Association of ERAP1, but not IL23R, with ankylosing spondylitis in a Han Chinese population

Abstract

Objective

The results of a recent genome-wide association study have shown that ERAP1 and IL23R are associated with ankylosing spondylitis (AS) in Caucasian populations from North America and the UK. Based on these findings, we undertook the current study to investigate whether single-nucleotide polymorphisms (SNPs) covering the genes ERAP1 and IL23R are associated with AS in a Han Chinese population.

Methods

A case–control study was performed in Han Chinese patients with AS (n = 527) and controls (n = 945) from Shanghai and Nanjing. All patients met the modified New York criteria for AS. The Sequenom iPlex platform was used to genotype cases and controls for 21 tag SNPs covering IL23R and 38 tag SNPs covering ERAP1. Statistical analysis was performed using the Cochran-Armitage test for trend.

Results

Multiple SNPs in ERAP1 were significantly associated with AS (for rs27980, P = 0.0048; for rs7711564, P = 0.0081). However, no association was observed between IL23R and AS (for all SNPs, P > 0.1). The nonsynonymous SNP in IL23R, rs11209026, widely thought to be the primary AS-associated SNP in IL23R in Europeans, was found not to be polymorphic in Chinese.

Conclusion

Our results demonstrate that genetic polymorphisms in ERAP1 are associated with AS in Han Chinese, suggesting a common pathogenic mechanism for the disease in Chinese and Caucasian populations, and that IL23R is not associated with AS in Chinese, indicating a difference in the mechanism of disease pathogenesis between Chinese and Caucasian populations. This may result from the fact that rs11209026, the nonsynonymous SNP in IL23R, is not polymorphic in Chinese patients, providing further evidence that rs11209026 is the key polymorphism associated with AS (and likely inflammatory bowel disease and psoriasis) in this gene.

Ankylosing spondylitis (AS) is a common inflammatory arthritis characterized by axial skeletal inflammation, enthesitis, and association with HLA–B27. Arthritis in AS leads to increased bone formation and consequent ankylosis, resulting in significant morbidity. The overall prevalence of AS in Chinese is 0.2–0.54% (1). The primary gene associated with AS is HLA–B27, a class I major histocompatibility complex (MHC) gene that is present in >90% of Chinese patients as compared with ∼5% of healthy Chinese (2). However, only 1–5% of HLA–B27 carriers develop AS, and it is thought that the presence of HLA–B27 is required for the inheritance of the disease. However, other genetic factors play a role in modifying its penetrance (3). In fact, the presence of HLA–B27 is responsible for only ∼40% of the familiality in AS (4). Other genes have been shown to be associated with AS in Chinese populations, including HLA–B60 (5) and IL1A/B (6).

Recent genome-wide association studies in European Caucasian populations have identified ERAP1 (the gene for endoplasmic reticulum aminopeptidase 1 [ERAP-1]) and IL23R (the gene for interleukin-23 receptor [IL-23R]) as being associated with AS (7), with the 2 genes responsible for 26% and 9% of the population-attributable risk in AS, respectively. ERAP1 has 2 known functions, either of which could explain its association with AS. ERAP1 is involved in peptide processing within the endoplasmic reticulum for class II MHC presentation (8, 9). The strong association between AS and HLA–B27 suggests that AS is primarily a class I MHC–mediated disease, and the association with ERAP1 may help to explain the mechanism by which HLA–B27 contributes to AS. ERAP1 has also been shown to cleave cell surface receptors for the proinflammatory cytokines IL-1 (IL-1RII) (10), IL-6 (IL-6Rα) (11), and tumor necrosis factor (TNF) (TNF receptor superfamily 1A) (12). This mechanism could therefore have proinflammatory effects through variations in ERAP1 causing loss of function.

IL23R encodes a critical cytokine receptor in the Th17 subset of T lymphocytes. These newly characterized CD4+ T cells were originally identified through their ability to secrete high levels of the proinflammatory cytokine IL-17 upon stimulation (13, 14). IL-23R is required for the differentiation and maintenance of Th17 populations (15–17). Variation in IL23R has also been demonstrated in both inflammatory bowel disease (IBD) (18, 19) and psoriasis (20). AS, IBD, and psoriasis are closely related clinically, and the shared association of these diseases with polymorphisms in IL23R is at least partly responsible for their co-occurrence. Through the involvement of IL-23, Th17 cells have been implicated in the pathology of a number of autoimmune diseases in murine models, including the experimental autoimmune encephalomyelitis model of multiple sclerosis (21) and the collagen-induced arthritis model of rheumatoid arthritis (RA) (22). The association of IL23R with AS suggests that Th17 cells may play a role in the pathogenesis of the disease. This is supported by evidence of elevated levels of circulating Th17 memory cells in the blood of patients with AS (23).

