Table 6 Gene–environment interaction between HLA-SE (none, any) and smoking status (never, past, current) in all rheumatoid arthritis (RA) groups and in groups stratified by serological status in the Nurses’ Health Studies Smoking HLA-SE status None Never None Past None Current Any Never Any Past Any Current Additive: AP1 (95% CI) p For interaction RERI Synergy index Multiplicative: p For interaction Additive: AP2 (95% CI) p For interaction RERI Synergy index Multiplicative: p For interaction All RA 81/111 92/110 32/46 85/77 99/60 38/27 Cases/controls OR* (95% CI) 1.00 (reference) 1.13 (0.74 to 1.73) 0.90 (0.51 to 1.59) 1.60 (1.03 to 2.49) 2.25 (1.43 to 3.52) 1.89 (1.03 to 3.47) OR{ (95% CI) 1.00 (reference) 1.03 (0.66 to 1.61) 0.90 (0.49 to 1.64) 1.67 (1.05 to 2.63) 2.23 (1.40 to 3.56) 1.91 (1.03 to 3.55) 0.48 0.51 0.23 (20.15 to 0.61) 0.24 (20.15 to 0.63) 0.23 0.22 0.52 (20.40 to 1.44) 0.54 (20.41 to 1.48) 1.71 (0.54 to 5.40) 1.77 (0.52 to 6.08) 0.21 (20.35 to 0.77) 0.18 (20.42 to 0.78) 0.47 0.55 0.39 (20.80 to 1.59) 0.35 (20.91 to 1.60) 1.80 (0.26 to 12.58) 1.62 (0.25 to 10.36) 0.41 0.58 Seropositive RA Cases/controls 42/111 50/110 15/46 54/77 71/60 27/27 0.04 0.24 0.12 0.28 OR{ (95% CI) 1.00 (reference) 1.11 (0.68 to 1.83) 0.90 (0.45 to 1.81) 1.78 (1.08 to 2.95) 3.03 (1.83 to 5.03) 2.71 (1.41 to 5.19) 0.38 (0.03 to 0.73) 1.14 (20.17 to 2.44) 2.27 (0.71 to 7.27) 0.38 (20.10 to 0.85) 1.02 (20.66 to 2.70) 2.49 (0.46 to 13.63) 39/111 42/110 17/46 31/11 28/60 11/27 0.91 0.93 0.86 0.85 Extended report Seronegative RA Cases/controls OR{ (95% CI) 1.00 (reference) 1.05 (0.62 to 1.77) 1.03 (0.52 to 2.06) 1.18 (0.70 to 2.09) 1.28 (0.71 to 2.33) 1.10 (0.49 to 2.48) 0.04 (20.68 to 0.74) 0.06 (21.31 to 1.08) 1.24 (0.02 to 67.47) 20.10 (21.22 to 1.02) 20.11 (20.88 to 0.99) 0.49 (0.00 to 2327.19) *Conditional logistic regression. {Conditional logistic regression, adjusting for parity, breast feeding and menstrual irregularity, and age at menarche, menopausal status and postmenopausal hormone use at diagnosis. {Unconditional logistic regression adjusting for year of birth, parity, breast feeding and menstrual irregularity, and age at menarche, menopausal status and postmenopausal hormone use at blood draw and at diagnosis. AP, attributable proportion due to interaction, an index of additive interactions between binary smoking variable and binary HLA-SE genotype; RERI, relative excess risk due to interaction. Evidence for gene–environment interaction between HLA-SE and smoking in seropositive RA risk was seen in patients with undifferentiated arthritis at the Leiden Early Arthritis Clinic. Among the participants who were HLA-SE positive, ever smoking significantly increased the OR for development of CCP-positive RA from 3.3 to 8.0 (p = 0.002 for multiplicative interaction)21; HLA-SE and ever smoking also increased the OR of CCP antibodies, with evidence for additive but not multi- plicative interaction.20 35 A Danish case-control study of HLA-SE interactions with RA epidemiological risk factors, did not demonstrate any significant multiplicative interaction for HLA-SE* smoking, however, HLA-SE homozygotes had a 52- fold increased risk of CCP-positive RA compared to non-carrier/ never smokers. Testing for additive interaction was not performed. In contrast, a significant HLA-SE* smoking interac- tion was demonstrated in the Iowa Women’s Health Study, an older Caucasian female cohort in which smoking was associated with increased risk of RA only among subjects who were HLA- SE negative, but not among subjects who were HLA-SE positive.36 The reasons for this discrepancy are unknown, although it may relate to the older age at RA onset or the small sample size (116 cases) in that cohort. A case-only analysis combining data from three large US populations was unable to confirm an interaction between HLA-SE alleles and cigarette smoking in relation to presence of CCP antibodies but smoking status was defined only as never/ever smoking.22 In one cohort with information on pack-years of smoking, there was an independent effect of smoking on the presence of CCP among heavy smokers (.20 pack-years). Strengths of this study include the prospective collection of exposure information prior to the onset of RA, the detailed smoking data collected every 2 years and availability of high- resolution HLA genotyping. Limitations include the lack of data on CCP antibody status, as most cases were diagnosed prior to Ann Rheum Dis 2010;69:54–60. doi:10.1136/ard.2008.102962 the widespread usage of this test, and absence of plasma samples for CCP testing in about half the cases. However, RF status was available from medical record reviews, and other gene–environ- ment interaction studies demonstrate similar relationships for RF-positive and CCP-positive phenotypes.13 19 The rate of seropositive RA in this study (60%) is similar to that reported from a large US registry study, the National Databank (n = 14 000) with patients recruited from rheumatology practices across the US.37 Limited generalisability of NHS a concern, as the NHS cohorts are comprised of middle to older aged women with high educational levels and with primarily Caucasian heritage. However, an advantage of similar ethnic background in genetic studies is a lower potential for population stratification. This study of gene–environment interactions in RA in a cohort of Caucasian US women demonstrates a significant additive and multiplicative interaction between the strongest genetic risk factor for RA, the HLA-SE, and heavy cigarette smoking .10 pack-years, the strongest environmental risk factor for RA, for seropositive but not seronegative RA. We demonstrate only additive interaction between smoking categorised as never/ever smoked or as three categories (never, past, current) with the HLA- SE and seropositive RA; however, if smoking is classified by dose (as pack-years), we demonstrate additive and multiplicative interaction for seropositive RA. This illustrates the importance of considering the dose effects of environmental and genetic factors in gene–environment interaction studies. Additionally, it lends evidence to the theory that seropositive and seronegative RA have different risk factors and pathogenic pathways. Acknowledgements: The authors gratefully acknowledge the participants in the NHS for their continuing participation. The authors also thank Gideon Aweh, Karen Corsano, Wei-Zi Ding, Lingsheng Dong and Brendan Keenan for their technical assistance. Funding: Supported by NIH grants R01 AR49880, CA87969, P60 AR047782, K24 AR0524-01 and BIRCWH K12 HD051959 (supported by NIMH, NIAID, NICHD and OD). 59