Ankylosing spondylitis (AS), the prototype disease in the spectrum of spondyloarthritides, is a chronic, systemic, inflammatory disease with a strong predilection for the axial skeleton [1]. AS is estimated to affect 0.1%–0.3% of the population and thus constitutes a significant health problem worldwide [2]. Currently, the pathogenesis of AS is poorly understood, but recent large-scale genetics and gene-expression profiling studies have identified some of the underlying mechanisms and pathways contributing to this disease [3,4].

A recent study identified multiple risk variants for AS through high-density genotyping of immune-related loci [5]. In that study, new loci associated with AS at genome-wide significance were observed in both in European and East Asian cohorts. These loci included rs12615545 (in the UBE2E3 gene), rs4676410 (in the GPR35 gene), rs1250550 (in the ZMIZ1 gene), rs2531875 (in the NOS2 gene), and rs7282490 (in the ICOSLG gene). These single-nucleotide polymorphisms (SNPs) showed diverse allele frequencies among people from different cohorts. In that study, the East Asian cohort consisted of 1,550 cases and 1,567 controls from the Chinese, Taiwanese, and Korean populations [5]. In the present study, we determined whether these susceptibility loci were associated with the risk of AS in the Han Chinese population [6-9].

Patient selection and clinical characteristics of AS in the study cohort

We recruited 848 Han Chinese patients who fulfilled the 1984 modified New York criteria for AS [10]. Among them, 660 patients were male, and 188 were female. AS was diagnosed by a qualified rheumatologist and sacroiliitis was confirmed by a qualified radiologist. These patients were consecutively recruited at Peking University Shenzhen Hospital, Shenzhen, Guangdong, China. The detailed clinical history included extraspinal manifestations, age at symptom onset, and family history of AS. A total of 1123 healthy controls were recruited from people undergoing medical examinations at our hospital. The study was approved by the Peking University Health Science Center, and the study design and final report conform to the Declaration of Helsinki. All the subjects gave written consent.

Bath AS indices

BASDAI and BASFI were applied to evaluate disease activity and physical function, respectively. In all patients, the BASDAI and BASFI scores were evaluated at the active stage of the disease. The modified Chinese versions of BASDAI and BASFI have good intra-class correlation and Cronbach’s alpha [11].

Genotyping

Genomic DNA from the AS patients and healthy controls was isolated from peripheral blood cells using the Innogent genomic DNA extraction kit (Innogent, Shenzhen, Guangdong, China), according to the manufacturer’s protocol. The genotypes of rs12615545, rs4676410, rs1250550, rs2531875, and rs7282490 were directly genotyped by HRMA using Light Cycler software (release 1.5.0, Roche, Indianapolis, IN, USA) with high sensitivity detection and auto-grouping after polymerase chain reaction (PCR) amplification. The sequences of the primers used in the current study are listed in Supplementary Table 1. PCR was performed in a volume of 10 μl containing 5 μl Ssofast^TM Evagreen R Supermix (BioRad, US), 0.5 μl of forward primer (10 μM), 0.5 μl of reverse primer (10 μM), 3 μl water, and 1 μl DNA (40 ng/μl). The auto-grouping and sequencing of the targeted SNPs are illustrated in figure 1.

Table 1: Genotype and allele frequencies of indicated polymorphisms in controls and patients with AS; AS: Ankylosing Spondylitis; OR: Odds Ratio; CI: Confidence Interval.

Figure 1: Auto-grouping of the indicated SNPs using LightCycler software (version 1.5.0) and the results of verification by DNA sequencing.

Statistical analysis

The Hardy–Weinberg equilibrium was assessed by the chi-square test with one degree of freedom. Differences in genotype and allele frequencies between the patient and control groups were analyzed by the chi-square test. Analysis of variance was used to compare the means of continuous variables (BASDAI, BASFI, and Bath Ankylosing Spondylitis Patient Global score) among AS patients with different genotypes. Multiple regression analysis was used to adjust for age, sex, and disease duration. The Bonferroni test was used to correct for multiple tests. P values less than 0.05 was considered significant.

Association between rs4676410/rs2531875 genetic polymorphisms and AS susceptibility

As shown in Table 1, the results of the Hardy–Weinberg equilibrium tests showed that allele and genotype frequencies of all the indicated polymorphisms in the studied population were stable. Among the five studied SNPs, the rs4676410T allele (p=0.0237; odds ratio (OR): 1.109; 95% confidence interval (CI): 1.014–1.213) and rs2531875G allele (p=0.0308; OR: 1.117; 95% CI: 1.01–1.234) were found to be associated with AS susceptibility. A significant association between the rs30187T allele and the risk of AS was also observed. Age at onset and gender had no influence on the allele frequencies of the selected polymorphisms (Tables 2 and 3).

Table 2: Genotype and allele frequencies of indicated polymorphisms in male or female AS patients.

Table 3: Genotype and allele frequencies of indicated polymorphisms in juvenile (<18) or adult (18) onset AS patients.

