HLA-B60 and B61 are strongly associated with ankylosing spondylitis in HLA-B27-negative Taiwan Chinese patients
J. C. C. Wei,
W. C. Tsai1,
H. S. Lin2,
C. Y. Tsai3 and
C. T. Chou3
Division of Allergy, Immunology and Rheumatology, Department of Internal Medicine and Institute of Medicine, Chung Shan Medical University, Taichung, 1 Department of Medicine, Kaohsiung Medical University Hospital, 2 Department of Nursing Management, Fooyin University, Kaohsiung and 3 Division of Allergy-Immunology-Rheumatology, Veterans General Hospital, Taipei, Taiwan.
Correspondence to: C. T. Chou, Division of Allergy-Immunology-Rheumatology, Veterans General Hospital-Taipei, No. 201, Sec. 2, Shpai Road, Beitou Chiu, Taipei, Taiwan 112. E-mail: ctchou{at}vghtpe.gov.tw
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Abstract
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Objectives. Carriage of HLA-B60 has been shown to increase the risk of ankylosing spondylitis (AS) in B27-positive Caucasian patients, but the association in B27-negative cases is less certain. This study assessed HLA class I gene associations in Chinese HLA-B27-negative AS patients.
Methods. Forty-one Chinese HLA-B27-negative AS patients fulfilling the modified New York diagnostic criteria for AS were recruited, and 11 383 HLA-B27-negative blood donors were used for comparison. HLA-A and -B typing was done with the microlymphocytotoxicity assay.
Results. Among the B27-negative AS patients, 21 were male and 20 were female. Of HLA-B alleles, only B60 and B61 significantly increased susceptibility to AS in HLA-B27-negative patients (P<0.001).
Conclusions. In Taiwan Chinese, carriage of B60 is increased in HLA-B27-negative AS patients. The association between B61 and HLA-B27-negative AS patients has not been reported previously. Whether the gene involved is HLA-B60 or B61 or another gene in linkage disequilibrium with these genes is unknown.
KEY WORDS: Ankylosing spondylitis, HLA-B27-negative, HLA-class I genes
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Introduction
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Ankylosing spondylitis (AS) is a seronegative spondyloarthropathy (SpA) that mainly affects the vertebrae and the peripheral joints. In 1973, the genetic marker HLA-B27 was found to play a key role in the occurrence of this disease [1, 2]. Previous epidemiological research has found that AS predominantly occurs in Caucasians and a small number of Native American tribes [3, 4]. The incidence of AS in Japan and Africa is considerably lower [57], probably because of the lower prevalence of HLA-B27. The frequency of B27 in the Chinese population is 49% [4, 7], while the prevalence of AS is about 0.20.3% (similar to that in Caucasians) [8, 9]. Although HLA-B27 is an important genetic marker, AS can occur in a small population with HLA-B27-negative individuals [4, 7].
There are few data regarding the involvement of the MHC in HLA-B27-negative AS. Early studies by Khan et al. showed that B7 was associated with HLA-B27-negative African-American AS patients and B16 (currently B38 and B39) was associated with HLA-B27-negative Caucasian AS patients [10, 11]. Yamaguchi demonstrated the association of B39 with HLA-B27-negative Japanese AS patients [12].
Several studies have suggested that HLA-B60 increases the risk of developing AS in both B27-positive and -negative individuals [1315]. Certain HLA-DR alleles are also thought to influence the phenotype of AS, with effects on age of disease onset, and presence of uveitis and peripheral arthritis [16]. Many recent immunogenetic studies have investigated the presence of non-B27 AS susceptibility genes, and other non-MHC genes [1720]. Epidemiological investigations also support AS being an oligogenic disease, with B27 the major but not sole risk factor [21].
We previously demonstrated that the prevalence of B27 in Chinese patients with AS was approximately 98% [22]. However, a small percentage of B27-negative individuals can develop definite AS with typical clinical and radiological findings. In this study, we addressed one major question. Do other class I genes increase the risk of AS in Chinese AS patients in an HLA-B27-negative population?
