ASSOCIATION OF SPECIFIC AMINO ACID SEQUENCE (QRRAA) OF HLA-DRB1*0405 WITH RHEUMATOID ARTHRITIS IN A TUNISIAN POPULATION

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1 ASSOCIATION OF SPECIFIC AMINO ACID SEQUENCE (QRRAA) OF HLA-DRB1*0405 WITH RHEUMATOID ARTHRITIS IN A TUNISIAN POPULATION T. DHAOUADI 1, I. SFAR 1, L. ABDELMOULA 2, R. BARDI 1, S. JENDOUBI-AYED 1, M. MAKHLOUF 1, H. AOUADI 1, T. BEN ABDALLAH 1, R. ZOUARI 2, K. AYED 1 AND Y. LAKHOUA-GORGI 1* 1 Laboratoire de Recherche en transplantation rénale et en immunopathologie, EPS Charles Nicolle, Tunis, Tunisia (LR01SP03). 2 Department of Rheumatology, EPS Charles Nicole, Tunis, Tunisia. * Auteur correspondant : Tél : / ; gorgi.yousr@gmail.com RESUME Le but de cette étude était d analyser le polymorphisme de L allèle HLA-DRB1 chez des malades Tunisiens atteints de polyarthrite rhumatoïde (PR) et d examiner son rôle dans la sévérité de la maladie. Le typage HLA-DRB1 et le sous-typage DRB1*04 et DRB1*01 a été réalisé chez 90 malades et 100 témoins normaux par PCR-SSP L allèle HLA-DRB1*04 était significativement plus fréquent chez les malades (51.1%) que chez les témoins (27%) [OR=2.83, p= ]. Le DRB1*0405 était le seul allèle associé à la PR (28.88% vs 6%) [OR=6.36, p= ]. Une baisse significative de la fréquence de l allèle DRB1*0701 était observée chez les malades (16.6%) comparativement aux témoins (36%) [p=0.0045]. Par ailleurs, la fréquence des malades portant l épitope partagé (EP) QRRAA, était légèrement augmentée par rapport aux témoins (37.8% vs 23%) [OR=2.03, p=0.039]. Nous n avons pas trouvé d association des facteurs rhumatoïdes, de l HLA-DR4 et de l allèle DRB1*04 avec les érosions osseuses et les manifestations extra-articulaires. Dans notre population, l EP QRRAA exprimé au niveau des allèles DRB1*04 est associé à la susceptibilité à la PR mais ne semble pas influencer la sévérité de la maladie, alors que l'allèle DRB1*0701 semble constituer un facteur protecteur. ABSTRACT This study aimed to investigate HLA-DRB1 alleles in rheumatoid arthritis (RA) patients from Tunisia and to examine the effect of these alleles on disease severity. HLA-DRB1 alleles and sub-typing of DRB1*04 and *01 were determined in 90 patients and 100 healthy controls, by PCR-SSP. HLA-DRB1*04 was significantly higher in patients (51.1%) than in controls (27%) [OR=2.83, p= ]. DRB1*0405 was found to be the unique DR4 allele associated with RA (28.88% vs 6%) [OR=6.36, p= ]. A significant decrease in the frequency of HLA-DRB1*0701 was observed in RA patients (16.66%) compared to controls (36%) [p=0.0026]. However, the frequency of patients carrying the shared epitope (SE) QRRAA, was slightly increased compared with controls (37.8 % vs 23%) [OR=2.03, p=0.039]. We found that the presence of rheumatoid factor, HLA-DR4 and HLA- DRB1*0405 were not significantly associated with bone erosions or the presence of extra-joint involvement. In our population, the SE (QRRAA) expressed in DRB1*04 alleles is related to the susceptibility to RA but it is not involved in RA severity in Tunisia, while DRB1*0701 might protect against this disease. Mots clés: Susceptibilité à la maladie, antigènes HLA-DR, polyarthrite rhumatoïde. Key words: disease susceptibility, HLA-DR antigens, rheumatoid arthritis. Archs. Inst. Pasteur Tunis, 2010, 87 (1-2) 53

2 A SSOCI AT ION OF SPECI F IC A M I NO ACI D SEQU E NCE (QR R A A) INTRODUCTION Rheumatoid arthritis (RA) is a chronic inflammatory joint disorder in which both environmental and genetic factors seem to be involved in its pathogenesis 1. RA is a complex polygenic disease of unknown etiology. Among genetic markers, the HLA-DR gene is the most important factor that influences both susceptibility and disease evolution. The use of molecular analysis had shown that some HLA-DRB1 alleles such as DRB1*0101, *0102, *0401, *0404, *0405, *0408, *1001 and *1402 were associated with RA, while other alleles such as *0402 or *0403 were not 2-4. The associated alleles share a conserved amino acid sequence at positions 70 to 74 in the third hyper