Association of MICA polymorphism with rheumatoid arthritis patients in Koreans

BRIEF COMMUNICATION

Association of MICA Polymorphism With Rheumatoid Arthritis Patients in Koreans Jee Won Mok, Yun Jong Lee, Jeong Yeon Kim, Eun Bong Lee, Yeong Wook Song, Myoung Hee Park, and Kyung Sook Park ABSTRACT: To investigate whether genetic variations of MICA are associated with susceptibility to rheumatoid arthritis (RA), the (GCT)n microsatellite polymorphism of the transmembrane domain was analyzed in 144 Korean patients with RA and in 297 unrelated healthy controls. The allele frequency of MICA*A9 significantly decreased in RA patients compared with controls (9.0% vs. 15.3%, odds ratio [OR] ⫽ 0.55, p ⫽ 0.0098, pc ⫽ 0.049), whereas the frequency of the MICA*A4 and MICA*A5.1 alleles tended to increase in RA patients (21.2% vs. 14.8%, OR ⫽ 1.55, p ⫽ 0.018, pc ⬎ 0.05; 20.5% vs. 15.0%, OR ⫽ 1.46, p ⫽ 0.0403, pc ⬎ 0.05). By subgroup analysis, the MICA*A4 allele significantly increased in seropositive RA patients versus controls (23.0% vs. 14.8%, OR ⫽ 1.69, p ⫽ 0.0082, pc ⫽ 0.041). ABBREVIATIONS RA rheumatoid arthritis HLA human leukocyte antigen MICA major histocompatibility complex class I

INTRODUCTION Rheumatoid arthritis (RA) is a chronic inflammatory joint disorder and is considered a multigenetic disorder with many genetic polymorphisms that contribute to the disease pathogenesis. It is known that almost all racial ethnic groups exhibit associations between the human leukocyte antigen DRB1 (HLA-DRB1) shared epitope From the Department of Biology (J.W.M, K.S.P.), Sungshin Women’s University, Seoul, Korea; and Departments of Internal Medicine (Y.J.L., J.Y.K., E.B.L, Y.W.S.) and Laboratory Medicine (M.H.P.), Medical Research Center, Seoul National University College of Medicine, Seoul, Korea. Address reprint requests to: Dr. Yeong Wook Song, Department of Internal Medicine, Seoul National University Hospital, 28 Yongun-dong, Chongno-gu, Seoul, Korea, 110-744; Tel: ⫹82 (2) 760-2234; Fax: ⫹82 (2) 762-9662; E-mail: [email protected]. Received April 16, 2003; revised September 3, 2003; accepted September 4, 2003. Human Immunology 64, 1190 –1194 (2003) © American Society for Histocompatibility and Immunogenetics, 2003 Published by Elsevier Inc.

HLA-DRB1*0405 was strongly associated with RA (pc ⫽ 0.0000008), and strong linkage disequilibrium was observed between HLA-DRB1*0405 and MICA*A4 alleles in controls (pc ⫽ 0.000004) as well as in RA patients (pc ⫽ 0.0012). In Korean patients, HLA-DRB1*0405 was primarily associated with RA and the weak association of RA with MICA*A4 was secondary to that with HLADRB1*0405. Additionally, MICA*A9 might have a weak protective effect on the susceptibility to RA in Koreans. Human Immunology 64, 1190 –1194 (2003). © American Society for Histocompatibility and Immunogenetics, 2003. Published by Elsevier Inc. KEYWORDS: MICA; polymorphism; rheumatoid arthritis

IDDM

chain-related gene A insulin-dependent diabetes mellitus

QKRAA or QRRAA and RA. Some studies have also suggested a role for non-HLA genes in RA pathogenesis, for example, tumor necrosis factor (TNF), T-cell receptor, interleukin-1 (IL-1) promoter, IL-3, IL-4, and the IL-10 receptor genes [1]. Major histocompatibility complex (MHC) class I chain-related gene A (MICA) is a cell stress-induced glycoprotein, which is expressed in fibroblast, epithelial cell lines, gastrointestinal epithelium, freshly isolated keratinocytes, endothelial cells, and monocytes. Although the role of MICA is not entirely clear, its localization in the human leukocyte antigen region and its reaction with ␥␦ T cells and natural killer cells suggest the association with autoimmune disease [2]. The (GCT)n microsatellite polymorphisms in the transmembrane region of MICA consists of four (A4), five (A5, 0198-8859/03/$–see front matter doi:10.1016/j.humimm.2003.09.010

