HLA Allele Prevalence in Disease-Modifying Antirheumatic Drugs-Responsive Enthesitis and/or Arthritis Not Fulfilling ASAS Criteria: Comparison with Psoriatic and Undifferentiated Spondyloarthritis

Spondyloarthritis (SpA) is a group of inflammatory rheumatic diseases characterized by common clinical features, such as inflammatory enthesitis, arthritis and/or back pain. SpA is strongly associated with human leukocyte antigen (HLA) class I allotype B27. Ankylosing spondylitis has historically been the SpA subgroup with one of the strongest, best-proven associations with HLA-B27. The remaining SpA subgroups, namely psoriatic arthritis (PsA), inflammatory bowel diseases-associated arthritis/spondylitis, reactive arthritis, and undifferentiated SpA (uSpA), have also been associated with HLA allotypes other than HLA-B27. In this retrospective study, we analyzed the association between the HLA class I and II haplotypes and the susceptibility to enthesitis and/or arthritis (E/A). Special attention was paid to E/A responding to disease-modifying antirheumatic drugs (DMARDs) not fulfilling ASAS classification criteria (ASAS−), as compared to ASAS+ forms including PsA and uSpA. The whole E/A group showed significant independent associations with HLA-A28(68), B27, Cw3, Cw12, and DQ1; taken singly, PsA was associated with HLA-B27 and DQ1, uSpA with HLA-B16(38,39) and B27, and E/A ASAS− with HLA-A28(68), Cw8, and Cw12. This study identified novel risk HLA allotypes for different SpA subgroups in an Italian population. HLA typing could aid the diagnosis and treatment of E/A subgroups, including DMARDS-responsive forms not fulfilling ASAS classification criteria.


Introduction
Spondyloarthritis (SpA) is a heterogeneous group of chronic inflammatory rheumatic diseases encompassing different clinical subgroups, namely ankylosing spondylitis (AS), psoriatic arthritis (PsA), inflammatory bowel diseases-associated arthritis/spondylitis (enteropathic arthritis), reactive arthritis, and undifferentiated SpA (uSpA). The latter includes patients with enthesitis and/or arthritis (E/A) fulfilling ASAS classification criteria [1] (E/A ASAS + ) but who cannot be included under the classification criteria of any of the single well-defined ASAS subgroups. SpA can also be clinically classified as axial, peripheral, or combined forms, depending on whether axial or peripheral joints or both [1][2][3] are the predominant sites affected by clinical manifestations.
In daily practice, the rheumatologist is very often faced with patients showing signs and symptoms consistent with E/A. Even in the presence of ultrasound-documented E/A, the diagnosis of SpA is not always straightforward when 2011 ASAS criteria are not fulfilled [1] (i.e., patients with only E/A; E/A ASAS − ), ASAS + patients' disease features do not satisfy any of the classification criteria related to SpA subgroups (uSpA), and/or patient-reported symptoms are not clearly inflammatory [7]. In these cases, the possibility of a mechanical/metabolic E/A cannot be ruled out [6]. Even so, a subgroup of E/A ASAS − patients can still respond efficiently to DMARDs. Due to this uncertainty, it would be helpful to rely on markers to (a) better define E/A ASAS − patients and/or (b) predict a good response to DMARDs and eventually to more advanced therapies [8].
The aim of this study was to investigate the HLA profile in a Caucasian cohort of patients affected by E/A, paying particular attention to the ASAS − as compared to the ASAS + subgroups, namely PsA and uSpA.

Patients and Methods
From 2013 to 2018, 113 consecutive patients with symptomatic peripheral E/A confirmed through ultrasound were recruited at the Rheumatology Research program Unit of the University of Bari. Patients with rheumatoid arthritis (fulfilling the 2010 ACR/EULAR classification criteria), as well as patients with AS, enteropathic, or reactive arthritis, were excluded. Patients with PsA were defined according to CASPAR criteria [19], while uSpA were defined according to the ASAS criteria for peripheral SpA in the absence of a more definite diagnosis (E/A ASAS + ). Patients with E/A, responding to DMARDs, but not fulfilling ASAS criteria, were defined as E/A ASAS − . For each patient, data related to gender, age, and age at the onset of first symptoms, and medical history, including the presence of cardiovascular risk, metabolic syndrome, other autoimmune diseases (systemic or organ specific), fibromyalgia and malignancies, were recorded. Response to DMARDs was assessed according to DAPSA criteria, considering a minimum improvement of 50% from baseline [20,21]. Controls included a total of 318 HLA-I and -II serotyped healthy donors (HD) (female to male ratio 1:1). The study was approved by the Ethics Committees of the University of Bari. All participants gave written informed consent to enrollment as part of a project to study HLA disease markers.

Statistical Analysis
Statistical analyses were performed using Statistical Package for Social Sciences (SPSS) software (v21 for Windows). The Mann-Whitney U test was used to compare the differences between groups for continuous variables. Comparisons between groups for nominal variables were performed using Fisher's exact test. Variables with statistically significant associations were analyzed by multivariate logistic regression, adjusting for gender as confounding variable. Multivariable logistic regression was performed to define the independent association between variables and SpA. For all tests, a p-value < 0.05 was taken to indicate statistical significance.

