Behçet s disease (BD) is a chronic inflammatory disorder

Association of MICA Gene and HLA-B*5101 with Behçet s Disease in Greece Kazuro Yabuki, 1,2 Nobuhisa Mizuki, 1,2 Masao Ota, 3 Yoshihiko Katsuyama, 3 Gerasimos Palimeris, 4 Caterina Stavropoulos, 5 Yvonni Koumantaki, 4 Marylin Spyropoulou, 5 Ernestini Giziaki, 4 Virginia Kaklamani, 6 Evangelia Kaklamani, 4 Hidetoshi Inoko, 2 and Shigeaki Ohno 1 PURPOSE. Behçet s disease (BD) is known to be associated with HLA-B51 in many different ethnic groups. Recently MICA, a member of a novel family of the human major histocompatibility complex (MHC) class I genes termed MIC (MHC class I chain-related genes), was identified near the HLA-B gene, and a triplet repeat microsatellite polymorphism was found in the transmembrane (TM) region. Because a strong association with BD of one particular MICA-TM allele, A6, was shown in a Japanese population, the present study was conducted to investigate microsatellite polymorphism in Greek patients with BD to know whether this association is generally observed in BD occurring in other populations. METHODS. Thirty-eight Greek patients with BD and 40 ethnically matched control subjects were examined for MICA microsatellite polymorphism using polymerase chain reaction (PCR) and subsequent automated fragment detection by fluorescent-based technology. RESULTS. Similar to the Japanese patients with BD, the phenotype frequency of the MICA-TM A6 allele was significantly increased in the Greek patients with BD (50.0% in control subjects versus 86.8% in BD cases), with an odds ratio (OR) of 6.60 (P 0.0012). The MICA-A6 allele was found in a high frequency both in males and females (weighted OR 6.68; P 0.0017). No association was found between the A6 allele and several disease features. A strong association exists between the MICA-TM A6 allele and the B*5101 allele in both the control subjects and patients with BD (weighted OR 44.39; P 0.0000023). CONCLUSIONS. This study revealed in Greek patients a strong association of BD with a particular MICA-TM allele, MICA-A6, providing insight into the molecular mechanism underlying the development of BD. (Invest Ophthalmol Vis Sci. 1999;40:1921 1926) Behçet s disease (BD) is a chronic inflammatory disorder with recurrent oral and genital ulcers, uveitis, and vasculitis, along with mucocutaneous, arthritic, and neurologic manifestations. 1 It exists worldwide but is found in a higher prevalence in Japan, China, and Korea and along the Silk Route to the countries of the Mediterranean. 2 We and others have presented evidence for an HLA association with BD, and From the 1 Department of Ophthalmology, Yokohama City University School of Medicine; the 2 Department of Genetic Information, Division of Molecular Life Science, Tokai University School of Medicine, Kanagawa; and the 3 Department of Legal Medicine, Shinshu University School of Medicine, Nagano, Japan; the 4 Department of Hygiene and Epidemiology, University of Athens Medical School; the 5 National Tissue Typing Center, George Gennimatas General Hospital, Athens, Greece; and the 6 Newton Wellesley Hospital, Department of Internal Medicine, Boston, Massachusetts. Supported by Grants-in-Aid 07041166 and 08457466 from the Ministry of Education, Science, Sports and Culture, Japan; a grant from the Ministry of Health and Welfare, Japan; and a research grant from Kanagawa Academy of Science and Technology. Submitted for publication November 24, 1998; revised February 22, 1999; accepted March 10, 1999. Proprietary interest category: N. Reprint requests: Shigeaki Ohno, Department of Ophthalmology, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawaku, Yokohama, Kanagawa 236-0004, Japan. HLA-B51 (HLA-B*51 at the DNA typing-defined allele level) was found to be the most strongly associated genetic marker in these populations. 