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TABLE 1 Allele and genotype frequencies of the MICA polymorphism in RA patients and controls Control (n ⫽ 297)

RA (n ⫽ 144)

Odds ratio [95% CI]

p Value

pc Value

88 (14.8%) 192 (32.3%) 89 (15.0%) 134 (22.6%) 91 (15.3%)

61 (21.2%) 86 (29.9%) 59 (20.5%) 56 (19.4%) 26 (9.0%)

1.55 [1.06–2.25] — 1.46 [1.0–2.14] — 0.55 [0.34–0.89]

0.018 NS 0.0403 NS 0.0098

NS NS NS NS 0.049

4 (1.3%) 80 (26.9%) 213 (71.7%)

5 (3.5%) 51 (35.4%) 88 (61.1%)

— — 0.62 [0.40–0.96]

NS NS 0.0248

NS NS NS

A5.1/A5.1 A5.1/Yb Y/Y

10 (3.4%) 69 (23.2%) 218 (73.4%)

12 (8.3%) 24 (16.7%) 97 (67.4%)

2.61 [1.02–6.71] — —

0.0247 NS NS

NS NS NS

A9/A9 A9/Zc Z/Z

11 (3.7%) 69 (23.2%) 217 (73.1%)

1 (0.7%) 24 (16.7%) 119 (82.6%)

— — 1.76 [1.03–2.99]

NS NS 0.0268

NS NS NS

Allele A4† A5 A5.1 A6 A9 Genotype A4/A4 A4/Xa X/X

Abbreviations: CI ⫽ confidence interval; NS ⫽ not significant; pc ⫽ probability value by Bonferroni correction; RA ⫽ rheumatoid arthritis. † Control versus seropositive RA: 14.8% vs. 22.7%, p ⫽ 0.0082, pc ⫽ 0.041, CI ⫽ 1.69 [1.12–2.54]. a Allele other than A4; b allele other than A5.1; c allele other than A9.

A5.1), six (A6), and nine (A9) triplet GCT repeats, which have been reported to be associated with many autoimmune diseases, including several arthritis syndromes (such as, spondylitis, Behcet’s disease, psoriatic arthritis, and juvenile idiopathic arthritis) [3– 8]. In addition, Singal et al. and Martinez et al. recently demonstrated that the MICA allele 6 might be a protective genetic marker in Caucasian RA patients [9, 10]. Therefore, we evaluated whether the (GCT)n microsatellite polymorphisms of MICA confer susceptibility to RA in Korean patients. MATERIALS AND METHODS One hundred forty-four RA patients (15 males, and 129 females) were enrolled at the Rheumatology Clinic, Seoul National University Hospital. Rheumatoid arthritis was diagnosed according to the criteria proposed by the American College of Rheumatology [11]. The mean age at onset was 40.7 years old (⫾ 14.7 years standard deviation [SD]; range, 12–70 years old) and the duration of disease was 8.01 ⫾ 9.1 years (range, 0.1– 8.1 years). One hundred eight patients were seropositive (75.0%) and 91 patients (63.2%) had joint erosions at the first visit. The control group consisted of 297 blood donors with the same ethnic background. DNA samples were extracted from peripheral mononuclear cells using the QIAamp blood kit (Qiagen, Va-

