Altered Antibody Response to Epstein-Barr Virus in Patients With Rheumatoid Arthritis and Healthy Subjects Predisposed to the Disease. A Twin Study.

Objectives: To study Epstein-Barr virus (EBV) antibody patterns in twin individuals with rheumatoid arthritis (RA) and their healthy co-twins, and to determine the heritability of antibody responses against the EBV encoded EBNA1 protein. Methods: Isotypes of EBNA1 antibodies were measured in 137 RA affected- and 150 healthy twin pairs. We estimated the effect of RA and RA predisposition, anti-citrullinated antibodies (ACPA), IgM rheumatoid factor (RF), the shared epitope (SE) and the PTPN22-T allele (PTPN22) on the level of EBNA1 antibodies. We also determined the heritability of EBNA1 antibody levels. Results: IgA-EBNA1 antibody levels were increased in twins from RA discordant twin pairs irrespective of RA, ACPA or IgM-RF status. The IgG-EBNA1 antibody level was elevated in healthy co-twins from RA discordant twin pairs but not in RA affected twins. The IgM-EBNA1 antibody level was elevated in both RA twins and their healthy co-twins. The effect of RA on the IgA-EBNA1 antibody level was reversed when SE was present and with no effect of PTPN22. The heritability of IgA-, IgG- and IgM-EBNA1 antibody level was 40.6, 65.5, and 54.3%, with no effect of environment shared by the twins. Conclusion: EBNA1 antibody levels are distinctively different between patients with RA and healthy subjects but also between relatives of RA strongly predisposed to RA and healthy subjects. The high level of IgA EBNA1 antibodies associated with RA and a family predisposition to RA is attributable to both genetics incl. the shared epitope and environmental variation.

White blood cell mitochondrial DNA copy number is decreased in rheumatoid arthritis and linked with risk factors. A twin study.

Low mitochondrial DNA copy number (mtDNA CN) has been associated with e.g. cancer, cardiovascular and autoimmune diseases. We aimed to study a potential association between mtDNA CN and rheumatoid arthritis (RA). The relative quantity of mitochondrial DNA compared to nuclear DNA was measured in peripheral white blood cells from 149 RA affected twin pairs and 1321 non-affected twin pairs. Multiple regression analysis including RA discordant twin pairs was performed in order to separate specific effects of RA and familial RA predisposition using non-RA affected twin pairs as reference group. In addition, we performed a twin pair level analysis including only RA discordant twin pairs evaluating the effect of cell type, auto antibodies and RA genetic risk factors. Both the RA twins and their non-affected co-twins had significantly lower mtDNA CN than non-affected twins (-28.7 and -23.1 mtDNA CN, respectively). Adjusting for cell count attenuated these differences (-23.1 mtDNA CN and -20.1 mtDNA CN respectively). Within RA discordant twin pairs PTPN22(T) positive RA twins had a significantly lower amount than their co-twins (-16.3 mtDNA CN). PTPN22(T) had no effect among twins from non-affected twin pairs. MtDNA CN is significantly lower in persons with established RA and in predisposed non-affected RA co-twins suggesting that mitochondrial variation may be involved in the RA disease pathways. Our results also suggest that the RA associated genetic risk factor, PTPN22(T), further decreases the mtDNA CN, but only in carriers with established RA.

Twin DNA Methylation Profiling Reveals Flare-Dependent Interferon Signature and B Cell Promoter Hypermethylation in Systemic Lupus Erythematosus.