2 6 However, it has not yet been clarified whether the HLA-B51 gene itself is the pathogenic gene related to BD or whether it is some other gene in linkage disequilibrium with HLA-B51. Although the cause and pathogenesis of BD are still uncertain, the onset of BD is believed to be triggered by the involvement of some external environmental factors in people with a particular genetic background. The mean age at onset is the third decade, children are rarely affected, and few neonatal cases have been reported. The main microscopic finding at most sites of active BD is immune-mediated occlusive vasculitis. At the cellular level, CD4 T cells are found in the perivascular inflammatory exudates, and Th1 cells respond to various stimuli to produce interleukin (IL)-2, interferon (IFN)-, and tumor necrosis factor- (TNF ). 7 In the recent studies, an increased number of T cells in the peripheral blood and the involved tissues, and the phenotypically distinct subset of T cells at the sites of inflammation were reported. 8 10 Furthermore, significant T-cell proliferative responses to mycobacterial 65-kDa heat shock protein peptides and their homologous peptides derived from the human 60-kDa heat shock protein were observed in patients with BD. 11,12 Therefore, BD is probably not a simple hereditary disease, and the onset of the Investigative Ophthalmology & Visual Science, August 1999, Vol. 40, No. 9 Copyright Association for Research in Vision and Ophthalmology 1921

1922 Yabuki et al. IOVS, August 1999, Vol. 40, No. 9 disease may be triggered by some exogenous antigen(s) such as bacteria, virus, or some microorganism. Recently, a highly divergent MHC class I chain-related gene family, MIC, was identified within the class I region. 13 Among the five MIC genes so far identified, two genes, MICA and MICB, are functional genes and are located between the HLA-B and TNF genes. The MICA gene located only 46-kb centromeric of HLA-B 14 is a highly polymorphic member of this family and is mainly expressed in epithelial cells, keratinocytes and monocytes 15,16 in contrast to MHC class I genes, which are almost ubiquitously expressed. The characteristics include the absence of association with 2 -microglobulin ( 2 M), stable expression without conventional class I peptide ligands, and the absence of a CD8 binding site. 16,17 Expression of MICA is not affected by type I and II interferons, 16,17 known to upregulate markedly the level of typical MHC class I gene expression. However, notably, the 5 -end flanking region of the gene for MICA includes putative heat shock elements similar to those of HSP70 genes, and MICA mrnas are augmented in heat shock stressed epithelial cells. 16,17 In the recent study, MICA molecule was found to be recognized by particular T cells expressing diverse V 1 T-cell receptors extracted from intestinal epithelium tumors. 18 It is suggested that MICA molecule may play an important role as a selfantigen, stress-induced, and may broadly regulate protective responses by V 1 T cells in the epithelium. 18 During nucleotide sequence analysis of the transmembrane (TM) region of the MICA gene, we found a triplet repeat microsatellite polymorphism of GCT (alanine). 