Cardiovascular Disease Factor 15 and in the 9p21.3 Region in Patients with Rheumatoid Arthritis and and Growth Differentiation CD40 Polymorphisms of the Genes Encoding

Objective. Genes or gene products associated with coronary artery disease in the general population were analyzed in rheumatoid arthritis (RA) patients with atherothrombotic manifestations (ATM). Methods. A cross-sectional study of 681 individuals (498 women; 183 men) with RA (American College of Rheumatology criteria), a mean age of 60.6 ± 13.2 years, and mean disease duration of 15.5 ± 12.6 years who were consecutively recruited and followed for 6 years. The prevalence of ATM [i.e., myocardial infarction, angina pectoris with intervention, deep vein thrombosis/pulmonary embolism (DVT/PE), and/or stroke/transient ischemic attack (TIA)] was recorded. Polymorphisms were analyzed in the genes coding for growth differentiation factor 15 (GDF15)/monocyte inhibitory cytokine-1 (MIC-1; rs1058587), CD40 (rs1535045 and rs3765459), and the 9p21.3 locus (rs1333049). Controls were randomly selected (n = 687; matched for age and sex). Results. The distribution of genotypes of GDF15/MIC-1 differed significantly between patients with RA and controls (chi-squared = 6.40, 2 df, p = 0.041). ATM were associated with polymorphism of the GDF15/MIC-1 G allele (OR 2.21, 95% CI 1.17–4.18), and with CC genotype of the 9p21.3 locus (rs1333049; OR 1.92, 95% CI 1.15–3.19). Stroke/TIA in women was associated with GDF15/MIC-1 GG genotype (OR 3.75, 95% CI 1.06–13.33), while stroke/TIA in men was associated with CD40 homozygous major alleles (OR 6.48, 95% CI 1.31–32.0 and OR 2.78, 95% CI 0.78–9.91, respectively). DVT/PE was associated with polymorphism in the GDF15/MIC-1 gene (rs1058587) minor allele (OR 3.53, 95% CI 1.30-9.58). Conclusion. The gene polymorphisms analyzed were associated with different ATM in RA. The GDF15/MIC-1 gene polymorphism was also associated with RA per se, suggesting a common etiology for RA and ATM. (First Release April 15 2012; J Rheumatol 2012;39:939–45; doi:10.3899/ jrheum.111336) Key Indexing Terms: RHEUMATOID ARTHRITIS ATHEROTHROMBOTIC DISEASE 9p21.3 LOCUS GENE POLYMORPHISM GROWTH DIFFERENTIATION FACTOR 15 CD40 From the Departments of Public Health and Clinical Medicine/Rheumatology, Umeå University, Umeå, Sweden. Supported by grants from the Swedish Research Council (K2010-52X-20307-04-3), the Swedish Rheumatism Association, the King Gustav Vth 80-year Foundation, and the Swedish COMBINE program. L. Ärlestig, BSc; S. Rantapää-Dahlqvist, MD, PhD, Professor, Departments of Public Health and Clinical Medicine/Rheumatology, Umeå University. Address correspondence to Dr. S. Rantapää-Dahlqvist, Department of Public Health and Clinical Medicine, Rheumatology, University Hospital, SE-901 85 Umeå, Sweden. E-mail: [email protected] Full Release Article. For details see Reprints/Permissions at jrheum.org Accepted for publication January 24, 2012. An increased morbidity and mortality due to cardiovascular disease (CVD) among individuals with rheumatoid arthritis (RA) has been identified in several studies1,2,3. The etiological and pathogenic mechanisms are not well defined, but the inflammatory activity in patients with RA is important for the development of CVD4. Risk factors for CVD in patients with RA show a pattern different from that of the general population5,6 [with the exception of hypertension (HTN)], one that is highly prevalent in RA7. However, other traditional risk factors for CVD, such as age, being male, current smoking, and hypertriglyceridemia, have also been shown to affect CVD in patients with RA in prospective studies8. In a previous study we reported an association between plasminogen activator inhibitor type 1 gene and ischemic heart disease (IHD) in patients with RA9. Several studies have shown an increased risk of a CV event and/or mortality in patients with RA carrying HLA-DRB1 shared-epitope alleles10,11,12,13. Further, associations have been shown between polymorphisms of the vasculature endothelial growth factor A gene14, the interleukin 6 gene15, the methylene tetrahydrofolate reductase