Supplementary: Genome-wide association study meta-analysis of chronic widespread pain: evidence for involvement of the 5p15.2 region

1 Department of Internal Medicine, Erasmus Medical Center Rotterdam, Rotterdam, The Netherlands 2 The Netherlands Genomics Initiative-sponsored Netherlands Consortium for Healthy Aging (NGI-NCHA), Leiden/Rotterdam, The Netherlands 3 Department of Epidemiology, Erasmus Medical Center Rotterdam, Rotterdam, The Netherlands 4 Laboratory of Neuroimmunology and Developmental Origins of Disease, University Medical Center Utrecht, The Netherlands 5 Icelandic Heart Association Research Institute, Kopavogur, Iceland 6 Aberdeen Pain Research Collaboration (Musculoskeletal Research), University of Aberdeen, Aberdeen, UK 7 Arthritis Research UK Epidemiology Unit, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK 8 Mental Health and Neurodegeneration Group, School Community Based Medicine, University of Manchester, Manchester, UK 9 Department of Medical Statistics and Bioinformatics, Section of Molecular Epidemiology, Leiden University Medical Centre, Leiden, The Netherlands 10 Clinical Epidemiology Unit, Boston University School of Medicine, Boston, Massachusetts, USA 11 Department of Twin Research and Genetic Epidemiology, King’s College London, London, UK 12 Institute for Community Medicine, University of Greifswald, Greifswald, Germany 13 NIHR Musculoskeletal Biomedical Research Unit, University of Oxford, Oxford, UK 14 MRC Lifecourse Epidemiology Unit, University of Southampton, Southampton General Hospital, Southampton, UK 15 School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand 16 Molecular Nociception Group, University College London, London, UK 17 Intramural Research Program, Laboratory of Epidemiology, Demography, and Biometry, National Institute on Aging, Bethesda, Maryland, USA 18 Department of Anesthesiology, Erasmus Medical Center Rotterdam, Rotterdam, The Netherlands 19 Aberdeen Pain Research Collaboration (Epidemiology Group), University of Aberdeen, Aberdeen, UK 20 Department of Rheumatology, Leiden University Medical Center, Leiden, The Netherlands 21 Department of Clinical Epidemiology, Leiden University Medical Center, Leiden, The Netherlands 22 Centre for Integrated Genomic Medical Research, University of Manchester, Manchester, UK 23 Department of Clinical Genetics, Erasmus Medical Center Rotterdam, Rotterdam, The Netherlands 24 Medical Research Institute, University of Dundee, Dundee, UK 25 Department of Medicine, University of Iceland, Reykjavik, Iceland 26 Institute of Functional Genomics, Ernst Moritz Arndt University Greifswald, University of Greifswald, Greifswald, Germany 27 NIHR Biomedical Research Unit, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford, UK

extension of the cohort was initiated in which 3,932 subjects were included, aged 45-54 years (out of 6,057 invited), called Rotterdam Study III (RS-III). The participants were all examined in some detail at baseline. They were interviewed at home and then had an extensive set of examinations in a specially built research facility in the centre of their district. These examinations were repeated every 3-4 years in characteristics that could change over time. The participants in the Rotterdam Study are followed for a variety of diseases that are frequent in the elderly. Informed consent was obtained from each participant, and the medical ethics committee of the Erasmus Medical Center Rotterdam approved the study. In Rotterdam, the participants completed the same pain homunculus as the subjects of ERF. Individuals were categorised as CWP cases when they report joint pain in the left side of the body, in the right side of the body, above waist, below waist, and in the axial skeleton. Subjects not being a CWP case were categorised as controls, but subjects using pain medication were excluded from the control group.
The TwinsUK study. The TwinsUK cohort (www.twinsuk.ac.uk) is a British adult twin registry shown to be representative of singleton populations and the United Kingdom population. 5687 females aged between the ages of 16-88 completed questionnaires related to chronic widespread pain between 2002 -2008. These questionnaires asked the participants about any pain in muscles, bones or joints lasting at least one week in the past three months.