19 Microsatellite polymorphism was investigated in Japanese patients with BD, and a strong association of six GCT repetitions (MICA-A6 allele) with BD was found. Thus, the MICA gene was considered a strong candidate gene controlling the susceptibility to BD based on its chromosomal localization, its predicted immunologic function as a ligand of V 1 T cells, its restricted and heat shock induced expression in epithelial cells, and a strong association of this particular MICA-TM allele, MICA-A6, with BD. In this study, to investigate whether there is the same MICA association with BD in different ethnic groups, microsatellite polymorphism in the MICA gene was analyzed in 38 Greek patients with BD and compared with that in 40 ethnically matched healthy control subjects. MATERIALS AND METHODS Subjects Patients and healthy control subjects included in this study were selected from subjects enrolled in a large ongoing case control study designed to investigate the risk factors in BD. Thirty-eight Greek patients with BD (27 men and 11 women), who fulfilled the ISG (International Study Group) diagnostic criteria for Behçet s disease 20 based on interview and clinical findings after a controlled protocol, were included, as well as 40 healthy control subjects (21 males, 19 females), unrelated to each other or to the patients and matched to the patients in ethnic origin and age 5 years. Only white people of Greek ancestry were accepted as either patients or control subjects. Patients with BD were seen as outpatients for a period of 1 year, in an outpatient rheumatology clinic in the Athens metropolitan area and were clinically examined by an experienced rheumatologist and ophthalmologist. The characteristics of the TABLE 1. BD Patient Characteristics Sex Male 27 71 Female 11 29 Age at onset 11 20 18.4 21 30 44.8 31 40 26.3 40 10.5 Clinical Data Uveitis Positive 76 Oral ulcers Positive 100 Genital ulcers Positive 74 Erythema nodosum Positive 49 Vasculitis Positive 49 Arthritis of peripheral joints Positive 46 Central nervous system involvement Positive 30 Pathergy skin test Positive 38 patients with BD are presented in Table 1. The patients ages ranged from 20 to 59 years (mean, 37.5 10.6 years). The age of the control subjects ranged from 19 to 68 years (mean, 36.5 12.6 years). All patients and control subjects agreed to a blood examination conducted according to the guidelines of the Declaration of Helsinki. Serologic HLA Class I Typing Serologic HLA class I typing was performed using peripheral blood lymphocytes by a standard microlymphocytotoxicity technique. 21 HLA-DNA typing for the B5 group was performed in all B51 patients and control subjects by the polymerase chain reaction sequence specific oligonucleotide (PCR-SSO) method, using the unique B5 substitutions at nucleotide position 92 in exon 3. Two PCR-amplification and seven specific probes were adopted to identify the B*5101, B*5102, B*5103, and B*5104 alleles. All primers and SSOs were provided by the 12th International Histocompatibility Workshop. 22 Analysis of Triplet Repeat Polymorphism in the Transmembrane Region of the MICA Gene For analysis of microsatellite repeat polymorphism in the TM region of the MICA gene, PCR primers flanking the TM region were designed. 19 The forward primer was labeled at the 5 end with 6-FAM (PE Biosystems, Foster City, CA), and PCR was performed according to a protocol described before. 19 To determine the number of triplet repeats in the TM region of the MICA gene, the amplified products were denatured for 5 minutes at 100 C, mixed with formamide containing a stop buffer, and electrophoresed on 6% polyacrylamide gels containing 8 M urea in an automated DNA sequencer (model 373A; PE Biosystems). The number of microsatellite repeats was estimated automatically using software (Genescan 672; PE Biosystems) and the local Southern method with a size marker of 350 TAMRA (PE Biosystems) as well as the PCR products of the B-cell lines used as standard size markers that had been determined for the triplet repeat polymorphisms by nucleotidesequence determination, as described before. 19 Statistical Analysis MICA gene and phenotype frequencies were estimated by direct counting. Statistical analysis was performed by the Man- %

