Rheumatology International

, Volume 28, Issue 7, pp 685–691

Polymorphisms of the serotonin-2A receptor and catechol-O-methyltransferase genes: a study on fibromyalgia susceptibility

Authors

    • Department of Physical Medicine and RehabilitationOndokuzmayis University School of Medicine
  • Sezgin Gunes
    • Department of Medical Biology and GeneticsSchool of Medicine, Ondokuzmayis University
  • Omer Boke
    • Department of PsychiatrySchool of Medicine, Ondokuzmayis University
  • Gamze Alayli
    • Department of Physical Medicine and RehabilitationOndokuzmayis University School of Medicine
  • Nurten Kara
    • Department of Medical Biology and GeneticsSchool of Medicine, Ondokuzmayis University
  • Hasan Bagci
    • Department of Medical Biology and GeneticsSchool of Medicine, Ondokuzmayis University
  • Ferhan Canturk
    • Department of Physical Medicine and RehabilitationOndokuzmayis University School of Medicine
Original Article

DOI: 10.1007/s00296-008-0525-8

Cite this article as:
Tander, B., Gunes, S., Boke, O. et al. Rheumatol Int (2008) 28: 685. doi:10.1007/s00296-008-0525-8

Abstract

Genetic and environmental factors are thought to play roles in the etiopathology of fibromyalgia syndrome (FMS). The objective of this study was to determine the potential effects of single nucleotide polymorphisms (SNPs) in catechol-O-methyltransferase (COMT) (rs4680) and 5-hydroxytryptamine (serotonin) 2A (5-HT2A) receptor (rs6313 and rs6311) genes on susceptibility to FMS. One hundred seventy-one women (80 FMS, 91 control) were enrolled in the study. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used for the genotyping analyses. Genotype and allele frequencies were calculated by the chi-square test. Beck depression inventory, state and trait anxiety inventory and symptom checklist-90 revised (SCL-90-R) tests were applied to both patients and controls. There were no observed differences in the frequencies of alleles and genotypes between patients and controls for the COMT, and the two 5-HT2A receptor gene polymorphisms (P > 0.05). Our results suggest that the investigated polymorphisms seem not to be the susceptibility factors in etiology of FMS.

Keywords

FibromyalgiaGene polymorphism102T/C5-HT2A−1438G/A

Introduction

Fibromyalgia syndrome (FMS) is a chronic disorder that is estimated to affect 2–4% of the population; it is characterized by diffuse musculoskeletal pain, sleep disturbance, fatigue, stiffness, and presence of multiple tender points [1, 2]. Investigators have shown that psychiatric disorders such as depression, anxiety, and hypochondriasis are related to FMS [3, 4]. The pathogenesis of FMS is unknown, although it has been postulated that various psychiatric abnormalities that develop in a context of both genetic and environmental factors may play significant roles [5]. Recently, some authors reported a strong familial aggregation in FMS [6, 7]. Researchers studied roles of polymorphism of the genes in the serotoninergic, catecholaminergic, and dopaminergic systems in the etiology of FMS [811].

Serotonin (5-hydroxytryptamine, 5-HT) is a key neurotransmitter in the central nervous system. It plays role in sleep cycle, pain responses, vasoconstriction, depression, and anxiety. The effect of serotonin is mediated by different 5-HT receptor subtypes [12]. The 5-HT2A receptor is located in cortex, caudate nucleus, and all the intestines, and it may play a role in the etiology of several neuropsychiatric diseases and pain perception [13]. Human 5-HT2A is mapped to chromosome 13q14-21 [14]. Recently, a silent polymorphism in the 5-HT2A receptor gene was identified, which is defined by a T to C transition at position 102, and a novel G to A base change at position −1438 of the promoter region has been detected, which is in a very strong linkage disequilibrium with the 102T/C polymorphism of 5-HT2A [15]. Although the 102T/C polymorphism does not result in alteration of the amino-acid sequence of the protein, a strong association was found between 102T/C polymorphism and some psychiatric illnesses such as mood disorder and schizophrenia [16, 17]. There are conflicting results regarding the association of this polymorphism with FMS [8, 18, 19]. There is no report about the relationship between −1438G/A promoter polymorphism of 5-HT2A receptor gene and FMS up to now. Furthermore, it was reported that the −1438G/A polymorphisms in the 5-HT2A promoter region may be the susceptibility gene in patients with obsessive-compulsive disorder (OCD) and seasonal affective disorder [20, 21].

