Despoina Dimopoulou

Investigation of juvenile idiopathic arthritis susceptibility loci: results from a Greek population.

The strategy of studying the putative role of RA susceptibility genetic factors in the development of juvenile idiopathic arthritis (JIA), an autoimmune disease characterized by persistent chronic arthritis, has been proven highly successful so far. Moreover, accumulated evidence indicates that an ethnic heterogeneity of genetic factors exists for rheumatic disorders. We investigated whether five single nucleotide polymorphisms (SNPs), previously found to be associated with JIA in various populations so far, are also associated with JIA in Greece. The sample set consisted of 128 Caucasian JIA patients and 221 healthy controls from Northern Greece. Five Single Nucleotide Polymorphisms (SNPs) markers, namely TRAF1/C5 rs10818488, PTPN22 rs2476601, STAT4 rs7574865, CD247 rs1773560 and PTPN2 rs7234029 SNPs were genotyped in a case-control study with Restriction Fragment Length Polymorphisms (RFLPs) or Taqman primer-probe sets. This study demonstrated for the first time in a Greek population that the PTPN22, TRAF1/C5 and CD247 polymorphisms examined are associated with an increased susceptibility to JIA, thus suggesting that the respective risk alleles may confer susceptibility to clinically distinct disorders. However, our results did not demonstrate any association of STAT4 and PTPN2 SNPs with the disease in our population, thus highlighting the importance of comparative studies in different ethnic populations.

Infliximab as a treatment option for patients with rheumatoid arthritis and primary biliary cirrhosis

Rheumatoid arthritis (RA) is a chronic inflammatory systemic disease which commonly requires treatment with biologic agents targeting various inflammatory pathways. Tumor necrosis factor alpha is a proinflammatory cytokine which plays a pivotal role not only in the pathogenesis of RA but also in other autoimmune diseases such as primary biliary cirrhosis. The co-existence of more than one autoimmune disorder in the same individual is very challenging in the daily practice as therapy strategies applicable to one disease setting may cause clinical and/or biochemical relapse of the other clinical entity. As a result, treatment options able to control different diseases are highly desirable among rheumatologists and other specialties. In that respect, we present a case of a 61-year-old female patient with RA and concomitant primary biliary cirrhosis with poor clinical response to conventional disease-modifying drugs for RA. The introduction of tumor necrosis factor alpha antagonist infliximab led to significant clinical improvement of RA and to stabilization of liver function. In this case review study, we discuss aspects of pathophysiology of primary biliary cirrhosis associated with tumor necrosis alpha and we review the available data of similar published cases.

Low disease activity—irrespective of serologic status at baseline—associated with reduction of corticosteroid dose and number of flares in patients with systemic lupus erythematosus treated with belimumab: A real-life observational study

BACKGROUND Low disease activity is a validated target of current systemic lupus erythematosus (SLE) therapy. The aim of this study was to assess the ability of belimumab to achieve low disease activity states in real-life settings. METHODS Multicentre prospective observational study of consecutive SLE patients receiving belimumab for at least 3 months, due to active disease refractory to at least one conventional immunosuppressant. Disease activity, including the recently defined lupus low disease activity state (LLDAS) and remission (clinical SLEDAI-2K = 0), accrual of organ damage, flares and side effects were documented. RESULTS Ninety-one patients were included [94.5% women, mean (SD) age 45.9 (12.5) years]. Most frequent manifestations were arthritis (76.7%), rash (72.5%), serologic activity (low C3/C4 and/or high anti-dsDNA; 54.9%), hair loss (47.2%) and mucosal ulcers (27.5%). Median (range) duration of treatment was 10.5 (3.0-42.1) months. Belimumab significantly decreased average SLEDAI-2K, physician global assessment (PGA) and daily prednisone dose over time, as early as 3 months after initiation, with over 20% of patients discontinuing corticosteroids. Although reduction in clinical (i.e., excluding serology) SLEDAI-2K was more pronounced in patients who were serologically active (from 8 to 1.5 at 12 months) as compared to serologically inactive (from 6 to 4) at baseline, attainment of LLDAS did not differ between the two groups and was reached by more than 40% of completer patients after 9-12 months. In addition, the number of flares and severe flares was reduced by 62% and 50%, respectively, during the first 12 months of treatment. Twenty patients (22.0%) discontinued treatment due to inadequate response and two due to side effects potentially related to the drug. CONCLUSIONS In real-life, belimumab is efficacious in achieving low disease activity in over 40% of unselected patients, in combination with reduction of corticosteroid dosage and number of flares. Both serologically active and inactive patients respond to the drug.

