LETTERS 1033 alleles are primarily a risk factor for anti–cyclic citrullinated peptide antibodies and are not an independent risk factor to develop rheumatoid arthritis. Arthritis Rheum 2006;54:1117–21. 5. Huizinga TW, Amos CI, van der Helm-van Mil AH, Chen W, van Gaalen FA, Jawaheer D, et al. Refining the complex rheumatoid arthritis phenotype based on specificity of the HLA–DRB1 shared epitope for antibodies to citrullinated proteins. Arthritis Rheum 2005;52:3433–8. DOI 10.1002/art.22555 Reply Figure 1. Sharp/van der Heijde scores (mean ⫾ SEM) in rheumatoid arthritis patients with TNFA ⫺308 genotype AA ⫹ AG and those with genotype GG, during 4 years of followup. P ⫽ 0.8, 0.7, 0.8, and 0.7, respectively, at baseline and at 1 year, 2 years, and 4 years of followup, by Mann-Whitney test. In our opinion, because the TNFA ⫺308 A allele is in linkage disequilibrium with HLA–DR3, which is reported to be associated with only anti-CCP–negative RA, and RA patients without anti-CCP antibodies generally experience a less severe disease course compared with RA patients who have anti-CCP antibodies, the analysis of the association between the TNFA ⫺308 A allele and the rate of joint destruction should be corrected for the presence or absence of anti-CCP antibodies. Therefore, we analyzed white RA patients who were included in the Leiden Early Arthritis Clinic, and we did not observe an association between the TNFA ⫺308 A allele and radiographic joint destruction in RA patients, neither in the presence nor in the absence of anti-CCP antibodies. A. H. van der Helm-van Mil F. A. Kurreeman R. E. Toes T. W. Huizinga Leiden University Medical Center Leiden, The Netherlands 1. Khanna D, Wu H, Park G, Gersuk V, Gold RH, Nepom GT, et al. Association of tumor necrosis factor ␣ polymorphism, but not the shared epitope, with increased radiographic progression in a seropositive rheumatoid arthritis inception cohort. Arthritis Rheum 2006;54:1105–16. 2. Irigoyen P, Lee AT, Wener MH, Li W, Kern M, Batliwalla F, et al. Regulation of anti–cyclic citrullinated peptide antibodies in rheumatoid arthritis: contrasting effects of HLA–DR3 and the shared epitope alleles. Arthritis Rheum 2005;52:3813–8. 3. Verpoort KN, van Gaalen FA, van der Helm-van Mil AH, Schreuder GM, Breedveld FC, Huizinga TW, et al. Association of HLA–DR3 with anti–cyclic citrullinated peptide antibody–negative rheumatoid arthritis. Arthritis Rheum 2005;52:3058–62. 4. Van der Helm-van Mil AH, Verpoort KN, Breedveld FC, Huizinga TW, Toes ER, de Vries RR. The HLA–DRB1 shared epitope To the Editor: Dr. van der Helm-van Mil et al suggest that our analyses of the relationships of the rate of joint destruction with the TNFA ⫺308 A allele and with the SE should be corrected for the presence or absence of anti-CCP antibodies in our RF-positive early RA cohort. They base this on their finding that the TNFA ⫺308 A allele was not associated with the Sharp/van der Heijde scores at baseline, 1, 2, and 4 years in their Leiden Early Arthritis cohort of 327 patients, 56% of whom were anti-CCP positive and ⬃55% of whom were RF positive (1). They assume, based on their data (1), that our finding of HLA–DRB1*0301 in 55% of our patients with the TNFA ⫺308 AA ⫹ AG genotype (versus 3% of those with the GG genotype) indicates that these patients would be anti-CCP negative and therefore should have less radiographic progression, because anti-CCP–negative RA is associated with less severe joint damage (2). Similarly, they reason that both the SE and RF are associated with anti-CCP antibodies, and anti-CCP antibodies are significantly associated with more rapid joint destruction; therefore, we should have found that the presence of the SE is associated with more rapid radiographic progression rather than less rapid progression, as