We describe here the results of a confirmation study that we undertook in order to further investigate the genetic basis of AS. Cases and controls were genotyped to determine whether polymorphisms in ERAP1 and IL23R contribute to AS susceptibility in a Han Chinese population.

PATIENTS AND METHODS

Subjects.

Unrelated AS patients (n = 527) were recruited from among patients attending outpatient clinics at several hospitals in Shanghai and Nanjing. All had been diagnosed as having AS, which was confirmed by a qualified rheumatologist. In all cases, sacroiliitis was confirmed by radiography, and each patient met the modified New York criteria for AS (24). Unrelated healthy, ethnically matched blood donors (n = 945) recruited from the Shanghai Blood Bank were included as controls.

Single-nucleotide polymorphism (SNP) selection.

To select the SNPs for this study, haplotype data on Chinese and Japanese subjects were obtained from the International HapMap Project (release 23, National Center for Biotechnology Information [NCBI] B36 assembly, dbSNP b36) for ERAP1 and IL23R and their surrounding areas. Tag SNPs covering these genomic regions were selected using Tagger in Haploview 4.0 and gave 100% coverage of both genes (r² ≥ 0.8). The markers rs27037 and rs27434 in ERAP1 and rs11209026 and rs11209032 in IL23R were forcibly included in the set of tag SNPs, since these polymorphisms were shown to be the most strongly associated with AS in a previous study (7). In total, 38 SNPs in ERAP1 and 21 SNPs in IL23R were selected.

Genotyping.

SNP genotyping was performed using the MassArray platform (Sequenom, San Diego, CA), which utilizes chip-based matrix-assisted laser desorption ionization–time-of-flight mass spectrometry technology. Polymerase chain reaction (PCR) and iPlex extension reactions were designed using MassArray Assay Design 3.1 in multiplex format. Genotyping was then performed according to the manufacturer's standard protocols. MassArray Typer 4.0 was used to read the extended mass and assign genotype calls. SNP rs4655690 could not be genotyped using the methods described above and, instead, was typed with a TaqMan method using a 7900HT genotyper according to the standard protocols of the manufacturer (Applied Biosystems, Foster City, CA). Four samples known to be heterozygous for rs11209026 were included in the genotyping to confirm the assay for this particular SNP.

Statistical analysis.

Association analysis was performed using the Cochran-Armitage test for trend and Plink 1.03 (online at http://pngu.mgh.harvard.edu/purcell/plink/) (25). SNPs with absence rates >0.1 were excluded, and an exact test for Hardy-Weinberg equilibrium was performed in controls. The SNP marker map used for analysis was NCBI dbSNP b128 (October 2007). SNP imputation was performed using MACH 1.0, with phased data from Chinese and Japanese individuals from the International HapMap Project (release 22) serving as the reference set of haplotypes. Association analysis for imputed data was performed using logistic regression of the probability scores for the imputed genotypes for SNPs within a 50-kb region 5′ and 3′ of the respective gene. SNPs were excluded if confidence regarding imputation accuracy was low (r² < 0.3 with flanking SNPs; quality score <0.95). Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated. Study power was determined using the Genetic Power Calculator (online at http://pngu.mgh.harvard.edu/∼purcell/gpc/cc2.html).

RESULTS

A total of 527 AS cases and 945 healthy controls were genotyped for polymorphisms in ERAP1 and IL23R using the Sequenom iPlex Gold platform. Some samples (n = 134) were excluded because of low genotyping rates (genotyping <90% completed), leaving 492 cases and 846 healthy controls in the final analysis. All markers genotyped were in Hardy-Weinberg equilibrium (P > 0.05). Eighty SNPs in ERAP1 and 50 SNPs in IL23R were included based on sufficient imputation accuracy (r² > 0.3 with flanking markers; quality score >95%).