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Materials and methods
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Cases and controls
Forty-one HLA-B27-negative subjects with AS, as defined by the modified New York criteria [23], were recruited from patients attending the Veterans General Hospital, Taiwan. All patients were assessed by a rheumatologist and had X-ray-confirmed at least bilateral class II sacroiliitis or unilateral class III sacroiliitis. No patient had psoriasis, Reiter's syndrome or inflammatory bowel diseases. As a control group, 11 383 blood samples from HLA-B27-negative healthy subjects (male:female ratio 1.6:1) were donated by the Chinese Blood Donation Association. All cases and controls were of Han Chinese ethnicity. This study was approved by the Ethical Committees of the Veterans General Hospital.
Determination of HLA-B27 and class I genes
Fifteen millilitres of venous blood per person was taken from both the AS patient group and the control group. HLA-B27 carriage was determined by flow cytometry [24]. The microlymphocytotoxicity method was used to genotype other HLA-A and -B genes (One Lambda, Canoga Park, CA, USA)
Statistical analysis
For the casecontrol study, differences in phenotype frequencies between patients and controls were assessed with the uncorrected
2 test. Bonferroni correction was performed for the 16 HLA alleles other than HLA-B60 with minor allele frequencies >10% for which the study had adequate statistical power [>80% power to detect an odds ratio (OR) of >2.5 with a two-tailed P value of <0.05]. Haplotype frequencies in the control samples were determined using the program Phase [25]. The presence of significant linkage disequilibrium was tested using Fisher's exact test, and its magnitude was assessed using Lewontin's standardized statistic (D'). This measures linkage disequilibrium on a scale of 0 (no disequilibrium) to 1 (complete linkage disequilibrium).
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Results
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Forty-one B27-negative Chinese patients with AS participated in this study (21 males, 20 females). Age ranged from 25 to 52 yr and age at onset was 1240 yr (mean 29.1 yr). Mean disease duration was 13.3 yr. The class I genes (A, B) present in each patient are shown in Table 1. In some cases only one allele was identified, which may indicate that the individual was either homozygous for that allele or (less likely) carried a previously unidentified allele. As the proportion of unidentified alleles is likely to be very low, these cases were considered to be homozygous for the genotyped allele in all further analyses. The HLA-A alleles in AS patients and controls are shown in Table 2. The phenotype frequency of HLA-A26 in the HLA-B27-negative AS patients (17.1%) was significantly greater than that in the controls [6.3%; OR = 3.1, 95% confidence interval (CI) 1.46.9, Puncorrected = 0.01, Pcorrected = not significant].
For the B alleles, HLA-B61 was present in 11 of the 41 patients (26.8%) compared with 213 of 11 383 controls (1.9%), giving an OR of 19.2 (95% CI 9.538.9, Puncorrected<0.001, Pcorrected<0.001). Carriage of HLA-B60 alleles was also increased in HLA-B27-negative AS patients (63.4 vs 38.0%; OR = 2.8, 95% CI 1.55.3, Puncorrected<0.001, Pcorrected<0.001) (Table 3).
The number of control haplotypes considered in the linkage disequilibrium calculations was 22 748, and the phase of 6976 (31%) of these could be determined with >90% certainty. Considering haplotypes determined with >90% certainty, no significant linkage disequilibrium was observed between HLA-A26 and either HLA-B60 or -B61 (D' = 0.04 and 0.02, P = 0.3 and 0.1 respectively). In the total data set, although statistically significant linkage disequilibrium was observed between HLA-A26 and HLA-B61 (D' = 0.04, P = 0.0001), the level of disequilibrium observed was close to zero and in the main these alleles segregate independently.
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Discussion
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The relationship between HLA class I genes and AS has been known for a long time, but it is thought that, in addition to B27, other class I and II genes may increase susceptibility to the development of AS [1316]. In the present study, we investigated class I genes in HLA-B27-negative AS patients and controls. To our knowledge, this is the largest such study reported to date. The frequency of B60 and B61 was significantly increased in our B27-negative AS patients. The association of B60 with AS was first reported by Robinson and colleagues, who found that B60 was increased in HLA-B27-positive AS patients [13]. Subsequently, Brown and colleagues demonstrated that B60 was associated with AS in HLA-B27-positive and -negative individuals [15]. It is unknown whether B60 and B61 are associated with AS in Taiwanese Chinese, which will be the subject of a future study.
