Rheumatoid arthritis (RA) is an inflammatory disease of unknown cause. The course of rheumatoid arthritis is ranging from mild to aggressive forms, the latter being very difficult to cope with. It has been shown that early diagnosis and treatment reduce joint destruction, and improve survival 1. Risk factors have been identified in groups of patients with different outcomes such as baseline radiographic joint changes, presence of rheumatoid factor (RF), specific human leukocyte antigens (HLA); HLA-DRB1 genotypes, high disease activity, high disability scores, and high levels of acute phase proteins 2. Antibodies to cyclic citrullinated peptide (anti-CCP) have been documented extensively over recent years as highly specific serological markers for rheumatoid arthritis, with important clinical implications for diagnosis and prognosis 3.

It has been suggested that the etiology of RA is due to an interaction between genetic and environmental factors. Genetic studies have demonstrated multiple HLA-DRB1 alleles encoding a conserved sequence at amino acid positions 70–74 are associated with susceptibility and severity of RA. This conserved sequence is commonly known as the shared epitope (SE). The role of the SE in the evolution of articular destruction has yielded conflicting results 45.

The present study is a cross-sectional analysis aimed to evaluate the significance of the presence of SE genes, defined as DRB1*01 or DRB1*04, in relation to anti-CCP antibodies, antikeratin antibody (AKA) and RF in individuals who developed RA. We focused on disease activity and joint damage, evaluated on radiographs, as outcome variables.

This study was carried out on 60 outpatients who fulfilled the American college of rheumatology criteria for RA. A written consent was obtained from the patients according to the Declaration of Helsinki prior to enrollment in the study. The study was approved by the Assiut University local ethical committee. Patients were subsequently treated with disease modifying antirheumatic drugs (usually methotrexate, sulphasalazine, or a combination of both). The patients were evaluated for age; sex; body mass index; disease duration; duration of morning stiffness; patients' assessment of pain (on a visual analogue scale); number of swollen and tender joints, disease activity score; presence or absence of nodules and extra-articular manifestations. Disease activity by the disease activity score (DAS28) 6. Global health and pain and visual analogue scale were assessed. Functional disability was evaluated using health assessment questionnaire 7. Hand, wrist, and foot radiographs were obtained, evaluated and scored using Larsen method 8.

Clinical and laboratory investigations were illustrated in Table 1. The median age of the patients was 50 years and 90% were women. According the positivity of anti-CCP antibodies, 50 (83.3%) patients were anti-CCP antibodies positive. CRP, RF and AKA were detected in 34 (56.7%), 43 (71.7%) and 31 (52%) of patients respectively.

Comparing anti-CCP positive with anti-CCP negative patients (Table 1); anti-CCP positive patients had a significantly higher frequency of high ESR, RF, ANA and AKA (P = 0.04, 0.02, 0.03 and 0.05) respectively.

The frequencies of HLA-DRB1 alleles in patients and control are shown in Table 2. There was a statistically significant increase in the frequency in DRB1*01, DRB1*02, DRB1*04 compared to control (P = 0.001, 0.05 and 005 respectively). In this study 45% had HLA-DRB1*01 and 35% of patients had one or two HLA-DRB1*04 alleles.

It was found that RF associated allele (DRB1* 0405) was present in of 80% of DRB1*04 positive patients. "Double dose" of DRB1*04 (homozygous) was found in 8/60 (13%) of our patients versus 3/100 (3%) in the control P = 0.05 (Data is not shown in the table).

Anti-CCP-antibody is a synthetic cyclic peptides containing citrulline. It is incorporated into newly proposed diagnostic criteria for rheumatoid arthritis, with higher sensitivity. The high predictive value of anti-CCP antibodies is indicating that citrullination and production of anti-CCP antibodies and RF are early processes in the development of RA 11. The present work revealed strong complementarities of RF to anti-CCP as 65% patients lacking RF found to be positive for anti-CCP. Some reports suggest that RF and anti-CCP should be combined to reach optimum diagnostic properties 12. Discrepancy can be explained by the fact that in the literature stratification for specificity was not performed, and therefore, the results of different studies are not comparable 13. The frequencies of ANA and AKA were 36%, 52% respectively which indicate that testing for this spectrum of auto antibodies carries little advantage over RF or anti-CCP in diagnosing RA. A significant association of AKA with Anti-CCP may relate to that citrulline (profilaggrin) appears to be an essential constituent of the antigenic determinants recognized by AKA 14.

In the present work, the sensitivity of anti-CCP was significantly higher than that of the RFs for prediction of grade of disease activity. This findings were supported by the data previously published 115.

It was reported that anti-CCP is an important predictor of radiological outcome. Preventing and diminishing joint damage is an important treatment goal in early rheumatoid arthritis 1.

