HLA-B*27 allele associated to Behçet s disease and to anterior uveitis in Moroccan patients

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1 Ann Biol Clin 2011; 69 (4): HLA-B*27 allele associated to Behçet s disease and to anterior uveitis in Moroccan patients HLA-B*27 allèle associé à la maladie de Behçet et à une uvéite antérieure chez les patients marocains Asmaa Radouane 1 Mounia Oudghiri 1 Abdelfettah Chakib 2 Abdallah Naya 1 Abderrahman Belhouari 3 Abdelouhab El Malki 4 Siham Bennani 4 1 Laboratoire de physiologie et génétique moléculaire, Faculté des Sciences, Université Hassan II Aïn Chock, Maârif, Casablanca, Maroc <oudghiri.m@menara.ma> <mouniaoudghiri@gmail.com> <m.oudghiri@fsac.ac.ma> 2 Hôpital Ibn Rochd, Casablanca, Maroc 3 Faculté des Sciences, Ben M Sik, Casablanca, Maroc 4 Institut Pasteur, Casablanca, Maroc Article received 7 January 2011, accepted 18 February 2011 Abstract. Human leukocyte antigen HLA-B51 is the most strongly associated gene with Behçet disease (BD) in different ethnic populations. We analyze the influence of HLA-B alleles in BD predisposition in Moroccan population and its association with clinical manifestations. The HLA-B phenotype frequencies were analyzed by serologic HLA class I typing and by polymerase chain reaction sequence-specific oligonucleotide (PCR-SSO) reverse dot blot hybridization in 120 unrelated Moroccan patients: all of whom fulfilled the international study group criteria for Behçet s disease, and in 112 ethnically matched healthy controls. Besides HLA-B*51 allele (20%), a significant increased frequency of the HLA-B*27 allele was found in Moroccans patients with Behçet s disease when compared to controls (13.3% of patients versus 2.7% of controls, chi square = 8.75, OR = 5.59, 95% IC [ ] and particularly in the patients who presented an anterior uveitis (25% vs. 5.5%, p < 0.005). Key words: association HLA, Behçet s Disease, HLA-B 27, Moroccan population, anterior uveitis Résumé. L implication du système HLA en particulier l allèle HLA-B51 dans le développement de la maladie de Behçet (MB) a été rapportée par différentes études et dans diverses ethnies. Pour analyser l influence du système HLA de classe I dans le développement de la MB dans la population marocaine et son association avec les manifestations cliniques, les fréquences alléliques ont été analysées dans un groupe de 120 patients marocains par biologie moléculaire (PCR-SSO). Tous les cas ont été colligés au Centre hospitalier universitaire Ibn Rochd, sélectionnés suivant les critères internationaux de la MB et comparés à un groupe de 112 individus contrôle de donneurs sains de même ethnie. L analyse statistique des résultats obtenus a montré, à côté de l expression significativement élevée du HLA-B51 (20 %), celle également de l allèle HLA-B27 (13,33 % des patients versus 2,67 % des contrôles, chi square = 8,75, OR = 5,59, 95% IC [1,58-19,75] et particulièrement chez les patients qui présentaient une uvéite antérieure (25 % vs 5,5 %, p < 0,005). Mots clés : association HLA-B27, maladie de Behçet, patients marocains, uvéite antérieure doi: /abc Behçet s disease (BD) is an inflammatory disorder of unknown cause, characterized by recurrent oral aphthous ulcers, genital ulcers, uveitis, and skin lesions [1]. A close association of the human leukocyte antigen HLA-B51 allele Reprints: M. Oudghiri with the disease suggests that genetic predisposition contributes to susceptibility to BD [2]. In addition, infections with agents such as herpes simplex virus [3, 4] and Streptococcus sanguis [5] have been implicated in the development of BD, although no specific infectious agent has been identified as its cause [6, 7]. To cite this article: Radouane A, Oudghiri M, Chakib A, Naya A, Belhouari A, El Malki A, Bennani S. HLA-B*27 allele associated to Behçet s disease and to anterior uveitis in Moroccan patients. Ann Biol Clin 2011; 69(4): doi: /abc