variable region (HVR3) of DRB1 chain. This so-called the RA shared epitope (SE) comprises the amino acid sequences QKRAA, QRRAA and RRRAA 5. Several incidence and prevalence studies of RA had been reported during the last decades, suggesting a considerable variation of disease occurrence among diverse populations. A higher prevalence had been reported in certain Native Americans 6, and a very low frequency of RA in some areas of rural Africa 7, 8. RA is a heterogeneous disease, and the distribution of DR4 subtypes varies among different populations 3, 9. Therefore, the association of RA with HLA-DR4 alleles undergoes an ethnic variation due the different baseline frequencies of these alleles in the studied populations. In those from Northern Europe and North America, RA is strongly associated with HLA-DRB1*0101, *0401 and *0404 alleles 9-11, while, in Mediterranean populations, RA is rather associated with HLA-DRB1*0405, *1001 and * In Asian patients (Korea, China, Japan), RA is associated with HLA-DRB1* These genetic disparities may determine differences in disease expression, and disease outcome of early RA. As far as we know, no study of human leukocyte antigen (HLA) had been done in North Africa. In the present study, we investigated the distribution of HLA-DRB1 alleles in RA patients and controls subjects from Tunisia. PATIENTS AND METHODS Patients and control subjects This study included 90 Tunisian RA patients and 100 healthy voluntary blood donors from the same geographic origin. Patients were visiting the Rheumatology Department of the Charles Nicolle University hospital in Tunis (Tunisia) and were diagnosed as RA patients according to the revised criteria of the American College of Rheumatology (formerly, the American Rheumatism Association) 24. The disease activity score in 28 joints (DAS28) was used to measure the disease activity in RA patients 25. Seventy five percent of patients were women. Mean ± SD age at onset and disease timespan were ± (15-75) and 11.8 ± 8.23 (1-18) years, respectively. Controls were healthy subjects matched for age, sex and ethnicity. None of the healthy subjects had any evidence of autoimmune diseases or a disease with a known HLA association. All patients and controls gave informed consent to participate in the study, and the Ethics committee of Charles Nicolle Hospital approved the study. Methods Genomic DNA was extracted from peripheral blood using salting-out method 26. HLA-DRB1 alleles were studied by molecular procedures at both low and high resolution. Low and high resolution typing were carried out by a polymerase chain reaction with specific sequence primers (PCR-SSP) (One Lambda, Inc. Kittridge Street, Canoga Park, USA) following the manufacturer s instructions. Statistical Methods Univariate analysis was performed using chi-square test or fisher s exact test for small numbers (Epiinfo Stat 6.04 program CDC, Atlanta). Probability (p) values were corrected for the number of tested alleles (pc). Values < 0.05 were considered to be statistically significant. Frequencies of genotypes, alleles and phenotypes were analyzed by chi-square test. In order to evaluate the strength of associations, the odds ratios (OR) together with 95% confidence intervals (CI) were calculated. RESULTS The study consisted of 70 females and 20 males, with female to male ratio of 3.5. Radiographic examination revealed bone erosive disease in only 29 (32.2%) patients. 13 patients had extra-articular manifestations (14.4%) while 77 (85.6%) had jointlimited RA (Table I). 66 (73%) patients had a seropositive RA defined by the presence of rheumatoid factor. 5 4 Archs. Inst. Pasteur Tunis, 2010

