The relationship between ASIC3 gene polymorphism and ﬁ bromyalgia syndrome

Objectives: Fibromyalgia syndrome (FMS) is a chronic pain syndrome characterized by widespread body pain over a long period, the cause of which is not yet clearly known. FMS patients usually have high pain sensitivity. We aimed to investigate whether rs4148855 and rs2288646 polymorphisms of acid-sensing ion channel 3 ( ASIC3 ), one of the factors contributing to pain, cause a predisposition to FMS in the Turkish population. Methods: ASIC3 gene rs4148855 and rs2288646 polymorphisms in DNA samples obtained from blood samples of 175 patients with FMS and 176 healthy individuals were analyzed by real-time polymerase chain reaction (RT-PCR) using a hydrolysis probe. Statistical data were obtained by chi-square ( χ 2 ) test and logistic regression analysis. Results: No signi ﬁ cant association was found between ASIC3 gene rs4148855 and rs2288646 polymorphisms and the Turkish population ’ s FMS group and control group (p>0.05). Conclusions: As a result, no signi ﬁ cant association was found between the genotype and allele distributions of ASIC3 polymorphism (rs4148855 and rs2288646) in patients with FMS compared to controlsin the Turkish population. Further studies are needed to elucidate the relationship between ion channels and FMS to elucidate the mechanisms of FMS.


Introduction
Fibromyalgia syndrome (FMS) is a chronic pain syndrome characterized by long-lasting widespread body pain and tenderness in at least 11 of 18 specific areas identified.FMS is associated with fatigue, sleep disturbance, somatic syndromes, psychological disorders, disability and impaired quality of life [1].The prevalence of FMS in the general population is 2.9-4.7 %.Although FMS can be seen in all ages and genders, it usually affects women aged 40-60 [2,3].Gender and advanced age are risk factors for FMS [4].Physical and mechanical traumas and stress factors in the social environment are influential in the emergence of FMS [5].Although the etiology of FMS has been attributed to infection, genetic and autoimmune factors, the mechanism is not fully known.Micro-muscle trauma, immunologic, psychological, neuro-hormonal, central nervous system, and environmental factors have been suggested to play a role in the development of the disease [6,7].It has been suggested that widespread chronic pain in FMS patients is caused by genetic factors affecting peripheral and central pain mechanisms [8].The classification of FMS as neuropathic pain and its connection with pathophysiology is becoming increasingly important [9].Despite many studies on FMS, its pathophysiology still needs to be clarified.In addition, according to genetic studies, there is a predisposition to FMS.Several pain-related genes have been identified, and there is evidence that many polymorphisms are associated with FMS [10].
ASIC3 are sodium channels activated by protons from outside the cell and are a member of acid-sensitive channel proteins structurally related to epithelial sodium channel proteins [11].ASIC3 is predominantly expressed in both the central and peripheral nervous systems and is activated by cellular proteins [12][13][14].They are the most sensitive acid sensors with high expression in peripheral nerves [15,16].In studies on rats, peripheral ASIC3 channels are the primary sensors of acidic pain in inflammatory conditions [17].ASIC3 regulates myocardial ischemic pain, muscle pressure reflex, and possibly unstable autonomic regulation.Acidosis in muscle tissue is implicated in chronic muscle pain [12,18].ASIC3 ion channels activate muscle pain receptors and cause chronic muscle pain [19,20].
Therefore, in our study, we examined the relationship between ASIC3 gene polymorphisms (rs4148855 and rs2288646), which is one of the subunits of ASICs, which is important in pain perception, and there is no study in this area in our country, and FMS.This gene is located in the 7q35-36.1 region of the chromosome and is also localized in the intron region of ASIC3 in the first SNP rs4148855 we investigated.Nucleotide polymorphism occurs with the conversion ofin this 110 bp (base pair) long region to GTC.The other one, the SNP rs2288646 region, is also located in the exon region of ASIC3, and a single nucleotide polymorphism occurs when the adenine (A) base in this 106bplong region converts to the guanine (G) base.As a result of this polymorphism, the presence of the G allele in the exon region resulted in a splicing-reducing effect, and the presence of the A allele resulted in a splicing-enhancing impact [21].
In addition, genetic variants of ASIC3, rs4148855 and rs2288646, were found to be associated with diseases such as pain, hypertension, anxiety and insulin resistance.

Study population
This study consisted of 175 patients with FMS and 176 healthy controls admitted to Sivas Cumhuriyet University Faculty of Medicine, Department of Physical Medicine and Rehabilitation, and Sivas Numune Hospital, Physical Therapy Clinic.Patients who had no previous history of FMS and were diagnosed with FMS for the first time were included in the study, and the patients were from the same ethnic group and geographical region.The control group consisted of individuals admitted to the same hospitals, were similar to the selected patient group in terms of age and gender, were not diagnosed with FMS, and did not have any other disease.The diagnosis of FMS was made by a physician according to the American College of Rheumatology classification criteria [22].Various questions such as name, surname, age, gender, height, weight, occupation, sleep disturbance, fatigue, headache, morning fatigue, dry mouth, leg numbness, dry eyes, difficulty concentrating, feeling of swelling in soft tissues, family history of FMS were filled in the questionnaire in question-answer form.Our study was approved by Sivas Cumhuriyet University Clinical Research Ethics Committee (Date: 12.05.2015,Decision no: 2015-05/01) and was conducted by the Declaration of Helsinki.

