There is current debate on whether nr-axSpA is a different disease from AS [8,9], an early form of AS [10,11] or whether both are two expressions of the same disease [12,13]. In the present study, we analysed the clinical and demographic characteristics, laboratory markers and SNPs associated with the susceptibility to develop AS, Ps, PsA and IBD in 62 patients of the University Hospital of Basurto (Bilbao, Spain), with the aim of determining whether these two entities, apart from sharing clinical characteristics, had a common genetic background.
It was observed that the patients with AS had a lower symptom onset age, shorter diagnostic time and longer duration of the disease compared to the patients with nr-axSpA (Table 2). This could be due to the fact that AS can be more aggressive than nr-axSpA, with a faster radiographic progression, which would imply an earlier symptom onset and diagnosis compared to nr-axSpA. Regarding peripheral manifestations (arthritis, enthesitis, dactylitis), our data confirm those obtained in other studies related to the prevalence of enthesitis in the two patient groups [10-12]. However, the prevalence of peripheral arthritis and dactylitis was greater in AS patients [10-12] (Table 2). With respect to extra-articular manifestations, we found a greater prevalence of uveitis among the patients with AS, as has been reported in other studies , which could be due to the fact that uveitis is associated with the duration of the disease, which is longer in AS  (Table 2). Nevertheless, the prevalence of Ps and IBD is greater among individuals diagnosed with nr-axSpA, which is in contrast to what has been reported in the literature to date [10-12] (Table 2).
The BASMI index is statistically higher among individuals diagnosed with AS [11,12,25,32] (Table 2), due to the decreased mobility of the axial skeleton as a consequence of the formation of syndesmophytes and even bone bonds and changes in the SIJs . The BASFI and BASDAI indices, which measure the clinical activity, damage and functional deterioration, would be expected to be higher in individuals with AS, given their greater severity and progression [10,11,32]; however, in the present study, higher values of these indices were found in those individuals with nr-axSpA (Table 2). In this study, high levels of CRP were more frequent in patients with AS (Table 2), which could be due to the fact that axSpA patients with high CRP values tend to progress more rapidly to radiographic sacroiliitis .
Regarding the gender of the patients, our results confirmed the predominance of males among the individuals diagnosed with AS (77.55%) (Table 2), which could be due to the fact that these patients have a faster progression than females, as well as more structural changes, which in turn, may cause a more severe disability . Another possible influencing factor is physical activity, which, in the case of men usually involves greater mechanic stress, which would increase the inflammatory activity . However, in the group of nr-axSpA patients, the proportion of women (46.15%) was very similar to that of men (53.85%) (Table 2), as has been described in other studies conducted in European and North American cohorts [8,11,12]. The explanation suggested for the differential prevalence between the two entities in terms of gender is that women have a lower radiographic damage and a slower progression to a radiographic state, remaining in the non-radiographic form for longer periods of time , suggesting the possible differential role of hormones in the formation of new bone material in patients with SpA.
Some studies carried out in relatives and twins indicate that the genetic component has a fundamental role in the immunopathogenesis of SpA, although the primary trigger is still unknown . Allele HLA-B27, which has been traditionally granted considerable relevance, could contribute to up to 25% of the total inheritance of AS . In the present study, HLA-B27 showed a significantly greater frequency among the patients with AS compared to those with nr-axSpA (87.8% vs 38.5%), which is in line with the results obtained in other studies [12,28,53-55] (Table 2). Furthermore, this greater frequency of HLA-B27 in the AS group confirms the hypothesis that associates the presence of HLA-B27 with a lower onset age that characterize the AS and an earlier diagnosis  (Table 2).
Apart from allele HLA-B27, 6 additional alleles of the HLA-B gene were found in the patients of this study (HLA-B39, HLA-B40, HLA-B47, HLA-B55, HLA-B56 and HLA-B07). Alleles HLA-B39, HLA-B40 and HLA-B47 have been previously associated with AS [65-67] and, in the case of HLA-B39, with PsA . However, allele HLA-B07 is associated with a decrease in the risk of developing AS  and alleles HLA-B55 and HLA-B56 are not associated with AS. Although the proportion of individuals with other alleles of the HLA-B gene different from HLA-B27 is greater in the group with nr-axSpA with respect to the AS group (61.5% vs 12.2%), both entities share some alleles (HLA-B40, HLA-B55 and HLA-B56). According to these data, it can be stated that there is a predominance of allele HLA-B27 in patients with AS (87.8%); however, the patients with nr-axSpA showed a greater heterogeneity regarding the alleles of the HLA-B gene.
GWAS studies have revealed a considerable number of genes or gene regions that contribute to the susceptibility to develop AS. In our study, we analysed the allele frequencies of 28 risk SNPs, and found no statistically significant differences between the AS and nr-axSpA patients. It was observed that 75% of the individuals of both groups shared a large number of risk alleles (60%) (Figure 1). These results indicate that both pathological entities have a common genetic background, at least at the level of risk SNPs.
At the genotype level, no statistically significant differences were found between the two groups of patients for risk SNPs located in genes involved in the pathogenesis of AS. Some SNPs showed similar genotypes in most of the individuals of the two groups, e.g., ERAP1 (rs30187), ERAP2 (rs10045403), IL-23R (rs11209026), GPR25 (rs41299637), and in intergenic region 2p15 (rs10865331) (Figure 2). Outside of the MHC, it is worth highlighting the ERAP1 gene for its strong association with the susceptibility to develop AS. ERAP1 is an aminopeptidase involved in the cleavage of peptides to a length of 8-9 amino acids, in order for the new peptide segments to be presented by MHC class I molecules, such as HLA-B27 . The SNPs located in the ERAP1 gene only appear to be associated with AS in HLA-B27(+) and HLA-B40(+) patients [65,67]. The ERAP2 is another aminopeptidase, whose association with AS has been described in HLA-B27(+) and HLA-B27(-) patients . The high frequency of risk SNPs of the ERAP-1 and ERAP-2 genes found in the analysed patients could suggest the alteration of the correct functioning of both genes , thus influencing the supply of optimum peptides for MHC class I molecules, such as HLA-B27, which could explain their relationship with the development of SpA [48,70].