OBJECTIVE: Systemic lupus erythematosus (SLE) has limited monozygotic twin concordance, implying a role for pathogenic factors other than genetic variation, such as epigenetic changes. Using the disease-discordant twin model, we investigated genome-wide DNA methylation changes in sorted CD4+ T cells, monocytes, granulocytes, and B cells in twin pairs with at least 1 SLE-affected twin. METHODS: Peripheral blood obtained from 15 SLE-affected twin pairs (6 monozygotic and 9 dizygotic) was processed using density-gradient centrifugation for the granulocyte fraction. CD4+ T cells, monocytes, and B cells were further isolated using magnetic beads. Genome-wide DNA methylation was analyzed using Infinium HumanMethylation450K BeadChips. When comparing probes from SLE-affected twins and co-twins, differential DNA methylation was considered statistically significant when the P value was less than 0.01 and biologically relevant when the median DNA methylation difference was >7%. Findings were validated by pyrosequencing and replicated in an independent case-control sample. RESULTS: In paired analyses of twins discordant for SLE restricted to the gene promoter and start region, we identified 55, 327, 247, and 1,628 genes with differentially methylated CpGs in CD4+ T cells, monocytes, granulocytes, and B cells, respectively. All cell types displayed marked hypomethylation in interferon-regulated genes, such as IFI44L, PARP9, and IFITM1, which was more pronounced in twins who experienced a disease flare within the past 2 years. In contrast to what was observed in the other cell types, differentially methylated CpGs in B cells were predominantly hypermethylated, and the most important upstream regulators included TNF and EP300. CONCLUSION: Hypomethylation of interferon-regulated genes occurs in all major cellular compartments in SLE-affected twins. The observed B cell promoter hypermethylation is a novel finding with potential significance in SLE pathogenesis.

Incidence of Chronic Persistent Rheumatoid Arthritis and the Impact of Smoking: A Historical Twin Cohort Study.

OBJECTIVE: To assess the incidence of chronic persistent rheumatoid arthritis (RA) in a population-based cohort of twins and to determine the impact of smoking. METHODS: In a historical cohort study on twins born in 1920 to 1982, we identified 157 cases of RA among 45,280 responders (response rate 80%). Information on smoking was obtained by questionnaire and interview. A mixed-effects Poisson regression model was used to estimate incidence rate ratios with age, sex, smoking duration, and smoking intensity as covariates. We used the SplitLexis procedure in the Epi R package to study a possible effect of period or cohort in addition to age on the variation of the incidence. RESULTS: The annual incidence of chronic persistent RA was 18.8 per 100,000 person-years, ages 15-73 years (females 25.2, males 12.0), increasing with age to a maximum at age 60 years in females and age 70 years in males. The incidence rate ratio among ever-smoking patients was 1.96 (95% confidence interval [95% CI] 1.43-3.76), 1.93 (95% CI 1.00-3.7) after 30 pack-years, and 1.034 (P < 0.001) per year of smoking, implying a doubling of risk after 20 years regardless of sex and smoking intensity. We did not detect significant period or cohort effects. CONCLUSION: The incidence of chronic persistent RA is lower than the incidence figures reported in inception cohorts. Smoking duration, but not intensity, doubled the risk of RA after 20 years of smoking in both sexes.

Differentially Methylated DNA Regions in Monozygotic Twin Pairs Discordant for Rheumatoid Arthritis: An Epigenome-Wide Study.

OBJECTIVES: In an explorative epigenome-wide association study (EWAS) to search for gene independent, differentially methylated DNA positions and regions (DMRs) associated with rheumatoid arthritis (RA) by studying monozygotic (MZ) twin pairs discordant for RA. METHODS: Genomic DNA was isolated from whole blood samples from 28 MZ twin pairs discordant for RA. DNA methylation was measured using the HumanMethylation450 BeadChips. Smoking, anti-cyclic citrullinated peptide antibodies, and immunosuppressive treatment were included as covariates. Pathway analysis was performed using GREAT. RESULTS: Smoking was significantly associated with hypomethylation of a DMR overlapping the promoter region of the RNF5 and the AGPAT1, which are implicated in inflammation and autoimmunity, whereas DMARD treatment induced hypermethylation of the same region. Additionally, the promotor region of both S100A6 and EFCAB4B were hypomethylated, and both genes have previously been associated with RA. We replicated several candidate genes identified in a previous EWAS in treatment-naïve RA singletons. Gene-set analysis indicated the involvement of immunologic signatures and cancer-related pathways in RA. CONCLUSION: We identified several differentially methylated regions associated with RA, which may represent environmental effects or consequences of the disease and plausible biological pathways pertinent to the pathogenesis of RA.

Low-field magnetic resonance imaging or combined ultrasonography and anti-cyclic citrullinated peptide antibody improve correct classification of individuals as established rheumatoid arthritis: results of a population-based, cross-sectional study.