2 HLA-B51 association with BD has been described in several ethnic groups [8-18]. However, the relative risk of HLA-B51 positive individuals to develop the disease follow the distribution of B51 allele around the world, although there are some communities, i.e. Inuit or North American Indian, who have high levels of HLA-B*51 and no reported disease. In most cases, the highest levels of HLA-B*51 follow the trade routes as does BD [19]. Furthermore, among the several B51 alleles (B* ), the B*5101 is the most frequently observed [20-24]. In a systematic review and meta-analysis of case-control genetic association studies; they showed the strength of the association between BD and HLA-B51/B5 and its consistency across populations of various ethnicities, this lends further support to this allele being a primary and causal risk determinant for BD [25]. In Moroccan patients, only two studies reported the distribution of HLA alleles. In one of them, both alleles HLA-B*15 and HLA-B*51 have been found associated with BD with a frequency more important in women than in men [26]. Results were compared to a control group from the big Casablanca city. In the other study [27], HLA-B*51, HLA-B 58 and HLA-B 72 were been found the alleles associated with BD. The HLA-B*15 was not detected in this study where the control group was originated from a general Moroccan population without ethnic origin distinction. The Moroccan population comprises different ethnic groups (Arabic speaking from west Morocco; Berber speaking from the north of Morocco or Berber speaking from the south, Jewish communities...and so). It is characterized by a great ethnic diversity. Several reports have shown that HLA gene frequencies correlate with geographically related population [28-34]. Each population exhibits some specific variants and some uncommon alleles. The choice of the control group is very important because of a great ethnic diversity of the Moroccan population. In other populations the HLA B*27 [35] and HLA B*52 were the alleles that been found to be significantly increased in BD. In the present study, we have analyzed the HLA-B polymorphism in a group of 120 Moroccan BD patients, by comparison with 112 healthy controls [34] with the same ethnic origin, from the big Casablanca city. The association with the clinical manifestations and HLA-B expression was also investigated. Patients and methods Patients The study included one hundred twenty Moroccan patients, coming from the Ibn Rochd s Hospital and Pasteur institute of Casablanca, all of whom fulfilled the international study group criteria for Behçet s disease (ISG+) with oral ulcers and two other criteria (recurrent genital ulceration, eye lesions, skin lesions and positive pathergy test). There were 84 males (70%) and 36 females (30%). Age ranged from 14 to 53 years (mean: 31 years, sex ratio: 2.3). The control group of one hundred and twelve unrelated healthy Moroccan that was included in this study was a group of blood donors who volunteered for this study all of them were originated from the big Casablanca city and were of the same ethnic origin like the patients group. All subjects were informed about the objectives and methods of the study and gave their consent. HLA typing Peripheral blood lymphocytes were isolated by using Ficoll density gradient separation. Genomic DNA was isolated from peripheral EDTA anti-coagulated whole blood using the Wizard Genomic DNA Purification Kit (Qiagen, QIAamp DNA Midi and Blood Midi Handbook). Generic HLA-B typing was performed by polymerase chain reaction sequence-specific oligonucleotide (PCR-SSO) reverse dot blot hybridization using the Dynal RELI SSO HLA-B test kits (INNO LiPA HLAB Update plus). Statistical analysis Allelic frequencies were determined by direct counting of the individuals carrying the allele considered. Odds ratios (OR) were calculated according to the Woolf s formula, and were given with 95% confidence interval [CI of 95%]. The distribution of the alleles was compared between patients and controls using Chi-square ( 2 ) test. And the p values were calculated using Fisher s exact test. p value was considered significant if it was < Results Clinical characteristics of the 120 Moroccan BD patients included in this study are summarized in table 1. All patients presented oral ulcers. The other clinical symptoms were genital ulcers (80%), skin lesions (70%), Pathergy test in 60%, arthritis in 55%, uveitis in 42.5% (anterior in 40% and posterior in 2.5%), neurological involvement in 5%, cardiovascular and gastrointestinal in 2,5% of the patients. The frequency of posterior uveitis in these patients was low. No data are available on other Moroccan studies of BD to compare. HLA-B allele frequencies in patients and controls are shown in table 2. There was no difference in the frequency of HLA-B*15 allele between patients and controls; 6.66% vs. 6.25%, (OR = 1.07, 95% CI [ ], p = 0.89). 420 Ann Biol Clin, vol. 69, n 4, juillet-août 2011

3 HLA-B27 association with Behçet s disease Table 1. Patients clinical characteristics. Manifestation Frequency Males Females (%) Oral ulcers Genital ulcers Skin lesion Pathergy test Arthritis Anterior uveitis Posterior uveitis Neurological involvement Cardiovascular disease Gastrointestinal lesions The frequency of HLA-B*27 was significantly higher in BD patients compared to controls; 13.33% vs. 2.67%, (OR = 5.59, 95% CI [ ], p = 0.003). Only one patient, but none of controls were B*27 homozygote. The HLA- B*51 frequency was significantly higher in patients group 20% vs. 8.92%, in controls (OR = 2.55, 95% CI [ ], p = 0.017). All patients and controls were B*51 heterozygote. The distribution of HLA-B allele according to sex in patients (table 3) showed a significant increase in frequencies of HLA-B*27 and B*51 alleles (15.47% vs. 2.56% and 22.61% vs. 7.69% respectively) for men. Finally, HLA-B associations were analyzed according to the some clinical manifestations of the disease. The results are summarized in table 4. Table 2. HLA-B allele frequencies in Moroccan patients with Behçet s disease (BD). Alleles Patients with BD n = 120 (%) Healthy controls n = 112 (%) Chi square P Value OR 95% Confidence Interval B*05 1 (0.83) 3 (2.67) B*07 3 (2.5) 6 (5.35) B*08 6 (5) 0 (0.0) B*13 1 (0.83) 1 (0.89) B*14 4 (3.33) 5 (4.46) B*15 8 (6.66) 7 (6.25) B*16 1 (0.83) 3 (2.67) B*17 1 (0.83) 3 (2.67) B*18 1 (0.83) 5 (4.46) B*21 3 (2.5) 4 (3.57) B*22 0 (0) 1 (0.89) B*27 16 (13.33) 3 (2.67) B*35 3 (2.5) 7 (6.25) B*37 0 (0) 2 (1.87) B*38 1 (0.83) 4 (3.57) B*39 3 (2.5) 1 (0.89) B*40 0 (0) 1 (0.89) B*41 4 (3.33) 3 (2.67) B*42 3 (2.5) 1 (0.89) B*44 9 (7.5) 10 (8.92) B*45 1 (0.83) 4 (3.57) B*49 4 (3.33) 5 (4.46) B*50 9 (7.5) 4 (3.57) B*51 24 (20) 10 (8.92) B*52 3 (2.5) 3 (2.67) B*53 1 (0.83) 3 (2.67) B*57 1/0.83) 2 (1.87) B*58 3 (2.5) 2 (1.87) B*59 1 (0.83) 0 (0.00) B*72 0 (0) 1 (0.89) B*78 1 (0.83) 1 (0.89) (3.33) 7 (6.25) Ann Biol Clin, vol. 69, n 4, juillet-août

