COMT rs4680 single nucleotide polymorphism, ethnicity, and age are associated with fibromyalgia in women

To investigate the influence of the catechol-O-methyltransferase enzyme (COMT) single nucleotide polymorphism (SNP) rs4680 (G/A) on fibromyalgia in women. In this observational study of case-control type 29 women with a diagnosis of FM (cases) and 31 healthy non-fibromyalgia women (controls). Sociodemographic and anthropometric data were collected, as well as data relating to Symptom Severity Scale and Generalized Pain Index and peripheral blood samples for DNA extraction; genotypic analyzes were performed by PCR-SSP. We observed that rs4680 AA genotype was more frequently observed in fibromyalgia than controls (p=0.02). The A allele was also more often present in the fibromyalgia participants than in their control peers (p = 0.03). There was a statistically significant association between race and FM sufferers, showing that those of white ethnicity had a 2.05 times greater chance of developing the syndrome than non-white individuals (p=0.03; CI 95% 0.93 – 4.53). A statistically significant correlation between age and FM was observed (rS=0.812, p=0.01). This study demonstrates that white women above the age of 45, who have the AA genotype or A-allele, presents a higher risk of developing FM, showing that this polymorphism of the COMT gene may be one of the risk factors for the fibromyalgia.


Introduction
The syndrome of fibromyalgia (FM) is typified by generalized chronic musculoskeletal pain and is also associated with symptoms of fatigue, sleep and mood disturbances, morning stiffness and psychological disorders (Wolfe et al., 1990). It is considered to be the second most common rheumatological disorder, affecting 2.5% of the Brazilian population (Senna et al., 2004) and approximately 2% of the world's population, primarily affecting women over 35 years of age (Wolfe et al., 1995).
The etiology of FM may be related to environmental, hormonal or genetic factors. Studies show that the pathophysiology of the syndrome is linked to abnormalities in the pain processing mechanism of the central nervous system, showing dysfunctions in neurotransmitters which can result in both under-activity of inhibitory pain pathways and hyperexcitability of pain-processing pathways. These abnormalities may be related to genetic predisposition, triggered by physical or psychological stress (Bradley & McKendree-Smith, 2002).
Studies have associated FM with a genetic susceptibility directly related to the serotonergic, dopaminergic and catecholaminergic systems. The catechol-O-methyltransferase enzyme (COMT) works by inactivating the catecholamines and mutations in the gene that encodes this protein, and has been shown to be related to the symptoms of FM (Gursoy et al., 2003).
The COMT gene may show genetic abnormalities, such as single nucleotide polymorphisms (SNPs) implicated in or related to chronic pain.
The most researched SNP of the COMT gene is Val158Met (G/A) rs4680, the literature showing that individuals carrying the A-allele have higher sensitivity to post-surgical pain 6 and, fibromyalgia (Barbosa et al., 2012). A study analyzing the polymorphism of serotonin receptors and the COMT gene in patients with fibromyalgia produced significant results compared with the control group, in which patients with fibromyalgia exhibited a higher frequency for the L/L genotype (Met158Met), demonstrating that individuals with this genotype may be more susceptible to this syndrome, as this genotype encodes an enzyme incapable of metabolizing catecholamines (Matsuda et al., 2010). However, other studies found no association of chronic generalized pain or other types of chronic musculoskeletal pain with SNP in the COMT gene (Borchers & Gershwin, 2015), illustrating that this investigation is necessary for a better understanding of the role of the polymorphism in the COMT gene in the origins and/or seriousness of FM.
The objective of the present study was to analyze the relationship between the SNP of the COMT Val158Met (G/A) rs4680 gene in women, with or without fibromyalgia.

Study Design and Ethical Procedures
The study was approved by the Human Research Ethics Committee at the Pitágoras University UNOPAR under protocol no. 1.579.152 and the individuals taking part in the study signed a Free and Informed Consent agreement.
The present study was quantitative, transversal and a case-control study (Thomas et al., 2012). A convenience sample was composed of women with FM, over 18 years old and clinically diagnosed with FM by a neurology specialist, following the 2011 guidelines from the American College of Rheumatology (ACR) (Wolfe et al., 2011). The control group was made up of women, over 18 years old, without FM, with no history of chronic pain.
Participants' sociodemographic and anthropometric data were collected, as well as data relating to physical activity, the Beck Depression and Anxiety Inventory, Visual Analog Scale (VAS) for pain, Symptom Severity Scale and Generalized Pain Index.
For the genotype analysis, peripheral blood samples were collected in a 5mL Vacutainer tube containing EDTA. DNA extraction was performed based on the Salting-out protocol described by John et al. (John et al., 1991).