IOVS, August 1999, Vol. 40, No. 9 MICA Gene and Behçet s Disease in Greek Patients 1923 TABLE 2. Gene Frequencies of Microsatellite Polymorphism in the Transmembrane Region (Exon 5) of the MICA Gene in BD Patients and Control Subjects Microsatellite Allele Amplified Product (bp) Controls (%) (n 80) Patients (%) (n 76) OR P Pc A4 179 14 (17.5) 5 (6.6) A5 182 8 (10.0) 7 (9.2) A5.1 183 22 (27.5) 10 (13.2) A6 185 23 (28.8) 49 (64.5) 4.50 0.000016 0.000080 A9 194 13 (16.3) 5 (6.6) tel Haenszel one-tailed and two-tailed tests. 23 The significance of the distribution of alleles between the patients with BD and normal control subjects was tested by the 2 method with the continuity correction and Fisher s exact probability test. Furthermore, P was corrected by multiplication by the number of microsatellite alleles or phenotypes (corrected P: Pc). If a cell frequency was zero, the odds ratio (OR) was calculated by first adding 0.5 to each cell frequency. 24 To control for the effect of certain factors, the Mantel Haenszel weighted OR was calculated. 23 RESULTS The gene frequencies of the microsatellite polymorphism in the TM region (exon 5) of the MICA gene are shown in Table 2. All five distinct alleles were found in the control subjects and patients with BD. The MICA-A6 allele was found at a significantly higher frequency among the patients with BD (28.8% in controls versus 64.5% in BD cases; OR 4.50; P 0.000016; Pc 0.000080). All the other alleles were found at a lower frequency (OR 1). Sixteen of the patients with BD (42.1%) and three of the control subjects (7.5%) were homozygotes for the A6 allele (OR 8.97; P 0.00046; Pc 0.0065; Table 3). The phenotype frequency of the MICA-A5 and -A6 heterozygote was relatively increased in the patients with BD, but in the other heterozygotes, carrying MICA-A6 on one chromosome was not common. Table 4 shows the phenotype frequencies of the microsatellite polymorphism in the TM region (exon 5) of the MICA gene. Of 38 patients with BD, 33 had the MICA-A6 allele in a homozygous or heterozygous way (86.8%), whereas 20 of 40 healthy control subjects had the A6 allele (50.0%). Thus, the MICA-A6 allele was found to be strongly associated with BD in this Greek sample (OR 6.60; P 0.0012; Pc 0.0059). The MICA-A6 allele was observed at a high frequency both in men (OR 5.23; P 0.015) and women (OR 11.1; P 0.020). Stratified analysis was used for the confounding effect of sex, with an OR of 6.68 (P 0.0017; Table 5). No association was found between the MICA-A6 allele and several disease features including uveitis and erythema nodosum, in which the ORs were 6.90 and 3.19, respectively, but were not statistically significant (data not shown). As is summarized in Table 6, the HLA-B*5101 allele was found in nine controls (22.5%) and in 30 BD cases (81.1%), whereas the MICA-A6 allele was found in 20 controls (50.0%) and in 33 BD cases (86.8%). Statistical analysis of these results revealed that the association of MICA-A6 with BD produced an OR of 6.60 (P 0.0012; 95% CI 1.92 24.24), whereas B*5101 produced an OR of 14.76 (P 0.00000028; 95% CI 4.33 to 53.28). As expected given the location of MICA, there was a strong linkage disequilibrium between the HLA-B antigens and TABLE 3. Genotypes of Microsatellite Polymorphism in the Transmembrane Region (Exon 5) of the MICA Gene in BD Patients and Control Subjects Microsatellite Allele Controls (%) (n 40) Patients (%) (n 38) OR P Pc A4-A4 1 (2.5) 0 (0.0) A4-A5.1 2 (5.0) 0 (0.0) A4-A6 6 (15.0) 3 (7.9) A4-A9 4 (10.0) 2 (5.3) A5-A5 0 (0.0) 0 (0.0) A5-A5.1 5 (12.5) 1 (2.6) A5-A6 1 (2.5) 6 (15.8) 7.31 0.054 NS A5-A9 2 (5.0) 0 (0.0) A5.1-A5.1 4 (10.0) 1 (2.6) A5.1-A6 7 (17.5) 6 (15.8) A5.1-A9 0 (0.0) 1 (2.6) A6-A6 3 (7.5) 16 (42.1) 8.97 0.00046 0.0065 A6-A9 3 (7.5) 2 (5.3) A9-A9 2 (5.0) 0 (0.0) NS, not significant.

