Clinico-immunological profile of seronegative axial spondyloarthritis with special emphasis on the role of human leukocyte antigen B27

INTRODUCTION

Seronegative spondyloarthritis (SpA) is characterized by inflammatory arthritis with overlapping features and is negative for all serological markers. The worldwide prevalence of SpA ranges from 0.2% to 1.6%.^[1] B27 association is with various classes of seronegative SpA, the majority including ankylosing spondylitis (AS) (75–90%). Other clinical entities under seronegative spondyloarthritis are also associated with B27 including 75-90 % of undifferentiated SpA, 20-50% in psoriatic arthritis (PsA), 30-60% of reactive arthritis (ReA) and 10-40% enteropathy related arthritis (EA).^[1] All the subtypes of SpA share the majority of clinical characteristics with only a few defining features that may differentiate between them. The main clinical features include back pain, axial/peripheral arthritis, and inflammatory bowel disease.

AS is the prototype and most common SpA with a very strong H+ association. Human leukocyte antigen B27 (HLA-B27) is now a diagnostic criterion in the Assessment in Spondyloarthritis International Society classification criteria (ASAS) for SpA.^[1] The prevalence of + in radiographic axial SpA ranges from 71% to 85% in various studies.^[2] The frequency of H+ in the control/healthy population ranged from 2% to 3.5% in various Indian studies.^[3,4]

However, HLA-B27 is not an essential criterion for diagnosis of SpA and very few studies are available comparing the differences between disease phenotype in H+ and H− groups. Even fewer studies are available in the eastern part of North India, where limiting factors include the lower socio-economic status of a relatively large fraction of the population and the lack of advanced diagnostic facilities for genetic studies.

This study aimed to describe the features of SpA in this region of India and also to study the clinical and immunological differences between H+ and H− patient groups.

MATERIALS AND METHODS

This is a retrospective observational and cross-sectional study conducted during the period between 2020 and 2022 in the University Grants Commission Advanced Immunodiagnostic Training and Research Center. Two hundred and eighteen patients with features of axial SpA were diagnosed by the physician/rheumatologist according to ASAS/European Spondyloarthropathy study group criteria in our outpatient department and were recruited in the study.^[5,6] Exclusion criteria included unavailability of clinical or HLA-B27 data. Detailed clinical history, family history, and radiological details were noted, other laboratory and immunological test details were recorded, and HLA-B27 was done by polymerase chain reaction (PCR)-sequence-specific primer method (BAG Diagnostics).

HLA-B27 allele-specific primers were added to purify deoxyribonucleic acid (DNA), and amplification was achieved only if the primers completely matched the target allele. The mismatched primers, due to the absence of HLA-B27, did not generate amplicons. Gel electrophoresis was done to separate and analyze the PCR products. Internal control band was seen in all cases and controls to indicate the integrity of the PCR reaction. In HLA-B27 positive cases, two bands were observed, the band of B27 had a length of 420bp and/or 85 bp. In HLA-B27 negative patients, only a band of internal control was observed.

Routine investigations and quantitative C-reactive protein (CRP) were performed (Immage 800, Beckman Coulter, USA). Immunological tests in the form of serum antinuclear antibody (ANA), double-stranded DNA (dsDNA), anti-Sjögren’s syndrome-related antigen A (anti-SSA), anti-Sjögren’s syndrome-related antigen B (anti-SSB), anti-Smith, anti-centromere, anti-Jo-1, anti-RNP, and anti-Scl-70 were done. ANA was done by indirect immunofluorescence and dsDNA by enzyme immunoassay. Specific markers were tested by dot blot immunoassay from AESKU diagnostic, Germany.

Ethical approval was taken from the Institutional Ethics Committee. To statistically compare between H+ and H− groups, the Chi-square test was done using the Statistical Package for the Social Sciences software version 21.

RESULTS

A total of 116 H+ and 102 H− patients were included in the present study. The male-female ratio was more skewed toward males (6:1) in H+ than H− (2:1). H+ patients were younger with a mean age of 28 years compared to mean age of 33 years in H−. Ten patients were younger than 16 years of age, out of which seven were H+ (70% of juvenile SpA) and three were H−(30% of juvenile SpA).

The clinical and radiological features of sacroiliitis were much more significant in H+ (83%) versus H− (20%) [Table 1].

Multiple joint pain was significantly more associated with H+ (57%) rather than H−(41%). Morning stiffness was also much more common in H+ (95%) than H− (62%) and this association was statistically significant.

Uveitis was significantly associated with HLA positive SpA (24%) when compared to HLA negative SpA (6%).

Other features such as spondylitis, skin lesions, photosensitivity, oral ulcers, gastritis, uveitis, and urinary tract infection (UTI) were more common in H+ than in H−, but this was not statistically significant. Few patients of H+ had a history of infections such as tuberculosis (six patients) and Human immunodeficiency virus (HIV) (one patient) while H− had no such history of infections. Genetic associations in the form of a positive family history of SpA and related features were observed in both groups but it was slightly more common in H+ than H− group.

Other autoantibodies were also sometimes associated with SpA such as ANA (seen in one HLA-B27 negative patient) and RA factor (seen in two patients each in both HLA groups). Anti-cyclic citrullinated peptide (Anti-CCP) was positive in 3% of HLA-B27 negative SpA.

