Abstract
Objective. Investigators from the Australo-Anglo-American Spondyloarthritis Consortium have reported additional genes associated with ankylosing spondylitis (AS) susceptibility including IL1R2, ANTXR2, and gene deserts at 2p15 and 21q22. We evaluated these new candidate genes in a large cohort of Korean patients with AS.
Methods. A group of 1164 patients with AS and 752 healthy controls were enrolled for our study. Eight single-nucleotide polymorphisms (SNP) were analyzed to define genetic association with AS by MassARRAY system.
Results. Significant positive associations of AS with endoplasmic reticulum aminopeptidase 1 SNP, rs27037 (p = 1.31 × 10−4), and rs27434 (p = 4.59 × 10−6), were observed. The rs10865331 of gene desert at 2p15 also showed a significant association with AS (p = 4.63 × 10−5).
Conclusion. This is the first confirmation in a nonwhite population that genetic polymorphisms of rs27037, rs27434, and rs10865331 are associated with AS, implicating common pathogenetic mechanisms in Korean and white patients with AS.
Ankylosing spondylitis (AS) is a chronic inflammatory dis order primarily involving the sacroiliac joints and spine. It usually affects young people, and multiple genetic factors are implicated in conferring susceptibility to AS. HLA-B27 remains the major susceptibility gene, and is present in 80%–95% of AS cases. Several theories to explain the HLA-B27 association have been proposed, but the pathogenesis and mechanisms of its association with AS are not well defined1,2,3.
In 2007, an association study was published, reporting findings of 14,500 nonsynonymous single-nucleotide polymorphisms (SNP) in 1500 white controls and 1000 white patients with AS4. As expected, MHC genes showed the strongest association with AS, but 2 non-MHC genes, endoplasmic reticulum aminopeptidase 1 (ERAP1) and interleukin 23 receptor (IL-23R) also showed strong association. Recently, the Australo-Anglo-American Spondyloarthritis Consortium (TASC) reported that additional genes are implicated in AS susceptibility, including IL1R2, ANTXR2, and gene deserts at 2p15 and 21q225. It has now been agreed that 5 genes/genetic regions are definitively associated with AS (MHC, IL23R, ERAP1, 2p15, and 21q22)6. But these studies were performed exclusively in cohorts of white European descent, and only limited information has been reported from Asian countries7,8.
We have reported that ERAP1 is associated with AS in Koreans, while IL-23R is not8,9. In our current study, we evaluate the new candidate genes in a large cohort of Koreans with AS and healthy controls.
MATERIALS AND METHODS
Subjects
A total of 1164 patients with AS and 752 ethnically matched, healthy controls were enrolled for this study. All cases and controls were native Koreans, and AS cases satisfied the modified New York criteria10. Clinical information was collected systematically and informed consent was obtained from all participants. The ethics committee of Hanyang University in Korea approved our study.
Healthy controls were screened by questionnaire to exclude those with a personal or familial history of arthritis or spondyloarthropathy. Eighty-eight percent of the AS cases were male, age of symptom onset was 22.8 ± 8.9 years (mean ± SD), and mean age at study entry was 35.3 ± 9.8 years. Of these AS cases, 96.5% were HLA-B27-positive (n = 1078/1117), 21.4% (243/1140) had juvenile-onset AS (age at symptom onset < 16 yrs), 24.7% (n = 278/1128) had uveitis, and 41.7% (n = 465/1116) had peripheral arthritis. The control population included 658 men (87.6%) with age of 31.4 ± 7.3 years.
Methods
Peripheral blood from cases and controls was collected in EDTA tubes, and genomic DNA was isolated using a Puregene blood DNA kit (Qiagen). A total of 1916 Korean samples were genotyped for 8 SNP. The MassARRAY® system (Sequenom, San Diego, CA, USA) was used to genotype each study participant in a 2-well reaction using Assay Designer 3.1; SNP 1–7 (rs10865331, rs3734523, rs4672495, rs2310173, rs2242944, rs27434, and rs4333130) were run as a multiplex reaction, and SNP 8 (rs27037) was run as a separate uniplex reaction. The reactions were processed following standard protocols for iPLEX™ chemistry, with the following exceptions: the concentration of shrimp alkaline phosphatase in the cleanup reaction was increased by 25%, and incubation at 37°C was increased by 15 min. Upon completion of the extension reaction, the products were cleaned, spotted onto SpectroChip II™ arrays, and scanned using a MassARRAY Compact Analyzer (Sequenom). Genotypes were determined using MassARRAY Typer v.4.0 software.