Full description of the Replication Cohorts
1958BC. The National Child Development Study, also known as the 1958 British Birth Cohort Study is a large, on-going, prospective cohort study of all children born in England, Scotland, and Wales during one week of March 1958. Detailed methods have been reported previously (Power et al, 2006). Approximately 17,000 participants were recruited at birth and have subsequently been followed up at ages 7, 11, 16, 23, 33, 42 and 45 years. At age 45 years a biomedical survey collected information on health-related factors including the presence of pain.
The sample for the current study was 8,572 individuals who responded to a self-complete pain questionnaire at 45yrs (pain was not joint specific), sent in advance of a nurse interview, and who provided blood samples for genetic analysis.
The AGES Study. Age Gene/Environment Susceptibility Reykjavik (AGES-Reykjavik) Study. The Reykjavik Study cohort originally comprised a random sample of 30,795 men and women born in 1907-1935 and living in Reykjavik in 1967. A total of 19,381 people attended, resulting in 71% recruitment rate. The study sample was divided into six groups by birth year and birth date within month. One group was designated for longitudinal follow up and was examined in all stages. One group was designated a control group and was not included in examinations until 1991. Other groups were invited to participate in specific stages of the study. Between 2002 and2006, the AGES-Reykjavik study re-examined 5764 survivors of the original cohort who had participated before in the Reykjavik Study. Participants came in a fasting state to the clinic and answered questionnaires related to chronic widespread pain. Informed consent was obtained from all participants. Subjects were asked whether they had pain lasting at least one month in the past 12 months. Questions were asked specifically for hand and wrist, hip, knee, shoulder, feet, toes, ankles and back. The AGES Reykjavik Study GWAS was approved by the intra-mural research program of the National Institute on Aging, by the Iceland National Bioethics Committee (VSN: 00-063) and Data Protection Authority.

The Chingford Study.
The Chingford Study was established in 1989 when 1003 women, aged 44-67 years, derived from the register of a large general practice in Chingford, North London, were recruited to a prospective population-based longitudinal study of osteoarthritis and osteoporosis. In this study, data on joint and spinal pain, collected as part of the year 4 follow-up visit, was used. Subjects were asked whether they had pain in hand, knee, hip, feet and back during the last year. When they had pain, they were asked for how many days the pain lasted during the last month.
The DSDBAC Study. In 1983, 6542 healthy individuals aged between 42 and 92 years old resident in Newcastle and Greater Manchester were recruited into a longitudinal population-based study of cognition in healthy old age. Pain manikin data was collected via postal questionnaire on subjects remaining in the cohort in 2007, and additionally, subjects were asked whether they have had pain for more than 3 months. Pain was not asked joint specific.
The EPIFUND Study. EPIFUND is a prospective population-based study of functional disorders. Participants, aged 25-65 years old, were recruited from three primary care registers in the North-west of England. Pain manikin data was collected via a postal questionnaire at baseline and at two follow ups. Additionally to the manikins, subjects were asked whether they have had pain for more than 3 months. Pain was not asked joint specific. DNA was collected using buccal swab sampling from 1189 subjects who participated in all three phases.
The FOA Study. The Framingham Osteoarthritis Study is a population-based multigenerational cohort study of over 3500 participants, and is a sub-study of the larger Framingham Heart Study (FHS). In this study, we focused on study participants with information on widespread pain (collected in FOA) and genetic data (collected in FHS). All participants completed a pain homunculus to report the sites in the body having pain, aching, or stiffness on most days. Individuals were categorised as CWP cases when they report joint pain in the left side of the body, in the right side of the body, above waist, below waist, and in the axial skeleton.
Subjects not being a CWP case were categorised as controls, but subjects using pain medication were excluded from the control group.
The GARP Study. The GARP study from Leiden, the Netherlands, consists of 192 sibling pairs concordant for clinical and radiographically (K/L score) confirmed OA at two or more joint sites among hand, spine (cervical or lumbar), knee or hip 20 . Written informed consent was obtained from each subject as approved by the ethical committees of the Leiden University Medical Center. We recorded pain in the GARP questionnaire by asking the question: Have you had pain in and around your joints lasting most days of the last month? Patients could choose: hands (left and/ or right), hips (left and/ or right), knees (left and/ or right), back (cervical, thoracic or lumbar region), shoulders (left and/or right) and other sites as specified. When patients indicated that they had pain in the hands, they could specify the locations in the hand in a drawing.
When a patient indicated pain in two sections of two contralateral limbs and in the axial skeleton the patient is defined as a case of CWP. For controls, we used 925 randomly chosen Rotterdam Study participants.