According the prediction models reported by, Combe et al 2, for instance, variables reflecting disease activity also contribute to the prediction of joint damage. In this study, anti-CCP gave statistically significant differences between the positive and negative group with radiological damage. These data are comparable with the findings of other groups using a similar approach 151617. In our study serum anti-CCP added some prognostic information to ESR, CRP and RF, and it was the most sensitive predictor of joint damage, following ESR. These findings were in agreement with earlier reports 4, but other studies mentioned that anti-CCP was the only significant predictor, together with C reactive protein 18. Vencovsky et al reported that the combination RF, CRP and anti-CCP2 antibodies revealed 10 times higher radiological damage 17 and early erosions 19. However it was needed to be emphasized that the results based on dichotomization not useful in everyday clinical practice. So, we investigated whether appropriate cut off could be determined to identify those patients with the most aggressive radiological course.

Genetic studies have demonstrated that HLA alleles have been implicated in a number of chronic inflammatory diseases. RA has been associated with the SE of HLA-DRB1, which includes DRB1*04 and DRB1*01 alleles 19. In the present study we found a discrepant relationship between RF and anti-CCP2 with the presence of the SE gene. Also our results do not support the opinion that there is a direct association between SE gene carriage and the occurrence of antibodies directed to CCP (or RFs) leading to the development of RA, but rather suggest that there is a synergistic interaction between these factors. In contrast Berglin et al reported that patients with early RA, had significant association between anti-CCP antibodies and expression of DRB1*0401/0101 5. The conversion of arginine to citrulline in HLA-DRB1*0401 transgenic mice has been demonstrated to significantly increase activation of CD4+ T cells 20, so that individuals carrying the SE genes may have more sustained T- and B-cell responses to citrullinated antigens than non carriers 13. The presence of citrulline within the HLA binding peptide enhances the peptide-MHC affinity and leads to the activation of CD4+ T cells during presentation of citrullinated antigens 20. This specific T-cell dependent immune response to citrullinated peptides may contribute to the occurrence of RA. A quite contradictory suggestion is that HLA antigens do not predispose to the autoimmune disease per se but rather fail to provide protection. Abnormal T-cell regulation associated with certain HLA haplotypes leads to the loss of self-tolerance followed by polyclonal activation of T and B cells and the subsequent production of auto antibodies 21.

In the present work there was a statistically significant difference observed between carriage of the DRB1*04 and DRB1*01 alleles with radiological damage and DRB1*04 with disease activity, these findings were in agreement with those previously reported 1317. Recent genome wide scanning studies using micro satellite loci have confirmed that there is strong linkage between this region with susceptibility and severity of RA 2223. Moreover Petersson et al 4 reported that DRB1*04 SE double gene dose is associated with disease severity in RA. In contrast Eberhardt et al 24 did not find the genotypes to be strongly associated with disease severity after two and five years.

The role of the SE in the evolution of articular destruction has yielded conflicting results; however, in most studies the presence of the SE is associated with increased joint destruction 1422. Others revealed an association between HLA-DRB1*04 and erosive disease 25, Goronzy et al 25, and Petersson et al 4 considered homozygosity for HLA-DRB1*04 as a major predictor of the development of erosions. In Caucasians of Northern European origin, the double dose of DRB1*04 SE alleles (0401/0401 genotype) was associated with radiographic signs of progressive joint damage 26. A strong concordance was revealed between our study and that in East Asian populations, in which DRB1*0401 is rare and DRB1*0405 is the most frequent RA associated HLA-DRB1 genotype. The latter allele has also been reported to be associated with an increased risk for extra-articular manifestations of RA 27. In contrast, others reported that the presence of shared epitope in single or double dose did not provide any predictive information on the development of joint damage and was not related to functional outcome 28. In another study, by Mattey et al 16, reported that a predictive value of shared epitopes for radiological changes was restricted to RF negative patients. These discrepancies may reflect variability in the relative frequencies of HLA-DRB1*04 in different populations. Possibly, further studies on larger numbers of patients might solve this issue.

In the Present work the combination between anti-CCP, and the HLA-DR-B1*04 gave more identification of the grade of disease activity than a single test alone. The presence of RF, the SE, and anti-CCP2 antibodies yielded a 10 times higher average expectancy rate for a high radiological progression rate compared with the absence of the three parameters 1516.

Anti-CCP2 is superior to RF for the detection of RA and provided predictive information on joint destruction and disease activity. The presence of RA associated antibodies and/or the SE genes are indicative for a poorer radiological outcome and higher grade of activity. Thus, we shall need new strategies, both in research and in clinical practice, where we may now possess a new means for analyzing the risk of RA in individuals who will have different needs to acquire such information.

The authors declare that they have no competing interests.

NAF participated in study design, data acquisition and critical manuscript revision. AME-E participated in study design, performing laboratory investigations other than HLA, and critical manuscript revision. EM participated in study design, HLA analysis, analysis and interpretation of data, performed the statistical analysis, wrote the manuscript, and performing manuscript drafting and submission. RMB participated in study design, HLA analysis, analysis and interpretation of data, and critical manuscript revision. HBH participated in HLA analysis and manuscript revision. SA participated in study design, data acquisition, and critical manuscript revision. MM participated in study design and data acquisition. H-AGR participated in laboratory investigations other than HLA and manuscript revision. FA participated in study design, data acquisition, and critical manuscript revision. All authors read and approved the final manuscript.