Study population
Between August 1995 and June 1997, all inhabitants aged 20 years and older in Nord-Trøndelag county in Norway (n = 92,936) were invited to participate in the Nord-Trøndelag Health Survey ("Helseundersøkelsen i Nord-Trøndelag"= HUNT). In brief, two questionnaires including > 200 health-related questions were administrated to the participants. The first questionnaire (Q1) was enclosed with the invitation letter and delivered during attendance at the health examination. The second questionnaire (Q2) was filled in after the examination and returned by mail.
Chronic MSCs
The HUNT study included questions about musculoskeletal symptoms adopted from the Standardized Nordic Questionnaire [6], which has previously been evaluated and found to give reliable estimates for low back pain [7], and for upper limb and neck discomfort [6–8]. Information about pain in other parts of the body has not been validated. All participants were asked: "Have you during the last year continuously for at least 3 months had pain and/or stiffness in muscles and joints?" Individuals who answered "yes" were defined as having chronic MSCs and these were asked to mark the localization of this pain (neck, shoulders, elbows, wrist/hands, chest/abdomen, upper back, low back, hips, knees, and/or ankles/feet). Those who responded "no" to the screening questions concerning chronic MSCs were defined as controls.
We also identified individuals with "chronic widespread pain" defined as axial skeletal pain (pain in the neck, chest/abdomen, upper back or lower back) and pain above the waist (neck, shoulders, elbows, wrist/hands, chest/abdomen or upper back) and below the waist (lower back, hips, knees, or ankles/feet). The participants were not asked to distinguish between pain in the left and the right side of the body and, consequently, we could not use the 1990 American College of Rheumatology (ACR) definition of chronic widespread pain.
Genotyping of the COMT locus
Blood sampling was done whenever subjects attended, and details for the procedure and the content of the HUNT 2 biobank are described elsewhere [9].
DNA for genotyping was extracted from peripheral blood leukocytes from whole blood or blood clots stored in the HUNT 2 biobank, using the Puregene kit (Gentra Systems Inc.) manually or with an Autopure LS (Gentra Systems Inc.). Laboratory technicians were blinded to the answer of the question about MSCs. COMT genotypes were determined using the LightCycler (Roche Diagnostics Scandinavia AB, Bromma, Sweden) fluorescence resonance energy transfer method [10]. Polymerase chain reaction (PCR) amplifications were performed in 20 μl reactions on a LightCycler System, using 2 μl genomic DNA and the LightCycler-FastStart DNA Master Hybridization Probes kit (Roche Diagnostics Scandinavia AB, Bromma, Sweden). PCR primers (Eurogentec, Seraing, Belgium) and fluorescence labeled probes (PROLIGO, Paris, France) used are described elsewhere [11]. Based on melting curve profiles, participants were classified as having Val/Val, Val/Met, or Met/Met genotypes. Details on PCR and melting curve conditions are available on request.
Participation
Out of the 92,936 invited individuals, a total of 64,787 subjects (70%) answered the first question about chronic MSC in Q1. Details of the non-participants are described elsewhere [7, 12, 13].
In the HUNT 2 biobank a total of 62,664 DNA samples are stored. At the time of HUNT 2, participants were not sufficiently informed about possible genetic DNA-based research. Therefore, an extensive information campaign about functional genomic research was performed in 2002. Each surviving adult HUNT 2 participant (n = 61,426) received an information folder and a personal letter asking for re-consent to include genetic research. In total, 1185 (1.9%) persons withdrew their consent [9, 12]. Out of the remaining group of 60,241 participants, COMT gene polymorphism analyses were performed in a sample of 3048 individuals. Approximately 70% of these were selected completely at random, and the remaining 30% had been randomly selected as age-matched controls to a diabetic population, and as a consequence, these were somewhat older than the HUNT population as a whole. Out of the 3048 individuals, a total of 3017 (98%) subjects also had responded to the questions about chronic MSCs.
Ethics
The study was approved by the Regional Committee for Ethics in Medical Research, and by the Norwegian Data Inspectorate.
Statistical analysis
Differences between continuous variables were tested with analyses of variance (one-way ANOVA) and dichotomous variables by the chi-square test. Analyses used two-tailed estimation of significance, and due to multiple numbers of comparisons, p < 0.01 was considered to be statistically significance (adjustment with Bonferroni method). Overall, our sample of 1529 individuals with chronic MSCs and 1488 controls had power to detect a 6% difference in prevalence of chronic MSCs between genotypes with 95% certainty and 90% power. For the groups with low number of individuals, e.g. pain in chest/abdomen, the study had 80% power to detect a 5% difference in prevalence with 95% certainty.
Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS), version 13.0 (SPSS Inc, Chicago).