gene16 and the tumor necrosis factor (TNF)-308 gene17, and IHD, myocardial infarction (MI), and CVD, respectively. An association has been found between polymorphism in the lymphotoxin A gene and the risk of an MI18. In our study of patients with RA, polymorphisms in genes of Rheumatology The Journal on August 14, 2017 Published by www.jrheum.org Downloaded from reported to be associated with manifestations of CVD in the general population were analyzed. Two of the genes, i.e., growth differentiation factor 15 (GDF15)/macrophage inhibitory cytokine 1 (MIC-1) and CD40, have been described as having proinflammatory properties. One single-nucleotide polymorphism (SNP), rs1333049, on chromosome 9p21.3 was identified in several genome-wide association studies to be associated with coronary heart disease in the general population19,20,21,22. This association has been confirmed in a number of subsequent studies23 and has also been confirmed in the population from northern Sweden24. GDF15/MIC-1, a member of the transforming growth factor-ß superfamily, was first described as being expressed by activated macrophages25. It is considered a regulator of macrophage activation and a target for p53, which is involved in the injury response to DNA damage26. Elevated serum concentrations of GDF15 were found in different types of CVD, such as angina pectoris, MI, coronary revascularization, heart failure, stroke, and in death from a cardiovascular event27,28. A polymorphism in exon 2 designated H6D, rs1058587, causes an amino acid change from histidine to aspartic acid. The D variant is not recognized by an antibody that is conformationdependent29, suggesting that the protein conformation has altered and that it may function differently. High serum levels of GDF15 and presence of the D allele were associated with an earlier onset of RA and were predictive of early erosive disease30. CD40 is a cell-surface receptor involved in immunoregulatory signaling when interacting with its ligand, CD40L. The receptor is expressed on B lymphocytes, fibroblasts, anti gen-presenting cells, and endothelial and epithelial cells. The receptor is essential for T cell-dependent immunoglobulin class-switching and the development of memory B cells31. The analyzed polymorphisms, rs1535045 and rs3765459, are located in the first and last introns and are both associated with less coronary artery calcification32. In our study, the GDF15 (rs1058587), CD40 (rs1535045 and rs3765459), and 9p21.3 (rs1333049) polymorphisms were analyzed in patients with RA from northern Sweden in connection with the development of atherothrombotic disease. MATERIALS AND METHODS Study groups. Patients with RA (American College of Rheumatology criteria; n = 723)33 from northern Sweden were consecutively included into the study during the first 4 months of 1995, 2000, 2001, and 2002. Of these, 681 patients (498 women and 183 men) donated blood samples for DNA analysis. A thorough survey of all the available patient records from onset of RA until inclusion in the study was performed retrospectively according to a structured registration form. All patients were followed prospectively from the inclusion date for 6 years. Atherothrombotic manifestations (ATM) after the onset of RA were registered retrospectively according to the following definitions: an MI was diagnosed when changes on electrocardiograms, according to the Minnesota code34, were assessed by a clinical physiologist or cardiologist concurrent with the typical enzymatic pattern and/or verified on echocardiographic examination; IHD was included in addition to an MI when there was severe verified angina pectoris with coronary artery bypass graft surgery and percutaneous coronary intervention; stroke was recorded when an intracerebral hemorrhage or infarction had been diagnosed following computerized tomo graphy or magnetic resonance imaging, or when a typical clinical picture with neurological deficits had persisted for more than 24 h; a transient ischemic attack (TIA) was recorded in those cases in which a focal neurological deficit of presumed ischemic origin had persisted for < 24 