The Hertfordshire Cohort Study (HCS) is a cohort study of men and women born in
Hertfordshire, UK during 1931-39 and still living there in adult life. Approximately 3000 participants were recruited in the late 1990s and have subsequently been followed by clinic visit (East Herts only) and postal clinical outcomes questionnaire (all). The sample for the current study was drawn from individuals who completed a pain questionnaire at using a mannequin to report site of pain, and who had previously provided blood samples for genetic analysis. Individuals were categorised having CWP if they reported having pain for at least three months in a detailed pain questionnaire which corresponded pain in the left and right sides of the body, pain both above and below the waist and back pain (pain was not joint specific). Individuals who did not report such pain but reported use of analgesics were excluded from the analysis. All other individuals were categorized as controls resulting in 90 cases and 2117 controls.
The SHIP Study. The SHIP cohort (http://www.medizin.unigreifswald.de/cm/fv/ship.html) is a prospective, population based cohort study among 4,308 subjects aged ≥20 years from the West Pomerania, Germany. The study was designed to assess prevalence and incidence of risk factors, subclinical disorders and clinical diseases and to investigate associations among them using extensive medical assessments. In this study, we focused on participants for whom complete phenotypic, genotypic, and genealogical information was available. Informed consent was obtained from each participant, and the medical ethics committee of University of Greifswald approved the study. Subjects were asked to complete questionnaires related to joint pain. These questionnaires asked about pain during the last week, regarding the back, elbow, foot, arms, hands, hip, knee, neck, shoulder, head and facial pain. We decided to exclude head and facial pain, not being joint-related pain. Because no duration was asked for, the pain prevalence in SHIP is one of the highest among the included cohorts.

Genotyping, Quality Control and Imputation
The following sample quality control (QC) criteria were applied in the GWAS of RS-I, RS-II, RS-III and ERF: sample call rate >97.5%, gender mismatch with typed X-linked markers, evidence for DNA contamination in the samples using the mean of the autosomal heterozygosity >0.33, exclusion of duplicates or first-degree relatives estimated by pairwise IBD, exclusion of ethnic outliers (>4 SD from population mean using multidimensional scaling (MDS) analysis with 4 principal components (PCs)), and exclusion of samples with missing pain data, age and/or BMI.
In the GWAS of TwinsUK, normalised intensity data was pooled, and genotypes were called on the basis of the Illuminus algorithm [1]. No calls were assigned if the most likely call was less than a posterior probability of 0.95. Validation of pooling was done by visual inspection of 100 random, shared SNPs for overt batch effects; none were observed. SNPs that had a low call rate (≤90%), Hardy-Weinberg p-values<10 −6 and minor allele frequencies < 1% were excluded.
Samples with call rates <95% were removed.
Genotype imputation was used to evaluate the association of one and the same SNP across samples typed on different genotyping platforms. Genotypes were imputed for all polymorphic SNPs (minor allele frequency >0.01) using either MACH [2] or IMPUTE [3] software, based upon phased autosomal chromosomes of the HapMap CEU Phase II panel (release 22, build 36), orientated on the positive strand. Imputation QC metrics from MACH and IMPUTE were used for filtering out SNPs with low-quality data.

Stage 1 GWAS Meta-Analysis
The estimated inflation factors were 1.176, 1.014, 1.008, 1.006, and 0.989 for ERF, RS-I, RS-II, RS-III, and TwinsUK respectively. SNPs with a minor allele frequency <0.05, a MACH r 2hat <0.30, or a SNPTEST proper_info <0.40 were excluded from the meta-analysis. We obtained the combined results of the 2,224,068 autosomal SNPs, pooling the effect sizes by means of a fixed effects inverse variance meta-analysis as implemented in METAL. Estimated heterogeneity variance and forest plots were generated using the Comprehensive Meta-Analysis[4] software.
Regional association plots of the meta-analysis results were obtained with LocusZoom [5].

