Ankylosing spondylitis (AS) is a polygenic chronic inflammatory disease predominantly affecting the axial skeleton which leads to the new bone formation and disability. However, the link between new bone formation and inflammation in AS has not yet been fully understood [1, 2]. Furthermore, it is clearly demonstrated that new bone formation shows great variance in AS patients so that some patients never develop syndesmophytes throughout the entire disease span, whereas some rapidly develop spinal fusion [3, 4]. The extent of new bone formation is thought to be determined primarily by genetic factors with the contribution of environmental and acquired factors [5]. Despite growing evidence in the last decade for a genetic predisposition of the new bone formation, controversy remains for the individual genetic determinants of radiographic severity in AS. Recent genome-wide association studies have provided new insights into the AS pathogenesis, such as identification of new susceptibility genes other than HLA-B27 [6-8]. The strongest associations for AS susceptibility were identified in ERAP1 and IL-23R genes, contributing to 26% and 9% of AS risk, respectively [6]. ERAP1 gene product has an important role in antigen processing and regulates trimming of N-terminally extended peptides to their final epitopes, which are then presented by the major histocompatibility complex class I molecules [9, 10]. IL-23R gene encodes the receptor of IL-23, the key cytokine in the differentiation of naive CD4+ T cells into IL-17 producing T helper cells [11]. Polymorphisms in both genes have potential implications on inflammatory pathways in AS [12]. A strong association between the prostaglandin E receptor 4 (PTGER4) gene and AS susceptibility has also been reported [8]. The receptor encoded by this gene can activate T-cell factor signaling, mediate PGE2-induced expression of early growth response 1, and regulate the level and stability of cyclooxygenase-2 mRNA [13]. As a component of the “mechanostat” which regulates the anabolic bone response to physical stress at sites such as the enthesis, PTGER4 single nucleotide polymorphisms (SNP) may have a role in the new bone formation of AS [8].

The associations of these gene SNPs with AS have been well-established and replicated in multiple studies [14-17]. However, the contribution of these genes to radiographic severity/progression in AS has been investigated in only three studies in which radiographic severity or progression has been defined vaguely and differently [17-19]. Radiographic progression in AS is a very slow process [3, 4] in which determinants remain unclear. Therefore, in the present study, patients with AS were divided into two highly distinct groups: 1. Mild-AS defined as no evidence of axial radiographic progression following at least 10 years of disease duration, 2. Severe-AS defined as the presence of multiple bridging or syndesmophytes regardless of the disease duration. The primary aim of the study was to investigate the possible associations between ERAP1, IL23R, and PTGER4 gene polymorphisms which are linked to alterations in inflammatory pathways and new bone formation, and radiographic severity of AS. A second objective was to identify clinical characteristics associated with severe radiographic AS.

Radiographic damage in AS is a slow process, and its extent and rate varies among patients [22]. However, it is currently unknown which patients are more likely to develop significant radiographic damage of the spine and which patients are not. This is the first study to examine the association of inflammation (ERAP1 and IL23R) and new bone formation (PTGER4) gene variants with radiographic severity of AS. We found that the risk allele frequencies of ERAP1 rs27044 and rs30187, IL23R rs11209032 and PTGER4 rs10440635 SNPs were high in AS patients but not associated with the spinal radiographic severity. Male gender and coxofemoral joint involvement were significant clinical determinants associated with severe radiographic AS.

The association between ERAP1 rs27044 and rs30187, IL23R rs11209032 and PTGER4 rs10440635 SNPs and susceptibility to AS has been previously reported in numerous studies [7, 8, 15-17, 23]. On the other hand, a few studies with different radiographic severity definitions addressed the association of these SNPs with radiographic severity of AS [17-19]. A Taiwanese study of AS patients (HLA-B27 positivity ~92%) categorized as syndesmophyte (+) and (-) reported that ERAP1 rs27044 G allele (OR=1.59) and rs30187 T allele (OR=1.63) were modestly associated with syndesmophyte formation [19]. Another study conducted in Chinese Han AS patients showed that PTGER4 rs10440635 AA genotype was more frequent in severe patients compared to healthy controls (severe AS definition: surgery requirement within the first 10 years of the disease due to inability to stand upright, look straight ahead, or compression of the viscera due to kyphosis) [17]. However, genotype or allele frequencies of severe and normal type of AS were not compared in that study [17]. Differences in ethnicities and radiographic severity definitions might explain the discrepancy in our study. In the Taiwanese study, the mean mSASSS of the syndesmophyte (+) and (-) groups were not reported, and the normal group patients in the Han Chinese study may also have syndesmophytes as implied by the mSASSS scores of groups (severe vs. normal: 36.4±20.7 vs. 7.71±1.86) [17, 19]. A recent study investigating the association of SNPs in genes involved in antigen presentation with radiographic severity and progression reported that ERAP1 rs30187 SNP was associated with the baseline mSASSS in univariate analysis [18]. However, none of the examined SNPs of ERAP1gene were associated with radiographic progression (increase of mSASSS ≥1 unit per year) [18]. Although this is indirectly consistent with our findings, due to the slowly progressive nature of the disease, studies with longer follow-up would be more informative for differentiation of progressors and non-progressors [18]. For the IL23R polymorphisms, a French study reported an association between IL23R rs11209026 SNP and radiographic sacroiliitis in AS patients without addressing vertebral changes [24], whereas a Portugese study found no association between IL23R SNPs and functional status [14].

