Repeated analysis of individual samples during the study period showed stable values. vs 53.9%, p=0.01), while the frequency of the T variant was similar. HLA-SE, combined with smoking, was significantly related to all combinations of anti-CCP and RF isotypes in patients with RA. No such relationships were found for the first-degree relatives. Conclusions All anti-CCP and RF isotypes analysed occurred more commonly in unaffected first-degree relatives from multicase families than in controls, but with different isotype distribution from patients with RA. Introduction A genetic component of susceptibility to rheumatoid arthritis (RA) has long been suggested by data from twin and familial studies.1 2 Twin studies have estimated the inheritability of RA to be between 50% and 60%,3 although the role of genetic factors has been questioned.4 Consistent associations implicate a role for genes located in the human leucocyte antigen (HLA) region in the risk of developing RA.5 Linkage studies support a role for the HLA locus in genetic susceptibility to RA.6 A large number of genes have, in genome-wide association studies, been particularly associated with anti-citrullinated protein/peptide antibody (ACPA) positive patients with RA.7 8 Another robust association with RA is the R620W polymorphism of the locus.9 10 The association between the T variant of the gene and RA was more evident in patients seropositive for rheumatoid factors (RFs) and/or ACPAs.11C13 We have previously shown that a combination of antibodies against CCP with either HLA-shared epitope (SE) or the T variant of analysed before the onset of symptoms of disease strongly predicted future onset of RA with a high relative risk of developing RA.14 15 Smoking is one of the aetiological factors identified as a risk factor for RA.16 Data from a number of studies have suggested that a combination of HLA-SE alleles and exposure to tobacco interact in the development of ACPA-positive RA.17 18 In addition to the previously identified IgG isotype, we have shown that ACPAs of either the IgA or IgM isotype predate the onset of RA by a number of years.19 20 All three isotypes were also significantly increased after development of the disease. In addition, ACPAs have been shown to contribute NPI64 to progression of arthritis in collagen-induced arthritis in mice, appearing 7 days after immunisation before clinical disease.21 Taken together, these results suggest a pathogenic role for ACPAs in the development of RA. In a previous study of native North Americans with a high prevalence of severe RA, ACPAs of different isotypes were present with increased frequency in the unaffected relatives of that cohort.22 In another study of first-degree relatives of patients with RA, the frequency of RFs and/or anti-CCP IgG increased to 16%.23 The aim of the present study was to analyse the isotypes of ACPAs and RFs in unaffected first-degree relatives of patients with RA from multicase families from northern Sweden in relation to HLA-SE alleles and polymorphism and smoking habits. Materials and methods Familial clustering of RA was identified using a questionnaire distributed to patients with RA attending departments of rheumatology in the four northern-most counties of Sweden. All of the reported relatives from families who wished to participate were interviewed, by a second NPI64 questionnaire, TRAF7 about symptoms and signs of joint disease. Affected relatives were evaluated clinically by a rheumatologist and inspection of their medical records, and personal interviews were used to confirm the diagnosis of RA (defined by the ACR 1987 criteria).24 Sixty-one multicase families, with a total of 196 individuals with RA, were willing to participate. From 51 of these families, 157 first-degree relatives (59.9% female) who reported no symptoms of inflammatory joint disease on a questionnaire evaluated by us were willing to donate blood samples. These 51 families included between two and 11 affected members with a total of 163 RA cases; 71.8% were female. Of NPI64 the patients with RA, 62.6% had erosive disease and 21.5% had extra-articular manifestations. IgG, IgA and IgM anti-CCP isotypes were determined in.