Association Between Polymorphisms of DRD2, COMT, DBH, and MAO-A Genes and Migraine Susceptibility

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INTRODUCTION
M igraine, the most common neurological disease, seriously threatens the health of people all over the world, and has some symptoms including headache, nausea, vomiting, fatigue, irritability, and nervousness. Patients with migraine are generally aged from 25 to 50, and the risk of migraine in females is 3 times higher than that in males. [1][2][3] Genetic factors have been found to be involved in the etiology of the disease. [4][5][6][7] Dopamine, a hormone and neurotransmitter of catecholamine and phenethylamine, plays an important role in the repair of nervous system in human brain and associates with decreased dopamine activities. Several diseases in nervous system are associated with dysfunctions of dopamine system, including migraine and Parkinson disease. Dopamine receptor D2 (DRD2) gene locates on chromosome 11q22. 2-22.3, and its most studied single nucleotide polymorphisms (SNPs) include rs1799732 and rs6275. The former, a deletion polymorphism (À141C Ins/Del), correlates with reduced DRD2 expression, and the latter is a synonymous polymorphism locating in exon 7 of DRD2 gene.
Isoenzymes monoamine oxidases A (MAO), involved in catabolism of monoamine neurotransmitters, catalyzes oxidative deamination and participates in functional regulation of cell structures. 8,9 MAO gene can be divided into 2 subtypes of MAO-A and MAO-B according to different distributions and autoimmune features of cells. 10,11 MAO-A gene locates on chromosome Xpll.23, and has a high affinity to endogenous neurotransmitters, and one of its most studied SNP, VNTR, contains a 30 bp long repeated sequence. Catechol-O-methyltransferase (COMT) is an enzyme inactivating catecholamines. The COMT gene functional polymorphism rs4680 can affect the enzyme activities. 12 DBH gene, with 12 exons and a length of 23 kb, locates on chromosome 9q34. 13 The SNP rs7239728 in the promoter region of DBH gene is associated with phenotypic variations in plasma.
Genetic factors have been implicated in enzyme activities, and they, to some extent, can result in DNA damage, and finally cause the occurrence of diseases. Several studies have investigated the relationship between genetic polymorphisms of DRD2, COMT, DBH, and MAO-A genes and migraine susceptibility. [14][15][16][17][18][19][20][21][22][23][24] But the results are conflicting rather than conclusive. Our meta-analysis combining 3138 cases and 4126 controls aims to provide a more precise estimation of the association. Pooled odds ratio (OR) was the main outcome of this metaanalysis.

Search Strategy and Inclusion Criteria
We searched Pubmed, CNKI, and Embase for relevant studies using the combination of the items ''DRD2'' or ''COMT'' or ''DBH'' or ''MAO-A,'' ''polymorphism,'' and ''migraine.'' All eligible studies evaluating the association between polymorphisms of the 4 genes and migraine susceptibility were selected according to the following criteria: with a case-control design; stating sufficient data for calculating pooled ORs with 95% confidence intervals (95% CIs). Studies were precluded if they were case-only studies, duplicates or with unrelated titles and abstracts. As all analyses were performed based on previous published researches, the ethical approval and patient consent are not required.

Data Extraction
Two investigators independently extracted requisite data from all eligible studies according to the identical criteria. The extracted data included: the name of first author, publication year, ethnicity, country of origin, numbers of cases and controls, genotyping methods, genotype frequencies, and P-value for Hardy-Weinberg equilibrium (HWE) in control group. Inconsistent data were discussed between the 2 investigators until reaching a consensus.

Quality Assessment
Assessment of the methodological quality of observational studies was done independently by 2 investigators. A risk-ofbias score modified from a previous meta-analysis 25 was used (Table S1, http://links.lww.com/MD/A513). The score has 4 domains: information bias: ascertainment of cases and controls, assessment of genotyping assay, confounding bias: population stratification and common confounding variables were evaluated, selective reporting of outcomes, HWE was assessed among controls. The full score was 21. Studies awarded at least 14 scores were defined as low-bias studies.

Statistical Analysis
Pooled ORs with 95% CIs were utilized to evaluate the relationship between polymorphisms of the 4 genes and migraine susceptibility. Heterogeneity among included studies was detected by Q test and I2 metric. Pooled ORs were calculated with a fixed-effects model when P-value >0.05 and I 2 < 50%, which indicated low possibility of heterogeneity; otherwise, a random-effects model was used. Publication bias was examined by Begg funnel plot and Egger test. HWE was checked in control groups by x 2 test. Sensitivity analysis was conducted, by removing the independent studies (one at a time) and reestimating the pooled ORs, to test the stability and robustness of the combined estimates. Statistical analysis was performed using STATA version 12.0 (Stata Corporation, College Station, TX).

Study Characteristics
As displayed in Figure 1, a total of 133 articles were identified from databases, and 122 of them were precluded for duplicates, unrelated titles and abstracts, case-only studies, and obvious irrelevance. Finally, 11 papers were included into our meta-analysis. 14,17,21,23,24,26 -31 All these studies had a low risk of bias, with the total score ranging from 14 to 17. The main characteristics of included studies are displayed in Table 1.

Meta-Analysis
Lack of an association between polymorphisms in DRD2, DBH, MAO-A and migraine As shown in Table 2, DRD2 rs1799732 was not associated with migraine risk when assuming a homozygote (OR ¼ 11.55,

Decreased Migraine Risk and COMT rs4680
In a meta-analysis of all studies concerning COMT rs4680 and risk of migraine, carriers of the AA genotype versus carriers of GG þ GA genotypes had 24% decreased migraine risk (AA vs. GG þ GA: OR ¼ 0.76, 95% CI ¼ 0.60-0.97, P Het > 0.642, I 2 ¼ 0). In subgroup analysis, the AA genotype versus the GG þ GA genotypes was 0.75-fold less likely to develop the disease in Caucasian populations (AA vs. GG þ GA:   Figure 2. No signals of relationship were seen in other genetic models tested ( Table 2).

