ORIGINAL ARTICLE
Pain sensitivity in fibromyalgia is associated with catechol-O-methyltransferase (COMT) gene M. Martínez-Jauand1, C. Sitges1, V. Rodríguez1,2, A. Picornell1,2, M. Ramon1,2, D. Buskila3, P. Montoya1 1 University Institute of Health Sciences Research (IUNICS), University of Balearic Islands, Palma, Spain 2 Department of Biology, University of Balearic Islands, Palma, Spain 3 Division of Internal Medicine, Ben-Gurion University of the Negev, Israel
Correspondence Pedro Montoya E-mail:
[email protected] Funding sources Research was supported by grants from the Regional Government of the Balearic Islands (Acciones Especiales #AAEE0027/08), Spanish Ministry of Education (grant #AP2008-03742 awarded to M.M.J.) and Spanish Ministry of Science and Innovation and European regional development funds (grant #PSI201019372 awarded to P.M.), and La Marato TV3 Foundation. Conflicts of interest None declared. Accepted for publication 26 March 2012 doi:10.1002/j.1532-2149.2012.00153.x
Abstract Background: Recent evidence suggests that genetic factors might contribute to individual differences in pain sensitivity, risk for developing clinical pain conditions and efficacy of pain treatments. The purpose of the present study was to investigate the relationship of three common haplotypes of COMT gene affecting the metabolism of catecholamines on pain sensitivity in patients with fibromyalgia (FM). Methods: One hundred and thirteen FM patients and 65 age-matched healthy volunteers participated in the study. We genotyped four single-nucleotide polymorphisms (SNPs) (rs6269, rs4633, rs4818 and rs4680 or Val158Met) and identified haplotypes previously designated as low (LPS), average (APS) and high pain sensitivity (HPS). Thermal, pressure and touch thresholds were also examined using a quantitative sensory testing protocol. Results: The frequency of genetic variations associated with low COMT enzyme activity was significantly higher in FM patients than in healthy volunteers. FM patients were more sensitive to experimental pain than healthy volunteers and, in particular, FM individuals with the met/met genotype (Val158Met SNP) or the HPS-APS haplotypes showing higher sensitivity to thermal and pressure pain stimuli than patients carrying the LPS haplotype or val alleles (Val158Met SNP). No differences due to genotype or haplotypes were found on non-painful touch thresholds. Conclusions: According with previous research, our findings revealed that haplotypes of the COMT gene and genotypes of the Val158Met polymorphism play a key role on pain sensitivity in FM patients.
1. Introduction Fibromyalgia (FM) is an idiopathic diffuse pain syndrome characterized by lowered pain thresholds and other pain-related symptoms such as fatigue, anxiety and depression (Wolfe et al., 1990; Wolfe and Häuser, 2011). The prevalence of FM in the general population is around 3–5% (Gran, 2003) and 90% of affected individuals are women (Wolfe et al., 1995; Carmona et al., 2001). Our understanding of FM has made significant advances over the past decades and the current concept views FM as the result of central nervous system malfunction resulting in amplification
of pain transmission and interpretation (Gracely et al., 2002). Moreover, although the aetiology of FM is still unknown, a growing number of studies have focused on genetic contributors of FM and other chronic pain states, suggesting that both genetic and environmental factors might play a relevant role in the origin and development of FM (Buskila et al., 2007; Bradley, 2009). Single-nucleotide polymorphisms (SNPs) in the gene encoding for the catecholamine-O-methyltransferase (COMT) enzyme have been extensively analysed in association with pain perception and FM (Diatchenko et al., 2005, 2006; Vargas-Alarcón et al., 2007; Finan
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COMT gene, pain sensitivity and fibromyalgia
What’s already known about this topic? • Polymorphisms in the gene encoding for the COMT enzyme are associated with pain sensitivity in healthy controls. • These polymorphisms have been discussed as possible biomarkers for the development of fibromyalgia. What does this study add? • The study provides empirical evidence that differences in pain sensitivity between fibromyalgia patients and healthy controls are modulated by differences in the COMT gene. et al., 2010; Barbosa et al., 2012). Thus, for instance, the Val158Met (rs4680) is a functional SNP that substitutes the amino acid valine for methionine at codon 158 and determines the thermostability of the COMT enzyme involved in the metabolism of catecholamines (Lotta et al., 1995; Männistö and Kaakkola, 1999). The met/met genotype of the COMT Val158Met polymorphism has been associated with higher sensitivity in response to pain stimuli (Zubieta et al., 2003; Diatchenko et al., 2006; Jensen et al., 2009), as well as with high risk for the development of FM (Gürsoy et al., 2003; García-Fructuoso et al., 2006; Matsuda et al., 2010; Barbosa et al., 2012). Nevertheless, it seems that Val158Met SNP alone cannot account for pressure and thermal pain sensitivity (Kim et al., 2004) and that other SNPs (rs6269, rs4633, and rs4818) along the COMT gene locus may interact with Val158Met SNP to determine enzyme activity and to modulate pain sensitivity in healthy individuals (Diatchenko et al., 2006). Thus, for instance, a recent study by Diatchenko et al. (2005, 2006) have defined three genetic variations or haplotypes that determine COMT enzymatic activity and account for approximately 11% of the variability in responses to experimental pain. The aim of the current study was to examine differences on pain sensitivity and other pain-related symptoms in FM patients due to three common haplotypes of the COMT gene and the Val158Met polymorphism. For this purpose, four SNPs located in the central region of the COMT gene (rs6269, rs4633, rs4818 and rs4680 or Val158Met) were genotyped and ‘low’ (LPS), ‘average’ (APS) and ‘high pain sensitivity’ (HPS) haplotypes were identified according to previous reports (Diatchenko et al., 2005). We hypothesized that patients with met/met genotype (Val158Met polymorphism) and APS or HPS haplotype would display higher pain sensitivity than val homozygotes and heterozygotes, or carriers of LPS haplotype. 2