Τhe genetics of juvenile idiopathic arthritis: Searching for new susceptibility loci

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.

Autoimmune Thrombotic Thrombocytopenic Purpura: Two Rare Cases Associated with Juvenile Idiopathic Arthritis and Multiple Sclerosis

Secondary thrombotic microangiopathies are associated with several underlying conditions, with most of them being resolved after the treatment of background disease. Thrombotic thrombocytopenic purpura (TTP) is a rare microangiopathy presenting with anemia, thrombocytopenia, and neurological deficits, occurring most often in various autoimmune diseases due to inhibition of ADAMTS13 by autoantibodies, as well as in pregnant women with or without an autoimmune substrate. In this article, we report two newly diagnosed TTP cases, who have not been published so far. The first is a 27-year-old woman with a history of polyarticular rheumatoid factor negative juvenile idiopathic arthritis, who presented with thrombocytopenia, anemia, schistocytes on blood smear, headache, and active arthritis. Originally she was treated successfully with plasma exchange, intravenous prednisone, and vincristine, and a few months after the TTP episode, she was commenced on rituximab, resulting in remission of primary disease and no relapse of TTP. The second case refers to a 29-year-old pregnant woman complaining of dizziness and fatigue with microangiopathic hemolytic anemia. She was treated with plasma exchanges, intravenous prednisolone, and INN human normal immunoglobulin with full remission of the TTP episode. Six and half years later, she was diagnosed with multiple sclerosis and was commenced on interferon beta-1 alpha, with no recurrent episode of TTP. These cases broaden the spectrum of autoimmune disorders manifested or complicated clinically by TTP. Furthermore, biological agents such as rituximab appear to be an effective treatment option for refractory cases of TTP related to systemic rheumatic disease, indicating an alternative therapeutic solution in persistent cases of this disorder.

THU0015 Investigation of juvenile idiopathic arthritis (JIA) in greece: new susceptibility LOCI

Background Juvenile 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. Objectives We 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. Methods The 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 Fishers exact test. Bioinformatic analysis was performed using BlastP, Pymol and Maestro and Desmond (Schrodinger Inc.). Results A 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. Conclusions This 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 369:767–78. Hinks et al (2010). Ann Rheum Dis 69:1049–53. Hinks et al. (2012). Ann Rheum Dis 71:1117–21. Hinks et al (2013). Nat Genet 45: 664–9. Vazgiourakis et al (2007). Lupus 16:867–74. Disclosure of Interest None declared

AB0442 Real-life experience with belimumab in systemic lupus erythematosus (SLE): control of disease activity and flares in a multicenter cohort