we reported. We did not report the anti-CCP status of our cohort, because it was not available when we planned and began our observational study in 1992 and 1993. In response to the letter from van der Helm-van Mil and colleagues, Specialty Laboratories (Valencia, CA), which has kindly performed serologic studies and maintained a frozen specimen bank for our study, was able to locate frozen specimens from 122 of the 189 patients analyzed in the TNFA ⫺308 polymorphism study (3); specimens from the remaining 67 patients were not readily accessible. There were no statistically significant differences in radiographic progression rates (total Sharp score), joint space narrowing scores, or erosion scores between the 122 anti-CCP– positive patients analyzed and the 67 excluded patients (P ⫽ 0.47–0.88), nor in the presence of the TNFA ⫺308 A allele, the presence of the SE, age at onset, and sex between these 2 subsets (P ⫽ 0.36–0.81). Specialty Laboratories performed anti-CCP antibody assays in the 122 specimens (test no. 3133: cyclic citrullinated peptide IgG antibody. Online at http://www.specialtylabs.com/ tests/details.asp?id⫽3133). One hundred eight specimens (88.5%) are anti-CCP positive, which is consistent with the estimate of van der Helm-van Mil et al that 82–87% of RFpositive patients with RA are also anti-CCP antibody positive. Among the 122-patient subset, 90 (74%) have the SE, and 14 of the 122 specimens are DRB1*0301 positive. The presence 1034 of DRB1*0301 is associated with anti-CCP antibody positivity (10 of 14; P ⫽ 0.056 for the association), contrasting with the reported association of anti-CCP antibody negativity with DRB1*0301-positive patients in the Leiden cohort (54 of 93) (1). The presence of the SE is associated with the presence of anti-CCP antibody; 85 of 90 patients with the SE are also anti-CCP positive (P ⫽ 0.002). The TNFA ⫺308 AA ⫹ AG genotype (29 of the 122-patient subset) is associated with the presence of anti-CCP antibodies (22 of 29; P ⫽ 0.022) and also with DRB1*0301 (13 of 29; P ⬍ 0.0001), as we originally reported (3) with the 189-patient cohort (27 of 49 patients with the TNFA ⫺308 AA ⫹ AG haplotype had DRB1*0301; P ⬍ 0.0001). Linear regression modeling of the unweighted total Sharp scores as a function of age at symptom onset, female sex, TNFA ⫺308 A or not, and anti-CCP antibody status showed a significant positive relationship with TNFA ⫺308 AA ⫹ AG (coefficient 2.63, standard error 0.69, P ⬍ 0.0001) and a marginal relationship with female sex (P ⫽ 0.095) but not with the presence of anti-CCP antibodies (P ⫽ 0.23; model R2 ⫽ 0.13). A similar analysis weighted by the number of radiographic observations for each patient increased the significance of the relationship with female sex (P ⬍ 0.0001) and TNFA ⫺308 AA ⫹ AG presence (coefficient 2.74, standard error 0.34, P ⬍ 0.0001), and a positive relationship with anti-CCP antibody was now evident (coefficient 1.29, standard error 0.47, P ⫽ 0.006; model R2 ⫽ 0.13). Age at onset was not significant in either analysis. Thus, the analysis of anti-CCP antibody status in a 122-patient subset of 189 RF-positive patients with early RA in our original report supports some of the findings in the Leiden early RA cohort (88.5% of our RF-positive patients are also anti-CCP antibody positive; 94% of SE-positive patients are also anti-CCP antibody positive), but does not support others (only 29% of HLA–DRB1*0301–positive patients are antiCCP antibody negative). Both weighted and unweighted linear regression analyses support the significant association of TNFA ⫺308 AA ⫹ AG with the rate of radiographic damage, with the contribution of anti-CCP antibodies reaching significance only in the weighted analysis. The addition of anti-CCP antibody does not explain our finding that