M. Martínez-Jauand et al.
2. Methods 2.1 Participants One hundred and thirteen patients diagnosed with FM (mean age 51.1 ⫾ 8.8) and 65 healthy volunteers (mean age 49.7 ⫾ 10.4) with comparable sociodemographic characteristics and without a history of chronic pain were recruited from different health centres and Fibromyalgia Patients Associations. All participants were female Caucasians and unrelated. Subjects were excluded from the study if they were pregnant or had any neurologic disease. Patients were included in the study if they fulfilled the classification criteria of the American College of Rheumatology (Wolfe et al., 1990) for FM, with a minimum of 11 tender points (of a total of 18 specific tender points), and had pain as their dominant symptom. At the time of recruitment, participants were verbally informed about the details of the study and provided written consent. The study was in accordance with the Declaration of Helsinki (1991) and was approved by the Ethics Committee of the Balearic Islands (Spain).
2.2 Assessment of socio-demographic and clinical pain characteristics All participants underwent an extensive medical and psychological assessment, including clinical pain characteristics through self-report questionnaires and a semi-structured clinical interview. The Beck Depression Inventory (Beck et al., 1961), the State-Trait Anxiety Inventory (Spielberger et al., 1970), the West Haven–Yale Multidimensional Pain Inventory (WHYMPI; Kerns et al., 1985) and the Edinburgh Handedness Inventory (Oldfield, 1971) were completed. Data about pain and quality of life were also obtained from a semi-structured interview, in which participants were also asked about their history of menopausal transition or days from last menstrual cycle in order to control possible hormonal influences on pain sensitivity (Martin, 2009).
2.3 Pain and touch threshold measurement 2.3.1 Touch sensitivity Touch sensitivity was assessed at the following locations of the non-dominant body side: (1) ventral surface of the wrist, (2) at the elbow and (3) at the index fingertip. For this purpose, a kit of von Frey monofilaments (Somedic Sales AB, Hörby Sweden) consisting of 17 nylon hairs with increasing diameters
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(0.14–1.01 mm), constant lengths and nominal force ranging from 26 mg to 110 g (manufacturer’s data) were used. They were applied by pressing the filament at a 90° angle against the skin until it was bent. The filament was held for 1.5 s and then removed. Subjects were instructed to answer ‘yes’ when a touch stimulus was perceived. The procedure starts with a thick filament and depending if it was felt or not, thicker or thinner filaments were applied. The detection threshold for mechanical stimuli at one specific body location was defined as the lowest pressure perceived by the subject in three consecutive trials. Responses with a delay greater than 3 s were considered as invalid and the trial was repeated. The order for testing the three body locations was counterbalanced across the subjects. 2.3.2 Pressure pain thresholds Pressure pain thresholds were assessed with a digital dynamometer using a flat rubber tip (1 cm2; Force One, Wagner Instruments, Greenwich, CT USA) at three body locations: (1) elbow over forearm muscles attach to lateral epycondile (tender point location as defined by the American College of Rheumatology; Wolfe et al., 1990), (2) medial area of the ventral surface of the wrist and (3) index fingertip. The last two body locations (wrist and fingertip) were chosen as pain-free control sites in FM patients. The pressure pain threshold was defined as the amount of pressure in newtons (N) at which subjects perceived the stimulus as painful. The maximal force allowed was 60 N at the elbow, 65 N at the wrist and 70 N at the fingertip. Subjects were excluded from this assessment procedure if they referred tunnel carpal syndrome on the wrist or epicondylitis at the elbow. 2.3.3 Heat pain thresholds Measures of heat pain threshold were obtained with a computer controlled contact thermal stimulator (Cold/ warm plate AHP-301CPV, Teca, Schubert, IL, USA). Subjects were instructed to put the index fingertip (from distal phalanges) on the thermal plate. Temperature increased from a slightly lukewarm temperature of 37 °C at a mean rate of 0.2 °C/s until a maximum of 52 °C. Thermal pain threshold was defined as the temperature (°C) at which subjects first perceived heat pain. 2.3.4 Cold pain tolerance For the assessment of cold pain tolerance, a thermal plate was set at a constant temperature of 1 °C. Par-
COMT gene, pain sensitivity and fibromyalgia
ticipants were instructed to keep the fingertip in contact with the plate base until the perception was unbearable. During the task, participants were instructed to rate current pain by using a 0–100 numerical rating scale (NRS; 0 representing ‘no pain’, and 100 the ‘maximal pain imaginable’) every 10 s. A pain score was computed by calculating the mean change score between two consecutive NRS values. Cold pain tolerance time was defined by the time that participants were able to tolerate the cold stimulus (maximal testing time: 150 s). Pain thresholds (cold, heat and pressure) were assessed once bilaterally following a randomized order, and values from right and left sides were averaged. Touch thresholds were evaluated three times at the non-dominant body side. Different orders of pain and touch threshold tests were designed avoiding the consecutive presentation of two different thermal stimuli (heat and cold). Subjects were assessed by the same person in order to avoid experimenter bias, and stayed in the lab alone with the experimenter during the assessment. For statistical analyses, pain and touch thresholds data were previously transformed to Z scores.