DNA isolation
A 4 mL blood sample was collected from healthy control subjects and FMS patients in sterile citrated blood tubes.Genomic DNA isolation was performed manually using the precipitation method at high salt concentrations when the blood samples reached the laboratory [23].

Statistical analysis
The relationship between fibromyalgia and ASIC3 gene polymorphism, age, gender, height, weight, occupation, sleep disturbance, fatigue, headache, morning fatigue, dry mouth, leg numbness, dry eyes, difficulty concentrating, soft tissue swelling, and family history of fibromyalgia was determined using the Chi-square (χ 2 ) test (bias value α=0.05).Risk estimates for fibromyalgia occurrence were determined by applying a logistic regression test.The degree of association was described as 95 % CI (confidence interval) OR (Odds Ratio).Statistical data analysis was performed using the Statistical Package for the Social Sciences (SPSS) program (Version 22).

Demographic data and statistical analysis of the people included in the study
Demographic information and statistical findings of patients diagnosed with FMS and healthy individuals are given in Table 1.According to the data, 10 (5.7 %) of the patients were male, and 165 (94.3 %) were female.Among the controls, 10 (5.1 %) were male, and 166 (94.9 %) were female.The mean ages of patients and controls were 40.33 ± 16.30 and 39.69 ± 17.27 years for males and 43.89 ± 9.76 and 44.05 ± 8.87 years for females, respectively.

Comparison of ASIC3 polymorphism (rs4148855 and rs2288646) genotypes and allele frequencies in study groups
The statistical analysis of the association of rs4148855 polymorphism of the ASIC3 gene with genotype and allele distributions in FMS patients and healthy individuals in the The statistical analysis of the relationship between rs2288646 polymorphism of the ASIC3 gene and genotype and allele distributions of FMS patients and healthy individuals in the control group is given in Table 4.In the statistics of ASIC3 (rs2288646), the genotypes of 176 individuals in the control group were determined as 2 GG, 4 as AG, and 170 as AA.Genotypes of 175 individuals in the FMS patient group were found to be 0 as GG, 6 as AG, and 170 as AA.In the analysis of ASIC3 polymorphism with the χ 2 method, no statistically significant correlation was found when FMS patients and controls were evaluated in terms of G and A alleles (χ 2 : 0.28 p: 0.597, Raw OR: 1.33 % 95 % CI: 0.42-4.37).When patients and controls were evaluated in terms of ASIC3 (rs2288646), the analysis of GG and AG using the χ 2 method was not significant (χ 2 : 2.40 p: 0.121 Raw OR: 0.67 % 95 % CI: 0.38-1.17).The analysis of GG and AG with the χ 2 method was not found significant (χ 2 : 1.98 p: 0.160 Raw OR: 0.99 % 95 % CI: 0.97-1.00).The analysis of GG and AG + AA using the χ 2 method was insignificant (χ 2 : 2.00 p: 0.157 Raw OR: 0.99 % 95 % CI: 0.97-1.00).

Gene variants between ASIC3 polymorphism (rs4148855) and fibromyalgia syndromes
In patients with Fibromyalgia given in Table 5, ASIC3 polymorphism (rs4148855) with sleep disturbance, fatigue, headache, dry mouth, numbness in legs, dry eyes, difficulty concentrating, swelling in soft tissues and familial history of fibromyalgia were statistically evaluated by χ 2 method.No significant difference was found between them.However, when morning fatigue was analyzed by the χ 2 method, a meaningful relationship was obtained between gene variants.(χ 2 : 7.74, p: 0.02).

Gene variants between ASIC3 polymorphism (rs2288646) and fibromyalgia syndromes
In patients with Fibromyalgia given in Table 6, no significant difference was found between gene variants when the ASIC3 polymorphism (rs2288646) and clinical parameters were evaluated statistically by the χ 2 method.

