The Polymorphism of Regulatory Region of NOS3 Gene (rs2070744, Genotype CC) Protect Patients from Chronic Migraine

Aims: There is evidence that endothelial nitric oxide synthase has a role in migraine pathophysiology. In our research, the role of SNP rs2070744 (c.-813C>T) in promoter region of NOS3 gene in the episodic and chronic forms of migraine is considered. Place Study: University Headache Clinic June 2012 November 2014 and Department of Genetics, Faculty of Biology of Lomonosov Moscow State University between October 2013 and March 2016. Methodology: The study included 138 patients with migraine (44 with chronic and 96 with episodic migraine). The control group included 348 unexamined subjects. Genotypes were determined using real-time PCR with allelic discrimination test. Statistical processing was performed using Fisher test and Pearson's chi-squared test. Results: Our study evaluated the link of CC genotype of rs2070744 with migraine (Fisher’s p=0.026) and episodic migraine (Fisher’s p=0.022). Conclusion: Genotype CC of SNP rs2070744 in the regulatory region of NOS3 gene is more specific for episodic migraine and may prevents the chronification of migraine.


INTRODUCTION
Migraine affects 14.7% of people worldwide causes more years lost to disability than any other neurologic disorder [1]. A nationwide population-based survey of headache disorders in Russia revealed a considerably higher 1-year prevalence of migraine (20.3%) and an unusually high prevalence (10.5%) of headache occurring on ≥15 days/month [2].
Genetic predisposition to migraine is well-known and proven by epidemiological genetic studies [3]. The study of the genome in families of patients with hemiplegic migraine revealed five types of migraine with monogenic inheritance (familial hemiplegic migraine I, II, III, IV, V). However, these forms are extremely rare and are not involved in the development of common migraine with aura or without aura [4].
Vascular hypothesis, specifically endothelial dysfunction, is currently discussed as a shared mechanism for migraine and neurovascular disorders [5,6].
Nitric oxide (NO) is an important mediator of vasodilation in the intra-and extracranial vessels. This small molecule easily penetrates the membranes of vascular smooth muscle cell, and activates soluble guanylate cyclase and cGMP (cyclic guanosine monophosphate) synthesis, leading to vasodilation. NO also promotes the release of CALCA (calcitonin related polypeptide alpha, also known as CGRP -calcitonin generelated peptide), which is a powerful vasodilator. The SNPs (single nucleotide polymorphism) in gene coding endothelial NO synthase (NOS3) may influence the pathogenesis of migraine. Some investigators suggest the role of the polymorphisms in NOS3 gene in the pathogenesis of migraine or in the formation of clinical characteristics of migraine (aura) [7][8][9]. Other investigators have noted the absence of such associations for single SNP [10][11][12][13][14]. Haplotype association analysis of multiple SNPs in NOS3 gene shows positive results [11,13]. These studies examined the association of the polymorphisms in NOS3 gene with migraine, migraine with or without aura. But search of association of polymorphism in the NOS3 gene with chronic and episodic migraine was not carried out earlier.
The objective of our study was to determine the effect of a single nucleotide polymorphism rs2070744 (c.-813C>T), which is located in the promoter region of NOS3 gene, in the episodic and chronic forms of migraine.

Patients
The retrospective study included 138 patients with migraine living in Moscow and Moscow region, with the average age of 41.6 ± 12.5 years old (44 with chronic and 94 with episodic migraine). All those patients primarily applied to -Familial or sporadic hemiplegic migraine, according to the ICHD-III; -Other significant medical conditions except migraine; -Actual chronic migraine risk factor (current evidence of depression based on a Beck Depression Inventory (BDI) total score > 19, sleep disorders, stressful life events, caffeine overuse); -Any prophylactic treatment for migraine for at least 3 months prior to the study. The control group included 348 unexamined subjects (population control), inclusion criteriaplace of residence in Moscow and Moscow region. Patients and controls were age-matched. Migraine was diagnosed by experienced neurologists based on the criteria of the ICHD-III. The patients were questioned regarding the disease duration, the type of migraine, the frequency of migraine attacks (for the last 3 months), drugs used, underwent a clinical neurological examination and blood sampling for genotyping The study was approved by the local ethics committee and all subjects gave informed consent to participate in the study. The study was conducted according to the Declaration of Helsinki Principles.