2.4 Genotyping DNA was extracted from buccal cells using standard phenol-chloroform extraction. The rs4680 (Val158Met), rs6269, rs4633 and rs4818 SNPs of the haplotype were genotyped by real-time polymerase chain reaction (PCR) method (Mobascher et al., 2010). For this purpose, 6.75 ng of genomic DNA were amplified in a total volume of 10 mL with 0.5 mL of allele probes TaqMan® SNP Genotyping Assays (Applied Biosystems, Foster City, CA, USA) and 5 mL of LightCycler® 480 Probes Master (Roche Diagnostics SL, Barcelona, Spain). The parameters for the amplification condition were: 10 min at 95 °C (ramp rate 4.40 °C/s), followed by 50 cycles at 92 °C for 15 s (ramp rate 2.20 °C/s) and at 60 °C for 1 min and 10 s (ramp rate 2.20 °C/s). Allelic discrimination analysis was performed on the LightCycler® 480 Real-Time PCR System using the software LightCycler® 480 (LC480) (Roche Diagnostics). Genotypes were classified based on endpoint genotyping (fluorescence) profiles. All genotypes were determined twice. Reference DNA samples and negative controls were included to ensure the accuracy of the SNP genotyping assay. Pairwise linkage disequilibrium estimations (LD, D’) between SNPs and haplotype reconstruction were performed with Haploview software 4.2 (Broad Institute of Massachusetts, Cambridge, MA, USA).
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COMT gene, pain sensitivity and fibromyalgia
To determine the Val158Met genotypes, the method for restriction fragment length polymorphisms described by Birklein et al. (2008) was additionally used. For this purpose, a 109-base pair PCR product was generated in 35 cycles with a primer annealing temperature at 58 °C. Each reaction contained 50 ng of template DNA, DyNAZyme IX reaction buffer (Finnzymes, Madrid, Spain), 0.2 mM of each dNTP, 1 U DyNAZyme II DNA Taq polymerase (Finnzymes) and 0.1 nM of each primer, previously published (Zubieta et al., 2003) in a total volume of 50 mL. Negative controls were included. The amount of 30 mL of the PCR products was digested in a volume with 1 ¥ BSA, 1 ¥ NEB buffer 4 and 5 U NlaIII (New England Biolabs Inc., Ipswich, MA, USA) at 37 °C during 3 h, and followed by a 3–4% agarose gel electrophoresis. The val homozygotes (87 and 22 base pairs), met homozygotes (69, 18 and 22 base pairs) and val/met heterozygotes (87, 69, 22 and 18 base pairs) were visualized by ethidium bromide staining. In the case of discrepancy between the two methods for genotyping the rs4680, samples were sequenced by using Big Dye® Terminator Cycle Sequencing kit v. 3.1 (Applied Biosystems) and ABI PRISM® 3100 Genetic Analyser (Applied Biosystems). In the present study, discrepant results were found in 3.1% of the samples for rs4680 genotyping.
2.5 Data analyses The Hardy–Weinberg equilibrium was tested for each polymorphism. Allele and genotype frequencies were estimated by gene counting and tested by using chisquare test. To test the effects of the COMT gene on clinical data and sensitivity measures, participants were divided in low (carriers of only APS and/or HPS haplotypes) and high (carriers of at least one LPS haplotype) COMT activity. In addition, the effects of Val158Met genotypes in FM patients were analysed by grouping the participants into met homozygotes and val carriers (val homozygotes and val/met heterozygotes). Socio-demographic and questionnaire data were analysed by using analyses of variance (ANOVAs) and chi-square test. Differences on sensitivity measures were assessed by using ANOVAs with the betweensubject factors GROUP (FM patients vs. healthy subjects) and HAPLOTYPE (LPS carriers vs. HPS-APS carriers). For testing the effects of Val158Met on sensitivity measures, ANOVAs were performed only by using the between-subject factor GENOTYPE (met/met vs. val carriers) in FM patients. ANOVAs also included the within-subject factor BODY LOCATION (elbow vs. wrist vs. finger) for testing the effects on pressure pain 4
M. Martínez-Jauand et al.
and touch thresholds, and MODALITY (heat vs. cold) for thermal pain thresholds. Post hoc tests were carried out with Bonferroni adjusted multiple pairwise mean comparisons included in the SPSS package (SPSS Inc., Chicago, IL, USA). In order to control for the effects of depression, anxiety, age and medication on sensitivity measures, additional ANOVAs were also carried out using them as covariates. For all ANOVAs, degrees of freedom were corrected using the Greenhouse–Geisser epsilon. The statistical significant level was set at p < 0.05.