BACKGROUND: The aim of the present study was to evaluate the accuracy of two approaches using magnetic resonance imaging (MRI) or combined ultrasonography (US) and anti-cyclic citrullinated peptide antibody (ACPA) for diagnosis and classification of individuals with established rheumatoid arthritis (RA). METHODS: In 53 individuals from a population-based, cross-sectional study, historic fulfilment of the American College of Rheumatology (ACR) 1987 criteria ("classification") or RA diagnosed by a rheumatologist ("diagnosis") were used as standard references. The sensitivity, specificity and Area under Curve for Receiver Operating Characteristics curves (ROC-area: (sensitivity + specificity)/2) were calculated for "current fulfilment of the ACR 1987 criteria" (list format), "adapted ACR 1987 criteria" (list format, substituting IgM rheumatoid factor with ACPA and clinical joint swelling and erosions on radiography with synovitis and erosions detected by US on a semi-quantitative scale), and RA MRI scoring System (RAMRIS) scores on low-field MRI in the unilateral hand. RESULTS: For the ACR 1987 criteria the ROC-area was 75% (sensitivity/specificity = 50%/100%) (with "classification" as standard reference) and 69% (44%/94%) (with "diagnosis" as standard reference), while for the adapted ACR 1987 criteria it was 86% (75%/97%) (classification) and 82% (72%/91%) (diagnosis). For RAMRIS synovitis score in metacarpophalangeal (MCP) joints only (cut-off ≥5), the ROC-area (sensitivity/specificity) was 78% (62%/94%) (classification) and 85% (69%/100%) (diagnosis), while for the total synovitis score of MCP joints plus wrist (cut-off ≥10) it was 78% (62%/94%) (both classification and diagnosis). CONCLUSIONS: Compared with the ACR 1987 criteria, low-field MRI alone or adapted criteria incorporating US and ACPA increased the correct classification and diagnosis of RA.

Newborn infant characteristics and risk of future rheumatoid arthritis: a twin-control study.

Low birth weight has been proposed as a risk factor for rheumatoid arthritis (RA). The twin-control study design provides an opportunity to investigate the significance of potential prenatal determinants for adult morbidity by accounting for maternal characteristics and early environmental and genetic factors. We investigated the association between birth weight and RA in a sample of 42 twin pairs discordant for rheumatoid arthritis in which valid information on birth weight, birth length, and order was available from midwife records. Difference plot and conditional logistic regression were used to investigate the relationship between RA and birth weight or birth order adjusting for birth length and sex. The intra-pairwise birth weight differences, i.e., RA twin minus co-twin, ranged from -750 to 1,100 g, mean 78 g (95 % CI -13 to 70), 146 g (95 % CI (-36 to 329) in monozygotic, 32 g (95 % CI -90 to 154) in dizygotic, same sex and 69 g (95 % CI -122 to 260) in dizygotic, opposite sex twin pairs. The odds ratio for birth weight as risk factor for RA was 1.00 (95 % CI 0.997-1.003) when adjusting for birth length, birth order, and sex, irrespective of ACPA status. The odds ratio for developing RA as first born twin was 2.33 (95 % CI 0.97-5.60) when adjusting for birth length, birth weight, and sex, irrespective of ACPA status. In this twin-control study, birth weight was not associated with the development of RA in adult life. Being born first may predispose to RA.

On the origin of rheumatoid arthritis: the impact of environment and genes--a population based twin study.

