Juvenile idiopathic arthritis (JIA) is an autoimmune disease that is characterized by persistent chronic arthritis and affected by genetic and environmental factors. Different genetic variations have been reported as risk factors for JIA. However, given that many results could not be replicated in individuals of different ancestral origin, it was assumed that heterogeneous genetic factors are involved in this disease. In the present study, we analyzed three single nucleotide polymorphisms (SNPs), namely PTPRC (rs10919563), TYK2 (rs34536443) and PRKCQ (rs4750316), which were found to be associated with JIA in previous studies. We also investigated whether the intron-4 located 27-bp VNTR of endothelial nitric oxide synthase (eNOS), is associated with risk for JIA in Greece. In total, 125 JIA patients and 221 healthy controls from northern Greece were included in the study as a sample set. Samples were then analyzed, and genotyped for the three SNPs with TaqMan primer-probe sets, using a Real-Time PCR platform (ViiA™ 7 Real-Time PCR system), while eNOS VNTR polymorphism was genotyped by PCR. Statistical analysis was performed using a GraphPad Prism statistical program. The χ2 test was used to examine differences of genotype and allele frequencies between patients and controls. Statistical significance was defined by using the two-tailed P<0.05 test. Bioinformatics analysis was conducted by using BlastP, Pymol, Maestro and Desmond. In the case-control association study performed, eNOS only was found to be associated with JIA. Genotype a/a and allele ‘a’ were found in a higher frequency in JIA patients than in controls [p<0.0001, odds ratio (OR)=0.15, 95% confidence intervals (CI): 0.065–0.37; and p<0.0001, OR=0.34, 95% CI: 0.23–0.49, respectively]. No associations with JIA were detected for TYK2, PTPRC or PRKCQ. Aiming to investigate the structural consequences and the structure/function relationships accompanying the Pro1104 to Ala (rs34536443) mutation on TYK2 protein, bioinformatics analysis was performed. Combining three-dimensional (3D)-modeling and molecular dynamics simulations we identified changes in structural flexibility, affecting the functionality of the kinase domain of TYK2. To the best of our knowledge, this is the first time that eNOS VNTR polymorphism is associated with susceptibility to JIA, suggesting a differential role of allele ‘a’ in various complex diseases. The current data emphasize the importance of comparative studies in populations of a different ancestral background towards the clarification of the role of specific alleles in the development of JIA.

Background: The programmed cell death protein-1 (PD-1) and its ligands (PD-L 1 and 2) suppress immune responses, thus promoting self-tolerance. Among the immunomodulatory cells, acting through the PD-1 pathway, are the B-regulatory cells (Bregs). The role of the PD-1 pathway in Juvenile Idiopathic Arthritis (JIA) has not been adequately studied. Aims of the study: To investigate the immunophenotypic profile of T- and B-cells and the activity of the PD-1 pathway in JIA patients. More specifically, we will examine the levels of: a) the soluble form of PD-1 (sPD-1), b) Bregs; and the expression levels of: c) PD-1 on CD4+ and CD8+ T-cells, d) PD-L1 on Bregs and CD19+ B-cells, in blood and synovial fluid samples, at various stages of the disease (onset, relapse, remission, on or off treatment). The above biomarkers will be investigated for correlation with JIA activity. Methods: A case-control study of JIA patients (expected number: 60) and healthy controls (n: 20). Total expected number of samples: 100 of peripheral blood, 120 of serum (solely for soluble markers) and 60 of synovial fluid. The patients’ demographic data and treatment will be recorded. JIA will be classified according to the ILAR and the recently proposed PReS/PRINTO criteria. JIA activity will be assessed using the JADAS-10 tool. The biomarkers will be determined using multiparametric-polychromatic flow cytometry (quintuple fluorescence protocol) and immunoenzymatic assay ELISA. Anticipated benefits: Further elucidation of the immunophenotypic expression and variation of the abovementioned molecules and cells during active inflammation and remission in JIA. Thereby, the present study is expected to contribute to: a) the modern research and understanding of the confirmed immune dysfunction at the cellular level, which leads to the development of serious autoimmune diseases in childhood, such as JIA, and b) the search for biomarkers that could be targets of early “intelligent” treatment and thereby could support the implementation of precision-medicine. The early diagnosis and targeted treatment of JIA are crucial for the maintenance of normal physical functioning and the psychosocial balance of the still growing adolescent/child.

BackgroundJuvenile idiopathic arthritis (JIA) is an autoimmune disease characterized by persistent chronic arthritis, in which both genetic and environmental components are involved [1]. Different genetic variations have been reported as risk factors for JIA, but a difficulty of the replication of results in different ethnic backgrounds indicates the existence of an ethnic heterogeneity of genetic factors for JIA.ObjectivesWe sought to validate three single nucleotide polymorphisms (SNPs), namely PTPRC (rs10919563), TYK2 (rs34536443) and PRKCQ (rs4750316), previously found to be associated with JIA [2–4], and to investigate whether the 27-bp VNTR polymorphism on intron 4 of eNOS, which is associated with various autoimmune diseases so far [5], is associated with risk for JIA in Greece.MethodsThe sample set consisted of 125 JIA patients and 221 healthy controls from Northern Greece. Genotyping of the three SNPs was performed with Taqman primer-probe sets, using a Real-Time PCR platform (Applied Biosystems, ViiA™ 7 Real-Time PCR System), while eNOS VNTR polymorphism was genotyped by PCR. Odds ratios (OR) and 95% confidence intervals (CI) were calculated and the statistical difference in allele distribution was assessed by means of x2 test or Fisher's exact test. Bioinformatic analysis was performed using BlastP, Pymol and Maestro and Desmond (Schrodinger Inc.).ResultsA case–control association study was conducted enrolling 4 successfully genotyped markers. eNOS only was found to be associated with JIA. Genotype a/a and allele “a” were more common in individuals with JIA than in controls (p<0.0001, OR=0.15, 95% CI 0.065–0.37 and p<0.0001, OR=0.34, 95% CI 0.23–0.49, respectively). No associations with JIA were detected for TYK2, PTPRC or PRKCQ. Aiming to investigate the structural consequences and the structure/function relationships accompanying the Pro1104 to Ala (rs34536443) mutation on TYK2 protein, bioinformatics analysis was performed. Combining 3D-modeling and Molecular Dynamics simulations we have noted changes in structural flexibility, affecting the functionality of the kinase domain of TYK2.ConclusionsThis study demonstrated for the first time that eNOS VNTR polymorphism is associated with susceptibility to JIA, thus suggesting that the risk allele “a” may confer susceptibility to clinically distinct disorders. Apart from the previously reported evidence for the role of PTPRC rs10919563, PRKCQ rs4750316 and TYK2 rs34536443 in an increased risk for JIA, our results demonstrate no association of these genes with JIA in the Greek population. However, the lack of association of PTPRC SNP with JIA is in line with previous data reported from cohorts in US and Australia. Taken together, the results highlight the importance of comparative studies in different populations, considering that replication of previously identified markers is paramount to determine which SNPs represent true risk loci, thus pointing towards key disease pathways which warrant further study.References Ravelli and Martini (2007). Lancet 369:7...