lencia, CA, USA). Polymerase chain reaction (PCR) primers for exon 5 containing the polymorphic site were designed as reported previously [7]. PCR amplification was performed using 200-ng DNA, 10-pmol of each primer, 0.2-mM dNTPs, 1.5-mM MgCl2, 50-mM TrisHCl, and 1.0-U Taq DNA polymerase (Bioneer, Seoul, Korea). Amplified products were mixed with denaturing solution (95% formamide, 20-mM EDTA, 0.05% bromophenol blue) and heated at 95 °C for 15 minutes. The denatured mixtures were electrophoresed on 6% polyacrylamide gel with 8-M urea and stained with silver. Representative samples were confirmed using ABI310 DNA sequencer (PE Applied Biosystem, Foster City, CA, USA). To analyze the association of MICA and HLA-DR4 with RA, presence of HLA-DRB1*04 alleles was also tested. After amplification of DRB1*04 alleles with group-specific primers, single strand conformation polymorphism (SSCP) analysis was performed according to the method described previously [12]. DNA samples for HLA-DRB1 typing were available in the whole RA patients and 156 controls. Comparisons of the allele and genotype frequencies of patients and controls were performed using the chisquare test or Fisher’s exact test, where applicable. Probability value (p value) was corrected (pc) by the number of comparisons made and pc was considered significant when ⱕ 0.05.

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TABLE 2 Distribution of HLA-DRB 1 alleles in study patients

DRB1*04 (⫹) DRB1*04 alleles frequency *0401 *0403 *0404 *0405 *0406 *0407 *0408 *0410

Control (n ⫽ 156)

RA (n ⫽ 144)

p or pc Value

52 (33.3%)

85 (59.0%)

p ⫽ 0.000008

2 (0.6%) 12 (3.8%) 4 (1.3%) 49 (15.7%) 25 (8.0%) 2 (0.6%) 2 (0.6%) 8 (2.6%)

2 (0.7%) 21 (7.3%) 7 (2.4%) 26 (43.8%) 7 (2.4%) 0 (0.0%) 3 (1.0%) 4 (1.4%)

NS NS NS pc ⫽ 0.0000008 NS NS NS NS

Association with MICA allele

MICA*A4†

pc ⫽ 0.000004 in control and pc ⫽ 0.0012 in RA patients. Abbreviations: HLA ⫽ human leukocyte antigen; MICA ⫽ major histocompatibility complex class I chain-related gene A; NS ⫽ not significant; pc ⫽ probability value by Bonferroni correction; RA ⫽ rheumatoid arthritis. †

RESULTS All alleles and genotypes were observed in the RA and control groups. Table 1 illustrates the distribution of (GCT)n microsatellite polymorphisms in both groups. When compared with the controls, MICA*A9 was significantly lower in RA patients in terms of allele frequency (9.0% vs. 15.3%, odds ratio [OR] ⫽ 0.55; 95% confidence interval [CI] ⫽ 0.34 – 0.89, pc ⫽ 0.049). The MICA*A4 and MICA*A5.1 alleles tended to be higher in RA patients, but this were not statistically significant (21.2% vs. 14.8%, OR ⫽ 1.55, 95% CI ⫽ 1.06-2.25, p ⫽ 0.018, pc ⬎ 0.05; 20.5% vs. 15.0%, OR ⫽ 1.46, 95% CI ⫽ 0.99 –2.14, p ⫽ 0.403, pc ⬎ 0.05). When different MICA genotypes were analyzed in the RA and control groups, MICA*A5.1/5.1 homozygosity was insignificantly elevated in RA patients (8.3% vs. 3.4%, OR ⫽ 2.61, 95% CI ⫽ 1.02– 6.71, p ⫽ 0.0247, pc ⬎ 0.05). By subgroup analysis, the MICA*A4 allele was significantly higher in seropositive RA patients (23.0% vs. 14.8%, OR ⫽ 1.69, 95% CI ⫽ 1.12–2.54, p ⫽ 0.0082, pc ⫽