Molecular Genetic Testing and Data Analysis
The Statistical processing was performed using Fisher test and Pearson's chi-squared test available in software WinPePi.
For reconstruction of signaling pathways the program PathwayStudio 10.0 with ResNet12 data base (Elsevier) was used.

RESULTS
The frequencies of genotypes and alleles of rs2070744 substitution in patients with migraine and in the control sample are presented in Table 2.
The significant differences between patients with migraine and control subjects are obtained. For the study SNP the significance of recessive model of inheritance is observed ( Table 3). The significant differences between patients with chronic migraine and control subjects not found (Two-tailed Fisher's P = 0.447). No significant differences between patients with chronic and episodic migraine found (Two-tailed Fisher's P = 0.478). Significant association found with episodic migraine, recessive model of inheritance (Table 4).

DISCUSSION
The polymorphism of NOS3 gene encoding an enzyme, that synthesizes NO in endothelial cells, considered as a possible genetic risk factor for migraine [16]. Early studies showed that migraine patients more often had a headache after the administration of nitroglycerin than patients without migraine [17]. NO initiates a slow pathological reaction that leads to headaches; it is also due to the fact that migraine patients are hypersensitive to exogenous and endogenous NO [18].
Also, the artificial increase in NO concentration can increase the activity of NO synthase in nociceptive trigeminal neurons [19,20]. At the level of the trigeminal system, neuronal NOS controls production of NO, as a result of the activation of the ejection of CGRP from the trigeminal fibers, this in turn, causes vasodilation. At the level of the blood vessels, CGRP appears to activate endothelial NO-synthase, as a result, this leads to production of NO and relaxes of vascular smooth muscle [21][22][23][24].
The NOS3 gene (nitric oxide synthase 3 (endothelial cell)) is located on chromosome 7q36, it consists of 29 exons (Gene ID: 4846). The NOS3 protein constitutively synthesize nitric oxide by the reaction of converting L-arginine to L-citrulline, resulting in a five-electron transfer, carried out using nicotinamide adenine dinucleotide phosphate (NADP) [25]. The enzyme acts as a homodimer, which may be functionally divided into two main areas: the Cterminal reductase domain and the N-terminal oxygenase domain [22]. Catalytic activity of NOS3 requires the presence of heme and cofactors: tetrahydrobiopterin, flavinadeninedinucleotide, flavinmononucleotide and calmodulin [26]. Production of nitric oxide is regulated by changes in the expression or activity of the enzyme NOS3, or by changing in the suitability of activating endogenous cofactors or inhibitory molecules [27,28].
In several studies the associations of NOS3 gene polymorphisms with migraine have shown. A group of researchers from Italy showed that the genotype AspAsp (rs1799983, p.Glu298Asp) met 3 times more frequently in patients with migraine with aura, compared with migraine without aura, and 2 times more often than the control [29]. The increase of the frequency of the minor allele A (rs3918166) in patients with migraine with aura, compared with migraine without aura, was shown by Investigators from USA [9]. When studying 5 polymorphic sites (rs2070744, rs1799983, 27 bp VNTR in intron 4, rs3918226 and rs743506) in patients from Brazil was found that genotype GA (rs743506) is more common in controls than in patients while haplotype "CCa-Glu-G" and "CCb-Glu-G" (rs2070744 -rs1799983 -27 bp VNTR -rs3918226 -rs743506) predominant in patients with migraine with aura compared with migraine without aura [13].
But there were no the studies dedicated to the association of the polymorphism of the NOS3 gene with chronic or episodic migraine. Allele C decreases the promoter activity of NOS3 gene [30]. What is role of allele C of NOS3 gene in the pathogenesis of migraine? To evaluate the effect of allele C in chronic or episodic migraine, we have reconstructed, according to the literature, the signaling pathways, describing the activation of transcription of endothelial nitric oxide synthase and its role in the development of major migraine symptoms: vasodilation and pain (Fig. 1).

CONCLUSION
Thus, genotype CC of SNP rs2070744 in the regulatory region of NOS3 gene decreases the promoter activity of NOS3 gene. This genotype is more specific for episodic migraine and may prevent the chronification of migraine.

CONSENT
This research used de-identified administrative data obtained from University Headache Clinic; informed consent was not required.

ETHICAL APPROVAL
The study was conducted in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki.