����Migraine is a debilitating neurological disorder affecting about10% of world��s population [1]. It can be broadly classified into twotypes- Migraine without aura ��MO�� and Migraine with aura ��MA����
����It is a complex disorder involving interplay between genes andenvironmental factors. The exact pathophysiology of migraine isstill unclear. However, the gender biasness in the incidence ofmigraine has paved the way for hormonal theory. Fluctuatinghormone ��especially estrogen�� levels in women are considered to bethe major contributing factors. It is observed that both estrogenwithdrawal as well as high estrogen levels increase migraine risk inwomen [2]. Various genes of estrogen pathway are involved inestrogen signaling and its downstream effects. Hence, a detailedunderstanding of the polymorphisms and their functional effectsmay provide useful insights in the field of migraine pathophysiology.
����CYP19A1 ��cytochrome P450, family 19, subfamily A, polypeptide1�� gene encodes aromatase enzyme which is involved in thefinal step of estrogen synthesis. Varying estrogen levels areassociated with polymorphisms in the 39 UTR ��rs10046 C.Tand rs4646 G.T�� of this gene [3,4]. However, there is a singlestudy on role of rs10046 polymorphism in migraine [5].
����Estrogen imparts its genomic effects through its receptors. Themost common are the ligand gated receptors ��ESRa encoded byESR1 gene and ESRb encoded by ESR2 gene���� Many studies inmigraine have focused on ESR1 gene polymorphisms. Intronicpolymorphisms [rs2234693 ��ESR1 PvuIIC.T�� and rs9340799��ESR1 XbaIA.G��] are associated with serum estradiol concentrations[6,7]. The rs2234693 has been previously studied [8]
����whereas the other intronic polymorphism ��rs9340799�� has not yetbeen explored in migraine. Reports on synonymous exonicpolymorphisms [rs1801132 ��C325G�� and rs2228480 ��G594A��] ofESR1with migraine are conflicting [8,9,10,11,12]. Two of theabove polymorphisms ��rs2234693 and rs1801132�� have also beenstudied by our group previously [13]. ESR2 promoter polymorphism��rs1271572�� may influence transcriptional factor bindingand gene expression [14]. On the other hand, exonic polymorphism��rs1256049�� results in a silent change which may affectmRNA folding, transcription and stability [15]. These polymor-phisms have been found to be associated with comorbid diseaseslike cardiovascular disorder [15]. However, no group has studiedthese polymorphisms in migraine.
����The present study was undertaken to explore the role ofpolymorphisms in genes of estrogen pathway in migrainesusceptibility in Northern Indian population. The CYP19A1��rs10046 and rs4646���� ESR1 ��rs2234693, rs1801132, rs2228480and rs9340799�� and ESR2 ��rs1271572 and rs1256049�� polymorphismswere selected for the present study. The conflicting reportson ESR1 gene polymorphisms and lack of reports on CYP19A1 andESR2 gene polymorphisms led us to validate the results in areplicative cohort and finally pooling the results by meta analysis.
����Results
����The genotypic and allelic frequencies of the study subjects areshown in Tables S1, S2, S3, S4, S5, S6, S7, and S8. All the studiedpolymorphisms followed Hardy Wienberg equilibrium in thecontrol population.
����CYP19A1 gene polymorphisms ��rs10046 and rs4646��CYP19A1 rs10046 polymorphism. In the primary cohort,the frequencies of heterozygous ��CT�� and variant ��TT�� genotypeswere significantly higher ��p,0.001�� in migraine patients ascompared to healthy controls ��HC���� In case of CT, subgroupanalysis showed significant association in migraine without aura��MO�� only. Further subgroup analysis on the basis of gender alsoyielded statistically significant results in migraine and MO for boththe genders. However, the results could be replicated in femalesonly. Significant associations were observed for TT genotype inboth migraines without and with aura ��MA���� Sub stratification onthe basis of gender yielded significant results in females only. Allthe significant results were replicated. Similarly, we foundstatistically significant p values ��pcorr = 0.01�� on applying Fisher��smethod. Mantel �C Haenszel test odds ratios confirmed the risk ofheterozygous and variant genotypes in migraine susceptibility��Table 1����
����At allelic level, significant results were seen with migraine as wellas subgroups in the primary cohort except male MA ��data notshown���� All the results were replicated except for males. Onpooling the data, significance was retained in all these subgroups��pcorr = 0.01�� showing risk of variant allele. Carrier analysis usingdominant model showed highly significant results with migraineand its subgroups. However significance could not be obtained inmale patients ��Table 1����
����CYP19A1 rs4646 polymorphism. In the primary cohort, theheterozygous ��GT�� genotype did not show significant associationswith migraine or its clinical subgroups ��data not shown���� However,on gender stratification, female migraine patients ��p= 0.022;OR= 0.544�� and female MA patients ��p= 0.015; OR= 0.427��showed protective effect. We were able to replicate the results. Onpooling, significant results were obtained in both the subgroupsshowing an overall protective effect of the genotype ��Table 2����
����At allelic level, protective effect of the variant T allele wasevident in MA patients in both the cohorts. Similar associationswere observed for female migraine and female MA patients.
����Furthermore, we found highly significant associations on pooling.
����In addition, dominant model also showed significant results forfemale migraine patients and female MA patients in both thecohorts as well as on pooling ��Table 2����
����CYP19A1: LD and Haplotype Analysis. On LD analysis, D9value of 0.4637 was observed, suggesting a moderate linkagedisequilibrium. The frequency of rs10046T-rs4646G haplotypewas significantly higher in patients of both primary ��37.7%�� andreplicative ��33.1%�� cohorts as compared to HC ��20.0%���� impartingsignificant results. Similar results were obtained for the rs10046Trs4646Thaplotype. The pooling of data validated these results��Table 3���� On subgrouping, similar results were observed for MO.
����However, we did not find significant predominance of anyhaplotype groups in MA. Furthermore, stratification on the basisof gender again yielded significant results for rs10046T-rs4646Ghaplotype.
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