The IL-23R gene encodes for IL-23 receptor. IL-23 is a key cytokine involved in the differentiation of naive CD4+ T-lymphocytes into Th17 lymphocytes, which produce IL-17, IL-6, IL-22, TNF-α and other similar proinflammatory cytokines. Furthermore, other genes of the IL-23 route have been associated with AS, which shows that this route is an important pathway in the pathogenesis of AS [46,48,49]. There are polymorphisms of IL-23R, such as rs11209026, associated with AS , IBD  and Ps . This SNP has been found with a very high frequency among the patients of this study (Figure 2), which confirms the evidence that IL-23 and its entire pathogenic route are involved in the susceptibility to develop axSpA, granting inflammation a main role in the triggering of the disease.
GWAS studies have shown the association of SNPs of intergenic region 2p15 (rs10865331) with AS, although their role in the pathogenesis of AS is unknown. It has been hypothesised that this region has non-coding RNA species or protein-coding genes unknown to date, which could be involved in the susceptibility to develop AS . G-protein coupled receptor 25 (GPR25), which is associated with AS  is strongly expressed in memory T-cells and NK-cells and involved in the positive regulation of B-cell proliferation , suggesting a potential mechanism in different autoimmune diseases. Although it is still unknown how the SNPs located in 2p15 (rs10865331) and GPR25 (rs41299637) may influence the pathogenesis of AS, their high prevalence in our study could suggest a key role in the onset and development of axSpA (Figure 2).
The results obtained for SNPs located in genes (ERAP1, ERAP2, IL-23R, GPR25) and gene regions (2p15), along with the different HLA-B gene subtypes, demonstrate their importance in the pathogenesis of axSpA, probably with an essential role in the onset of both AS and nr-axSpA. Despite the fact that the individuals who suffer from different axSpA have common genotypes for some SNPs of genes that are important in the pathogenesis of AS, we observed great genetic heterogeneity within each group and between the two pathological entities, which demonstrates the complex nature of this type of diseases (Figure 3).
Moreover, given that AS, IBD and Ps could have a common pathogenic mechanism, we analysed 15 SNPs associated with the susceptibility to develop IBD, Ps and PsA, finding no statistically significant differences for any of these SNPs between the two groups of patients (AS and nr-axSpA). However, the combination of the risk genotypes of 2 SNPs located in the NOD2 gene associated with CD was found mainly in individuals with AS (AS: 30.7% vs nr-axSpA: 7.7%), which could indicate that these two SNPs are associated with a greater progression of this disease (Figures 4 and 5). The NOD2 gene was identified as the most relevant risk factor of CD , although its association with AS had not been described to date. NOD2 is an intracellular pathogen recognition receptor  and plays a role in the immune response to bacterial lipopolysaccharides, since it regulates the response of Th17 cells for the elimination of bacteria by inducing the secretion of cytokines IL-23 and IL-1B, which appear in individuals with CD who have a mutation in NOD2 . The results suggest that individuals with these two SNPs of the NOD2 gene will be at greater risk of developing intestinal lesions characteristic of IBD throughout their lives (Figure 4). Furthermore, the presence of these and other SNPs located in genes associated with AS and IBD would confirm the hypothesis about the relevance of intestinal dysbiosis in the genesis of axSpA .
Between 20-25% of the known inheritance of AS is attributed to allele HLA-B27, and 3-7% to SNPs identified in GWAS studies [35,36], thus approximately 70% of the inheritance of this pathology could be related to genetic variants that have not been described to date. Missing heritability is a common issue in complex genetic diseases, and it may be caused by multiple factors. It has also been suggested that general heritability is overestimated, with the probability that epigenetic factors, especially environmental factors (smoking and obesity), may be more relevant in the susceptibility to develop AS.
Smoking is an important environmental factor in the inflammatory process of rheumatic diseases, including SpA, and it is a risk factor of cardiovascular disease. In the present study, 66.1% of the patients were smokers or ex-smokers, with a higher proportion of smokers in the individuals diagnosed with AS (69.4%) with respect to those diagnosed with nr-axSpA (53.9%) (Table 2). This suggests that smoking could contribute to increasing the progression to the radiographic form of SpA , promoting spine damage and an earlier onset of the inflammatory back pain, which characterises AS .
Obesity and overweight are an increasing issue worldwide due to their influence on the development of metabolic, cardiovascular and rheumatic diseases, which increases the mortality and morbidity of patients [77,78]. In our study, it was observed that 61.3% of the patients were obese (BMI>29.99 kg/m2) or overweight (BMI>24.99 kg/m2), with BMI and the prevalence of obesity being similar between patients with AS and those with nr-axSpA (Table 2). Excess adipose tissue in overweight and obese individuals may have immunomodulating properties that affect the course of the disease , which could be associated with an increase in the production of proinflammatory cytokynes . Previous studies have reported a greater prevalence of obesity and overweight  and a significantly higher BMI in AS patients compared to the healthy population . It has been described that overweight and obese patients with AS have greater functional limitations, greater activity of the disease and a lower response to anti-TNF-α therapies [79,82], which would contribute to increasing physical inactivity and, thus, gaining weight [80,82]. The high prevalence of smoking and obesity or overweight among the individuals analysed in the present study would suggest the relationship between these life habits and the development of these diseases, with inflammation playing a key role in their triggering.