PCR-SSP
After DNA extraction, genotyping was performed for the G/A polymorphism of the COMT rs4680 gene, using the polymerase chain reaction with specific primers for polymorphic sequences (PCR-SSP). The reactions were performed in a final volume of 10 μL containing a 1x buffer, 2.5mM of MgCL2, 10mM of dNTPs, 10mM of each primer and 1.25μL Taq polymerase DNA. The following primers were used to amplify the target fragments: COMT-A, 5'-TggTggATTTCgCTggCA-3' and 5'-ACACCCATACAAgcaTTCATCAgTT-3'; and COMT-G, 5'gCATgCACACCTTgTCCTTCAC3' and 5'-TgAgCATAgAggCTAAgggACCAT-3'. Cycling was as follows: heating for 1 minute at 96°C, then at 96°C for 20 seconds, 70°C for 45 seconds for the first 5 cycles, followed by 21 cycles at 96°C for 25 seconds, 65°C for 50 seconds and 72°C for 30 seconds each and then 21cycles at 96°C for 30 seconds, 55°C for 60 seconds and 72°C for 90 seconds, followed by a final phase of 2 minutes at 20°C. For analysis of the results, 1% agarose gel electrophoresis was used, the size of the amplified protocol being 455 pb for the G-allele and 322 pb for the A-allele.

Statistical Analysis
Data was analyzed using the Statistical Package for Social Sciences (SPSS) program, version 21. Categorical variables were expressed in absolute and relative frequencies and continuous variables as means and standard deviations. The chi-squared test, Fischer's exact test and the odds ratio were used to check the association between race and genotype frequency with fibromyalgia and Kendall's τ coefficient for a correlation between fibromyalgia patients and age, with a confidence interval of 95% and a significance level of 5% being established (p<0.05).

Results
60 women took part in the study, 29 in the group diagnosed with fibromyalgia and 31 healthy women comprised the control group. The average age was 50.76 (±9.03) in the group with fibromyalgia and 70.32 (±6.86) in the control group, while 70% of the population were white and 68.3% exhibited some form of comorbidity. Regarding physical activity, only 22% of the participants reported practicing some type of physical activity on a regular basis. The group with the highest proportion among participants in the study had the GG genotype (58%) and the G-allele (75%), only 8.3% and 25% of women having the AA genotype and the A-allele, respectively. (Table 1). There were no statistically significant correlations in FM patients among the following variables: physical activity, visual analog scales, Beck depression and anxiety inventories, allele frequency and anthropometric data (p>0.05). However, a statistically significant association between the genotypes and FM was observed. The rs4680 AA genotype was more frequently observed in fibromyalgia than in controls [odds ratio (OR) (95%confidence interval, 95%CI) = 4.07 (1.37 -12.14; p=0.02). The A allele was also present in the fibromyalgia participants more often than in their non-fibromyalgia peers [OR (95% CI) = 4.03 (1.65 -10.27; p=0.03; Table 3).
There was a statistically significant association between race and FM sufferers, showing that those of white ethnicity had a 2.05 times greater chance of developing the syndrome than non-white individuals (p=0.03). (IC 95% 0.93 -4.53). A statistically significant correlation between age and FM was observed; the older patient, the greater the chance of developing the syndrome (rS=0.812, p=0.01).

Discussion
COMT is an enzyme involved in the process of breaking down catecholamines, which are the sympathetic neurotransmitters, through the transfer of a methyl group of S-adenosylmethionine to dopamine, noradrenaline and adrenaline in the synapses in the cerebral cortex, mainly affecting the metabolism of dopamine in the prefrontal cortex (Lachman et al., 1996), acting as a modulator of dopadrenergic, noradrenergic and adrenergic neurotransmission (Matsuda et al., 2017).
The SNP of the COMT rs4680 gene exhibits a transition of nucleotides in codon 158, resulting in a change in the protein's aminoacids. The Val/Val (G/G) genotype is related to a high level of enzyme activity, while the Met/Met (A/A) genotype is related to low activity and Val/Met (G/A) to an intermediate level of activity (Matsuda et al., 2017). Lower levels of activity of this enzyme lead to a significant reduction in the dopamine levels of the postsynaptic neurons (Lachman et al., 1996).
The present study demonstrates that 17.2% of patients with FM showed a frequency of the AA (Met/Met) genotype, while no participants in the control group were seen to have it. Regarding allele frequency, 38% of patients with FM had the According to Ablin and Buskila (2015), the reduction of synaptic tone related to the pathogenesis of chronic pain may be associated with the reduction of pain in FM patients. Thus several studies with genetic markers have been performed associating the metabolism of catecholamines with COMT in order to identify their role. found that the Met/Met (AA) genotype seems to affect brain response to pain after repeated painful stimuli.
In relation to the study limitations, the sample may have been insufficient to detect other interactions among the variables. It is possible to say that, for more assertive conclusions it is necessary to study a larger sample population and evaluate other polymorphisms of the gene combining an analysis of gene expression COMT, for further clarification. Our findings may be biologically meaningful and informative, and should be further investigated in other populations.
Concerning the clinical application of the results of the present study, the presence of association among fibromyalgia, the gene COMT, and the ethnicity and age, shows that the from these assessments it is possible to identify a genetic characteristic and some limitations of patients with this disease, thus aiding in a better therapeutic approach.

Conclusions
The present study demonstrates that white women above the age of 45, who have the AA genotype and the A-allele, have a higher risk of developing FM. Further studies are needed to confirm the present findings and it is necessary in the future researches a study with a larger sample population and evaluate other polymorphisms of the gene combining an analysis of gene expression COMT, for further clarification.