radiographic damage did not progress more rapidly in SE-positive patients. We agree with the Leiden findings that SE-positive anti-CCP–positive patients (n ⫽ 85; progression rates of median total Sharp score [TSS] 1.06; joint space narrowing [JSN] score 0.19; erosion score 0.66) tend to progress more rapidly than SE-negative anti-CCP–positive patients (n ⫽ 23; median TSS 0.40; JSN 0.07; erosion 0.42), but these differences are not significant by the Kruskal-Wallis test (P ⫽ 0.89, P ⫽ 0.62, and P ⫽ 0.77, respectively). The findings with respect to association of radiographic progression with SE status in our cohort remain perplexing (3). The differences between the findings in the 2 early RA cohorts probably are due to differences in the patient populations. We limited our study to RA patients with positive RF and active disease as judged by joint counts. The lack of RF-negative patients in our cohort probably influences our findings. Only 11% of our subjects were anti-CCP negative, but 44% of the Leiden cohort were anti-CCP negative, and about 45% were rheumatoid factor negative (1). The finding that only 29% of our HLA–DRB1*0301 patients were negative for LETTERS anti-CCP antibodies, versus negative anti-CCP antibodies in 58% of the Leiden HLA–DRB1*0301 patients may help explain the differences in the effect of the presence of the TNFA ⫺308 A allele in radiographic progression. Van der Helm-van Mil et al did not present a separate analysis of their approximately 180 RF-positive patients, although they report that analysis of their 182 anti-CCP antibody–positive patients showed no significant differences in Sharp/van der Heijde scores between those with and without the TNFA ⫺308 A allele. The relative distribution of the TNFA ⫺308 A/G alleles among their RF-positive and RF-negative and anti-CCP antibody–positive and anti-CCP antibody–negative subsets is not known. Perhaps our findings apply only to RF-positive patients. Alternatively, the sample sizes of our cohort and of the RF-positive subset of the Leiden cohort are probably marginal for determining the relationship of the TNFA ⫺308 A allele with radiographic progression in seropositive RA patients. Careful additional studies with larger cohorts of seropositive RA patients will be needed to explain the differences between our findings and those of the Leiden group, and to confirm or refute the proposed association of radiographic progression with this presumably important gene that encodes TNF␣. We thank Punchong Hanvivadhanakul and Michelle Kim from the University of California at Los Angeles for their assistance in identifying and preparing the specimens for assaying. Dinesh Khanna, MD, MSc University of Cincinnati Cincinnati, OH Hui Wu, MD Grace S. Park, MPH Richard H. Gold, MD Betty P. Tsao, PhD Harold E. Paulus, MD David Geffen School of Medicine at UCLA Los Angeles, CA Lony C. Lim, PhD Navanit Lal, MD Cabrini Delaney, BS Specialty Laboratories Valencia, CA John T. Sharp, MD University of Washington Seattle, WA 1. Verpoort KN, van Gaalen FA, van der Helm-van Mil AH, Schreuder GM, Breedveld FC, Huizinga TW, et al. Association of HLA– DR3 with anti–cyclic citrullinated peptide antibody–negative rheumatoid arthritis. Arthritis Rheum 2005;52:3058–62. 2. Huizinga TW, Amos CI, van der Helm-van Mil AH, Chen W, van Gaalen FA, Jawaheer D, et al. Refining the complex rheumatoid arthritis phenotype based on specificity of the HLA–DRB1 shared epitope for antibodies to citrullinated proteins. Arthritis Rheum 2005;52:3433–8. 3. Khanna D, Wu H, Park G, Gersuk V, Gold RH, Nepom JT, et al. Association of tumor necrosis factor ␣ polymorphism, but not the shared epitope, with increased radiographic progression in a seropositive rheumatoid arthritis inception cohort. Arthritis Rheum 2006;54:1105–16.