Catechol-O-methyltransferase (COMT) is an enzyme that inactivates catecholamines (dopamine, adrenaline, and noradrenaline) and thereby acting as a key modulator of dopaminergic neurotransmission [22]. Gene that codes COMT is located on 22q11. Various single nucleotide polymorphisms (SNPs) have been shown in COMT gene, and the best studied of them is rs4680 [9]. The COMT gene has been implicated in the pathogenesis of neuropsychiatric disorders such as OCD and schizophrenia [23, 24]. The relationship between the presence of FMS and COMT gene polymorphism has been investigated only in one study so far [9].

Fibromyalgia is a member of the family of the disorders called “affective spectrum disorder” (ASD). These disorders may have a common heritable pathophysiologic feature. Hudson et al. demonstrated that the forms of ASD frequently occur together within individuals (cooccurrence) and within families (coaggregation) further suggesting that these various forms might all share common etiologic factors and that are possibly heritable [25]. Fibromyalgia and affective disorders can be due to a common underlying abnormality, which according to the theories of depression, is most probably related to the catecholaminergic or serotonergic neurotransmission [26, 27].

Whether the FMS and some neuropsychiatric disorders share the same etiopathogenesis, or they are separate entities that coincidentally have the same alterations in gene loci are unknown. The aim of this study was to determine the potential effects of 102T/C and −1438G/A polymorphisms of the 5-HT2A receptor and COMT genotypes in patients with FMS and healthy controls in a Turkish population.

Materials and methods

Subjects

This study was designed as a case–control study. The study was conducted between May 2003 and July 2006 at the outpatient clinics of Physical Medicine and Rehabilitation Department. Eighty women who met the 1990 American College of Rheumatology criteria [1] for the diagnosis of FMS and 91 age-matched controls were enrolled.

Patients and controls were of the same ethnic origin and from the same geographic area (Black Sea Coastal Region). The approval from the Ethical Committee of Human Studies was obtained. Written informed consent was also obtained from all patients as well as the healthy volunteers.

Subjects were selected with the following criteria: (1) the symptoms of fibromyalgia for at least 1 year preceding the study, (2) age > 18 years, (3) at least 5 years of basic education, and (5) no history of another major physical or psychiatric illness that could interfere with the application of the psychological tests such as psychosis and mental retardation. Healthy controls with no history of chronic rheumatologic or psychiatric disease were matched for sex, age, and education.

All the patients had normal laboratory tests results regarding erythrocyte sedimentation rate, complete blood cell count, and liver function tests. The patients were questioned about their educational level, habits, social, and marital status. A visual analog scale (VAS) was used to measure perceived pain intensity in all subjects. Tender point examination was carried out by the exertion of a uniform amount of manual finger pressure, until fingernail bed blanches. The patients and controls were evaluated by a single psychiatrist (O.B.) according to Diagnostic and Statistical Manual of Mental Disorders (DSM IV TR) criteria, and patients who had any psychiatric diagnosis were excluded from the study to avoid genetical interference of psychiatric diagnosis and FMS. The patients and controls were also evaluated using psychiatric tests including the Beck depression inventory (BDI), revised symptom checklist-90 (SCL-90 R), and state and trait anxiety inventories (STAI)-I and -II. All patients were able to complete the questionnaires independently alone in a hospital room reserved for them.