3. Results 3.1 Differences on allelic and haplotype frequencies of the COMT gene The genotype and the allele frequencies for the individual SNPs and haplotypes of the COMT gene are summarized in Table 1. Frequency distribution for all SNPs was in accordance with Hardy–Weinberg equilibrium. No significant differences were observed between FM patients and healthy controls in the allelic frequencies. Nevertheless, the frequency of rs4680 (Val158Met) genotypes differed significantly between groups (c2 = 9.383; p = 0.009), indicating that met homozygotes (AA) were more frequent in FM patients (23.9%) than in healthy volunteers (7.7%). Genotype distribution of rs4633 also differed significantly between groups (c2 = 7.727; p = 0.021), showing that the TT genotype was more frequent in FM patients (25.9%) than in healthy controls (9.2%). The genotypes of the other two SNPs (rs4818 and rs6269) were distributed similarly in FM patients and controls. Five major diplotypes were identified with no significant differences on frequency distribution between FM patients and healthy controls (Supporting Information Table S1, online-only material). However, haplotypes frequency (HPS-APS vs. LPS) was different in FM patients versus healthy controls (c2 = 4.754; p = 0.029). Patients had higher frequencies of APS (ATCA) and HPS (ACCG) haplotypes than healthy controls (37.8% vs. 21.9%, respectively). The analysis of linkage disequilibrium between SNPs revealed that the strongest associations were found between rs4633 and rs4680 (D’ = 0.975; R2 = 0.884), and between rs4818 and rs6269 (D’ = 0.868; R2 = 0.708).
3.2 Differences on socio-demographic and clinical data due to the COMT gene Higher scores were found in FM patients than in healthy controls for depression [F(1,169) = 99.96;
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COMT gene, pain sensitivity and fibromyalgia
Table 1 Genotype and allele frequencies of rs6269, rs4633, rs4818 and rs4680 (Val158Met) SNPs of COMT gene in FM patients and healthy controls. Allele frequencies ⫾ SD
Genotype frequencies n (%) SNP rs6269 AA AG GG rs4633 CC CT TT rs4818 CC CG GG rs4680 (Val158Met) AA AG GG a
FM (n = 113)
Healthy (n = 65)
p-valuea
FM (n = 113)
Healthy (n = 65)
p-valuea
39 (34.8) 53 (47.3) 20 (17.9)
15 (23.1) 36 (55.4) 14 (21.5)
0.262
A G
0.584 ⫾ 0.033 0.416 ⫾ 0.033
0.508 ⫾ 0.044 0.492 ⫾ 0.044
0.159
33 (29.5) 50 (44.6) 29 (25.9)
20 (30.8) 39 (60.0) 6 (9.2)
0.021
C T
0.518 ⫾ 0.033 0.482 ⫾ 0.033
0.608 ⫾ 0.033 0.392 ⫾ 0.033
0.101
41 (36.9) 53 (47.3) 18 (16.1)
14 (21.5) 37 (56.9) 14 (21.5)
0.109
C G
0.603 ⫾ 0.033 0.397 ⫾ 0.033
0.500 ⫾ 0.044 0.500 ⫾ 0.044
0.060
27 (23.9) 52 (46.0) 34 (30.1)
5 (7.7) 43 (66.2) 17 (26.2)
0.009
A G
0.469 ⫾ 0.033 0.531 ⫾ 0.033
0.408 ⫾ 0.043 0.592 ⫾ 0.043
0.265
Chi-square test was used.
p < 0.001], anxiety trait [F(1,177) = 83.9; p < 0.001] and anxiety state [F(1,177) = 23.2; p < 0.001] (Supporting Information Table S2, online-only material). Table 3 displays socio-demographic and clinical data according to the COMT haplotypes. ANOVAs for COMT haplotype revealed only a significant effect for the ‘solicitous responses’ WHYMPI scale (p = 0.025), indicating that FM patients carrying APS and/or HPS haplotypes reported more solicitous responses than FM patients carrying the LPS haplotype. No effects of COMT haplotypes were found on anxiety and depression for FM patients or healthy controls. Table 2 displays socio-demographic and clinical data according to the Val158Met genotype (met homozygotes and val carriers) in FM patients. FM patients carrying the met/met genotype reported higher levels of anxiety trait [F(1,112) = 6.09; p = 0.015] and affective distress [WHYMPI-subscale; F(1,106) = 4.09; p = 0.046] as compared with FM patients carrying val alleles. No effects due to the Val158Met genotype were found on anxiety state [F(1,112) = 2.72; p = 0.102] or depression [F(1,108) = 0.732; p = 0.394].