4 Table 3. Frequency of HLA B alleles in patients and controls according to sex. HLA Males Females Alleles Frequencies (%) Frequencies (%) Patients with BD Controls P OR Patients with BD Controls P OR n = 84 n = 78 (CI 95%) n = 36 n = 34 (CI 95%) B* B* BD: Behçet s disease; OR: odd ratio; CI: confidence interval; n: number of patient or control; p: considered significant if it was < The frequency of the patients expressing the HLA-B*27 allele with the anterior uveitis manifestation was significantly higher compared to the patients of the same group without this manifestation (25% vs. 5.55% p = 0.002). But we did not find any association between the HLA B*27 allele and skin lesions or arthritis. The HLA B*51 allele was not associated with the various manifestation studied. Discussion The genetic association between BD and the expression of HLA-B51 allele was described for the first time in 1982 by Ohno [2] in the Japanese population. The antigen HLA B*51 was present in 57% of patients versus 16% in the general population (p < 0.001). The association has been since confirmed in many other people of different ethnic and geographic groups (English, French, Italian, Greek, Turkish, Tunisian, Israeli, Iranian, Saudi, Kuwaiti, Chinese, Korean, Taiwanese and Mexican). The frequency of HLA B*51 varies from 13 to 79% in patients, and is two to three times higher than that observed in controls, regardless of the prevalence of this allele in the general population. The relative risk (defined by the odds ratios) of BD related to the presence of the B*51 allele varies from 3 to 15 [24] and OR ranges varies by geographic locations [25]. Our data showed that HLA B*51 allele expressed in 25% of Moroccan BD patients and confers a relatively low risk (OR = 2.55) compared to that observed in most ethnic groups studied. Whereas, in eastern Mediterranean populations and the external-east, the odd ratio is usually greater than 10. It is 11.7 in Saudi Arabian [17], 10.9 in Palestinian and Jordanian [11], 11.5 in Greeks [23], 18.2 in Israelis [12], and 16 in Italian [18]. Interestingly, the predisposing effect of HLA B*51 in Moroccans calculated for our study group was OR = This confirms what has been found in Moroccan studies [26, 27] and in patients who are known to be genetically related to the Moroccan population: Portuguese patients (OR = 2.38) [35], Tunisian patients with (OR = 2.77) [36] and in Spanish patients with (OR = 2.7) [14] More surprising is our observation of a predisposition effect of HLA-B*27 to BD. This allele has never been incriminated in susceptibility to BD in Moroccan population so far [26, 27]. We found 13.33% of patients expressing HLA-B*27 vs. 2.67% of control group. The association of HLA-B27 and BD was described for Portuguese patients [35]. A Turkish study showed the existence of a weak association of BD and HLA-B*2702 allele [37]. In the other hand; 75% (12/16) of patients expressed HLA-B*27+ have anterior uveitis manifestations. The uveitis and systemic disease have been reported to be associated with the HLA- B*27 expression [38]. In this study we found that the HLA-BD expression may be associated with gender differences. As previously reported [16, 26, 30, 38], our results confirm that the prevalence of HLA B*27 and B*51 alleles were more frequent in males. It was also reported that the HLAB27 associated systemic disease developed earlier in males than in females (31 vs. 37 years) [38]. Consequently, at onset of acute anterior uveitis, HLA-B27 associated systemic disease was more frequent in males than in females. However over time, it was observed that males and females were at equal risk of developing a HLA-B27 associated systemic disease [38] after the onset of acute anterior uveitis. These HLA-disease associations were also described in other diseases, such as polyarthritis rheumatoid, multiple sclerosis psoriasis and leukemia [39-41]. HLA-B*52 who is a second sub-division of HLA B*5 is not associated with the disease. HLA B*5101 and HLA B*52 differ only in two amino-acids (position 63 and 67), localized in the site of peptide binding at the level of a pocket which welcomes the major anchored residue of the peptide (residue P2). These positions are strongly involved in the specificity and the affinity of the binding, and could have an important role in the presentation of a pathogenic antigen [42]. 422 Ann Biol Clin, vol. 69, n 4, juillet-août 2011

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