Based on the assumption of a population prevalence of disease of 0.2%, a significance level of 0.05, minor allele frequencies of 0.1–0.5, and a value for D′ of 0.9, the study had 80% power to detect an additive association (defined as an OR of 1.3–1.5 in heterozygotes).

SNP association findings for IL23R and ERAP1 are presented in Figures 1 and 2, respectively. More detailed findings along with statistical comparisons are presented in Supplementary Table 1 (available on the Arthritis & Rheumatism web site at http://www3.interscience.wiley.com/journal/76509746/home). For 2 markers in ERAP1, association with AS was observed at P values of ≤0.01, and for 5 additional markers in ERAP1, association was observed at P values of ≤0.05. Peak association was observed for marker rs27980 (OR 0.80 [95% CI 0.68–0.93], P = 0.0048). Moderate association was observed for rs27037 (OR 1.22 [95% CI 1.04–1.44], P = 0.012), which served as a confirmation marker in a previous study (7), while no association was observed for rs27434 (P = 0.14).

Figure 1. Case–control association findings for the IL23R locus. Distances are measured from the p-telomere (in basepairs), according to the National Center for Biotechnology Information B36 human genome assembly. Association results are reported as −log(P value); −log(P = 0.05) = 1.3. SNPs = single-nucleotide polymorphisms.

Figure 2. Case–control association findings for the ERAP1 locus. Distances are measured from the p-telomere (in basepairs), according to the National Center for Biotechnology Information B36 human genome assembly. Association results are reported as −log(P value); −log(P = 0.05) = 1.3. SNPs = single-nucleotide polymorphisms.

No association with AS was observed for genotyped markers in IL23R (for all SNPs, P > 0.1). The marker rs11209026 was confirmed to not be polymorphic in Han Chinese. All 4 samples known to be heterozygous for rs11209026 were correctly genotyped by Sequenom assay.

At IL23R, the strongest association between an imputed SNP and AS was observed for rs10889665 (P = 0.051). At ERAP1, the strongest imputed SNP association was observed for rs26492 (P = 0.01). Association (P < 0.05) was observed across this locus for markers extending 29 kb, from 96114867 to 96143831 bp (markers rs26492 to rs27619).

DISCUSSION

We undertook this case–control genotyping study to investigate the association between AS and polymorphisms in ERAP1 and IL23R in a Han Chinese population. Our results indicate that ERAP1 is associated with AS not only in Caucasians, but also in Han Chinese. To our knowledge, this is the first study to show that polymorphisms in ERAP1 are associated with AS in a non-Caucasian population, which suggests that the disease has a common pathogenic mechanism in Chinese and Caucasians.

The association of ERAP1 with AS can be plausibly explained based on its known biologic functions. Within the endoplasmic reticulum, ERAP-1 is involved in trimming peptides to optimal length for class I MHC presentation (26). As with AS in Caucasians, AS in Chinese is a class I MHC–mediated disease, with >90% of Chinese patients with AS carrying the HLA–B27 allele (1). Although the association of HLA–B27 with AS has been known for quite some time, the mechanism of how it increases the risk of developing AS remains a mystery. If the association of ERAP1 with AS is found to relate to its role in peptide presentation, this may help to explain the association of HLA–B27 with AS.

The association of ERAP1 with AS in Chinese indicates that ERAP1 plays a key role in disease pathogenesis in an ethnic group that is quite removed from the European Caucasian populations in which the association was first observed. This provides important confirmation of the major role of this gene in determining AS susceptibility. The association between ERAP1 and AS was previously demonstrated in a population of European Caucasian ancestry, in which 95% of B27-positive AS patients were carriers of B*2705 (27). In the current Chinese population, 81% of AS patients are B*2704 carriers (28). To our knowledge, no functional studies comparing interactions between B27 subtypes and ERAP1 have been reported, but based on the results of the current study and previous studies in European Caucasians, it appears that ERAP1 is associated with AS in populations with B27 subtypes that are predominantly either B*2704 or B*2705, a finding that should be taken into consideration when designing future functional analyses of the role of ERAP1 in AS susceptibility.