3 T. DH AOUA DI E T A L. Table I: Clinical and serological characteristics in RA patients. Number of patients 90 Mean age ± S.D. (years) ± Mean duration of disease ± S.D. (years) 11.8 ± Females / Males 70 / 20 Rheumatoid factor positive n (%) 66 (73) Extra joints manifestations n (%) 13 (14.4) Bone erosion n (%) 29 (32.2) DRB1 genotyping DRB1*04 was found in 46 (51.1%) patients compared with 27 (27%) in controls (p= , OR=2.83, 95%CI [ ]). Females with RA had a propensity to carry the DRB1*04 allele more than males (52.2% and 45% respectively). The difference was, however, not statistically significant. There were no significant differences between the patients and the controls in the frequencies of DRB1*01 (6.6% vs 17%), *1001 (11.1% vs 8%) and *1402 (2.2% vs 5%) (Table II). Eight (8.88%) were homozygous for DRB1*04 compared with none in the controls (fisher exact test p=0.002). A significant decrease in the frequency of HLA-DRB1*0701 was observed in RA patients (16.66%) compared to controls (36%) [p=0.0045]. Table II: HLA-DRB1 phenotype frequencies in Tunisian patients with RA and controls. DRB1 alleles Controls (%) Patients (%) n=100 n=90 *0101/* * * a * b * * * * * * * * * a: p=0.0006, OR=2.83, IC ( ) ; b: p=0.0026, OR=0.36, IC (0,17-074) DRB1*04 subtyping Among the 46 DRB1*04 positive patients, 26 carried the DRB1*04 allele (56.5%). Seven alleles of the DRB1*04 group were found in the probed populations. The most frequent DRB1*04 allele in the Tunisian controls was DRB1*0403 (13%) followed by *0405 (6%). The phenotype frequency of *0405 allele was significantly higher in RA patients than in controls (28.9% vs 6%, OR=6.36, 95%CI [ ], p= ). Whereas, DRB1*0403 allele was decreased in patients compared with controls (4.4% vs 13%, OR=3.21, 95%CI [ ], p=0.039), and *0402 was increased in patients compared with controls (12.22% vs 4%, OR=3.34, 95%CI [ ], p=0.036). The other DRB1*04 subtypes such as *0404, *0407 and *0408 were not associated with RA (Table III). Table III: DRB1*04 allele s frequencies in RA patients and controls. Controls Patients DR4 subtypes N (%) N (%) 0 * (14.8) * (23.4) a 13 (48.1) * (8.7) b 0 * (22.1) * (56.5) c 3 * * * Total 46 a: p=0.067, OR=3.34; b: p=0.0003, OR=9.75; c: p= , OR=4.55 Interestingly, DRB1*0401 allele was absent in both RA patients and controls. The SE (QRRAA of DRB1*0404, *0405, *0408 and *01), known to be the RA predisposing epitope, was found in 34 (37.7%) RA patients compared with 23 (23%) in controls (OR=2.03, 95%CI [ ], p=0.039). Whereas, the amino acid sequence QKRAA was absent in both patients and controls. We observed a gene dose effect in the association of the SE with RA. 28 of 46 DRB1*04 positive patients (60.86%) had the amino acid sequence QRRAA on the DRB1*04 allele, shared mainly by DRB1*0405 (Table IV). Thus in our population RA is linked to HLA-DRB1*04-QRRAA alleles. In another hand, the frequency of DRB1*04 was not significantly different between patients with extraarticular disease manifestations and patients with Tome 87 (1-2) 55

4 A SSOCI AT ION OF SPECI F IC A M I NO ACI D SEQU E NCE (QR R A A) joints-limited RA (10.8% and 18.8% respectively). Moreover, there were no significant differences in the frequency of DRB1*04 between the erosive and non-erosive forms of disease (32.6% and 31.8% respectively), and between seropositive RA patients and seronegative RA patients (80.4% and 65.9% respectively). Table IV: SE frequencies in RA patients and controls. RA N=90 Controls N=100 QRRAA 34 (37.7%) 23 (23%) RRRAA 10 8 QRRAA (homozygous) 6 0 DRB1*04-QRRAA 28 (60.8%) a 6 (22.2%) a: p=0.0015, OR=5.44 DISCUSSION Although the etiology of RA is unknown, there is a strong genetic association between HLA-DR alleles and RA predisposition. It had been reported that HLA class II genes account for 37% of the overall genetic susceptibility to RA 26. Our results indicated that: 1) DRB1*04 is the primary locus associated with RA; 2) DRB1*0405 individuals have the highest risk of developing RA (OR=6.36); 3) RA is linked to HLA-DRB1*04- QRRAA alleles; 4) we observed a gene dose effect in the association of the SE with RA; 5) DRB1*01 and DRB1*10 are not associated with RA; 6) DRB1*0401 is totally absent in both RA patients and controls and does not make any contribution to Tunisian RA. Seven alleles of the DRB1*04 group were found in the Tunisian population. DRB1*0402 represents about 23.9% (11/46) of the DR4 alleles observed in RA patients