1924 Yabuki et al. IOVS, August 1999, Vol. 40, No. 9 TABLE 4. Phenotype Frequencies of Microsatellite Alleles in the Transmembrane Region (Exon 5) of the MICA Gene in BD patients and Control Subjects Microsatellite Allele Amplified Product (bp) Controls (%) (n 40) Patients (%) (n 38) OR P Pc A4 179 13 (32.5) 5 (13.2) A5 182 8 (20.0) 7 (18.4) A5.1 183 18 (45.0) 9 (23.7) A6 185 20 (50.0) 33 (86.8) 6.60 0.0012 0.0059 A9 194 11 (27.5) 5 (13.2) the MICA microsatellite alleles in the Greek population. Notably, a strong association exists between B*5101 and MICA-A6 in both the control subjects and BD cases (weighted OR 44.39; P 0.0000023; Table 7). All the 30 patients with B*5101 also possess MICA-A6. Our examinations thus far have shown that B*5101 is completely linked to MICA-A6 even in the control subjects of any ethnic origin. In comparison, MICA-A6 is linked not only to B*5101 but also to other HLA-B alleles, including B44 and B52 (data not shown). To elucidate which one is the real pathogenic gene related to BD, HLA-B*5101 or MICA-A6, an association of MICA-A6 with BD stratified for the possible confounding effect of B*5101 was analyzed. However, no association was observed in this analysis (data not shown). In contrast, when an association of B*5101 with BD stratified for the possible confounding effect of A6 was analyzed, a statistically significant association of B*5101 was observed (weighted OR 12.22; P 0.00090; 95% CI 2.3 72.01; Table 8). DISCUSSION TABLE 5. Association of MICA-A6 and BD, Controlling for Sex MICA-A6 Male BD Female Controls BD Cases Controls BD Cases Present 11 23 9 10 Absent 10 4 10 1 OR 5.23 11.11 P 0.015 0.020 95% CI 1.13 25.94 1.03 281 Weighted OR 6.68; P 0.0017; 95% CI 1.88 24.55. In this study, we have investigated microsatellite polymorphism in the TM region of the MICA gene in the Greek patients with BD. As a result, similar to the Japanese patients with BD, one particular MICA-TM allele, MICA-A6, was found to be strongly associated with BD in the Greek sample. Thirty-three of 38 patients (86.8%) were homozygous or heterozygous for MICA-A6 (Table 3), and the MICA-A6 allele was found to be distinctly predominant in the patient group. This MICA-A6 association was widely observed in the Greek patients with BD, regardless of sex and several BD clinical features. Therefore, a specific haplotype(s) surely exists in patients with BD that differs from those in healthy control subjects, and the pathogenic gene related to BD is the MICA gene itself or another gene located very near the MICA gene, including HLA-B or its nearby NOB genes. 14 In a previous study we have presented evidence that the BD pathogenic gene is located in the 230-kb segment between the MICB and HLA-C genes. 25,26 The MICA gene resides in proximity to the HLA-B gene, only 46 kb from HLA-B, and there was a strong linkage disequilibrium between them. Namely, a strong linkage between the MICA-A6 and HLA- B*5101 alleles was observed in both patients and healthy control subjects (Table 7). Thus, the HLA-B*510 MICA-A6 haplotype is predominant in the BD patient group and the pathogenic gene responsible for the predisposition to BD should be located on this haplotype. However, the MICA-A6 allele is associated with not only HLA-B51 but also HLA-B44 and -B52, which were not increased in the patient group to any degree. If MICA-A6 is primarily involved in the pathogenesis of BD, the frequencies of the HLA-B44 and -B52 antigens should be higher, along with the increase of this MICA-A6 allele. Further, stratification analysis of the MICA-A6 patients with BD on the possible confounding effect of HLA-B*5101 and vice versa (Table 8) suggests that the significant increase of MICA-A6 in the patient group could be explained by linkage disequilibrium with HLA-B*5101 and that HLA-B*5101 is a primary susceptible locus for BD. The presence of HLA- B*5101 negative patients with BD can be explained by the influence of other genetic factor(s) and/or of various external environmental or infectious agent(s). However, the possibility of the primary involvement of the MICA gene in the development of BD cannot be fully excluded. The MICA-TM (MICA-A4, -A5, -A5.1, -A6, and -A9) alleles are defined by the number of the microsatellite repeats TABLE 6. Association of BD with MICA-A6 and B*5101 Controls (%) BD Cases (%) MICA-A6* Present 20 (50.0) 33 (86.8) Absent 20 (50.0) 5 (13.2) B*5101 Present 9 (22.5) 30 (81.1) Absent 31 (77.5) 7 (8.9) *OR 6.60; P 0.0012; 95% CI 1.92 24.24. OR 14.76; P 0.00000028; 95% CI 4.33 53.28.