3.3 Differences on pain and touch thresholds due to the COMT haplotypes Figure 1 displays mean values for pressure pain, thermal pain and touch thresholds in FM patients and healthy controls. 3.3.1 Pressure pain threshold The ANOVA for pressure pain thresholds revealed significant effects of GROUP [F(1,164) = 135.82;
p < 0.001] and GROUP ¥ BODY LOCATION [F(2,328) = 3.70; p = 0.028]. Post hoc analyses revealed lower pressure pain thresholds in FM patients as compared with healthy controls at the three body locations (elbow, wrist and fingertip; all p < 0.001; Fig. 1). In addition, significant effects of COMT HAPLOTYPE [F(1,162) = 8.49; p = 0.004] and GROUP ¥ HAPLOTYPE ¥ BODY LOCATION [F(2,324) = 3.57; p = 0.032] were yielded. Post hoc analyses indicated that HPS-APS carriers showed lower pressure pain thresholds at the elbow (p = 0.011) in comparison to LPS carriers only in FM patients, whereas HPS-APS carriers showed lower pain thresholds than LPS carriers at wrist (p = 0.024) and fingertip (p = 0.012) only in healthy controls (Fig. 2). The interaction effect remained significant after controlling for anxiety, depression, age and medication [F(2,270) = 4.2; p = 0.017]. 3.3.2 Thermal pain threshold ANOVA for thermal pain measures revealed a significant effect of GROUP [F(1,174) = 61.86; p < 0.001], indicating that FM patients displayed higher heat (p < 0.001) and cold pain (p < 0.001) sensitivity than healthy controls (Fig. 1). A significant interaction GROUP ¥ HAPLOTYPE ¥ THERMAL MODALITY [F(1,169) = 4.61; p = 0.033] was found. Post hoc comparisons revealed that HPSAPS carriers showed higher cold pain responsiveness (p < 0.010) in comparison to LPS carriers only in FM patients, whereas no differences were yielded in healthy controls or for heat pain thresholds (Fig. 3).
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Table 2 Socio-demographic and clinical characteristics of the two groups of fibromyalgia patients according with the COMT genotype (met homozygotes and val carriers).
Age (years) Mean ⫾ SD Range Education level (%) 12 years Medication (%) Antidepressants Anxiolytics Analgesic/muscle relaxants/NSAIDs Relatives with chronic pain (%) Pain intensity by 10-cm VAS (range 0–10) Mean ⫾ SD Duration of pain (years) Mean ⫾ SD Range Beck Depression Inventory Mean ⫾ SD Range State Anxiety Inventory Mean ⫾ SD Range Trait Anxiety Inventory Mean ⫾ SD Range WHYMPI (0–6), mean ⫾ SD Social support Affective distress Pain interference Pain intensity Life control Distracting responses Solicitous responses Punishing responses Household chores Outdoor work Social activities Activities away from home a
val carriers FM patients (n = 35)
met/met FM patients (n = 27)
50.5 ⫾ 8.9 27–71
52.9 ⫾ 8.6 40–71
0.238
ANOVAs were also computed to test the effects of the Val158Met genotype on pain and touch thresholds. In the case of healthy controls, only five subjects were classified as met homozygotes and, therefore, only results obtained in FM patients group were reported.
13.7 35.6 50.7
26.1 30.4 43.5
0.380
3.4.1 Pressure pain threshold
59.5 57.0 77.2
64.0 64.0 92.0
0.688 0.533 0.102
50.3
61.3
0.695
p-value
7.1 ⫾ 1.6
7.6 ⫾ 1.7
0.240
19.0 ⫾ 12.5 2–45
19.5 ⫾ 12.5 2–43
0.896
19.6 ⫾ 10.2 2–43
21.5 ⫾ 8.4 6–34
0.394
27.1 ⫾ 11.2 7–56
31.1 ⫾ 10.4 15–51
0.102
34.0 ⫾ 11.3 6–57
39.8 ⫾ 7.8 22–53
0.015
3.6 ⫾ 1.6 3.6 ⫾ 1.2 3.7 ⫾ 1.4 4.2 ⫾ 1.1 3.6 ⫾ 1.3 3.3 ⫾ 1.6 2.6 ⫾ 1.6 1.6 ⫾ 1.7 3.9 ⫾ 1.2 2.4 ⫾ 1.3 1.6 ⫾ 1.6 2.0 ⫾ 1.3
3.8 ⫾ 1.8 4.1 ⫾ 0.8 4.1 ⫾ 1.2 4.4 ⫾ 0.9 3.6 ⫾ 1.2 3.8 ⫾ 1.6 3.2 ⫾ 1.7 1.0 ⫾ 1.1 3.6 ⫾ 1.5 2.4 ⫾ 1.1 1.5 ⫾ 1.7 2.1 ⫾ 1.2
0.425 0.046 0.138 0.509 0.968 0.231 0.101 0.095 0.356 0.943 0.938 0.782
Chi-square test and ANOVAs were used.
