Dear Editor,

We read with interest the recent article “Smoking is a risk factor for anti-CCP antibodies only in RA patients that carry HLA-DRB1 Shared Epitope alleles” by Dr. Linn-Rasker and colleagues in Leiden.[1] The question of whether true gene-environment interaction exists between genotype (HLA-DRB1 shared epitope, SE) and an environmental exposure, cigarette smoking in this case, both of which are known to greatly increase the risk of developing RA, is central to our understanding of how these factors predispose to RA. Does the risk associated with having both of these risk factors exceed what would be expected if these risks were independent and additive, and how so? Case-only analyses, such as this, are an efficient and sound method for screening for gene-environment interactions, under the assumption of independence of exposure and genotype in the population.[2,3]

In a recent editorial, Ahlbom and Alfredsson explained the concepts of biological versus statistical interaction.[4,5] Biological interaction is seen when two risk factors are involved in the same pathway to development of disease; statistically this is seen as a departure from additivity of disease rates. For example, Padyukov and colleagues in Sweden have demonstrated that a greater than additive interaction exists for the effects of cigarette smoking and the presence of 0, 1 or 2 copies of the SE.[6] To do so, they calculated the attributable proportion (AP), the proportion of RA among those with both exposures that is attributable to an additive interaction between these risk factors.[7] They found that among current smokers with a double copy of the SE, the AP is 0.7, or 70%, (95% CI 0.4, 0.9).[6] Another way of statistically testing for gene-environment interaction is to create a cross-classified variable, or interaction term, in a logistic regression model, and test it for significance, pointing to a greater than multiplicative interaction that is significant.

In their study, Linn-Rasker and colleagues employed a case-only analysis of patients in the Leiden Early RA clinic to evaluate the interaction between smoking and HLA-SE in predicting the risk of Anti-CCP antibodies. Their findings are very interesting and suggest an interaction between the two exposures, but it does not appear that the presence of a gene-environment interaction was statistically tested. We have employed their data to test statistically for the presence of a gene-environment interaction, both greater than additive and greater than multiplicative. Using the data presented in table 2 of their article, the AP, or the proportion of RA cases positive for anti-CCP antibodies which is attributable to an interaction between cigarette smoking and the presence of the SE, is 0.5, or 50%. (Given our limited access to the data, we are not able to use a re-sampling technique to obtain a 95% confidence interval.) When a cross-classified variable (smoking x HLA-SE) is created and employed in a logistic regression model using the data in Table 2, the term is not significant for a multiplicative interaction (p=0.19). Employing the terminology suggested by Ahlbom and Alfredsson,[4,5] the fact that an additive, but not multiplicative, interaction exists is important and suggests that cigarette smoking and HLA SE lie on the same causal pathway to the development of anti-CCP antibodies and RA. Presenting such statistical analyses promotes translational research between epidemiologists and basic scientists.

Karen H. Costenbader, MD, MPH (A)
Lori B. Chibnik, MPH (A)
Lisa Mandl, MD, MPH (B)
Elizabeth W. Karlson, MD (A)

(A) Brigham and Women’s Hospital, Harvard Medical School, Boston, MA
(B) Hospital for Special Surgery, Weill Cornell Medical College, New York, NY

References

1. Linn-Rasker SP, van der Helm-van Mil AH, Van Gaalen FA, Kloppenburg M, de Vries R, le Cessie S, Breedveld FC, Toes RE, Huizinga TW. Smoking is a risk factor for anti-CCP antibodies only in RA patients that carry HLA-DRB1 Shared Epitope alleles. Ann Rheum Dis 2005.

2. Botto LD, Khoury MJ. Commentary: facing the challenge of gene- environment interaction: the two-by-four table and beyond. Am J Epidemiol 2001; 153:1016-20.

3. Gatto NM, Campbell UB, Rundle AG, Ahsan H. Further development of the case-only design for assessing gene-environment interaction: evaluation of and adjustment for bias. Int J Epidemiol 2004; 33:1014-24. 4.

Andersson T, Alfredsson L, Kallberg H, Zdravkovic S, Ahlbom A. Calculating measures of biological interaction. Eur J Epidemiol 2005; 20:575-9.

5. Ahlbom A, Alfredsson L. Interaction: A word with two meanings creates confusion. Eur J Epidemiol 2005; 20:563-4.

6. Padyukov L, Silva C, Stolt P, Alfredsson L, Klareskog L. A gene- environment interaction between smoking and shared epitope genes in HLA-DR provides a high risk of seropositive rheumatoid arthritis. Arthritis Rheum 2004; 50:3085-92.

