RAR‐related orphan receptor A: One gene with multiple functions related to migraine

Abstract Aims RAR‐related orphan receptor (RORA) involves in regulation of several biological processes including inflammation and circadian rhythm that probably are involved in migraine pathophysiology. In the current study, the association between RORA rs11639084 and rs4774388 variants and susceptibility to migraine were investigated in a sample of Iranian migraine patients for the first time. Methods In a case‐control study including 400 participants, 200 migraineurs and 200 healthy controls, genotyping of RORA rs4774388 and rs11639084 polymorphisms was performed using tetra‐primer amplification refractory mutation system–polymerase chain reaction (TP‐ARMS‐PCR). Results The distribution of rs4774388 C/T and T/T genotypes differed significantly between the studied groups. Moreover, an association was observed between rs4774388 and migraine under the recessive mode of inheritance (P = 0.002; OR = 1.89.; CI = 1.25‐2.87). The distribution of rs11639084 alleles and genotypes was not significantly different between migraineurs and healthy controls. Conclusion Current results suggest RORA, as a molecular link, may explain inflammation and circadian rhythm dysfunction in migraine. Further studies in different ethnicities are required to confirm the function of RORA in migraine development.

of this disorder may be affected by inflammation, vascular dysfunction, circadian rhythm, and several signaling pathways. [8][9][10] Evidence shows a positive association between the elevated inflammatory agents and migraine disorder. 11 Among these agents, cytokines and CD4 + T cells are considered to play an essential role in neurovascular inflammation. 12 Moreover, several findings suggested a sharp increase in plasma T-helper 17 (Th17) cells, a member of three subsets of CD4 + T cells, and effector cytokines levels including IL-4, IL-5, IL-6, IL-10, and IL-17A during neurovascular inflammation among migraine patients. [13][14][15] Th17 cells secrete IL-17 that induces chronic inflammatory disorders. [16][17][18][19] Remarkably, the above-mentioned cytokines, especially IL-10, play essential roles in Th17 differentiation. 20 Although the genetic basis of migraine is not clear yet, several susceptibility regions, including the 15q11-q13, have been suggested to be associated with migraine. 21 Retinoic acid receptor-related orphan receptor alpha (RORA) is one of the three ROR nuclear receptors (α, β, and γ) located at 15q22.2 and is expressed in several tissues, including brain, lungs, liver, skeletal muscle, kidney, and thymus. 10,22 RORA plays a key role in the regulation of Th17-cell differentiation, circadian rhythms, and inflammation. 23,24 Several studies have demonstrated that RORA could regulate numerous cytokine expressions and Th17 differentiation directly or indirectly. [24][25][26] Induction of RORA expression by TGFβ and IL-6 results in the differentiation of Th17 cells as well as expression of IL-17. 24 Moreover, RORA itself can trans-activate the expression of IL-6 which also plays a role in the Th17-cell differentiation. 25,26 Based on the above-mentioned considerations, we hypothesized that the RORA gene might be a probable candidate gene involved in migraine pathophysiology. Therefore, in this study, for the first time, we aimed to investigate the association of RORA rs11639084 and rs4774388 variants with migraine susceptibility in Iranian patients. REC.1397.702), and all participants or their parents signed the informed written consent.

| Genotyping of RORA rs11639084 and rs4774388 variants
Genomic DNA was isolated from blood samples using the GeneAll according to the following cycling condition: initial denaturation at 94°C for 3 minutes, a subsequent series of 34 cycles of denaturation at 94°C for 30 seconds, annealing temperature as in Table 1 for 30 seconds, and extension at 72°C for 30 seconds. Final elongation was carried out at 72°C for 3 minutes. The primers were designed by using Primer1 online tool ( Table 2). 28 Amplified products were subjected to 2% agarose gel electrophoresis prepared in 0.5X TBE.
The rs4774388 C and T alleles generated a 197 and 273 bp PCR products, respectively, and the outer primers amplified a common 412 bp amplicon. The rs11639084 T and C alleles produced 215 and 162 bp amplification products, respectively, and the outer primers amplified a common321 bp product. For further genotype confirmation, samples were randomly selected and sequenced by using an ABI 3730 × l DNA analyzer (Macrogen, Korea).

| Statistical analysis
Genotype and allele frequencies and deviation from Hardy-Weinberg equilibrium (HWE) were assessed by a chi-squared test using SNPStats (available from http://bioin fo.iconc ologia.net/ SNPstats. The association between rs4774388 and rs11639084 and migraine was investigated in dominant and recessive inheritance modes. Odds ratios (ORs) with 95% confidence intervals (CI) were used to estimate the strength of association between the studied variants and migraine susceptibility. A P-value of less than 0.05 was considered to be statistically significant. Figure 1 represents the agarose gels for genotyping the studied polymorphisms. RORA rs4774388 genotypic distribution was in complete HWE in the controls and patients; however, the control group deviated from HWE regarding rs11639084. Table 3

| D ISCUSS I ON
Despite the remarkable role of genetic factors in migraine development, very few genes are documented as the intrinsic susceptible locus for this disease. 3 The advancements in our ability to identify these variants as well as the involved signaling pathways have led to an improved understanding of genomic landscapes in migraine and the novel therapeutic approaches.
In the present study, for the first time, we described that RORA rs4774388 is significantly related to the migraine susceptibility.  that migraine has a higher prevalence rate in asthma patients. 6,7 It may be more interesting when we know that high levels of IL-17 are associated with severe asthma. 40 Altogether, these observations further support the idea that migraine and MC-related disorders have a common immune mechanism, which may be through the

IL-17 function.
Besides the regulation of cytokine-mediated signaling pathway and inflammatory response, RORA seems to regulate several migraine-related pathways, including circadian rhythm, cellular response to hypoxia, nitric oxide biosynthesis, and VEGF production. [8][9][10]24,41 Several studies suggested that migraine attacks are under the control of the circadian rhythm and, as a result, occur more likely in the early morning. 42,43 Also, it is suggested that there is a bidirectional relationship between migraine and sleep disorders that more confirms the role of circadian rhythm impairment in this disease. 44   to know that RORA, as a transcription activator, can regulate the expressions of both BMAL1 and its repressor REV-ERBα. 22 Figure 4 shows a schematic overview of the possible roles of RORA in migraine pathogenesis.
Considering the crucial role of RORA in the regulation of inflammation and circadian rhythm, it would be a good therapeutic target in migraine treatment. Up to now, several natural and synthetic ligands are detected and also designed for RORA that harbor agonist or antagonist effects.
T0901317 and SR3335 serve as RORA modulators, which reduce the expressions of its target genes by suppressing the gene transcriptional function. 18 SR1001, as the derivative of T0901317, suppresses the differentiation of Th17 cells by selective targeting RORA, and hence, it may be used in the treatment of the Th17-mediated diseases. 51

| CON CLUS ION
Current results suggest RORA may serve as a molecular link between inflammation and circadian rhythm dysfunction in migraine.
Therefore, we recommend RORA as a new susceptible locus in migraine, which may offer new therapeutic intervention. Further research considering the role of RORA in migraine may enhance our understanding of the mechanisms underlying this gene.

ACK N OWLED G M ENT
The present article is financially supported by "Research Department of the School of Medicine Shahid Beheshti University of Medical Sciences" (Code: 15458).

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest in this work.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.