Background. The seronegative spondyloarthritis (SpA) are a group of chronic inflammatory diseases resulting from a complex interplay among genetic background (mainly represented by HLA-B27) and environmental factors, that leads to the activation of autoinflammation and the dysregulation of the immune-system.
In many cases, an early diagnosis and an appropriate monitoring of disease activity can be difficult because of the overlap of clinical features. Furthermore, because of the indices of inflammation, erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP), are in the normal range in at least half of SpA patients with a clear expression of disease activity, a delay in diagnosis and consequently in treatment in these patients has been documented. This imparts a tremendous symptomatic burden and loss of function in these patients during the productive years of life. For all these reasons, much attention is currently devoted to the identification of biochemical and genetic biomarkers to be used in the diagnosis as well as prognostic factors in evaluating the treatment effectiveness.
Among the genetic predisposing factors, a well-known role is that of HLA-B27, which contributes however to only 20–30% of the total heritability, whereas the whole major histocompatibility complex (MHC) accounts for about 40–50% of the genetic risk of developing SpA. This suggested that other genes are involved in pathogenetic mechanism. In fact, in addition to HLA-B27, a number of genetic factors in both, MHC and non-MHC locus, have been claimed to play a role in pathogenesis of SpA.
In this context, because of TNF-α is primarily involved in the propagation and perpetuation of inflammation in SpA, the study of TNF-α genetic is of great interest. Several polymorphisms (SNPs) in genes involved in TNF-α signalling, as TNFA, TNFSF15, TNFR1 and TRADD genes, have been identified as associated with SpA, even if results are controversial. Of great interest are also variants in MEFV gene, involved in the pathogenesis of the autoinflammatory disorder Familial Mediterranean Fever (FMF). Recent studies have shown that the SpA, and in particular the ankylosing spondylitis (AS), are very common among patients affected by FMF and that these patients can present with AS as a sole manifestation.
The present study, conducted in a cohort of 91 SpA patients and 223 controls, coming from a North-East Italian region, was aimed to identify biohumoral (biochemical and haematological) and genetic factors to support the diagnostic and prognostic (response to therapy) work-up of SpA diseases. In particular, in addition to biochemical and haematological indices, we investigated whether SNPs in the promoter region of TNFA, or SNPs in the autoinflammatory TNFRSF1A and MEFV genes, might concur with HLA-B27 in enhancing the risk of developing SpA disease and/or in predicting the response to anti-TNFα drugs.
Methods. The study population comprised 91 patients with a diagnosis of SpA (mean age ± standard deviation: 52.1 ± 12.5 years; 57 males, 34 females) and 223 blood donors (mean age ± standard deviation: 46 ± 11 years; 146 males, 77 females) coming from Veneto Region, a North-East Italian region. Among patients, 36 had a diagnosis of AS and 55 patients of psoriatic arthritis (PsA), which were based on New York and CASPAR criteria respectively. The protocol of this study was approved by the Local Institutional Ethic Committee of University-Hospital of Padua, Italy (Prot.n. 3024P/13), and all participants gave written informed consent before entering the study.
Demographic and physiological data, medical and familial history data were collected for each participant. Blood samples were collected and complete blood count, CRP, ESR, uric acid, prealbumin, alanina aminotransferase (ALT) and glucose were evaluated.
Direct sequencing of MEFV (exons 2,3,5 and 10) and TNFRSF1A (exons 2,3,4 and 6) genes were performed. HLA-B27 and TNFA polymorphisms (-1031T>C;-857C>T;-376G>A;-308G>A;-238G>A) were assayed by Real Time-PCR. HLA-CW6 allele presence was analysed by molecular genetic testing using a commercially available CE-IVD microarray. Statistical analysis was performed using STATA software (version 13.1).
Results. An higher number of circulating polymorphonuclear cells and higher CRP levels could be detected in SpA patients with respect to controls, and in PsA higher levels of ALT could be observed with respect not only to controls but also to AS. Anyway these indices were not highly elevated and often comprised within the reference intervals.
As expected, HLA-B27 was associated with AS (χ2=120.1; p<0.0001). Although a slightly higher frequency of HLA-CW6 carriers was observed among patients with AS (about 6%) or PsA (about 13%) with respect to controls (about 4%), the difference was not statistically significant.
Any single studied TNFA SNP was not associated with SpA diagnosis, nor with AS or PsA considered singly. The haplotypes deriving from the pairwise combinations of the five studied SNPs were also statistically inferred. The most frequent haplotypes in controls were selected as references, and only the haplotype -1031C/-308G was significantly associated with AS (p=0.015) exerting in this disease a protective role (Odds Ratio: 0.43; Confidence Interval 95%: 0.22-0.85).
Three SNPs were identified in TNFRSF1A gene and among them, only the R92Q (Minor Allele Frequency- MAF=0.034) and the c.625+10A>G (MAF=0.479) were selected for their potential functional implications. Both SNPs were not associated with the presence of SpA (χ2=1.073 and p=0.300 for R92Q; χ2=4.721 and p=0.094 for c.625+10A>G), but c.625+10A>G was associated with the response to anti-TNF therapy, assessed by BASDAI score lower /equal or higher than 4 at 10 months (p=0.031).
Twenty-one SNPs were identified in MEFV gene and among them, 10 with a known potential functional significance. Variant alleles were extremely rare in our population (MAF0.05).
Conclusions. In conclusion the results of this study indicate the relevant role of TNF-TNFR pathway genetics in the complex network causing SpA and conditioning response to therapy. TNFA was shown to be a predisposing factor for SpA, but mainly for AS, among Italian patients, while genetics of the autoinflammatory gene MEFV appears of no impact in this setting. The haplotype resulting from TNFA-1031C/-308G, potentially associated with lower TNF-α production, exerts a protective role in AS, while the TNFRSF1A c.625+10A>G polymorphism emerged as a potential predictor of response to anti- TNFα therapy