Association of B27-negative AS with B7 or other B7 cross-reactive group (B7, 13, 22, 27, 40, 41, 42, 47, 48, 81, 82) has been reported previously [26, 27]. Khan and Arnett found a high frequency of either B7, B22 or B40 in AS and Reiter's syndrome [28, 29]. Cedoz reported that B40 was associated with disease in a subset of undifferentiated SpA patients with prominent peripheral arthritis [26]. B60 and B61 are the major subtypes of B40 and they are closely related major histocompatibility complex class I molecules [30]; our study is the first study to investigate these two alleles (B60 and B61) separately. We observed independent associations of B60 and B61 with AS. Whilst the association of AS with B60 has been reported previously, the strong association of B61 with B27-negative AS has not previously been reported.
B60/61 may be associated with AS either directly or because of linkage disequilibrium with a further MHC gene. Significant differences in the structure of and peptide presentation by HLA- B60/61 compared with B27 suggest that the mechanisms of association of these HLA-B alleles with AS are different. The peptide binding motif of B60 is considerably different from that of B27 [31], making it unlikely that its effect is mediated by the presentation of similar arthritogenic peptides. The T-cell epitopes of B27 and B60 are quite similar, which would be consistent with molecular mimicry theories [32]. Considering the B7 CREG HLA-B alleles, B14, B15, B38, B39 and B73 but not B60 or B61 possess a cysteine residue at position 67, like HLA-B27 [33, 34]. Therefore, the association of these alleles with AS is unlikely to be due to the same theoretical mechanisms, implicating this amino acid as critical in the mechanism of association of B27 and AS [35, 36].
Unlike B locus alleles, alleles of the A locus have not previously been associated with AS. Although A26 was also associated with disease in our B27-negative AS patients, the association did not reach statistical significance after Bonferroni correction. Previous studies that have investigated HLA-A associations with AS have generally been too small to provide robust findings, but have suggested associations with HLA-A2 [37, 38] and -A9 [39]. We were not able to confirm these associations, but our study was not adequately powered to exclude other than a major genetic effect. Furthermore, in Sardinia, haplotype studies suggest that the HLA-A2 association in that country reflects differential linkage disequilibrium between this allele and HLA-B*2705 and B*2709 [40], which are themselves differentially associated with AS. This highlights the complexity of MHC disease association studies.
Our findings suggest that, in addition to B60, B61 may also play significant roles in the development of AS in Chinese people independently of HLA-B27. These findings strengthen the evidence supporting the existence of non-B27 genetic influences on susceptibility to AS.
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Acknowledgments
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The authors are grateful to Dr M. Brown for his critical review of this manuscript and assistance with the analysis.
The authors have declared no conflict of interest.
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References
|
---|
- Brewerton DA, Caffrey M, Hart FD et al. Ankylosing spondylitis and HLA-B27. Lancet 1973;1:9047.[ISI][Medline]
- Schlostein L, Terasaki PI, Bluestone R, Pearson CM. High association of an HLA antigen, BW27, with ankylosing spondylitis. N Engl J Med 1973;288:7046.[ISI][Medline]
- Khan MA, Van der Linden SM. Ankylosing spondylitis and other spondyloarthropathies. Rheumatol Clin North Am 1990;16:55179.
- Khan MA. Prevalence of HLA-B27 in world population. In: López-Larrea C, ed. HLA-B27 in the development of spondyloarthropathies. Austin (TX): R.G. Landes Company, 1997:95112.
- Khan MA, Braun WE, Kushner I. HLA B27 in ankylosing spondylitis: Differences in frequency and relative risk in American Blacks and Caucasians. J Rheumatol 1977;4(Suppl. 3):3943.