The interaction effect remained significant after controlling for anxiety, depression, age and medication [F(1,112) = 6.0; p = 0.015]. 3.3.3 Touch threshold ANOVA for touch thresholds revealed no significant main effects due to COMT HAPLOTYPE or its interaction with other factors. 6
3.4 Differences on pain and touch thresholds due to the Val158Met genotype
ANOVA revealed a significant effect due to Val158Met GENOTYPE [F(1,104) = 5.27; p = 0.024], showing that met/met FM patients displayed lower pressure pain thresholds than val carriers (Fig. 4). The effect of Val158Met genotype on pressure pain thresholds remained significant after controlling for anxiety, depression, age and medication [F(1,86) = 5.7; p = 0.019]. 3.4.2 Thermal pain threshold ANOVA revealed significant main effect of Val158Met GENOTYPE [F(1,110) = 4.69; p = 0.033] and interaction effects of MODALITY ¥ Val158Met GENOTYPE [F(1,110) = 5.38; p = 0.022]. Post hoc analyses of the interaction effect showed that met homozygotes had higher cold pain sensitivity than val carriers (p = 0.002; Fig. 4). Nevertheless, a non-significant trend of the interaction effect was yielded after controlling for anxiety, depression, age and medication [F(1,91) = 3.6; p = 0.060]. 3.4.3 Touch threshold ANOVA revealed no significant main effects due to Val158Met GENOTYPE or its interaction with other factors.
4. Discussion In the present study, we examined differences on pain sensitivity due to the presence of different SNPs in the gene encoding for the COMT enzyme in patients with FM. For this purpose, four SNPs (rs6269, rs4633, rs4818 and rs4680) from the central region of the COMT gene were determined, and three genetic variations or haplotypes were defined in accordance with previous findings by Diatchenko et al. (2005) in healthy controls. Thus, the GCGG sequence was identified as the ‘low pain sensitivity’ haplotype and the ATCA and ACCG sequences as ‘average/high pain sensitivity’ haplotype.
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COMT gene, pain sensitivity and fibromyalgia
Table 3 Socio-demographic and clinical characteristics of fibromyalgia patients and healthy controls according with APS-HPS or LPS haplotypes of COMT gene. FM patients
Age (years) Mean ⫾ SD Range Education level (%) 12 years Medication (%) Antidepressants Anxiolytics Analgesic/relaxants/NSAIDs Relatives with chronic pain (%) Pain intensity by 10-cm VAS (range 0–10) Mean ⫾ SD Duration of pain (years) Mean ⫾ SD Range Beck Depression Inventory Mean ⫾ SD Range State Anxiety Inventory Mean ⫾ SD Range Trait Anxiety Inventory Mean ⫾ SD Range WHYMPI (0–6), mean ⫾ SD Social support Affective distress Pain interference Pain intensity Life control Distracting responses Solicitous responses Punishing responses Household chores Outdoor work Social activities Activities away from home a
Healthy controls a
APS-HPS
LPS
p-value
APS-HPS
LPS
p-valuea
53.6 ⫾ 7.8 40–71
50.1 ⫾ 9.1 27–70
0.040
50.6 ⫾ 10.7 35–68
48.3 ⫾ 10.4 27–68
0.510
21.6 24.3 54.1
12.5 39.3 48.2
0.247
30.0 30.0 40.0
24.2 27.3 27.9
0.888
61.5 56.4 76.9 69.2
59.3 59.3 84.7 67.2
0.826 0.775 0.328 0.826
7.1 21.4 7.1 16.7
14.3 23.8 4.8 16.7
0.484 0.855 0.732 0.100
7.6 ⫾ 1.7
6.9 ⫾ 1.7
0.083
–
–
20.9 ⫾ 12.9 2–45
17.8 ⫾ 12.0 3–57
0.222
– –
– –
19.6 ⫾ 9.3 6–35
20.0 ⫾ 9.8 2–43
0.845
5.7 ⫾ 4.7 0–15
7.5 ⫾ 5.6 0–26
29.7 ⫾ 11.6 9–55
17.4 ⫾ 10.8 7–56
0.329
19.4 ⫾ 11.7 0–43
20.5 ⫾ 9.4 3–42
36.8 ⫾ 11.5 9–57
34.1 ⫾ 10.5 6–57
0.209
20.6 ⫾ 8.8 22–53
21.3 ⫾ 8.4 8–47
3.7 ⫾ 1.8 3.8 ⫾ 1.1 4.0 ⫾ 1.3 4.3 ⫾ 1.4 3.8 ⫾ 1.5 3.8 ⫾ 1.6 3.2 ⫾ 1.7 1.1 ⫾ 1.3 3.6 ⫾ 1.5 2.3 ⫾ 1.1 1.4 ⫾ 1.6 1.9 ⫾ 2.1
3.6 ⫾ 1.5 3.6 ⫾ 1.1 3.6 ⫾ 1.3 4.2 ⫾ 1.0 3.7 ⫾ 1.2 3.2 ⫾ 1.7 2.4 ⫾ 1.6 1.7 ⫾ 1.7 4.0 ⫾ 1.2 2.4 ⫾ 1.3 1.6 ⫾ 1.6 2.1 ⫾ 1.2
0.746 0.617 0.229 0.669 0.236 0.086 0.025a 0.074 0.271 0.706 0.466 0.684
– – – – – – – – – – – –
– – – – – – – – – – – –
0.265 0.329 0.761
0.811
Chi-square test and ANOVAs were used.