Revised symptom checklist-90

The revised symptom checklist-90 (SCL-90 R) is a self-report instrument that has previously been used in fibromyalgia patients and healthy controls. It measures 90 items on a five-point scale of distress from 0 to 4 (none–extreme) and contains nine dimensions of psychological distress: somatization, obsessive-compulsive, interpersonal sensivity, depression, anxiety, hostility, phobic anxiety, paranoid ideation, psychoticism, as well as global severity index [28, 29]. A Turkish adaptation of the SCL-90-R was used [30].

The Beck depression inventory

The depression rate was assessed by the Beck depression inventory (BDI) in all patients and controls (0–63). The test consists of 21 questions scaled in a Likert format. The higher score shows increased depression of the subjects. It was shown to be valid and reliable in Turkish patients [31, 32].

State and trait anxiety inventories-I and -II

The state and trait anxiety inventories (STAI) consist of 44 items on which subjects rate themselves on four-point scales that assess either the intensity of their angry feelings or the frequency with which anger is experienced, expressed, suppressed, or controlled [33]. The scale has been validated in Turkish population studies with Cronbach’s α of subscales ranging between 0.69 and 0.90 [34].

The fibromyalgia impact questionnaire

The fibromyalgia impact questionnaire (FIQ) is widely used in FMS patients to evaluate both the clinical severity of the disease and the efficacy of different treatments, and has been found to be valid and reliable in Turkish fibromyalgia patients [35, 36].

DNA extraction

DNA was isolated from the heparinized 4 ml peripheral blood samples from all subjects using the ‘salting out’ method [37]. The 102T/C, −1438G/A of 5-HT2A receptor gene, and G/A polymorphism in COMT gene were identified from the National Center for Biotechnology Information LocusLink database [38]. The genotypes for restriction fragment length polymorphisms (RFLP) of the 5-HT2A receptor gene and COMT gene were determined by polymerase chain reaction (PCR-RFLP) (Techne Gradient, Cambridge, UK) and enzymatic digestion of the products with MspI and NlaIII restriction enzymes.

Genotyping of 5-HT2A (rs6313, rs6311) receptor gene polymorphisms

The −1438G/A (rs6311) polymorphism in the promoter region of 5-HT2A receptor gene was determined by PCR-RFLP as described by Arranz et al. [15]. The following primers (Iontec, Bursa, Turkey) were used in PCR reactions: 5'-AAG CTC CAA GGT AGC AAC AGC-3' forward and 5'-AAC CAA CTT ATT TCC TAC CAC –3' reverse. PCR was carried out in a total volume of 25 μl with 50 ng DNA, 20 pmol of each primer, 1.5 mM MgCl2, and 1.0 U Taq polymerase (MBI, Fermentas, Lithuania). The amplification conditions were initial denaturation at 95°C for 2°min; amplification was performed by 35 cycles of denaturation at 95°C for 60 s, annealing at 61°C for 45 s, and extension at 72°C for 45 s. The reaction was terminated by final extension at 72°C for 7 min.

The 102T/C (rs6313) polymorphism of the 5-HT2A receptor gene was performed as described previously [21]. A 342-bp region of the 5-HT2A receptor gene was amplified using primers (Iontec, Bursa, Turkey) SG1 5'-TCT GCT ACA AGT TCT GGC TT-3' (forward) and SG2 5'-CTG CAG CTT TTT CTC TAG GG –3' (reversed). PCR reactions contained 20 pmol of each primers, 0.2 mM dNTPs, 1.5 mM MgCl2, 50 ng DNA, and 1.0 U Taq polymerase (MBI, Fermentas, Lithuania) in a total volume of 25 μl. The PCR program was initial denaturation at 95°C for 3 min and 35 cycles of denaturation at 95°C for 45 s, annealing at 60°C for 45 s and extension at 72°C for 60 s. The reaction was terminated by final extention at 72°C for 5 min. Seven microliters of amplification products of both fragments were then digested with 10 U of MspI (MBI, Fermentas, Lithuania) overnight at 37°C. All the digested products were resolved on 2.5% agarose gel. Four hundred and sixty-eight base pair PCR products of −1438G/A polymorphism were cut into 244-bp and 224-bp fragments by endonuclease digestion. The genotype was designated as A when the restriction site was absent and as G when the restriction site was present. The three possible genotypes of 102T/C polymorphism distinguished by three distinct banding patterns, depending on the presence or absence of the MspI restriction site: C/C (342 bp), T/C (342, 215, and 126 bp), and C/C (215 and 126 bp); these were analyzed on a 2.5% agarose gel stained with ethidium bromide.