Discussion
FMS is known to cause widespread skeletal-muscular pain, fatigue, sleep disturbance, depression, headache, morning fatigue, and bowel dysfunction [2,24].In Turkey, the prevalence of FMS among 20-64 year-olds was 3.6 % [25].In this study, the age range of patients diagnosed with fibromyalgia (n=175) was 20-62 years.The age ranges of other studies conducted in Turkey, and our current study is similar.FMS symptoms are usually observed in women and are found in the age range of 30-50 years [26].In our study, 165 of 175 FMS patients were female.There are biomarker studies for FMS, but no specific biomarker has been identified yet [10].
According to a study, high serum prolidase activities were observed in FMS [27].In research by Taş et al., the relationship between FMS and potassium ion channels was investigated, and determined that the level of potassium voltage-gated channel subfamily H member 2 (KCNH2) was low in plasma [28].In a study by Koçak et al., serum cathepsin S (CatS) and cystatin C (CysC) levels were evaluated in FMS, and both were found to be elevated [29].In addition, a significant correlation was observed for calcitonin gene-related peptide (CGRP), calcitonin receptor-like receptor (CLR), and receptor component protein (RCP) levels in FMS [30].In a study conducted by Zontul et al. within the scope of artificial intelligence with FMS, classification was performed with support vector machines and achieved high success [31].
Today, it is suggested that the susceptibility to chronic pain and the response to pain sensitivity are related to the individual's genetic structure.In recent genetic studies, the fact that pain varies from person to person is explained by genetic polymorphism [7].ASIC3 is expressed in peripheral sensory neurons and is essential in pain perception and acid-induced hyperalgesia [32].Our study examined the relationship between the rs4148855 and rs2288646 polymorphisms of the ASIC3 gene, one of the subunits of ASICs, which is essential in pain perception and which has not been studied in the Turkish population, and FMS.Yu-Linko et al. investigated the relationship between ASIC3 gene polymorphisms (rs2288646 and rs4148855) and blood pressure in a Taiwanese population.They found that individuals carrying the rs2288646-A allele (AA + AG genotype) were associated with high blood pressure [33].In another study, the relationship between the rs1042717 polymorphism of the beta-2 adrenergic receptor gene between patients with FMS and healthy individuals was examined, and no significant association was found [34].In a study by Kaydok et al., the relationship between estrogen receptor 1 (ESR1) gene polymorphisms (rs2228480 and rs2295190) and FMS was investigated.As a result of the evaluation of the relationship between rs2295190 polymorphism and FMS, it was found that patients with CG and GG genotypes had a lower risk of FMS than CC genotypes.No significant difference was observed between rs2228480 polymorphism and FMS [35].It has been reported that polymorphism of transient receptor potential vanilloid 2 (TRPV2), an ion channel in FMS, makes FMS more susceptible.TRPV3 has been suggested to increase the occurrence of FMS symptoms [36].In a study examining polymorphisms of the sodium voltage-gated channel alpha subunit 9 (SCN9A) gene, which encodes voltage-gated sodium channel, in FMS, rs6754031 polymorphism was found to be significantly significant [37].In a study by Yu et al., an FMS mouse model was created, electroacupuncture was applied, and the ASIC3 receptor was examined.It was found that mechanical hyperalgesia decreased in the mechanism involving ASIC3.It was also found that hyperalgesia and nociceptive signaling decreased in mice in which the ASIC3 gene was silenced [38].Hung et al. suggested that ASIC3 is activated by lysophosphatidylcholine (16:0) in chronic hyperalgesia caused by repeated and intermittent sound stress [39].In a mouse model of FMS, therapeutic ultrasound treatment activated ASIC3 in muscle afferents and produced an analgesic effect [40].ASIC3 expression levels have been reported to be increased in patients with chronic fatigue syndrome [41].This study determined the genotype distribution of ASIC3 gene (rs4148855 and rs2288646) polymorphisms in patients with FMS and healthy control subjects by RT-PCR.In terms of rs2288646, 169 (96.6 %) FMS patients were found to have GG, 6 (3.4 %) AG, and 0 (0 %) AA genotypes.Among the controls, 170 (96.6 %) had GG, 4 (2.3 %) AG, and 2 (1.1 %) AA genotypes.Our study observed no statistically significant difference between ASIC3 gene (rs4148855 and rs2288646) polymorphisms in patients with FMS and control subjects.
In this study, the number of male patients with FMS was not the same as the number of female patients.When ASIC3 gene polymorphisms are evaluated, the effect of these gene polymorphisms on protein level cannot be investigated due to the need for a project budget.These are the limitations of our study.

Conclusions
In the Turkish population, no significant association was found between the genotype and allele distributions of ASIC3 polymorphism (rs4148855 and rs2288646) in patients with FMS compared to controls.Further studies are needed to elucidate the relationship between ion channels and FMS to elucidate the mechanisms of FMS.

Table  :
Analysis of ASIC (rs and rs) genotypes of the study groups.

Table  :
Genotype and allele frequency distribution of rs polymorphism of ASIC gene in the study groups.

Table  :
Genotype and allele frequency distribution of rs polymorphism of ASIC gene in the study groups.
n, number of individuals.p<. determined as statistically significant.χ  , chi-square test; OR, odds ratio; CI, confidence interval.

Table  :
ASIC polymorphism (rs) genotype distributions in terms of fibromyalgia syndromes.

Table  :
ASIC polymorphism (rs) genotype distributions in terms of fibromyalgia syndromes.
n, number of individuals.p<. determined as statistically significant.χ  , chi-square test