Consistent with previous reports on radiographic severity [25-27], we found that male gender and coxofemoral joint involvement were associated with more severe axial disease. In contrast to our previous data [28], we could not demonstrate any association between HLA-B27 positivity and radiographic severity in this cohort which might be due to the differences in severity categorization. In the prospective OASIS cohort, HLA-B27 positive male AS patients had more severe radiographic progression [29]. On the other hand, HLA-B27 was not associated with radiographic severity in the TASC and SPARCC cohorts in which the HLA-B27 negativity was low [30]. As previously reported by us and others from Turkey, HLA-B27 positivity in Turkish AS patients is relatively low (~70%) in comparison to other Caucasian populations [28, 31]. Patient selection criteria for radiographic severity and the prevalance of HLA-B27 in the study population may have implications on the associations found.

Lastly, there are several hypotheses that ankylosis starts from entheseal inflammation sites [32, 33]. However, in examination of mSASSS scores and ERAP, IL23R and PTGER4 SNPs’ genotypes of patient who had enthesitis and who did not, we could not observe any significant difference.

Clear differentiation of mild and severe radiographic AS groups is an important stratification to study the effect of genes on axial new bone formation in a disease where the rate of progression is not uniform among patients. This differentiation represents the two extremes of the radiographic severity of AS incorporating time into the equation, therefore enhancing the possibility of determining the factors that may have impact on new bone formation. Thus, the present study is the first to evaluate the effect of these SNPs on axial radiographic severity of AS in such a well-separated group of AS patients.

This study has also some limitations worth noting. First, the sample size was relatively small. However, when we compared the genotype distributions of our entire cohorts, and mild and severe groups with another Turkish AS cohort (HLA-B27 [+] 66.7%) evaluating the association of ERAP1 polymorphisms with AS susceptibility, we found that ERAP1 rs27044 and rs30187 genotype distributions were quite similar to ours [15]. Second, patients in the mild group had relatively shorter disease duration (mean 14.1±4.8 years) than in the severe group and it may be assumed that some of the patients in the mild group could potentially progress to the severe group over time. However, the only long-term study addressing the natural disease course of AS reported that only 26% of the AS patients with mild functional loss after ten years of disease duration experienced heavy functional loss in the follow-up [22]. Likewise, 81% of the patients who had severe spinal restriction after 36 years of follow-up were already severely restricted within the first 10 years, suggesting that a predictable pattern of ankylosing spondylitis emerges within the first 10 years of the disease. Moreover, one of the most important predictors of future vertebral damage is prevalent syndesmophytes at baseline [3, 18, 34]. Accordingly, progression from the mild to the severe group in the next 10 years seems unlikely in our mild-AS patients who had no syndesmophytes within the first 10 years of the disease. Lastly, some of the medications may have an influence on the radiographic severity of AS. However, within the current therapeutic options only long-term NSAIDs or TNFi have been shown to retard radiographic progression to some extent [35-38]. Although we could not retrieve the total NSAID duration and pattern (continous or on-demand) data, previous and current NSAID exposure in both groups were not different. Additionally, TNFi use was significantly less frequent in the mild group that selection bias due to medication effect is unlikely.

Radiographic severity is a marker of long-term damage in patients with AS. Current concepts consider new bone formation as a pathological response to inflammation-mediated injury. However, there are controversial data either supporting or rebuting this hypothesis [1, 2, 39, 40]. Although patients in the severe group had a higher inflammatory burden evident with the higher CRP and TNFi use, we could not find any association between three important SNPs in genes (ERA1P and IL23R) associated with inflammation, and one SNP in PTGER4 gene associated with new bone formation. Further studies are required to clarify the genetic determinants of radiographic progression in AS, and the functionality of these genetic variants.

This study has demonstrated that radiographic severity of AS that is substantially genetically-determined, is not associated with ERAP1, IL23R and PTGER4 gene variants or HLA-B27.