Results
In this retrospective study, 113 patients with E/A were enrolled, of which 73 ASAS + (54 patients with PsA, 19 patients with uSpA), and 40 ASAS − , responsive to conventional DMARDS. The clinical characteristics of the study population are described in Table 1. The female to male ratio in the whole E/A cohort (wE/A) was 4:1, mean age ± SD was 53.8 ± 11.4, and mean age at the onset of the first symptoms was 44.5 ± 12.7. The mean body mass index (BMI) was 26.7 ± 4.4. No individuals in the uSpA or E/A ASAS − groups had cutaneous psoriasis (Table 1). In addition, the HD group female to male ratio was 1:1 with a mean age ± SD of 34.82 ± 12.21. As expected, the item "family history of psoriasis" was statistically higher in the PsA than in the uSpA (p < 0.001) or ASAS − (p < 0.001) subgroups (Table 2). Variables such as "peripheral and axial arthritis," "smoking," "hypertension," "cardiovascular risk," "metabolic syndrome," "autoimmune thyroiditis," "fibromyalgia," and "malignancy" were similarly distributed between subgroups (Table 2). Interestingly, significant differences in age at the onset of symptoms were found between PsA and ASAS − (p = 0.03), being higher in ASAS − . As expected, the HLA-B27 distribution percentage in the wE/A group (p = 0.001) and its subgroups, PsA (p = 0.001) and uSpA (p < 0.001), was significantly higher than in the HD group (Table 3).

Discussion
To the best of our knowledge, this is the first study to investigate the association between HLA class I and class II alleles and E/A ASAS − (vs. ASAS + PsA and uSpA) in a cohort of Caucasian patients (n = 113) as compared to an HLA-typed HD cohort (n = 318).
While previous studies investigated the association between PsA [22] and HLA-B27 only, our analysis was focused on alleles other than HLA-B27 in E/A patients and its subgroups, PsA, uSpA, and ASAS − .
In addition to HLA-B27, in the wE/A cohort, our data revealed a significant association with HLA-A28(68), Cw3, Cw12, and DQ1. All these associations, except the one with HLA-Cw12, were independent of HLA-B27, as demonstrated by multivariable regression analysis. Regarding HLA-Cw12, it was not possible to establish the interdependence with HLA-B27, because this allele was not present in any HD.
Interestingly, we found that both HLA-A28(68) and Cw12 were also associated with ASAS − but not with PsA or uSpA, suggesting that these alleles can confer susceptibility to ASAS − . While HLA-A28, with its splits 68 and 69, has been demonstrated to be significantly associated with B27 risk-related diseases, including AS [23], reactive arthritis [23], juvenile chronic polyarthropathy [23], and intermediate uveitis [24], only a minimal predisposition to cutaneous psoriasis has been reported for HLA-Cw12 [25,26].
ASAS − , which was the only E/A subgroup not associated with HLA-B27, presented an additional association with HLA-Cw8, never previously found to be associated with E/A or its subgroups.
Regarding HLA-Cw3 and HLA-DQ1, both of which favor wE/A, the former was reported to have a statistically high prevalence in other immune-mediated diseases such as rheumatoid arthritis-associated vasculitis [27,28], while HLA-DQ1 was shown to be a predisposing factor for autoimmune uveitis, the most common extra-articular manifestation of SpA, in a cohort of Caucasian Italian patients [29]. Furthermore, HLA-DQ1 and HLA-DQ2 have been associated with the presence of anti-Ro/SSA antibodies in both Sjögren's syndrome [30] and systemic lupus erythematosus patients [31].
In our study population, HLA-DQ1, along with HLA-B27, was also associated with PsA. The prevalence of PsA patients positive for HLA-B27 in our investigation was 13%, comparable to the 12% reported by Paladini et al. [22] in an Italian Caucasian cohort.
Several studies have shown that HLA-Cw6 is strongly associated with cutaneous psoriasis, but not with PsA [32,33]. The lack of an established diagnosis of cutaneous psoriasis in 51% of our PsA group can explain the absence of this subgroup association with HLA-Cw6.
A major study limitation is the lack of longitudinal observations that could establish whether ASAS − forms could eventually evolve into any other E/A subgroup, although the peculiar association of ASAS − with HLA-Cw8 makes this possibility unlikely.
The role of gender in influencing some HLA associations deserves some comment in that certain HLA allotypes, found to be significantly associated with wE/A (HLA-DQ3), uSpA (HLA-Cw7) or ASAS − (HLA-B15(62)) by Fisher's exact test, lost their significant association when "gender" was included as covariate in the multivariate analyses. These findings suggest that gender had an impact on the expression of certain HLA alleles in the E/A groups in our study, in agreement with previous studies showing gender-related HLA differences in different autoimmune diseases [39][40][41][42].
Finally, our work paves the way for further investigation aimed at genotyping the HLA alleles found in this study to be associated with uSpA or ASAS − .

Conclusions
Our study revealed that HLA-B27 and HLA-B16 (38,39) are significantly more frequent in uSpA patients, while HLA-A28(68), HLA-Cw8, and HLA-Cw12 were found to be associated with ASAS − in the general population in Italy. These alleles can thus be associated with a diagnosis of ASAS − , and HLA typing may contribute to correct clinical management, as well as to identifying family members at risk, in particular those patients with enthesitis and/or arthritis who do not meet the criteria for a uSpA diagnosis. These findings warrant confirmation in further large, multicenter cohort studies.
Funding: This work was supported by a grant from the University of Bari Medical School 2020. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Institutional Review Board Statement: All procedures performed in studies involving human participants were conducted in accordance with the ethical standards of the institutional and/or national research committee (Ethical Committee of the University of Bari Medical School, no. A31580/DS) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy restrictions.