Genotyping of COMT (rs4680) gene polymorphism

Amplification of the 185-bp fragment encompassing NlaIII polymorphic site was amplified using primers sense SG3 5'-GGA GTC GGG GGC CTACTG GT-3' and antisense SG4 5'-GGC CCT TTT TCC AGG TCT GAC A-3' (reversed). PCR reactions contained 20 pmol of each primers, 1 mM MgCl2, 100 mM of each dNTP, 50 ng DNA, and 1.25 U Taq polymerase (MBI, Fermentas, Lithuania). Following initial denaturation at 94°C for 3 min, amplification was performed by 35 cycles of denaturation at 94°C for 60 s, annealing at 60°C for 60 s, and extension at 72°C for 60 s. The reaction was terminated by extension at 72°C for 7 min. Ten microliters of the PCR products was digested with 5 U of NlaIII (MBI, Fermentas, Lithuania) at 37°C [9]. All the digested products were resolved on 3.5% agarose (Prona) gel. The three possible genotypes of COMT G/A polymorphism were distinguished by three distinct banding patterns, depending on the presence or absence of the NlaIII restriction site: G/G (114 bp, 36 bp and 35 bp), A/G (114 bp, 96 bp, 36 bp, 35 bp and 18 bp) and A/A (96 bp, 36 bp, 35 bp and 18 bp).

Statistical analysis

Analysis of data was performed using the computer software SPSS 12.0 (SPSS Inc., Chicago, IL, USA). The normality of the variables was evaluated by the Kolmogorov–Smirnov test. The genotype and allelic frequencies were expressed as a percentage of the total number of genotypes or alleles. Odds ratios (OR) and 95% confidence intervals (CIs) were used for the assessment of risk factors. 5-HT2A receptor gene and COMT genotypes in patients were compared with those in controls using a chi-square test (two-sided) according to Hardy–Weinberg equilibrium. The expected values were compared with those observed in controls and cases by the chi-square test contingency tables. A probability of less than 0.05 was required for statistical significance.

Results

The demographical characteristics of the patients are shown in Table 1. The groups were similar according to demographic data and the level of education (P > 0.05). The mean years of education indicate that ours was a well-educated sample.
Table 1

Demographic characteristics of fibromiyalgia patients and controls

 

Fibromyalgia patients (n = 80)

Controls (n = 91)

P

Age (year)

39.58 ± 9.38

37.28 ±  9.15

>0.05

BMI (kg/m2)

24.91 ± 4.57

23.56 ± 4.18

>0.05

Marital status (%)

 Married

67 (83.8)

55 (60.4)

 

 Single

11 (13.8)

31 (34.0)

 

 Widow

2 (2.5)

5 (5.6)

 

Occupational status (%)

 Housewife

47 (58.7)

29 (31.8)

 

 Working

27 (33.7)

60 (65.9)

 

 Retired

6 (7.6)

2 (2.3)

 

 Student

   

Duration of education (year)

10.51 ± 4.07 (5–15)

11.24 ± 4.65 (5–15)

>0.05

Values are mean ± SD

BMI body mass index

Mean duration of the disease was 3.69 ± 4.08 years. All patients had at least 1-year duration of the disorder. The mean VAS score and number of tender points were 7.26 ± 1.60 cm and 14.23 ± 2.39, respectively. The mean FIQ score was 61.25 ± 10.22 in FMS group. The psychopathological data assessed with the BDI, SCL-90-R, STAI-I, and STAI-II are shown in Table 2. There was no difference between the patients with FMS and control subjects according to these tests (P > 0.05).
Table 2