Sequenom iPLEX and Taqman Allelic Discrimination genotyping
Genotypes for CHINGFORD, EPIFUND, and HCS were generated using Sequenom iPLEX genotyping and Taqman Allelic Discrimination genotyping. Genomic DNA was extracted from samples of peripheral venous blood according to standard procedures. 1-2 ng genomic DNA was dispensed into 384-wells plates using a Caliper Sciclone ALH3000 pipetting robot (Caliper LS, Mountain View, CA, USA).
For Sequenom iPLEX genotyping, multiplex PCR assays were designed using Assay Designer on the website (https://mysequenom.com/tools/genotyping/default.aspx). For this, sequences containing the SNP site and at least 100 bp of flanking sequence on either side of the SNP were used. Briefly, 2 ng genomic DNA was amplified in a 5 ul reaction containing 1 × Taq PCR buffer (Sequenom), 2 mM MgCl2, 500 uM each dNTP, 100 nM each PCR primer, 0.5 U Taq (Sequenom). The reaction was incubated at 94°C for 4 minutes followed by 45 cycles of 94°C for 20 seconds, 56°C for 30 seconds, 72°C for 1 minute, followed by 3 minutes at 72°C. Excess dNTPs were then removed from the reaction by incubation with 0.3 U shrimp alkaline phosphatase (Sequenom) at 37°C for 40 minutes followed by 5 minutes at 85°C to deactivate the enzyme.
Single primer extension over the SNP was carried out in a final concentration of between 0.731 uM and 1.462 uM for each extension primer (depending on the mass of the probe), iPLEX termination mix (Sequenom), 10x iPLEX Buffer Plus and iPLEX enzyme (Sequenom) and cycled using the following program; 94°C for 30 seconds followed by 94°C for 5 seconds, 5 cycles of 52°C for 5 seconds, and 80°C for 5 seconds, the last three steps were repeated 40 times, then 72°C for 3 minutes. The reaction was then desalted by addition of 6 mg clear resin (Sequenom) followed by mixing (15 minutes) and centrifugation (5 min, 3,000rpm) to settle the contents of the tube. The extension product was then spotted onto a 384 well spectroCHIP using the SEQUENOM MassARRAY Nanodispenser RS1000 before analysis on the MassARRAY Compact System (Sequenom). Data collection was performed using SpectroACQUIRE 3.3.1.13 and clustering was called using TYPER Analyzer 4.0.3.18 (Sequenom). Additionally to ensure data quality genotypes for each subject were also checked manually.
For Taqman Allelic Discrimination genotyping (Applied Biosystems Inc., Foster City, CA, USA), all SNP assays were available at www.appliedbiosystems.com as pre-designed assays. The PCR reaction mixture included 1-2 ng of genomic DNA in a 2 μl volume and the following reagents: FAM and VIC probes (200 nM), primers (0.9 uM), 2x Taqman PCR master mix (Applied Biosystems Inc., Foster City, CA, USA). Reagents were dispensed in a 384-well plate using the Deerac Equator NS808 (Deerac Fluidics, Dublin, Ireland). PCR cycling reaction were performed in 384 wells PCR plates in an ABI 9700 PCR system (Applied Biosystems Inc., Foster City, CA, USA) and consisted of initial denaturation for 15 minutes at 95° C, and 40 cycles with denaturation of 15 seconds at 95° C and annealing and extension for 60 seconds at 60° C. Results were analysed by the ABI Taqman 7900HT using the sequence detection system 2.22 software (Applied Biosystems Inc., Foster City, CA, USA).

RNA isolation and real-time PCR for mRNA quantitation
Total RNA was isolated with the Trizol (Invitrogen, Paisley, UK) method and 1 μg of total RNA was used to synthesize cDNA with SuperScript Reverse Transcriptase (Invitrogen; carrageenan experiment) or iScript TM Select cDNA Synthesis Kit (Invitrogen; CFA experiment) using random hexamers.         I  I  I  I  I  I  I  I  I  I   GARP  I  I  I  I  I  I  G  I  I  G   HCS  G  G  G  NG  NG  NG  NG  G  NG  G   SHIP  I  I  G  I  I  I  G  I I G     pharmacists. We would like to thank Dr. Tobias A. Knoch, Luc V. de Zeeuw, Anis Abuseiris, and