- Brown MA, Jepson A, Young A, Whittle HC, Greenwood BM, Wordsworth BP. Ankylosing spondylitis in West Africansevidence for a non-HLA-B27 protective effect. Ann Rheum Dis 1997;56:6870.[Abstract/Free Full Text]
- Gonzalez-Roces S, Alvarez MV, Gonzalez S et al. HLA-B27 polymorphism and worldwide susceptibility to ankylosing spondylitis. Tissue Antigens 1997;49:11612.[ISI][Medline]
- Chou CT, Pai L, Chang DM, Lee CF, Liang MH, Schumacher HR. Prevalence of rheumatic disease in Taiwan: a population-based study of urban, suburban, rural difference. J Rheumatol 1994;21:3026.[ISI][Medline]
- Beasley RP, Bennett PH. Low prevalence of rheumatoid arthritis in Chinese. J Rheumatol 1983;10(Suppl.):115.
- Khan MA, Kushner I, Braun WE. A subgroup of ankylosing spondylitis associated with HLA-B7 in American blacks. Arthritis Rheum 1978;21:52830.[ISI][Medline]
- Khan MA, Kushner I, Braun WE. B27-negative HLA-Bw16 in ankylosing spondylitis. Lancet 1978;1:13701.
- Yamaguchi A, Tsuchiya N, Mitsui H et al. Association of HLA-B39 with HLA-B27-negative ankylosing spondylitis and pauciarticular juvenile rheumatoid arthritis in Japanese patients. Arthritis Rheum 1995;38:16727.[ISI][Medline]
- Robinson WP, van der Linden SM, Khan MA et al. HLA-Bw60 increase susceptibility to ankylosing spondylitis in HLA-B27+ patients. Arthritis Rheum 1989;32:113541.[ISI][Medline]
- Brown MA, Kennedy LG, MacGregor AJ et al. Susceptibility to ankylosing spondylitis in twins: the role of genes, HLA and the environment. Arthritis Rheum 1997;40:18238.[ISI][Medline]
- Brown MA, Pile KD, Kennedy LG et al. HLA class I associations of ankylosing spondylitis in the white population in the United Kingdom. Ann Rheum Dis 1996;55:26870.[Abstract]
- Brown MA, Kennedy LG, Darke C et al. The effect of HLA-DR genes on susceptibility to and severity of ankylosing spondylitis. Arthritis Rheum 1998;41:4605.[CrossRef][ISI][Medline]
- Fraile A, Nieto A, Beraún Y, Vinasco J, Matarán L, Martín J. Tumor necrosis factor gene polymorphisms in ankylosing spondylitis. Tissue Antigens 1998;51:38690.[ISI][Medline]
- Maksymowych WP, Suarez-Almazor M, Chou CT, Russell AS. Polymorphism in the LMP2 gene influences susceptibility to extraspinal disease in HLA-B27-positive individuals with ankylosing spondylitis. Ann Rheum Dis 1995;54:3214.[Abstract]
- Brown MA, Edwards S, Hoyle E et al. Polymorphisms of the CYP2D6 gene increase susceptibility to ankylosing spondylitis. Hum Mol Genet 2000;9:15636.[Abstract/Free Full Text]
- Laval SH, Timms A, Edwards S et al. Whole-genome screening in ankylosing spondylitis: Evidence of non-MHC genetic-susceptibility loci. Am J Hum Genet 2001;68:91826.[CrossRef][ISI][Medline]
- Brown MA, Laval SH, Brophy S, Calin A. Recurrence risk modelling of the genetic susceptibility to ankylosing spondylitis. Ann Rheum Dis 2000;59:8836.[Abstract/Free Full Text]
- Feltkamp TEW, Mardjuadi A, Huang F, Chou CT. Spondyloarthropathies in eastern Asia. Curr Opin Rheumatol 2001;13:28590.[CrossRef][ISI][Medline]
- Van der Linden S, Valkenburg HA, Cats A. Evaluation of diagnostic criteria for ankylosing spondylitis: A proposal for modification of the New York criteria. Arthritis Rheum 1984;27:3618.[ISI][Medline]
- Chou CT, Tsai YF, Lin J et al. The detection of HLA-B27 antigen by immuno-magnetic separation with enzyme-linked immunosorbent assay (IMS-ELISA)-A new method to flowcytometry measurement. J Immunol Methods 2001;255:1522.[CrossRef][ISI][Medline]
- Stephens M, Smith NJ, Donnelly P. A new statistical method for haplotype reconstruction from population data. Am J Hum Genet 2001;68:97889.[CrossRef][ISI][Medline]
- Cedoz JP, Wendling D, Viel JF. The B7 cross reactive group and spondyloarthropathies: an epidemiological approach. J Rheumatol 1995;22:188490.[ISI][Medline]
- Khan MA. B7 CREG and ankylosing spondylitis. Br J Rheumatol 1983;22(Suppl. 2):12933.