Our data indicated that allelic frequencies of all four SNPs were distributed in a similar way in FM patients and healthy controls, and that genotypes assumed the Hardy–Weinberg Equilibrium. Moreover, the genotype distribution in FM patients and healthy controls was similar to those reported in previous studies (Gürsoy et al., 2003; Kim et al., 2004; García-Fructuoso et al., 2006; Vargas-Alarcón et al., 2007; Matsuda et al., 2010). Nevertheless, we found that the HPS-APS haplotypes were more frequent in FM patients than in healthy controls. In this sense, our
results are in accordance with previous reports showing a strong association between the HPS haplotype and high scores on the Fibromyalgia Impact Questionnaire (Vargas-Alarcón et al., 2007), and that these haplotypes might be associated with increased risk of developing chronic pain disorders (Diatchenko et al., 2005). FM patients were overall more sensitive to experimental pain than healthy volunteers. We also observed that FM patients with the HPS-APS haplotypes were more sensitive to pressure stimuli at the elbow and reported higher pain responsiveness to cold
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Figure 1 Pain sensitivity and touch thresholds in FM patients and healthy controls groups. FM patients had the greatest pain responsiveness at the different modalities and body locations: pressure pain assessed at the elbow, wrist and fingertip (A); and heat and cold thermal pain (B). No significant differences were found on touch thresholds between groups (C). Data are expressed as means of Z scores ⫾ SD. Significant differences between groups are marked with asterisks (***p < 0.001).
stimuli than FM patients with the LPS haplotype. These findings are in line with previous studies showing that healthy individuals with HPS-APS haplotypes are much more sensitive to painful stimuli than subjects with LPS haplotypes (Diatchenko et al., 2005, 2006). Our findings are also in agreement with previous evidence suggesting that FM patients are especially sensitive to pressure stimulation at the elbow (Wolfe et al., 1990; Geisser et al., 2003) and that they display higher sensitivity for cold than for heat stimuli (Hurtig et al., 2001). Thus, it could be that genetic polymorphisms that reduce COMT expression can preferentially modulate mechanical and cold pain sensitivity in FM. To our knowledge, this is the first study showing a significant link between COMT haplotypes and sensitivity to experimental pain in FM. One possible explanation for the enhanced pain sensitivity to pressure and cold stimuli in FM patients with HPS and APS haplotypes could be linked to a reduced enzymatic activity of catecholamines. In this sense, it has been shown that HPS and APS haplotypes provide lower COMT activity compared with the LPS haplotype (Diatchenko et al., 2005). Our findings also appear to be in accordance with previous studies showing enhanced pain sensitivity in response to mechanical and thermal stimuli after administration
of COMT enzyme inhibitors (Nackley et al., 2007), or activation of b2 and b3-adrenergic receptors in rats (Khasar et al., 1999; Aley et al., 2001). A further interesting result of the present study was that the percentage of met homozygotes for the Val158Met polymorphism of COMT gene was significantly higher in FM patients than in healthy individuals. These findings are in accordance with previous data showing higher frequencies of met carriers in FM patients for Spanish (García-Fructuoso et al., 2006), Turkish (Gürsoy et al., 2003) and Brazilian populations (Matsuda et al., 2010; Barbosa et al., 2012). Our data are also consistent with previous studies showing that FM patients with met alleles reported the most severe pain symptoms (García-Fructuoso et al., 2006; Finan et al., 2011; Barbosa et al., 2012) and higher number of painful tender points than FM patients with val alleles (Cohen et al., 2009). Moreover, we observed that FM patients carrying a met/met genotype were more sensitive to pressure pain and cold pain than val carriers. The relationship between the Val1158Met polymorphism of the COMT and pain sensitivity has been consistently examined in healthy volunteers, showing that carriers of the met/ met genotype report the most intense pain (Zubieta et al., 2003; Diatchenko et al., 2006; Jensen et al.,
Figure 2 Mean and standard deviations (SD) for pressure pain thresholds assessed at the elbow, wrist and fingertip in FM patients and healthy controls categorized by the COMT haplotypes. Pressure pain threshold at the elbow was lower for FM patients carrying the HPSAPS haplotypes as compared with patients carrying the LPS haplotype. Pressure pain thresholds assessed at the wrist and fingertip differed significantly between HPS-APS and LPS healthy carriers, with no differences in FM patients group. Significant differences between groups are marked with asterisks (*p < 0.05).
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Figure 3 Mean thermal pain sensitivity in FM patients and healthy controls categorized by the COMT haplotypes. FM patients carrying the HPSAPS haplotypes had lower cold pain tolerance in comparison to LPS carriers with no differences observed in healthy controls. No differences were found for heat pain thresholds. Data are expressed as means of Z scores ⫾ SD. Significant differences between genotype groups are marked with asterisks (**p < 0.01).