7. Rothman KJ, Greenland S, Walker AM. Concepts of interaction. Am J Epidemiol 1980; 112:467-70.

Dear Editor,

We found the study by Elkayam et al (1) of great interest, as it sheds more light on the complex scenario of anti cyclic citrullinated peptide antibodies (anti-CCP), originally described as highly specific for rheumatoid arthritis (RA). Indeed, it appears that an immune response to these proteins is initiated (though it’s not yet clear how sustained) in several other conditions. It is not surprising that tuberculosis (TB) and RA may share serological markers like rheumatoid factor and, now, anti- CCP. The cytokine profile of these inflammatory diseases is, in fact, largely overlapping: type 1 cytokines and TNF-α are important in the defense against mycobacteria infection and pathogenesis of TB granuloma as well as in the induction and maintainance of RA synovial inflammation. The association of anti-CCP with other rheumatological and autoimmune diseases is less granted, but it’s being reported increasingly (2, 3).

We ourselves have evaluated the occurrence of anti-CCP in 483 sera of patients with rhematological (psoriatic and reactive arthritis, primary and erosive osteoarthritis) and autoimmune diseases (connective tissue diseases, autoimmune liver disease, celiac disease). Anti-CCP were tested using the commercial ELISA kit Diastat (Axis-Shield, Dundee, Scotland, UK) with sera diluted 1:100, according to the manufacturer’s instructions. They were globally detected in 76 sera (16%). The highest prevalence was found, as expected, in RA (53 out of 89, 60%). As also expected, the antibodies were found in a proportion of patients with psoriatic arthritis (2 out of 23, 9%), but they were positive also in erosive osteoarthritis (2/35, 5%), connective tissue diseases (4/42, 9%) and, especially, in autoimmune liver diseases (autoimmune hepatitis and primary biliary cirrhosis), in which we found 14 positive cases out of 182 (8%) (4). As in TB (1), no correlation was found between anti-CCP seropositivity and symptoms or signs of arthritis in autoimmune cases. High titres (≥ 20 with the ELISA kit used in our laboratory) were present in 75% RA positive sera, but also in 62% positive sera from non-RA patients. Anti-CCP, as detected by this commercially available ELISA, do not seem to be entirely specific for RA and the results of this test should be critically evaluated in the clinical setting. This consideration holds particularly true for overlap syndromes or in early RA, where anti-CCP have been included in prediction models used to select patients for more aggressive therapeutic approach (5).

In fact, in a recent study, by Gao et al (6) it’s been established that the positive predictive value of anti-CCP alone (the probability of fulfilling the ACR criteria for RA) was of little help to accurately predict RA. As the exact nature of the target antigen(s) used in the ELISA kit is still unknown, it’s entirely possible that non-RA-associated anti- CCP do bind to citrullinated proteins, that are different from those recognized by RA sera. If future studies will better characterize the nature of the antigens involved in the different anti-CCP associated diseases and when more specific tests will be set up, only then anti-CCP will deserve a unique place in the diagnosis and management of RA.

Reference

1. Elkayam O, Segal R, Lidgi M, Caspi D. Positive anti-cyclic citrullinated proteins and rheumatoid factor during active lung tubercolosis. Ann Rheum Dis 2005 Dec 16; (Epub ahead of print).

2. Hoffman IE, Peene I, Cebecauer L, Isenberg D, Huizinga TW, Union A et al. Presence of rheumatoid factor and antibodies to citrullinated peptides in systemic lupus erythematosus. Ann Rheum Dis. 2005;64:330-2.

3. Vander Cruyssen B, Hoffman IE, Zmierczak H, Van den Berghe M, Kruithof E, De Rycke et al. Anti-citrullinated peptide antibodies may occur in patients with psoriatic arthritis. Ann Rheum Dis. 2005;64:1145-9.

4. Fusconi M, Vannini A, Dall’Aglio AC, Pappas G, Cassani F, Ballardini G et al. Anti-cyclic citrullinated peptide antibodies in type 1 autoimmune hepatitis. Aliment Pharmacol Ther 2005;22:951-5.

5. Visser H, le Cessie S, Vos K, Breedveld FC, Hazes JMW. How to diagnose rheumatoid arthritis early: A prediction model for persistent (erosive) arthritis. Arthritis Rheum 2002;46:557-65.

6. Gao IK, Haas-Wöhrle A, Mueller KG, Lorenz H-M, Fiehn C. Determination of anti-CCP antibodies in patients with suspected rheumatoid arthritis: does it help to predict the diagnosis before referral to a rheumatologist? Ann Rheum Dis 2005;64:1516-7.

Dear Editor,

I read with interest the recent study describing clinical characteristics associated with antibodies that bind fragments of the Mi-2 beta antigen1 and it raises the question of how to best define autoantibodies. For economic reasons, ELISAs are taking over much of the immunology testing in rheumatology practice today. ELISAs are being used despite their often limited validation and the many concerns regarding the sensitivity, specificity and reproducibility of these approaches.2 There is considerable evidence that ELISA assays may not be measuring the same antibody populations that have traditionally been detected by immunodiffusion, indirect immunofluorescence or immunoprecipitation methods – the classic approaches that originally defined the clinical usefulness of most autoantibodies in systemic rheumatic conditions. Many groups have recognized this problem, and coordinated efforts similar to those of the Diabetes Antibody Standardisation Program to improve the sensitivity, specificity and comparability of the measurement of autoantibodies associated with type 1 diabetes (http://www.idsoc.org/committees/antibody/dasphome.html) should be encouraged in other fields.