- Khan MA, Kushner I, Braun WE. A subtype of ankylosing spondylitis associated with HLA B7 in American blacks. Arthritis Rheum 1978;21:52830.[ISI][Medline]
- Arnett FC, Hochberg FC, Bias WB. Cross reactive HLA antigens in B27-negative Reiter's syndrome and sacroiliitis. Johns Hopkins Med J 1977;141:1937.[ISI][Medline]
- Arnett KL, Parham P. HLA class I nucleotide sequences. Tissue Antigens 1995;46:21757.[ISI][Medline]
- Falk K, Rotzschke O, Takiguchi M et al. Peptide motifs of HLA-B58, B60, B61 and B62 molecules. Immunogenetics 1995;41:1658.[ISI][Medline]
- Lopez D, Garcia-Hoyo R, Lopez-de-Castro JA. Clonal analysis of alloreactive T cell responses against the closely related B*2705 and B*2703 subtypes. Implications for HLA-B27 association to spondyloarthropathy. J Immunol 1994;152:555771.[Abstract/Free Full Text]
- Taurog JD, El-Zaatari FAK. In vitro mutagenesis of HLA-B27. Substitution of an unpaired cysteine residue in the alpha 1 domain causes loss of antibody-defined epitopes. J Clin Invest 1988;82:98792.[ISI][Medline]
- Maclean L, Macey M, Lowdell M et al. Sulphydryl reactivity of the HLA-B27 epitope: Accessibility of the free cysteine residue in the
1 domain causes loss of antibody-defined epitopes. Ann Rheum Dis 1992;51:45660.[Abstract]
- Archer JR, Whelan MA, Badakere SS, McLean IL, Archer IV, Winrow VR. Effect of a free sulphydryl group on expression of HLA-B27 specificity. Scand J Rheumatol 1990;87(Suppl.):4450.
- Allen RL, OCallaghan CA, McMichael AJ, Bowness P. Cutting edge: HLA-B27 can form a novel beta 2-microglobulin-free heavy chain homodimer structure. J Immunol 1999;162:50458.[Abstract/Free Full Text]
- Bosak V, Kolostova K, Mateicka F, Rovensky J. MHC genes in ankylosing spondylitis. J Rheumatol 2000;27(Suppl. 59):25.
- La Nasa G, Mathieu A, Mulargia M et al. Association of the HLA-A2, CW2, B27, S31, DR2 haplotype with ankylosing spondylitis. A possible role of non-B27 factors in the disease. Dis Markers 1993;11:191203.[ISI][Medline]
- de Juan MD, Reta A, Cancio J, Belzunegui J, Cuadrado E. HLA-A*9, a probable secondary susceptibility marker to ankylosing spondylitis in Basque patients. Tissue Antigens 1999;53:1616.[CrossRef][ISI][Medline]
- Fiorillo MT, Cauli A, Carcassi C et al. Two distinctive HLA haplotypes harbor the B27 alleles negatively or positively associated with ankylosing spondylitis in Sardinia: implications for disease pathogenesis. Arthritis Rheum 2003;48:13859.[CrossRef][ISI][Medline]
Submitted 17 November 2003;
revised version accepted 12 March 2004.