Background Data on the efficacy of belimumab in SLE mainly originate from large randomized clinical trials, whereas reports from real-life clinical practice are lacking. Objectives To describe the clinical experience from the use of belimumab in Greece since the approval of the drug. Methods Multicentre observational study of patients receiving belimumab, with documentation of disease activity (SLEDAI-2K index), achievement of low disease activity states [remission (SLEDAI-2K=0) and lupus low disease activity state (LLDAS)], accrual of irreversible damage (SLICC damage index, SDI), number and severity of flares, and side-effects. Analyses were performed at quarterly intervals and only patients with at least 3 months of follow-up were included in the study. Results A total of 56 patients were included [53 women (94.6%), mean (SD) age 46.3 (12.7) years]. Evidence of serologic activity (low C3/C4 and/or high anti-ds DNA) was evident in 30 patients (53.5%). Most frequent manifestations were arthritis (82.1%), inflammatory rash (73.2%), active hair loss (57.1%), mucosal ulcers (26.8%) and leukopenia (10.7%). Median (range) duration of follow-up was 9.1 (2.9 - 34.6) months. We observed a significant decrease in the SLEDAI-2K, physician global assessment (PGA) and daily prednisone dose over time, starting as early as 3 months after belimumab initiation (Table 1). This effect was significantly more pronounced in patients who were serologically active (SA) at baseline, even after exclusion of the serologic component of the SLEDAI [median (range) clinical SLEDAI-2K for SA patients: 7 (1–24) at baseline vs. 2 (0–16) at 6 months and 2 (0–16) at 12 months, p<0.0001 and p=0.013, respectively; for serologically inactive patients: 6 (2–23) at baseline vs. 6 (0–14) at 6 months and 5 (0–18) at 12 months, p=0.017 and p=0.024, respectively]. For patients with ≥12 months of follow-up (n=20), belimumab treatment resulted in a significant decrease in flare rate [median (range) total number of flares for the 12 months before and after belimumab treatment, 3 (0–7) and 0 (0–2), respectively, p<0.0001). 10 patients (17.8%) discontinued belimumab due to inefficacy after a median (range) 7.1 (5.5 - 20.4) months of therapy and 5 patients discontinued due to planned pregnancy. There were no drug discontinuations due to side-effects. Conclusions In real-life clinical settings, belimumab is efficacious in controlling disease activity of SLE and permitting tapering of glucocorticoid dose. Similar to data from RCTs, this effect seems to be more pronounced in serologically active patients. Disclosure of Interest None declared

AutoimmuneThrombotic Thrombocytopenic Purpura

Secondary thrombotic microangiopathies are associated with several underlying conditions, with most of them being resolved after the treatment of background disease. Thrombotic thrombocytopenic purpura (TTP) is a rare microangiopathy presenting with anemia, thrombocytopenia, and neurological deficits, occurring most often in various autoimmune diseases due to inhibition of ADAMTS13 by autoantibodies, as well as in pregnant women with or without an autoimmune substrate. In this article, we report two newly diagnosed TTP cases, who have not been published so far. The first is a 27-year-old woman with a history of polyarticular rheumatoid factor negative juvenile idiopathic arthritis, who presented with thrombocytopenia, anemia, schistocytes on blood smear, headache, and active arthritis. Originally she was treated successfully with plasma exchange, intravenous prednisone, and vincristine, and a few months after the TTP episode, she was commenced on rituximab, resulting in remission of primary disease and no relapse of TTP. The second case refers to a 29-year-old pregnant woman complaining of dizziness and fatigue with microangiopathic hemolytic anemia. She was treated with plasma exchanges, intravenous prednisolone, and INN human normal immunoglobulin with full remission of the TTP episode. Six and half years later, she was diagnosed with multiple sclerosis and was commenced on interferon beta-1 alpha, with no recurrent episode of TTP. These cases broaden the spectrum of autoimmune disorders manifested or complicated clinically by TTP. Furthermore, biological agents such as rituximab appear to be an effective treatment option for refractory cases of TTP related to systemic rheumatic disease, indicating an alternative therapeutic solution in persistent cases of this disorder.

AutoimmuneThrombotic Thrombocytopenic Purpura: Two Rare Cases Associated with Juvenile Idiopathic Arthritis and Multiple Sclerosis.

