 The Authors.Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.

Table 2 .
VDR and GC genotypes of patients with migraine and healthy controls.

Table 3 .
Age at onset of migraine according to the VDR and GC genotypes.
Bold values indicate statistically significant of p-values.