and 14.8% (4/27) in controls. However this increase did not reach significance. DRB1*0402 is also the most frequent among Jewish populations and is not associated with RA in Jewish and other populations 11, 27. DRB1*0402 encodes the motif 70DERAA74. This motif could be considered as protective 28, 29. DRB1*0403, *0406 and *0407 are not frequent in European populations 11, 30, 31. These alleles encode the motif 70 QRRAE 74. None of them was found to be associated with an increased risk for RA, even in populations in which they are frequent 14, 32. In agreement with this observation the frequency of these alleles in our study was decreased in RA patients as compared to controls. DRB1*0405 is very common in countries surrounding the Mediterranean sea 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 and in Asia 21, 22, 23. It is relatively frequent in our data but rare among Northern European populations 27, 33. This allele encodes the motif 70 QRRAA 74 which is associated with RA in both Asian and Mediterranean populations 14, 34, 35. DRB1*01 encodes the motif 70 QRRAA 74. DRB1*0101 is a very common allele in Northern European and North American populations 9, 10, 11. The association of this allele with RA had been confirmed by many studies 14, 16, 28. Whereas DRB1*0102 is weakly associated with mild RA in some Mediterranean populations 3, 12, 13. In addition the relatively rare DRB1*1001, which had been described in association with RA in a study performed in Hispanics 14, 16, is not a susceptible allele for RA in our study. In numerous populations, HLA-DRB1 alleles that share a common amino acid sequence in their third hypervariable region (HVR3) at positions 70 to 74 of the DRß1 chain are associated with RA, including DRB1*0401, *0404, *0405, *0408, *0101, *0102, *1001 and *1402. This observation led to the SE hypothesis 5. Our study, found a significant association between SE and RA, since the proportion of subjects carrying at least 1 copy of DRB1-SE encoding alleles was significantly increased in RA patients as compared to controls. The other DRB1 alleles, namely DRB1*0101, *0102, *1001 and *1402 were not associated with RA in Tunisian. Interestingly, we found the HLA-DRB1*0405 to be the most prevalent allele encoding the SE in Tunisian population. Our results confirm those that had been found in other ethnic groups all around Mediterranean areas 12, 14. In Caucasians (Northern Europeans) the main alleles associated with RA are DRB1*0401, *0404 and *0101 and the dose of HLA- DRB1-SE alleles were associated with a more severe disease in most RA patients 36. These studies also suggested a significant association of the SE with disease severity and outcome 37. However several authors had reported a hierarchy of disease susceptibility alleles, with a more emphasized association with DRB1*0401 and *0404 and a higher frequency of these alleles in patients with a more severe disease 9, 15, 29, 38. From the above data it might be supposed that RA should be mild among populations in which the SE alleles encoding DR4 are neither *0401 nor *0404. Our data are consistent with this hypothesis, as RA had been shown to be less severe in Mediterranean populations 38, 39 and that these alleles are very rare or absent in our population. In addition, homozygous such as DRB1*0404/* Archs. Inst. Pasteur Tunis, 2010

5 T. DH AOUA DI E T A L. genotype appears to confer a high risk for disease severity 7, 17, 18, 40. Although, we observed a gene dose effect in the association of the SE with RA patients, SE in double dose did not influence the RA severity. The influence of alleles encoding SE on disease severity or disease outcome is variable and ethnically related. While studies in Japanese and white Americans indicate an influence of these alleles on joint damage, evidence from Hispanic, French and Blacks failed to show such influence 15, 39, 40. These conflicting results may be pointed to the ethnic background, social factors and the low prevalence of DRB1*0401 allele among these patients. In our study