BACKGROUND: Rheumatoid arthritis (RA) is an autoimmune disease with a complex origin. Previous studies have reported heritability estimates on RA at about 60%. Only 16% of the genetic background of the disease has been disclosed so far. The purpose of the present investigation was to provide an optimized estimate on the heritability of RA and to study the recurrence risk in a nationwide Caucasian twin population. METHODS AND FINDINGS: In a mail survey addressed to 56.707 twin individuals, RA was reported by 479 individuals, mean age 52 (range 16-73). Respondents underwent an interview and clinical examination. Ascertainment probability was 80%. RA was confirmed in 162 twin individuals yielding a prevalence at 0.37% (95% CI 0.31-0.43). The mean discordance time was 19 years (range 0-57). The concordance was 9.1% (95% CI 1.9 to 24.3) in MZ, 6.4% (95% CI 2.1 to 14.3) in DZss. The increased relative risk of attracting RA conditioned on having an affected cotwin compared to the background population risk was 24.6 to 35.4 in MZ twins and 17.3 to 31.6 in DZss twins. The correlation coefficients were 0.60 (0.33 to 0.78) in monozygotic (MZ) and 0.55 (0.33 to 0.72) in dizygotic same sexed (DZss) pairs. Twelve percent (95% CI 0-76%) of the phenotypic variance in the liability to RA was due to additive genetic effects, 50% (95% CI 0-72%) to shared environmental effects and 38% (95% CI 17-61%) to non-shared environmental effects. CONCLUSIONS: This study emphasizes that family factors are important for the development of RA. Although genetic effectors are important, shared and non-shared environmental triggers and/or epigenetic stochastic events seem to be even more significant. However, it should be borne in mind that the genetic and non-genetic components may not be the same across disease subsets.

The impact of genes on the occurrence of autoantibodies in rheumatoid arthritis. A study on disease discordant twin pairs.

OBJECTIVE: To assess the genetic effect on the occurrence of rheumatoid arthritis (RA) associated autoantibodies. METHODS: A co-twin control study of 27 monozygotic (MZ) and 51 dizygotic same sexed (DZss) RA discordant twins. OUTCOME MEASURES: The probandwise concordance rate of anti-keratin antibodies (AKA), anti-cyclic citrullinated peptide antibodies (ACPA), IgA- and IgM rheumatoid factor (IgA-RF and IgM-RF). The odds ratio for these autoantibodies based on both conditional and unconditional logistic regression adjusting for the two major genetic risk factors as well as smoking. RESULTS: The probandwise concordance rates (95% CI) of ACPA, AKA, IgM-RF and IgA-RF were 78.6 (55.4-92.4), 16.7 (0.6-58.4), 30.0 (7.3-60.6), 42.1 (14.5-71.1) in MZ twins and 25.0 (10.3-44.4), 0.0 (0.0-27.7), 10.5 (1.4-31.5) and 22.2 (6.8-45.0) in DZss twins. In twin pairs discordant for both RA and autoantibodies the OR of ACPA, AKA, IgM-RF and IgA-RF was 5 (0.5-236.5) 9 (1.3-394.5) 272 (3.5-593.2) and 10 (1.4-434.0) in MZ twin pairs and 17 (4.4-146.1) 20 (3.2-828.0) 33 (5.5-1342.4) and 577 (7.4-1149.2) in DZss twin pairs. In multiple logistic regression analysis on ACPA, the MZ/DZ OR was 21.1 (3.3-213.5) when adjusting for age, sex, ever smoking, PTPN22 1858 T-allele, Shared Epitope (SE) and SE-smoking interaction. CONCLUSION: There is a genetic contribution to ACPA generation independent of both SE and PTPN22 1858 T-allele. Environmental factors may trigger the expression of IgA-RF, ACPA and AKA in healthy persons who are predisposed to RA.

Prevalence of rheumatoid arthritis in the southern part of denmark.

The aim of the present study was to estimate the prevalence of rheumatoid arthritis in the southern part of Denmark. Using a screening questionnaire, telephone interview, register data, and a clinical examination cases were ascertained from a random sample of 4995 individuals over the age of 15. As case definition we used the original and modified 1987 American College of Rheumatology classification criteria. The overall point prevalence was 0.26% (95% confidence interval: 0.13-0.39) in the total sample and 0.35% (95% confidence interval: 0.17-0.52) among the responders; the cumulative prevalence was 0.75% (95% confidence interval: 0.52-0.97) in the total sample and 0.92% (95% confidence interval: 0.62-1.21) among the responders.The cumulative prevalence was higher than in other studies combining the results of a survey with register data. The point prevalence was underestimated due to low participation rate in the clinical examination and remission among the participants.

Mannose-binding lectin gene polymorphisms are associated with disease activity and physical disability in untreated, anti-cyclic citrullinated peptide-positive patients with early rheumatoid arthritis.

