A Combination of Autoantibodies to Cyclic Citrullinated Peptide (CCP) and HLA-DRB1 Locus Antigens is Strongly Associated with Future Onset of Rheumatoid Arthritis

Ewa Berglin; Leonid Padyukov; Ulf Sundin; Göran Hallmans; Hans Stenlund; Walther J van Venrooij; Lars Klareskog; Solbritt Rantapää Dahlqvist


Arthritis Res Ther. 2004;6(4) 

In This Article

Materials and Methods

A nested case–control study was performed within the Northern Sweden Health and Disease Study (NSHDS) and the Maternity cohort of Northern Sweden. All adult individuals of the county of Västerbotten were invited to participate; consequently, the cohorts are population-based and no individual was excluded. The NDHDS cohort consists of three subcohorts, which, together with conditions for recruitment into the cohorts and the collection and storage of blood samples, have previously been described in detail.[3] The registry of patients who fulfilled the American College of Rheumatology classification criteria for RA[1] and who attended the Department of Rheumatology, University Hospital, Umeå (the only medical centre for rheumatology in the county of Västerbotten), and with a known date of onset of symptoms or signs of joint disease, was coanalysed with the registers of the cohorts from the Blood Bank for Västerbotten located in Umeå. At the time of the study, the median duration of disease since the diagnosis of RA was 3.0 years (interquartile range [IQR] 1.8–5.8 years). Eighty-six individuals were identified from the cohorts as having donated blood samples before the onset of symptoms or signs of joint disease. Samples from three individuals were not available. Of the remaining 83 individuals (referred to here as 'prepatients'), blood samples for DNA analysis were available only from the NSHDS cohort, resulting in 59 prepatients (45 women and 14 men); the Maternity cohort did not include collection of samples for DNA analysis. Power calculations showed that two controls per patient would be sufficient, based on pretest probability of our previous results of HLA-DR4 frequencies in patients and controls from this area.[5] Therefore, we selected for genetic analysis two controls (out of the four who were previously analysed for antibody titres[3]) for every prepatient. The controls were randomly selected from the same subcohorts as the original cases within the NSHDS cohort and matched for sex, for age at the time of blood sampling, and for area of residence (rural or urban). The mean age of the prepatients at the time of blood sampling was 53 years (range 31–67 years) and of the controls, 53 years (range 30–67 years). The median sampling time before onset of symptoms of joint disease was 2.0 years (IQR 0.9–3.9 years). The antedating time for the samples was calculated to the onset of any symptoms of RA in all prepatients. Additional samples were collected from the prepatients at their first visit to the early-arthritis clinic (n = 52), i.e. when RA was diagnosed. On average, the diagnosis of RA was established 7.1 ± 2.8 (SD) months after the first symptoms of joint disease. The mean age at the onset of disease was 56.6 years, range 34–68 years. The Ethics Committee approved this study at the University Hospital, Umeå, and the blood donors to the Blood Bank had given their written informed consent.

HLA-DRB1 genotyping was performed using polymerase chain reaction sequence-specific primers from DR low-resolution kit and DRB1*04 subtyping kit (Olerup SSP AB, Saltsjöbaden, Sweden). The SE genes were defined as DRB1*0404 and DRB1*0401. Samples for DNA analysis from one prepatient and three controls were not available, and HLA typing of one prepatient and two controls was unsuccessful. Consequently, results of HLA typing were available from 57 prepatients and 112 controls.

The anti-CCP2 (Mark2) antibodies and the RFs were determined using enzyme-linked immunoassays as previously described.[3]

The chi-square test was used for testing differences in frequencies of categorical data between groups. The sensitivity and specificity of SE gene carriage both separately and in combination with anti-CCP antibodies and RFs were calculated. Logistic regression analyses were used to estimate the odds ratio (OR) for the presence of SE gene carriage separately and in combination with anti-CCP antibodies or RFs as predictors for RA. The OR was calculated with 95% confidence intervals (CI). All P values are two-sided, and P values equal to or less than 0.05 were considered statistically significant. The calculations were performed using the SPSS package for Windows (version 11.0; SPSS, Chicago, IL, USA).


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