Association between rs4676410 and iridocyclitis in AS patients

We further investigated whether the above two genetic polymorphisms were associated with the complications (uveitis and IBD) and clinical phenotypes (BASDAI and BASFI) of AS. As shown in Table 4, an association was found between the GPR35 polymorphism rs4676410 and iridocyclitis. We did not find any association between rs4676410/rs2531875 polymorphisms and IBD (Tables 5). When the combinatorial effects were considered using a two-locus model (rs4676410 and rs2531875), a borderline association (p=0.0459) was obtained between the polymorphisms and BASDAI scores (Table 6).

Table 4: Genotype and allele frequencies of rs4676410 and rs2531875 in AS patients with or without iridocyclitis; AS: Ankylosing Spondylitis; OR: Odds Ratio; CI: Confidence Interval.

Table 5: Genotype and allele frequencies of rs4552569 in AS patients with or without IBD; AS: Ankylosing Spondylitis; IBD: Inflammatory Bowel Disease; OR: Odds Ratio; CI: Confidence Interval.

Table 6: Difference in BASDAI and BASFI scores among AS patients stratified by rs4676410 and rs2531875 genotypes.

No associations of the selected genetic polymorphisms with HLA-B27 (+) AS patients

We compared HLA-B27 (+) subjects and normal controls in terms of the indicated genetic variants, but did not observe a significant association (Table 7). In HLA-B27 (+) AS patients, we examined whether the complications of AS were associated with rs4676410 and rs2531875; our results showed no statistically significant association after Bonferroni correction (data not shown here). Our results also revealed no association of the rs4676410 and rs2531875 genotypes with the BASDAI and BASFI scores among HLA-B27 (+) AS patients (Table 8).

Table 7: Genotype and allele frequencies of the indicated polymorphisms in AS patients who were positive or negative for HLA-B27.

Table 8: Difference in the BASDAI and BASFI scores among HLA-B27–positive AS patients stratified by rs4676410 and rs2531875 genotypes.

There are distinct genotypic distributions, linkage disequilibria, and haplotype blocks between Caucasians and Asians. Replication studies are needed to validate the association of newly identified susceptibility loci with the risk of AS in different populations. Here we first found the associations between rs4676410/rs2531875 polymorphisms and risk of AS in Han Chinese population. Furthermore, we observed an association between rs4676410 polymorphism and iridocyclitis, which was also a novel finding. The allele frequencies of rs12615545, rs4676410, rs1250550, and rs2531875 were similar to those reported in an East Asian cohort (Table 9) [5], while our data of rs7282490 had a marked deviation from that report. This difference requires further validation in different East Asian populations.

Table 9: Comparison of allele frequencies of indicated polymorphisms in different studies; a. International Genetics of Ankylosing Spondylitis C (Cortes A et al.)

A previous study also showed that rs4676410 was associated with early-onset IBD susceptibility [12], while it was found to be assoicated with uveitis in AS patients in the present study. rs4676410 lies within the GPR35 (G protein-coupled receptor 35) gene, which encodes a G protein-coupled receptor. High expression of GPR35 is found in immune and gastrointestinal tissues. In human invariant natural killer T cells, the specific activation of GPR35 by selective receptor agonists is functionally correlated with a significant reduction in interleukin-4 (IL-4) release [13]. IL-4 is a cytokine that induces the differentiation of naive helper T cells to Th2 cells and is significantly elevated in AS patients, as compared with that in controls [14]. Furthermore, another SNP in GPR35 is associated with primary sclerosing cholangitis and ulcerative colitis [15]. Other genes in the LD block of rs4676410 include CAPN10, KIF1A, and RNPEPL1. The effect of the rs4676410 genotype on the expression of these genes should be examined in the future.

The SNP rs2531875 (17q11) is located in the NOS2 (nitric oxide synthase 2) gene, which encodes a nitric oxide synthase expressed in the liver and is inducible by a combination of lipopolysaccharide and certain cytokines. Inducible nitric oxide synthase (iNOS) activation and increased NO production have been found to contribute to the pathogenesis of osteoporosis in AS patients [16]. Furthermore, lymphocytic infiltration and iNOS expression and activity were detected in duodenal and colonic mucosa from patients with AS [17]. Whether the different genotypes of rs2531875 have an impact on the expression or activity of NOS2 needs to be determined.

We also performed an association study between rs4676410/rs2531875 and disease severity. Although no association was found between these two polymorphisms and BASDAI/BASFI scores, we found increased BASDAI scores in the high-risk group, which was a combination of AS patients with the TT genotype of rs4676410, GG genotype of rs2531875, or heterozygous genotypes at both loci. These further indicated the involvement of these two polymorphisms in the development of AS.

In summary, the rs4676410 and rs2531875 genetic polymorphisms may be susceptibility factors for AS development in Han Chinese. An association between the rs4676410 T allele and iridocyclitis was also observed.

This work was supported by the General Program of Natural Science Foundation of Guangdong Province (S201201000815) and Fundamental Research Plan of Shenzhen City (JCYJ20120831143519567 & JCYJ20140416144209741).