h; deep vein thrombosis/pulmonary embolism (DVT/PE) was recorded when the diagnosis had been verified objectively (e.g., phlebography, sonography, scintigraphy, and/or arteriography) or when clinical signs combined with pulmonary radio graphy, electrocardiography, and laboratory changes resulted in fulltime treatment with warfarin. HTN was defined as treatment for it. At entry into the study, 83 patients had been affected, of whom 51 had been affected by IHD (an MI and/or angina pectoris with intervention), 26 by stroke/TIA, 12 by DVT/PE. Six of those patients had 2 of these ATM. Total accumulated disease activity was analyzed from retrospective data by calculation of the number of joints affected by arthritis, erythrocyte sedimentation rate (mm/h), and the physician’s global assessment as described by Baecklund, et al35. Treatments (≥ 6 months) with corticosteroids, diseasemodifying antirheumatic drugs (DMARD), anti-TNF treatment, and statins were registered. The 6-year followup information was derived from the register of the National Board of Health in Sweden to obtain data on hospital inpatient care and/or death with a diagnosis of a new event, e.g., MI (ICD-10; I21.0-9, I22.0-1, I22.8-9.), angina pectoris with intervention (Z 951 or 955 or FNG V9250, 9251, 9253), and/or stroke/TIA (ICD-10; I61.0-6, I61.8-9, I62.9, I63.0-6, I63.8-9 and I64), and DVT/PE (I26, I80.2). These outcomes have been validated in another study with 96% concordance36. A population-based group of 696 individuals from the county of Västerbotten who were participating in the Northern Sweden World Health Organization Multinational Monitoring of Trends and Determinants in Cardiovascular Disease (WHO MONICA) project were identified in the Medical Biobank of Northern Sweden and used as controls. They were randomly selected on the basis of age (women 60 ± 12 years and men 64 ± 10 years) and sex (513 women and 183 men). Among the controls, 42% of the women and 50% of the men had a history of smoking, and 43% of the women and 17% of the men had HTN. DNA extraction and assays. Genomic DNA was extracted from buffy-coat cells using standard methods. The 5’-nuclease allelic discrimination assays used for genotyping the polymorphisms rs1535045 and rs3765459 in the CD40 gene, rs1333049 at chromosome 9p21.3, and rs1058587 in GDF15 (C to G; also designated as H6D) were supplied by Applied Biosystems (Foster City, CA, USA). The manufacturer’s instructions were used for analyzing the SNP on an ABI Prism 7900HT sequence detector system (Applied Biosystems). For identification of the genotypes the SDS 2.1 software was used (Applied Biosystems). Anticitrullinated protein antibodies (ACPA) were analyzed using an ELISA (cutoff value 5 units/ml) from Axis Shield Diagnostics (Dundee, UK). Rheumatoid factors (RF) of the IgM isotype were measured with an ELISA (cutoff value 20 units/ml) from ORG 522M (Orgentec Diagnostika GmbH, Mainz, Germany). Statistics. The chi-squared test was used for testing categorical data between groups and logistic regression analyses were used to estimate OR for predicting variables for the dependent variable. For analysis of continuous data, the independent t-test was used. All p values are 2-sided, and p values ≤ 0.05 were considered statistically significant. Calculations were performed using SPSS 18.0 software (Chicago, IL, USA). Any adjustments in the multiple logistic analyses were chosen based on knowledge from previous studies. Selection of genes for analysis was based on a preformed hypothesis and no compensation was made for multiple testing. Based on previously published frequencies of the genes, the power in our cohort to detect significant differences (p < 0.05) for 9p21.3 types was 67% and for GDF15 polymorphisms was 70%. Linkage disequilibrium (D’ and r2 values) calculated for CD40 SNP were derived using Haploview 4.2 software. 940 The Journal of Rheumatology 2012; 39:5; doi:10.3899/jrheum.111336 Personal non-commercial use only. The Journal of Rheumatology Copyright © 2012. All rights reserved. of Rheumatology The Journal on August 14, 2017 Published by www.jrheum.org Downloaded from