Statistical analysis
The frequencies and OR of the alleles and genotypes of all polymorphic SNP were calculated with a 95% CI, and a chi-squared test was used to compare the results between AS subjects and controls. Bonferroni correction was applied for multiple comparisons. The observed allelic frequencies for these SNP satisfied the Hardy-Weinberg equilibrium in controls.
RESULTS
In total, 1916 subjects (1164 patients with AS and 752 controls) were genotyped. Among the 8 SNP, rs3734523 and rs4333130 SNP were rare in the Korean population (Table 1). Therefore, these SNP were excluded from analysis. Significant positive association of AS with 2 SNP of ERAP1, rs27037 (p = 1.3 × 10−4) and rs27434 (p = 4.6 × 10−6), were observed. SNP rs10865331 at chromosome 2p15 was also found to be significantly associated with AS (p = 4.6 × 10−5; Table 2).
When we analyzed the clinical symptoms among the AS cases, the rs27434 (OR 1.4, 95% CI 1.1–1.7, p = 1.5 × 10−3) was significantly associated with juvenile-onset AS compared with adult-onset AS. There were no significant differences between AS cases with peripheral arthritis and those without peripheral arthritis, nor with AS cases with and without uveitis (Table 3).
DISCUSSION
We present 2 principal findings concerning the genetics of Korean AS in our case-control study. First, significant positive associations of AS with ERAP1 SNP rs27037 and rs27434 were observed. Second, we demonstrated that rs10865331 at the chromosome 2p15 intergenic region was significantly associated with AS in Koreans. With completion of the Human Genome Project and the International HapMap Projects, genome-wide association studies (GWAS) are offering an efficient and effective method of discovering genetic associations with human diseases. Hundreds of loci for over 40 diseases have already been identified, producing new associations as well as confirming previous ones11. GWAS have ushered in major advances in AS genetics as well. In addition to confirming the well established MHC association with AS, at least 6 other loci have been found to be associated with AS. Many of these findings were seen in studies of white patients of European ancestry who had AS.
We have confirmed that certain SNP of ERAP1 are associated with AS in Koreans, while other genes including IL-1, CARD15, TLR-4, and IL-23R are not associated with AS in this population8,9,12,13. ERAP1 SNP rs27044 and rs30187 showed significant association with AS, but SNP rs17482078, rs10050860, and rs2287987 showed no association with AS in Korea8. Davidson, et al reported that in the Chinese Han population, ERAP1 polymorphisms were associated with AS, but SNP across IL23R were not7. When 38 SNP of ERAP1 were analyzed, rs27037 showed significant association with AS (p = 0.012), but 27434 did not (p = 0.14). In our study, these 2 SNP of ERAP1 were significantly associated with AS, and some additional SNP of ERAP1 also demonstrated strong association with AS among Asians. ERAP1 is involved in trimming of peptides in the endoplasmic reticulum prior to HLA Class I presentation. ERAP1 also binds directly to the extracellular domain of tumor necrosis factor receptor (TNFR)1 in vitro and promotes its interleukin 1ß-mediated ectodomain cleavage to generate TNFR1. But functional analysis of the polymorphic forms of ERAP has not been determined yet6,14. ERAP1 is not associated with inflammatory bowel diseases that have clinical links to the spectrum of spondyloarthropathies such as AS4. Further studies of ERAP1 in AS among Asians will be valuable because the prevalence of inflammatory bowel disease in Korean AS is very low.
The TASC GWAS demonstrated strong association of an intergenic region on chromosome 2p15 with AS5. No genes are known to be encoded at this locus, but TASC identified tags from a single long noncoding RNA transcript in AS cases and controls originating from this narrow locus (23 kb)5. Further research is required to fully characterize the noncoding RNA involved.
This is the first report on AS among Asians to demonstrate association with the 2p15 gene desert, which has been significantly associated with AS cases among whites. Except for ERAP1 and 2p15, there were no associations with AS in the Korean population.
Acknowledgment
We are grateful to Matt A. Brown for assistance with drafting the manuscript.
Footnotes
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Supported by Hanyang University, Korea, in program year 2010 (HY-2010-I).
- Accepted for publication September 1, 2010.