2009), reduced responses of the m-opioid endogenous system to sustained pain (Zubieta et al., 2003) and increased activation of pain-related brain areas (anterior and midcingulate cortex) during the processing of painful laser stimuli (Mobascher et al., 2010). In a similar way, it has been shown that chronic pain patients with met/met or val/met genotypes display stronger brain activity elicited by painful stimuli than val homozygotes (Vossen et al., 2010). In this sense, it is possible that this functional polymorphism of the COMT gene could be associated with enhanced responses to pressure and thermal pain stimuli requiring a temporal integration at the central nervous system (Zubieta et al., 2003; Diatchenko et al., 2006). Thus, it could be that genetic polymorphisms that reduce COMT expression can preferentially modulate mechanical and cold pain sensitivity in FM. The met158 allele of the COMT gene has been also linked to high risk for chronic pain syndromes such as migraine (Erdal et al., 2001), back pain (Vossen et al., 2010), headache (Hagen et al., 2006a) and temporomandibular joint disorder (Diatchenko et al., 2005). Moreover, it has been reported that COMT polymorphisms and pain catastrophizing are the best predictors of clinical pain ratings in post-operative shoulder pain (George et al., 2008). The Val158Met polymorphism of COMT appears to influence pain sensitivity through central mechanisms such as reduced responses in m-opioid endogenous system to sustained pain, higher receptor binding and enhanced brain processing of sensory and affective pain components in met carriers (Zubieta et al., 2003; Mobascher et al., 2010). The effects of the Val158Met polymorphism on the m-opioid
COMT gene, pain sensitivity and fibromyalgia
receptor system is also supported by studies in cancer pain patients, showing that met/met and val/met patients needed lower doses of morphine than val/val patients (Rakvåg et al., 2005; Reyes-Gibby et al., 2007). Thus, it could be argued that characterization of these polymorphisms in FM and other chronic pain syndromes might be useful to establish the optimal pharmacological treatment for these patients. However, the role of this polymorphism in the development of clinical pain syndromes is still controversial. In this sense, some studies have found a lack of association between functional Val158Met polymorphism and other chronic pain syndromes (Hagen et al., 2006b; Hocking et al., 2010) in population-based studies and in neuropathic pain patients (Armero et al., 2005). Hence, further investigations with larger samples should clarify the role of Val158Met polymorphism on chronic pain complaints. Finally, we also found that FM patients with met/met genotype reported more anxiety trait and affective distress than val carriers. Several studies have related COMT Val158Met polymorphism with a higher risk of anxiety-related behaviours and anxiety traits in women (Eley et al., 2003; Enoch et al., 2003; Stein et al., 2005). Moreover, recent studies clearly showed that COMT variants moderate not only pain but also maladaptive coping processes in FM patients (Finan et al., 2010, 2011). Authors reported that met/met FM patients experienced a greater decline in positive affect on days when pain was elevated (Finan et al., 2010), and greater pain on days when pain catastrophizing was elevated than did either val/met or val/val individuals (Finan et al., 2011). Although a possible modulation
Figure 4 Pressure and thermal pain sensitivity categorized by the COMT Val158Met genotype in FM patients group. Met homozygotes had lower thresholds to pressure pain and lower cold pain tolerance as compared with val carriers. Data are expressed as means of Z scores ⫾ SD. Significant differences between genotype groups are marked with asterisks (**p < 0.01; * p < 0.05).
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of pain sensitivity through an altered emotional processing in met/met individuals cannot be completely excluded, we found in the present study that the effects of Val158Met genotype remained after controlling for anxiety, depression, age and medication. This study has several limitations that deserve mention. We found only five healthy subjects with the met/met genotype and, therefore, differences due to Val158Met genotypes were not explored in this group. In order to compare the differential effects of met alleles on FM and healthy controls, larger samples of patients and healthy controls should be examined in future research. It should be also noted that our findings were obtained on female adults and, therefore, no information about the modulatory effect of gender on the association between pain sensitivity and the COMT haplotypes or the Val158Met polymorphism could be explored. In summary, we found that the frequency of genetic variations associated with low COMT enzyme activity was higher in FM patients than in healthy controls, and that FM patients who possess those genetic combinations displayed an increased sensitivity to experimental pain. According with previous studies, our data suggest that decrease of COMT activity might contribute to the maintenance of pain symptoms in FM. Over the years, it has became obvious that FM is not a homogeneous condition and that different subtypes of patients might exist (Turk et al., 1996, 1998; Giesecke et al., 2003; Thieme et al., 2005). In this sense, our findings suggest that specific genetic variations of the COMT gene might have different consequences for responsiveness to experimental pain in FM, and might play a significant role in classifying FM patients. Acknowledgements We acknowledge the technical support provided by Mr. Josep Agustí Pablo Canaves and Ms. Joana María Buades from the University of Balearic Islands.
Author contributions MMJ was involved in designing the study, performing the experiments, genotyping, data analyses, and wrote the first draft of the manuscript. CS was involved in statistical data analyses and contributed to the preparation of the manuscript. VR was involved in genotyping and preparing the manuscript. AP and MR were involved in designing the study and preparing the manuscript. DB was involved in the preparation of the manuscript. PM was involved in designing the study, statistical data analyses and contributed to the preparation of the manuscript. All authors read and approved the final manuscript.
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Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1. Haplotype frequencies for COMT gene in FM patients and healthy controls. Table S2. Sociodemographic and clinical characteristics of FM patients and healthy controls. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.
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