The results of psychiatric tests in in fibromyalgia patients and control subjects

 

Fibromyalgia patients (n = 80)

Controls (n = 91)

P values

BDI

14.98 ± 8.90

8.08 ± 6.25

>0.05

SCL-90-R

 Global severity index

0.96 ± 0.68

0.49 ± 0.40

>0.05

 Somatization

1.38 ± 0.82

0.69 ± 0.61

>0.05

 Obsessive-compulsive

1.25 ± 0.83

0.79 ± 0.65

>0.05

 Interpersonal sensivity

0.84 ± 0.65

0.57 ± 0.51

>0.05

 Depression

1.04 ± 0.81

0.61 ± 0.56

>0.05

 Anxiety

0.92 ± 0.76

0.44 ± 0.44

>0.05

 Hostility

0.83 ± 0.73

0.35 ± 0.44

>0.05

 Phobic anxiety

0.53 ± 0.59

0.23 ± 0.34

>0.05

 Paranoid ideation

0.86 ± 0.78

0.49 ± 0.49

>0.05

 Psychoticism

0.48 ± 0.60

0.20 ± 0.31

>0.05

STAI-I

45.92 ± 9.75

40.04 ± 8.39

>0.05

STAI-II

48.25 ± 9.75

43.20 ± 8.65

>0.05

Values are mean ± SD

BDI beck depression inventory, SCL-90-R symptom checklist 90 revised, STAI-I state and trait anxiety inventory

Patients’ genotypes

The genotype distributions were in Hardy–Weinberg equilibrium among FMS subjects and controls. Table 3 shows the distribution of −1438G/A (A/A, G/A, G/G), 102T/C (C/C, C/T, T/T), and COMT (A/A, A/G, G/G) genotypes in the study groups. The association analysis showed no overall differences in genotype distributions when patients with FMS were compared to controls (Table 3). There were no differences in allele frequencies for −1438G/A (P = 0.16), 102T/C (P = 0.17), and COMT (P = 0.96) genotypes between FMS patients and control subjects (Table 3).
Table 3

Distribution of overall genotypes and allele frequencies in fibromyalgia patients and control subjects

Genotype

Fibromyalgia patients n = 80 (%)

Controls n = 91 (%)

OR (95% CI)

χ2

P

1438G/A (rs6311)

 A/A

23 (28.7)

34 (37.3)

1.00 (reference)

0.42

1.00

 G/A

30 (37.5)

35 (38.4)

1.27 (0.62–2.60)

2.49

0.52

 G/G

27 (33.8)

22 (24.3)

1.81 (0.84–3.93)

 

0.13

 Allele frequency

  G

0.48

0.55

1.44 (0.94–2.21)

 

0.16

  A

0.52

0.45

   

102T/C (rs6313)

 C/C

27 (33.8)

20 (21.9)

1.00 (reference)

2.57

1.00

 C/T

31 (38.7)

42 (46.3)

0.56 (0.27–1.18)

2.00

0.13

 T/T

22 (27.5)

29 (31.8)

0.54 (0.25–1.21)

 

0.13

 Allele frequency

  T

0.53

0.45

0.72 (0.47–1.11)

 

0.17

  C

0.47

0.55

   

COMT (rs4680)

 A/A

22 (27.5)

27 (29.7)

1.00 (reference)

0.15

1.00

 A/G

32 (40.0)

34 (37.4)

1.16 (0.55–2.43)

0.02

0.70

 G/G

26 (32.5)

30 (32.9)

1.06 (0.49–2.30)

 

0.88

 Allele frequency

  A

0.48

0.48

1.04 (0.68–1.58)

 

0.96

  G

0.52

0.52

   