Important examples in the literature also show that antibodies raised against a given antigen may not interact with the target protein in the same way that spontaneously occurring autoantibodies do, and it is possible that such differences have important implications regarding the origin and pathologic significance of human autoantibodies. Prime examples are anti-Jo-1 autoantibodies directed against histidyl-tRNA synthetase. These myositis-specific autoantibodies were originally defined by immunodiffusion and immunoprecipitation of the target protein in a complex with its cognate tRNA and were found in all cases to inhibit the function of the target antigen.3 Later studies in mice suggested that antibodies could easily be induced that bind histidyl-tRNA synthetase, however, they were completely different in their disease association, epitope specificity, capacity to immunoprecipitate the target antigen with its cognate tRNA, and their ability to inhibit the function of the antigenic enzyme.4 Should such different antibodies also be called anti-Jo-1 autoantibodies? I think not and suggest that they should rather be referred to simply as antibodies that bind histidyl-tRNA synthetase or its peptide fragments by the methodology used.

I believe that this issue is a widespread one and a clarification of such antibody nomenclature would enhance our understanding and appreciation of the limitations that different methodologies impose. Until and unless data are generated to demonstrate that a new method of measuring a particular autoantibody gives equivalent results to a previously validated approach, I believe that it would be desirable that the “new antibody” should be given a new name in conjunction with the methodology used to detect that antibody. Such nomenclature would potentially minimize confusion in the literature regarding the sensitivity, specificity, phenotypic associations and clinical use of such antibodies.

This brings us back to the study by Hengstman et al.1 Is it possible that the differences in clinical associations noted in this paper, as compared to those found in previous studies5, are due to the detection of different antibody populations by different methods? Antibodies that bind to fragments of the chromodomain helicase DNA binding protein 4 (the Mi-2 beta antigen) by ELISA may be quiet different than those which are positive by immunodiffusion or which immunoprecipitate the Mi-2 antigen in conjunction with the rest of the nucleosome remodeling and histone deacetylase complex - the original ways that anti-Mi-2 autoantibodies and their clinical associations were defined. It would have been useful for the authors to have compared these methods directly in this study to address this possibility. Until this possible methodologic difference is resolved, however, perhaps it would be best to not define such ELISA- binding antibodies as anti-Mi-2 autoantibodies, but rather as antibodies which bind fragments of the chromodomain helicase DNA binding protein 4 by ELISA.

References

(1) Hengstman GJ, Vree Egberts WT, Seelig HP et al. Clinical characteristics of patients with myositis and autoantibodies to different fragments of the Mi-2 beta antigen. Ann Rheum Dis 2006; 65(2):242-245.

(2) Fritzler MJ, Wiik A, Tan EM et al. A critical evaluation of enzyme immunoassay kits for detection of antinuclear autoantibodies of defined specificities. III. Comparative performance characteristics of academic and manufacturers' laboratories. J Rheumatol 2003; 30(11):2374- 2381.

(3) Mathews MB, Bernstein RM. Myositis autoantibody inhibits histidyl-tRNA synthetase: a model for autoimmunity. Nature 1983; 304:177- 179.

(4) Miller FW, Waite KA, Biswas T, Plotz PH. The role of an autoantigen, histidyl-tRNA synthetase, in the induction and maintenance of autoimmunity. Proc Natl Acad Sci U S A 1990; 87(24):9933-9937.

(5) Love LA, Leff RL, Fraser DD et al. A new approach to the classification of idiopathic inflammatory myopathy: myositis-specific autoantibodies define useful homogeneous patient groups. Medicine (Baltimore) 1991; 70(6):360-374.

Dear Editor,

Genetic factors not only plays an important role for development of reactive arthritis in patients with Chlamydia infection, but genetic factors also influence the susceptibility to such infections. Recently recurrent Chlamydia infection have been found to be associated with human leukocyte antigen variants (DRB1*03-DQB1*04 and DQB1*06). Again a G-C-C haplotype corresponding to variants at IL-10 (encoding interleukin-10 [IL-10]) promoter positions -1082, -819, and -592 has been found to be underrepresented in individuals with recurrent infection. These genetic associations are independent of nongenetic factors, including number of sex partners, race, sex, duration of follow-up, and human immunodeficiency virus type 1 seropositivity.

Consistent with the observed IL-10 association, cervical secretions in female adolescents without the IL-10 G-C-C haplotype have an elevated IL-10 concentrations after Chlamydia infection, which may reflect involvement of a Chlamydia-specific mechanism for genetically mediated, differential IL-10 expression in the genital tract. This important observation will lead to better understanding of aetiopathogenesis of Chlamydia infection and its sequelae such as reactive arthritis, which requires further study.

Reference:

1. Wang C,Tang J,Geisler WM,Crowley-Nowick PA,Wilson CM and Kaslow RA.J Infect Dis.2005 Apr 1;191(7):1084-92.