Secondary thrombotic microangiopathies are associated with several underlying conditions, with most of them being resolved after the treatment of background disease. Thrombotic thrombocytopenic purpura (TTP) is a rare microangiopathy presenting with anemia, thrombocytopenia, and neurological deficits, occurring most often in various autoimmune diseases due to inhibition of ADAMTS13 by autoantibodies, as well as in pregnant women with or without an autoimmune substrate. In this article, we report two newly diagnosed TTP cases, who have not been published so far. The first is a 27-year-old woman with a history of polyarticular rheumatoid factor negative juvenile idiopathic arthritis, who presented with thrombocytopenia, anemia, schistocytes on blood smear, headache, and active arthritis. Originally she was treated successfully with plasma exchange, intravenous prednisone, and vincristine, and a few months after the TTP episode, she was commenced on rituximab, resulting in remission of primary disease and no relapse of TTP. The second case refers to a 29-year-old pregnant woman complaining of dizziness and fatigue with microangiopathic hemolytic anemia. She was treated with plasma exchanges, intravenous prednisolone, and INN human normal immunoglobulin with full remission of the TTP episode. Six and half years later, she was diagnosed with multiple sclerosis and was commenced on interferon beta-1 alpha, with no recurrent episode of TTP. These cases broaden the spectrum of autoimmune disorders manifested or complicated clinically by TTP. Furthermore, biological agents such as rituximab appear to be an effective treatment option for refractory cases of TTP related to systemic rheumatic disease, indicating an alternative therapeutic solution in persistent cases of this disorder.

SAT0013 Juvenile Idiopathic Arthritis Association of PTPN22 rs2476601 SNP Is Specific To Females in A Greek Population

Background Juvenile idiopathic arthritis (JIA) is an autoimmune disease characterized by persistent chronic arthritis [1] and is believed to be influenced by both genetic and environmental factors. PTPN22 single nucleotide polymorphism (SNP) rs2476601 has been recently reported in an Australian study to be restricted to females and to not be observed in males [2]. Objectives Considering that a significant source of inconsistency amongst the literature on autoimmune disease susceptibility genes stems from an inability to replicate genetic findings across different racial or ethnic groups, we attempted to confirm the female-specific association of rs2476601 in a homogeneous Greek population. Methods The sample set consisted of 128 (70.3% female) Caucasian JIA patients and 221 (28.1% female) healthy controls from Northern Greece. Genotyping for the PTPN22 rs2476601 (XcmI) was performed by restriction fragment length polymorphism (RFLP) analysis [3]. Logistic regression or the Gart (Woolf) method were used to estimate ORs and 95% CIs. Analyses were performed using Stata v13 (StataCorp, College Station, TX, USA). Results The rs2476601 was genotyped in 128 Caucasian JIA patientsand 221 healthy controls from Northern Greece. Overall, PTPN22 was associated with increased risk of JIA in this Greek sample (OR =2.3, 95% CI 1.1 – 5.1, p=0.038). Sex-stratified analyses showed that, once again, the risk association was restricted to females (Female: OR =19.9, 95% CI 1.2 – 342, p=0.0016; Male: OR =1.1, 95% CI 0.3 – 3.1, p=0.94) confirming the prior findings. Given that the sex bias differs by JIA subtype, ideally we would have liked to test for these associations in individual subtypes; however, small sample size prevented us from performing these analyses with any statistical certainty. Conclusions This data demonstrated that the sex-specific pattern of association detected is broadly applicable to different populations, and provided further impetus to undertake studies to understand the crucial role of rs2476601 in the mechanisms leading to diseases development preferentially in females. Our work is of interest given the importance of comparative studies in populations of differing ethnic and/or racial origin in any attempt to conclusively define the genetic architecture of JIA and the magnitude of the effects of specific risk alleles globally. References Ravelli and Martini (2007). Lancet 369:767–778. Chiaroni-Clarke et al (2015). Genes Immun. 16:495–498. Eliopoulos et al (2010). Lupus 105:501–506. Disclosure of Interest None declared