OR odds ratio, CI confidence interval

Discussion

The ethiopathogenesis of FMS is multifactorial. A few groups investigated the association between gene polymorphism and FMS [18, 19, 27]. However, these studies were not specific for FMS and were associated with ASD or functional somatic disorders [27]. Almost all of the studies analyzing the relationship between FMS and genetic polymorphisms have been performed on heterogenous groups consisting of patients both with and without psychiatric symptoms. Thus, these studies eliminating the possible effects of psychological factors are lacking [8, 9, 19]. The results of the psychiatric tests of the patients and control subjects were statistically similar. This is also the first study investigating three genetic markers with FMS in patients without any measurable psychiatric symptoms. To assess the results of genetic association for both study and control groups, it is important to study ethically homogenous population, because using a genetically homogenous population leads to less potential bias in genetic case–control studies. Some investigators have studied FMS patients of both sexes and different ethnic origin [18, 39]. In contrast to these studies, the current study involved subjects from same ethnic origin and same sex.

Serotonin plays an important role in many neuropsychiatric disorders by regulating serotonergic pathways [12]. The possible association of a serotonin transporter gene polymorphism has been frequently discussed in FMS patients as well. Therefore, serotonin gene dysfunction might be the common etiologic factor for the association of FMS and psychiatric problems [8, 19]. Some investigators supported the relationship between serotonin receptor genes and FMS [8, 18, 19]. Offenbaecher et al. showed that S/S genotype occurs more frequently in FMS group than in controls. The S/S subgroup showed higher mean levels of depression and psychological distress [19]. In the study by Cohen et al., a significant relationship has been shown between the anxiety and serotonin transporter gene polymorphism in FMS patients [39]. In another study, Gürsoy et al. found a significant correlation between T/T genotype of the 5-HT2A receptor gene and the subgroup of the SCL-90-R test in patients with FMS [8]. However, we did not detect any relationship between FMS and serotonine genes. Bondy et al. demonstrated that pain severity was significantly higher in T/T genotype of the 102T/C polymorphism of the 5-HT2A receptor gene in FMS patients than the controls [18]. However, in the current study, there was no significant difference between patients and controls in the silent 102T/C polymorphism of the 5-HT2A receptor gene in contrast to other studies [8, 18]. Recently, HTR3A and HTR3B genes have been shown to play significant roles in FMS [40]. The authors suggested that mutations of these genes represent the basis for the new pharmacologic treatment on the future.

The 5-HT2A receptor gene −1438G/A studies are also new and its association with FMS has not been evaluated yet. To the best of our knowledge, this is the first study investigating −1438G/A polymorphism of the 5-HT2A receptor gene in this patient group. The findings of this study do not support any association of FMS with −1438G/A polymorphism. We found no difference of the frequencies of G and A alleles between patients and controls.

Two studies have addressed the possible relationship between COMT gene and FMS [9, 41]. Zubieta et al. showed a positive relationship between COMT gene polymorphism and experience of pain [41]. However, findings in the current study indicated that COMT gene polymorphism is not significantly different in patients and controls. The A/A genotype was significantly lower in FMS patients according to Gürsoy et al. [9]. On the other hand, similar to our study, these authors found that the allele frequencies of COMT gene were not different between patients and controls [9].

Our sample sizes, patients and controls, respectively (80 and 91), are relatively larger than the three studies from Turkey (58 and 58) [8, 9, 42]. However, lack of blood catecholamine levels is the major limitation of this study. Additionally and in contrast with previous studies, this study is unique in that all subjects (patients and controls) had been examined thoroughly by the same psychiatrist (O.B., one of us). In this way, the effects of psychiatric disorders have been excluded. The current study suggests that two 5-HT2A receptor genes and COMT genes polymorphisms seem not to be associated with FMS. Thus, they do not seem to play a role in pathogenesis of this disorder. It is highly possible that multiple interactions of several different neurotransmitter systems and pathways are involved in FMS.

Acknowledgments

This study was supported by the Ondokuzmayis University Research Foundation (No. T-368).

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© Springer-Verlag 2008