Conversely, no association between IL23R and AS was observed. The lack of association with IL23R in Chinese patients with AS indicates a difference in the mechanism of disease pathogenesis between Caucasian and Han Chinese populations. In Caucasians, IL23R polymorphisms have been identified in Crohn's disease (18) and psoriasis (20), as well as in AS (7), suggesting that it may be a common susceptibility factor. However, we have confirmed that rs11209026, which is widely thought to be the causative polymorphism for disease susceptibility in Caucasians, is not polymorphic in Han Chinese. This is consistent with the fact that rs11209026 has been identified as the key IL23R polymorphism associated with AS.

There are a number of possible ways to explain why the association between IL23R and AS differs among ethnic groups. It is possible that there are different disease mechanisms in different populations, with AS developing in Chinese via a mechanism independent of IL23R, meaning that any polymorphisms in IL23R would be irrelevant to disease pathogenesis. This difference in mechanism could be due to association with a different gene also involved in the IL-23 signaling pathway, or AS may develop through other pathways in various populations.

The lack of association between IL23R and AS in Chinese may also be explained genetically, since rs11209026, which is thought to be the causative SNP for disease susceptibility in Europeans, is not polymorphic in Chinese. In a number of other diseases, the polymorphic status of SNPs has contributed to differing levels of association between ethnic groups. PTPN22 is strongly associated with RA in Caucasians (29); however, it has been shown that the C1858T SNP, which is thought to be the key SNP associated with the disease in European Caucasians, is not polymorphic in East Asians, explaining the lack of association of PTPN22 with RA in these populations (30). Similarly, the NOD2/CARD15 polymorphisms associated with Crohn's disease in European Caucasian populations are not polymorphic in Asians. More significantly, IL23R has been shown not to be associated with Crohn's disease in East Asians (31); this is also thought to result from the fact that rs11209026 is not polymorphic in East Asian populations, although it is polymorphic in Caucasians. Whether there is an association in East Asians between IL23R and ulcerative colitis or psoriasis, both diseases with which it is strongly associated in European Caucasians, is unknown.

The lack of association of IL23R with AS in Chinese patients and the fact that the causative SNP in Caucasians is not polymorphic in Han Chinese suggest that rs11209026 is the common SNP between the diseases in Caucasians; however, the gene does not play a role in disease pathogenesis in Asians. Alternatively, another disease-associated SNP, which is not polymorphic in Chinese, may be responsible for the association of IL23R with AS in European Caucasians. Further resequencing of the gene will be needed to make this determination.

A potential alternative explanation for the lack of association between IL23R and AS in Chinese could be that IL23R contributes to disease progression through a gene–environment interaction with a trigger not found in East Asia. However, this is unlikely to be the case, since ethnic Chinese people develop AS regardless of where they live, although it is possible that the putative environmental trigger involved is a cultural or dietary factor maintained even among Chinese people living abroad. Gene–gene interactions may also be a factor in the development of AS. If the pathogenesis of AS is dependent on interactions between IL23R and another gene that is not polymorphic in Chinese, no association of IL23R would be observed. However, this would be surprising given the strong association of IL23R SNPs with AS in Caucasians.

The results of this confirmation study highlight the value of utilizing the genetic diversity between different ethnic groups for gene mapping. Our findings suggest that there are significant differences in susceptibility genes between Han Chinese and European Caucasian populations, which could be exploited in an effort to distinguish true disease-associated polymorphisms from linkage disequilibrium effects. Additionally, it is possible that ethnic groups other than European Caucasians may have susceptibility genes that will provide further information about the pathogenesis of AS. Comprehensive gene mapping studies of East Asian cohorts are thus likely to be very valuable in understanding the genetic causes of rheumatic diseases.

Acknowledgements

We wish to thank the patients and controls for providing blood samples, and our colleagues Drs. Zhongwei Wang, Ting Li, Yiping Lin, and Chao Wu and Ms. Johanna Hadler, Ms Kathryn Addison, and Ms Karena Pryce for their assistance with DNA extraction and genotyping.

AUTHOR CONTRIBUTIONS

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. H. Xu had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Davidson, Wu, Liu, Wei, Cai, Sun, Duncan, Wang, Yu, A. Xu, Fu, Brown, H. Xu.

Acquisition of data. Davidson, Wu, Thomas, Duncan, Wang, Yu, Brown, H. Xu.

Analysis and interpretation of data. Davidson, Wu, Danoy, Duncan, Wang, Yu, Brown, H. Xu.