Sensitivity Analysis
Sensitivity analysis was conducted by excluding one single study at a time to observe alterations in whole results which had no substantial difference before and after the deletions, suggesting our meta-analysis results were stable and credible.

Publication Bias
The shape of the funnel plot seemed symmetrical (Fig. 3), implying negligible publication bias. Additionally, Egger test provided further statistical evidence for the absence of significant bias (P ¼ 0.748).

DISCUSSION
Although the pathophysiology of migraine remains incompletely understood, an effect conferred by dopamine was implicated almost 40 years ago. 32 Such influence is further illustrated by an observation that dopamine shows signs of hypersensitivity in patients with migraine and that dopamine receptors can mediate nociception, autonomic responses, and vascular tone. 33 Dopamine antagonists have reportedly been used to eliminate migraine-related symptoms and headache. 33 Evidence from a murine model suggests that dopamine receptors usually appear in the trigeminovascular pathway and that dopamine is able to inhibit nociceptive trigeminovascular transmission. 34 These reports highlight the central role of dopamine in migraine and make the dopamine-related genes such as DRD2, COMT, DBH, and MAO-A candidates.
Accumulating evidence supports a relationship between these genes and migraine development. A 7-transmembrane receptor protein of the dopamine pathway predominantly expressed in pars compacta of the substantia and neostriatum is encoded by the DRD2. 35 Genetic polymorphisms in this locus are known to have important functional consequences. Expression-based research in vitro revealed 2 times higher expression levels of DRD2 in individuals with Ins/Ins of rs1799732 than those with Del/Del. 36 Multiple studies in human models have also connected rs6275 to DRD2 transcript instability and reduced translational efficiency. 37 Several other polymorphisms in the gene, such as rs7131056 can directly affect the function of DRD2 product. 28 For rs7239728, a promoter polymorphism in DBH, has been linked to phenotypic variability in DBH activity in plasma. 38 Migraineurs versus controls have been reported to have significantly lower plasma norepinephrine levels. 39 The DBH catalyzes the dopamine-to-norepinephrine conversion, and reduced DBH activity in individuals with rs7239728 might be a cause of lower norepinephrine in migraneurs. 15 The VNTR polymorphism in the MAO-A gene is associated with higher enzyme expression of MAO-A. 40 The functional impact leads to increased MAO-A activity, a cause of hypermetabolism of amine neurotransmitters and decreased levels of serotonin, conditions previously implicated in migraine pathophysiology. 41 All these results point to the high possibility of a relationship between DRD2, DBH, MAO-A and risk of migraine.
Multiple epidemiological studies have investigated the association between migraine susceptibility and polymorphisms of the four genes DRD2, COMT, DBH, and MAO-A. The study by Ghosh et al 15 demonstrated no significant association between DRD2 rs6275 polymorphism and susceptibility to migraine. In another large-scale study, the researchers provided opposite results by showing an increased risk of migraine associated with DRD2 rs6275 and 1799732 polymorphisms. 14 Fernandez et al 27 found no significant relationship between DBH rs72393728 polymorphism and migraine susceptibility in their research. For the MAO-A-VNTR polymorphism, Ishii et al. 30 identified an increased risk of migraine. In addition, the study in Finns by Tammimaki and Mannisto 42 demonstrated some evidence for an increase in the migraine risk associated with COMT rs4680 polymorphism.
In our meta-analysis, there was no significant association between migraine susceptibility and 4 SNPs in 3 genes, including DRD2 rs1799732 and rs6275, DBH rs7239728, and MAO-A-VNTR. However, COMT rs4680 polymorphism was associated with a decreased risk of migraine, especially in Caucasians. There are abundant data supporting these findings. The rs4680 polymorphism results in a transition of valine (Val) to methionine (Met) at codon 158, and it is the amino acid substitution causes decreased thermostability and enzymatic activity and increased dopamine-degrading activity. [43][44][45] While the findings for DRD2 rs1799732 and rs6275, DBH rs7239728, and MAO-A-VNTR are inconsistent with the results from previous functional studies previously introduced in this section, the results identified for COMT rs4680 are in accordance with the published reports.  Due to some limitations, the present findings should be explained prudently. First, we demonstrated evidence for the absence of association for DRD2 rs1799732, DRD2 rs6275, DBH rs7239728, MAO-A-VNTR and the presence of association for COMT rs4680. These results may be caused by the limited data available for each polymorphism. Hence, we cannot exclude the probability that the association for the former 4 polymorphisms will be significant and that the association for the latter would be lost after the enlargement of the sample size. Second, we put equal emphasis on English and non-English publications during literature search. However, only those papers written in English were identified. In addition, no unpublished data were included. Thus, selection bias may have occurred, though there was no indication of significant bias in Begg funnel plot and Egger test. Third, we merely assessed the genetic effects on migraine risk, not considering environmental influence. Fourth, the results were based on unadjusted data, which might affect the accuracy of the results. Finally, subgroup analyses based on age, gender, and other potential confounding variables were not performed because of insufficient data.
In conclusion, our meta-analysis demonstrates a significant association between decreased risk of migraine and COMT rs4680 polymorphism, but no association for DRD2 rs1799732, DRD2 rs6275, DBH rs7239728, and MAO-A-VNTR. Largescale studies where gene-environment interactions are considered and adjusted effect is estimated are needed to determine the role of these dopamine-related genes in migraine, thus providing new insights into the mechanisms that underlie the disease pathogenesis.