OBJECTIVE: To study the association between polymorphisms in the mannose-binding lectin gene (MBL2) and disease activity, physical disability, and joint erosions in patients with newly diagnosed rheumatoid arthritis (RA). METHODS: Patients with early RA (n=158) not previously treated with disease modifying antirheumatic drugs, participating in a treatment trial (CIMESTRA study) were examined at inclusion for MBL2 pooled structural genotypes (O/O, A/O, A/A), regulatory MBL2 promoter polymorphism in position -221 (XX, XY, YY), anti-cyclic citrullinated peptide 2 antibodies (anti-CCP2), disease activity by Disease Activity Score-28 (DAS28 score), physical disability by Health Assessment Questionnaire (HAQ) score, and erosive changes in hands and feet (Sharp-van der Heijde score). RESULTS: Eight patients were homozygous MBL2 defective (O/O), 101 belonged to an intermediate group, and 49 were MBL2 high producers (YA/YA). Anti-CCP was present in 93 patients (59%). High scores of disease activity, C-reactive protein-based DAS28 (p=0.02), and physical disability by HAQ (p=0.01) were associated with high MBL2 expression genotypes in a gene-dose dependent way, but only in anti-CCP-positive patients. At this early stage of the disease there was no association with erosion score from radiographs. CONCLUSION: The results point to a synovitis-enhancing effect of MBL in anti-CCP-positive RA, whereas such an effect was not demonstrated for joint erosions.

Incidence of rheumatoid arthritis from 1995 to 2001: impact of ascertainment from multiple sources.

The aim of this study was to describe the mean incidence rate of rheumatoid arthritis over a 7-year period from 1995 to 2001 in a population in the southern part of Denmark, using the data from several sources. Cases fulfilling the 1987 American College of Rheumatology criteria for rheumatoid arthritis were identified at hospitals and private practising rheumatologists (referral centres), and in general practice. The observed incidence was 32/100,000 person-years (95% confidence interval 29-35). Using the ratio between the number of cases known only from general practice and the number known from general practice and referral centres, the estimated incidence was 35/100,000 person-years (95% confidence interval 32-38). We suggest that the estimated rate should be viewed as a plausible upper limit for the incidence of rheumatoid arthritis in the southern part of Denmark.

[The importance of physical activity and body mass index for body satisfaction]. / Betydningen af fysisk aktivitet og body mass index for tilfredshed med kropsbygningen.

INTRODUCTION: Body satisfaction has mostly been investigated among patients with psychiatric diseases. A few studies have found an association between physical activity, obesity and body satisfaction in the healthy population, but the focus has mostly been on the risk of developing diseases. The aim of the present study has been to study the association between body satisfaction, BMI, physical activity and chronic diseases. MATERIALS AND METHODS: A total of 15,252 women and 14,181 men born between 1953 and 1982 (both years included) and registered with The Danish Twin Registry answered a questionnaire which they were sent in 1994. We used the questions on chronic diseases, height and weight, physical activity and body satisfaction. Univariat analysis of body satisfaction and the other variables was carried out using chi2-tests and t-tests. Multivariat analysis was carried out with logistic regression. RESULTS: More men than women are satisfied with their bodies; BMI confers a negative association, while physical activity confers a positive association with body satisfaction. This satisfaction appears to some degree mediated by BMI. The presence of asthma and other lung symptoms and lower-back pain was negatively associated with body satisfaction. DISCUSSION: Thinness tends to be the ideal body image today and this may explain why more obese people are dissatisfied with their body. This ideal is more important for women s perception of their bodies. Physical activity and being healthy adds to body satisfaction.

Incidence of Rheumatoid Arthritis in the Southern part of Denmark from 1995 to 2001.

We estimated the incidence of rheumatoid arthritis in the southern part of Denmark from 1995 to 2001. At a rheumatology hospital serving a population of about 200 000 people over the age of 15, medical records were scrutinized. As case definition we used the tree and list format of 1987 American College of Rheumatology criteria for rheumatoid arthritis. The mean annual incidence rate per 100 000 person years was 40 in females, 21 in males, and 31 in females and males combined. The incidence of rheumatoid arthritis in Denmark is in accordance with recent studies from North America, the UK, and Northern European countries. The aetiology of rheumatoid arthritis is unknown but this study indicates that in these populations the exposure to non-genetic host and environmental aetiological factors is similar.

