Nicotinamide-N-Methyltransferase gene rs694539 variant and migraine risk

Background Migraine is a common neurovascular disorder affecting 10 to 20 % of the world population usually subdivided into migraine with auro (MA) and migraine without auro (MO). Homocysteine is involved in the pathophysiology of a number of neurological disorders. Elevated levels of homocysteine in the plasma is produced by the MTHFR gene rs 1801133 and rs 1801131 variants as well as the NNMT gene rs 694539 variant. Methods With the polymerase chain reaction-restriction fragment length polymorphism method developed recently in our laboratory, we were able to show an association between the NNMT gene rs694539 variant and migraine for the first time. Results Here we report the association of the Nicotinamide-N-methyltransferase gene (NNMT) rs694539 variant with migraine in a case–control study of 433 patients with migraine and 229 healthy controls (χ2 = 6.076, P = 0.048). After stratification, we were able only to show an association between the NNMT gene rs694539 variant and female patients with migraine on the genotype and allelic levels. However there was no association in male patients with migraine (χ2 = 1.054, P = 0.590). Conclusions Consequently our results clearly indicate that the NNMT gene rs694539 variant is a genetic risk factor for migraine.


Background
Migraine is a common and chronic neurovascular disorder affecting approximately 10-20 % of the world population. Clinically, It is sub-classified into migraine with aura (MA) and migraine without aura (MO) accompanied by severe recurrent headache attacks and associated symptoms such as nausea, vomiting, photo-and phonophobia [1].
Recently, we showed that the MTHFR C677T and A1298C variants were associated with migraine [1]. We also wanted to reveal whether the rs694539 variant of NNMT gene is associated with migraine. To do so we analyzed the allele and genotype frequencies of the NNMT gene rs694539 variant in 433 patients with migraine and 229 healthy controls.  [26]. The institutional review board approved the present study and informed consent was obtained from all subjects.

Genotyping
Genomic DNA was isolated from whole blood using the salting-out procedure [27]. Genotypes of the subjects were determined using a polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) method developed in our laboratory [28].

Statistical analysis
The Hardy-Weinberg equilibrium was verified for both cases and controls. Differences between cases and controls were determined using the χ2 test and Student's t test. Genotype and allele frequencies were calculated using the χ2 test. Odds ratio (OR) and confidence interval (95 % CI) were estimated using 2x2 cross-tabulation and a binary logistic regression model for age and gender. All statistical analyses were done using SPSS software package version 21.0. The P value <0.05 % was taken as significant.

Results and discussion
Here, we report an association between the rs694539 variant of NNMT gene with migraine for the first time (χ2 = 6.076, P = 0.048). The statistical power for all cases is 0.40 and for overall controls is 0.24 (Table 1). The individuals with AA genotype confer a 3.8-fold increased risk for migraine (χ2 = 5.372, P = 0.020, OR = 3.840, 95 % CI = 1.133-13.013). Moreover, the G allele showed a 3.8-fold protection against migraine (χ2 = 5.372, P = 0.020, OR = 0.260, 95 % CI = 0.077-0.883). Stratification analysis according to gender revealed that there was an association only in female patients with migraine. The female individuals with the AA genotype revealed a 4. However there was no association in male patients with migraine (χ2 = 1.054, P = 0.590). All patients with migraine and controls were in Hardy-Weinberg equilibrium (0.665 and 0.152, respectively) ( Table 1).
Distributions of genotypes GG, GA and AA were 62.6, 32.6 and 4.8 % in overall patients with migraine and 68.1, 30.6 and 1.3 % in overall controls respectively ( Table 1). The frequencies of G allele were 78.87 % in overall cases and 83.40 % in overall controls ( Table 1). The frequencies of A allele were 21.13 % in overall migraine patients and 16.59 % in overall controls (Table 1).
In the case control studies, a number of polymorphisms have been shown to be associated with migraine. Plasma urotensin-2 level and thr21met variant have been reported to be associated with migraine [29]. It has been also shown that the glutamatergic system is involved in the pathophysiology of migraine [30]. DNA methylation status of RAMP1 gene has been shown to be associated with migraine [31]. Rs4379368 in the C7orf10 gene and rs13208321 in the FHL5 gene have been reported to be associated with migraine [32]. Genetic variants in the SYNE1 and TNF genes have been shown to be related to menstrual migraine [33].
Recently genome-wide association studies have implicated neuronal, vascular, metalloproteinase and pain pathways in migraine [34].
More recently, GWASs have identified several single nucleotide polymorphisms associated with migraine pathophysiology [35][36][37] in genes and in regulatory regions of genes implicated in epigenetic processes, including MTDH, MEF2D and PRDM16. Metadherin (MTDH) is shown to be associated with nuclear factor B (NFB) and a HAT to promote the expression of NFB target genes [38]. Myocyte enhancer factor 2D (MEF2D) can target methyltransferase complexes to specific genes to make them available for gene expression [39]. MEF2 has recently been shown to be modulated through the glucocorticoid receptor [40], which may be one of the mechanisms by which stress hormones affect the epigenome. Finally, PR domain containing 16 (PRDM16) is implicated in positioning and removing specific chromatin modifications at enhancer regions of Notch target genes during olfactory neuron differentiation [41]. These studies indicate that some of the migraine GWAS hits may contribute to developing migraine through epigenetic alterations at their target genes [42].
However, global DNA methylation has not been reported in patients with migraine, elevated homocysteine levels were revealed to be associated with cognitive impairment [44,45].

Conclusions
In summary, the role of the rs694539 variant of the NNMT gene in migraine is unclear. Nonetheless, through dysregulation of epigenetics and/or elevated homocysteine levels or dysregulation of nicotinamide levels may result in migraine or particularly through