In the laboratory investigations performed, raised CRP titers were seen more commonly in H+ (83%) versus H− (20%) and this association was significant (P < 0.05).

DISCUSSION

The present study showed male predominance, but in H+ group, the male: female ratio was 5.8:1, and in H− group the male: female ratio was 2:1. Similar findings with greater male predominance in HLA-B27 positive SpA were seen in other studies by Zhang et al. and Yang et al.^[2,7,8] However, a study by Arévalo et al. showed male-female ratio of 3:1 with no difference between the two groups.^[9] Indian studies also showed male predominance in both groups.^[10] Aggarwal et al. also showed a male-female ratio of 5:1 similar to the present study^[11] [Table 2].

The present study showed no significant difference in the family history of SpA in both groups. Other studies also showed an association of family history in both groups but stronger with HLAB27.^[2,7,8] Study by Aggarwal et al. showed positive family history in 53% of patients.^[11] In HLAB27 negative patients, there may be other genetic associations leading to familial transmission. Parasannanavar et al. reported a study of HLA-B27 negative patients of SpA with significantly increased HLA B*07 positivity and decreased HLA B*40.^[12]

Our study showed H+ patients to be significantly younger than H− group. A similar result was reported by other studies also.^[2,7-9] An Indian study by Shougrakpam et al. showed slightly younger age in the HLA-B27 positive group (32 vs. 35 years).^[10]

Radiological features of sacroiliitis were significantly associated with H+ compared to H− patients. A study done by Chung et al. and Yang et al.^[7,8] showed likewise results and increased predominance of sacroiliitis in H+ patients; however, Zhang et al. found no difference in musculoskeletal disease in the two groups.^[2,7,8] Shougrakpam et al. showed significantly more axial skeleton involvement in HLA-B27 positive patients (86%).^[10]

Multiple joints involvement including peripheral joints and morning stiffness was significantly correlated with H+ in the present study (57% vs. 41% in H−). Similarly Yang et al. also reported increased peripheral arthritis (approx. 40%), hip joint involvement and lumbar spine involvement (when compared to thoracic or cervical) in H+.^[8] A study by Arévalo et al. and Chung et al. showed contradictory results with a higher prevalence of peripheral arthritis in HLA negative group.^[7,9] Zhang et al. showed no difference in joint involvement between the two groups. The present study shows a greater proportion of patients with multiple joint involvement compared to other Western studies showing 30%-50% of patients with peripheral arthritis.^[2] However, another Indian study showed that 62% of patients with peripheral joint involvement, although the difference was not significant.^[10] Aggarwal et al. reported an even higher proportion of peripheral arthritis (66%).^[11] It is possible, that patients in India are showing more frequent peripheral joint involvement including hip joint and knee joint.

Uveitis was more common in H+ (24%) and this association was also significant in the present study. Similarly, other studies also showed a significant association of H+ with uveitis. The prevalence of uveitis ranged from 10% to 38.5% in different studies and a meta-analysis showed pooled prevalence of 25.8%.^[2,13]

Other features such as spondylitis, skin lesions, photosensitivity, oral ulcers, gastritis, and UTI were more common in H+ than in H− but the association was not statistically significant. A study by Zhang et al. shows the association of psoriasis with absence of HLA-B27. Furthermore, the absence of HLA-B27 would understandably lead to a delay in diagnosis as in this study.^[2] Chung et al. reported a significant association of extra-articular manifestations with H− SpA.^[7]

Many studies have found H+ to be significantly more associated with family history when compared to H− patients.^[2] However, the present study showed slightly more patients with a family history in H+ than H− group; however, this was not statistically significant. Other studies showed lesser familial inheritance (approx. 22.5% in Yang et al., 11% in a study by Zhang et al.) in H− patients than in the present study (35%).^[2,8] This finding of increased familial clustering in the Indian population will have to be verified by larger studies.

Other autoantibodies such as anti-CCP (seen in 3% in H−), rheumatoid factor (2% in both groups), and ANA (1% in HLA-B27 negative patients) were seen. Minor levels of autoantibodies are also seen in healthy populations and this finding could be incidental. Still, the possibility of co-existence of other autoimmune diseases along with SpA cannot be ruled out in these patients. A study from India also reported the presence of autoantibodies in SpA patients.^[14]

Many Indian and Western studies have noted higher disease activity and higher CRP titers in H+ patients like the present study.^[10] Mishra and Singai reported 42% CRP positivity in their study.^[3]

Limitation of this study is that follow-up is not available, so the difference between the two groups in prognosis and response to treatment is not evaluated.

CONCLUSIONS

The present study is one of the very few Indian studies elaborating on the impact of HLA-B27 in the phenotype of axial SpA. The significant differences in the two groups of SpA based on HLA-B27 positivity include predominance of male gender, younger age, radiological features of sacroiliitis, morning stiffness, involvement of multiple joints, uveitis, and raised CRP in H+. Furthermore, H− patients also had a positive family history in 35% of patients, thus pointing toward other genetic associations apart from HLA-B27. More studies on larger sample sizes and follow-up are needed to confirm these findings as well as to search for other susceptibility genes.

As the spectrum of manifestations of SpA is quite variable ranging from musculoskeletal to extra-articular manifestations like uveitis, these patients present frequently in orthopedics for backache, and the role of the orthopedician is to keep the clinical features of seronegative SpA in mind, know what tests to perform and refer to a rheumatologist if diagnosed.