Twin study of genetic and aging effects on X chromosome inactivation.

To investigate the genetic influence on X chromosome inactivation and on age-related skewing of X inactivation, in particular, we analysed the X inactivation pattern (XIP) in peripheral blood cells from 118 young monozygotic (MZ) twin pairs (18-53 years), 82 elderly MZ twin pairs (55-94 years), 146 young dizygotic (DZ) twin pairs (20-54 years) and 112 elderly DZ twin pairs (64-95 years). Elderly twins had a higher frequency of skewed X inactivation (34%) than young twins (15%) (P<0.001). Our data suggest that the increase in skewing occurs after age 50-60 years. The intraclass correlation was 0.61 and 0.58 in young and elderly MZ twin pairs, and 0.08 and 0.09 in young and elderly DZ twin pairs. Biometric analysis showed that dominant genetic effects accounted for 63 and 58% of the variance of XIP in the young and elderly twin pairs, respectively. The dominant genetic effect and the shared environment for monochorionic MZ twins may explain the high intraclass correlation for the MZ twin pairs compared to the DZ twin pairs. We did not observe a significant decrease in the intraclass correlation in elderly MZ twins compared to young MZ twins, which would be expected if age-related skewing were due to stochastic factors. We conclude that the increased skewing with age implies that a genetically dependent selection of blood cells take place.

Validity of rheumatoid arthritis diagnoses in the Danish National Patient Registry.

Discharge diagnoses following hospital admissions in Denmark are recorded in the Danish National Patient Registry (NPR). Such routine hospitalization records may serve as useful research tools in epidemiological studies. The aim of the study was to provide measures of the validity and completeness of rheumatoid arthritis (RA) diagnoses recorded in the NPR. We identified medical records for 217 patients recorded as having RA in the NPR between 1977 and 2001. Using two definitions of RA (clinically confirmed RA and fulfilment of the American College of Rheumatology (ACR) 1987 diagnostic criteria for RA), a rheumatologist assessed the proportion of RA diagnoses recorded in the NPR that could be confirmed by scrutiny of the original medical records. The completeness of RA diagnoses in the NPR was estimated by a two-sample capture-recapture method. Overall, 59% of the 217 RA diagnoses in the NPR were confirmed by information in the medical records. However, major differences were seen according to characteristics of the underlying hospital registrations. Generally, RA diagnoses were most often confirmed for patients registered as inpatients and for patients with more than one hospital registration with RA. Specifically, only 42% of patients with one RA registration from a rheumatology department were confirmed as having RA. In contrast, 91% of patients treated at a rheumatology department and having three or more hospital registrations with RA were confirmed as having RA. The completeness of the NPR with respect to RA satisfying the ACR 1987 classification criteria was estimated to 26%. Our conclusion is that with careful attention to the limitations in the data, discharge diagnoses for patients with records of RA in the Danish NPR can be used for epidemiological research purposes; however, our findings prompt general carefulness when using non-audited registries for research in RA.

Relative importance of genetic effects in rheumatoid arthritis: historical cohort study of Danish nationwide twin population.

OBJECTIVE: To determine the relative importance of environmental and genetic effects in the development of rheumatoid arthritis. DESIGN: Historical cohort study with record linkage between a twin registry and the Danish discharge registry as well as the Danish national registry of deaths used to estimate completeness. SETTING: Two population based nationwide twin birth cohorts. PARTICIPANTS: 37 338 twins were sent a questionnaire about rheumatic diseases. Self reported rheumatoid arthritis was verified by clinical examination and from medical records. MAIN OUTCOME MEASURES: The probandwise concordance rate of rheumatoid arthritis in monozygotic and dizygotic twins. RESULTS: The response rate was 84.7%. Rheumatoid arthritis was verified in 13 monozygotic and 36 dizygotic twins. There were no concordant monozygotic twin pairs and two concordant dizygotic twin pairs. Based on capture-recapture methods the probability of ascertainment was 78.3%. The probandwise concordance rate was 0 (95% confidence interval 0 to 24.7) in monozygotic twins and 8.8 (1.9 to 23.7) in dizygotic twins. CONCLUSION: Genes are of minor importance in the development of rheumatoid arthritis.