0.041) than in controls. However, other subgroup analyses according to sex, joint erosion, or age at onset revealed no significant differences. In HLA-DRB1 analysis, RA was significantly associated with HLADRB1*04 and HLA-DRB1*0405 (p ⫽ 0.000008 and pc ⫽ 0.0000008, respectively; Table 2), which was consistent with a previous report about the association between HLA-DRB1 and Korean RA [13]. When linkage disequilibrium between HLA-DRB1*04 and MICA genes was analyzed, strong linkage disequilibrium was observed between HLA-DRB1*0405 and MICA*A4 in control (pc ⫽ 0.000004) as well as RA patients (pc ⫽ 0.0012). In seropositive patients, HLADRB1*0405 was significantly associated with MICA*A4 (pc ⫽ 0.004). However, MICA*A9 allele did not characterize an association with any HLA-DRB1*04 allele or shared epitopes (HLA-DRB1*0405, 0404, and 0401). To identify which of MICA and HLA-DRB1 indicated stronger association with RA, Svejgaard and Ry-

TABLE 3 Association of HLA-DRB1*0405 and MICA*A4 alleles with RA

A4 (⫹)

0405 (⫹)

Control (n ⫽ 156)

RA (n ⫽ 144)

19

37

p Value

Seropositive RA (n ⫽ 108) 32

0.0165 A4 (⫺)

0405 (⫺) 0405 (⫹)

26 7

19 31

0405 (⫹)

0405 (⫺) A4 (⫹)

104 19

57 37

0405 (⫺)

A4 (⫺) A4 (⫹)

7 26

31 19

A4 (⫺)

104

57

p Value 0.0057

13 21 0.0000003

0.0000031 42 32

0.0990

0.2675 21 13

0.0420

0.5794 42

Abbreviations: HLA ⫽ human leukocyte antigen; MICA ⫽ major histocompatibility complex class I chain-related gene A; RA ⫽ rheumatoid arthritis.

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der’s analysis was performed [14]. HLA-DRB1*0405 was primarily associated with RA, and the weak association of RA with MICA*A4 was secondary to that with HLA-DRB1*0405 (Table 3).

DRB1*0405 in the Korean population. But, to firmly establish the relationship between the MICA polymorphism and RA, further large-scale studies of RA patients are required in other populations.

DISCUSSION Although the function of MIC protein remains unconfirmed, genetic variations in MICA have been suggested to influence its structure and function. The A5.1 allele contains an additional nucleotide insertion (GGCT/ AGCC), which causes a frameshift mutation resulting in the premature termination of the transmembrane region [5]. Moreover, the affinity of MICA for NKG2D was found to be influenced by allelic differences in MICA [15]. From this point of view, MICA gene polymorphisms may affect the susceptibility of individuals to disease. A number of studies have attempted to evaluate associations between allelic variations of MICA and autoimmune diseases in different ethnic groups. And diseases such as ankylosing spondylitis, Behcet’s disease, psoriasis, insulin-dependent diabetes mellitus (IDDM), Addison’s disease, primary sclerosing cholangitis, Kawasaki disease, ulcerative colitis, and celiac disease have been reported to be either negatively or positively associated with the MICA gene [3– 6, 16 –21]. In the present study, the MICA*A9 allele was associated with RA and the MICA*A4 related to seropositive RA. It has been reported that MICA*A6 might confer protection against RA development in Caucasians [9, 10]. However, the frequency of MICA*A6 allele was not found to differ between RA patients and controls in our study. Rather the MICA*A9 allele was found to be negatively associated with RA and none of the HLADRB1 alleles were significantly associated with MICA*A9 in our study patients. It has previously been reported that the MICA*A9 allele might confer protection from IDDM and celiac disease in the Spanish population [22, 23]. We found that the frequency of MICA*A4 allele was elevated in seropositive RA patients. MICA*A4 has previously been demonstrated to be associated with many autoimmune diseases, such as, ankylosing spondylitis [3, 4], IDDM [17], celiac disease [22], HLA-B27 associated uveitis [24], and juvenile idiopathic arthritis [8]. However, in the case of ankylosing spondylitis, linkage disequilibrium with HLA-B27 and MICA was reported [25]. Our study indicated that MICA*A4 allele was significantly associated with HLADRB1*0405, and the association of RA with HLADRB1*0405 was mainly contributed to that with MICA*A4. The present study revealed a negative association between the MICA*A9 allele and RA, and linkage disequilibrium between MICA*A4 allele and HLA-

ACKNOWLEDGMENTS

This study was partly supported by a grant from Seoul National University.

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