IOVS, August 1999, Vol. 40, No. 9 MICA Gene and Behçet s Disease in Greek Patients 1925 in the TM region of the MICA gene, and MICA-A6 may not represent a unique MICA allele related to functional significance. In fact, four MICA alleles defined by genetic polymorphism in exons 2, 3, and 4, MICA003, MICA004, MICA006, and MICA009, share the same MICA-TM allele, MICA-A6, as a result of tight linkage. 27 One of these four MICA alleles sharing MICA-A6 presumably has a strong association with HLA-B51 and is possibly the real pathogenic gene for BD. In this respect, precise DNA typing in the extracellular domains (exons 2, 3, and 4) of the MICA gene is necessary and is now under investigation in our laboratory. Although V 1 T cells are of unknown function and no antigens recognized by them have been identified, they are believed to recognize self-antigens that may be stress-induced. The MICA molecule that is responsive to heat shock cell-stress was found to be recognized by V 1 T cells. Thus, two hypotheses can be set forth regarding the primary involvement of the MICA molecule with BD. First, after some bacterial infection, local immune response may be induced at the sites of infection, resulting in production of cytokines followed by stress-induced expression of MICA. Among many MICA alleles, MICA-A6 may tend to activate V 1 T cells more effectively through specific interaction with T cells, because of the presence of specific amino acids in the a1/a2 domains linked to MICA-A6 or because of a particular V 1 T-cell repertoire that can recognize MICA molecule with MICA-A6 in an efficient way, thus leading to the onset of BD. Second, after bacterial infection some bacterial components may have a specific role similar to that of superantigens in activation of the MICA molecule. In this model, a bacterial component may have specifically bound to MICA molecules with the MICA-A6 allele, induced its expression, and thus may have increased the MICA-A6 molecules that could activate V 1 T cells, triggering the unusual immune response responsible for the development of BD. In conclusion, we have investigated microsatellite polymorphism in the TM region of the MICA gene in a Greek sample and have found a strong association of a particular MICA-TM allele, MICA-A6, with BD. However, it is still uncertain which is the real pathogenic gene responsible for the development of BD, MICA-A6 or HLA-B*5101. In this respect, we have determined the genomic sequence covering the entire 1.8-Mb HLA class I region from the MICB gene to the HLA-F gene (including the MICB, MICA, HLA-B, HLA-C, HLA-E, HLA-A, HLA-G, and HLA-F genes) and have identified more than 700 microsatellite repeats in this region. It is necessary to TABLE 7. Association of MICA-A6 and B*5101, Controlling for Disease Status MICA-A6 Controls B*5101 BD Cases Present Absent Present Absent Present 9 11 30 3 Absent 0 20 0 4 OR 33.86 78.42 P 0.00061 0.00053 95% CI 1.06 633 3.47 1768.7 Weighted OR 44.39; P 0.0000023. TABLE 8. Association of B*5101 with BD Stratified for the Effect of MICA-A6 B*5101 analyze repeat polymorphisms at these microsatellite loci in patients with BD to determine precise localization of the pathogenic gene related to BD. Acknowledgments The authors thank Phaedon G. Kaklamanis for allowing us to use his patients and the patients who took part in this study for their cooperation. References Absence of MICA-A6 Presence of MICA-A6 Controls BD Cases Controls BD Cases Present 0 0 9 30 Absent 20 4 11 3 OR 4.55 12.22 P NS 0.00080 95% CI 0.09 261.6 2.37 71.99 Weighted OR 12.22; P 0.00090; 95% CI 2.36 72.01. NS, not significant. 1. Kaklamani VG, Vaiopoulos G, Kaklamanis PG. Behçet s disease. 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