the presence of DRB1*04-QRRAA alleles did not appear to influence the erosive course of the disease as there was no difference in the frequency of homozygous state of DRB1*04 between erosive and non erosive groups. Similarly, the presence of extra-joints manifestations was not associated with inheritance of DRB1 RA-predisposing alleles, which is in agreement with other reports 8, 11, 27. Inversely, a new study performed in Colombian population, confirmed the effect of HLA-DRB1 shared epitope on erosive disease 41. It is still unclear how these alleles of HLA class II operate to confer susceptibility to RA. The locus HLA-DRB1 is highly polymorphic, and this polymorphism influences the biochemical structure of antigens that can be bound by the HLA-DR molecule. The ß chain of HLA- DR molecule contains 5 binding pockets, P1, P4, P6, P7 and P9. The P4 pocket is critical in the control of the peptide binding specificity of the different HLA-DR molecules. SE includes amino acid residues positions 70, 71, 72 and 74 belonging to P4 pocket that is closely involved in direct T cell receptor (TCR) recognition. Antigenic peptides, with negatively charged residues, are efficiently bound by SE positive alleles 42. DRB1*04- QRRAA alleles have a positively charged P4 pocket, thus they can bind with higher affinity negative residues, and influence either the type of peptides that can be bound to HLA-DR molecule and TCR activation 43, 44. Our findings corroborate this hypothesis. A recent study showed up those residues of CDR3ß are critical for recognition of hucollp261/hla- DR4 by TCR affecting the conformation of the shared epitope expressed by DR alleles associated with RA susceptibility 45. Moreover, a Tunisian study had found an association of DRB1*04 allele in patients with undifferentiated arthritis 46. In another hand, HLA-DQ polymorphism was found to be susceptible to RA in another study in Tunisian population 47. As there is high linkage disequilibrium between HLA-DQ and HLA-DR genes, it is difficult to estimate independently the exact influence of each gene. In conclusion, our results suggest that susceptibility to developing RA in Tunisian is associated with presence at least of 1 DRB1-SE positive allele. DRB1*0405 was the most frequent allele among SE positive alleles in RA patients. However, RA is a polygenic disease and the SE hypothesis fails to fully explain the contribution of the HLA class II locus to RA predisposition, suggesting that other non-hla genes may be involved. Numerous studies are being performed now to identify the diagnostic markers and markers that predict disease severity. Further studies are needed to elucidate the molecular mechanisms involved in the association between HLA-DRB1 polymorphism and RA susceptibility and severity. REFERENCES 1- W.E. Ollier, B. Harrison and D. Symmons (2001). What is the natural history of rheumatoid arthritis? Best Pract. Res. Clin. Rheumatol., 15, P. Stastny (1976). Mixed lymphocyte culture in rheumatoid arthritis. J. Clin. Invest., 57, W.E. Ollier and W. Thomson (1992). Population genetics of rheumatoid arthritis. Rheum. Dis. Clin. North Am., 18, J. L. Newton, S.M. Harney, B.P. Words Worth and M.A. Brown (2004). A review of the MHC genetics of rheumatoid arthritis. Genes. Immun., 5, P.K. Gregersen, J. Silver and R.J. Winchester (1987). The shared epitope hypothesis: an approach to understanding the molecular genetics of susceptibility to rheumatoid arthritis. Arthritis. Rheum., 30, R.F. Willkens, G.T. Nepom, C.R. Marks, J.W. Nettles and B.S. Nepom (1991). Association of HLA Dw 16 with rheumatoid arthritis in Yakima Indians. Further evidence for the Shared Epitope hypothesis. Arthritis. Rheum., 34, A.J. Silman and M.C. Hochberg. Epidemiology of the rheumatic diseases 2 nd edition. Oxford University Press. 8- A.J. Silman, W. Ollier, S. Holligan, F. Birrell, A. Adebajo, M.C. Asuzu et al. (1993). Absence of rheumatoid arthritis in a rural Nigerian population. J. Rheumatol., 20, Tome 87 (1-2) 57

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