Tana Svitalkova

A3.30 Plasma level of HSP70 protein is increased in czech patients with idiopathic inflammatory myopathy

Background and Objectives Heat shock protein 70 (HSP70) plays an essential role as molecular chaperone by assisting the correct folding of nascent and stress-accumulated misfolded proteins, and preventing their aggregation. These proteins have a dual function depending on their intracellular or extracellular location. Intracellular HSP70 proteins have a protective, antiapoptotic role. Under normal conditions they act as molecular chaperones. Under a stress conditions, the expression of HSP70 genes is induced, which helps the cell to survive otherwise lethal condition. This is due to its important role in apoptosis. The extracellular HSP70 proteins, on the other hand, can act as mediators of immune response. They can elicit an immune response modulated either by the adaptive or innate immune system. Mechanisms of extracellular and intracellular action of Hsp70 is expected to be involved also in autoimmunity development. The aim of this study was to establish the plasma level of Hsp70 protein in patients suffering from IIM and find its possible relation to the clinical features of our patients. Materials and Methods We quantified plasma Hsp70 levels in a healthy control group (n = 37) and a group of 34 patient with IIM. Plasma Hsp70 protein was analysed in duplicates using a commercially available high-sensitivity sandwich enzyme-linked immunosorbent assay (ELISA), suitable for detection of Hsp70 protein in serum or plasma samples (Assay Designs EKS-715, USA). Statistical differences between the patients and control groups were established using Mann-Whitney U test. All the expression data are presented as medians with range. Results We have found that plasma levels of Hsp70 were significantly higher in patients with myositis than in healthy controls (0.173 [0.000 – 7.647] vs. 0.000 [0.000 – 1.008] ng/ml; p<0.01). However, plasma Hsp70 levels did not correlate with age, CRP, disease activity index (MYOACT), or other clinical features (muscle activity) of our patients. Conclusions In this study, we have found that the amount of Hsp70 protein in plasma is significantly higher in IIM patients when compared with healthy controls. Our results correlate with our former findings showing that mRNA levels of HSP70 genes were increased in myositis patients. Finally we can assume that the Hsp70 protein is possibly involved in the pathological mechanism of the myositis. Acknowledgement This study was supported by the Internal Grant Agency of the Ministry of Health in the Czech Republic [MZČR NT 13699].

A9.04 SNPS in the baff gene are associated with increased risk of anti-jo-1-positivity and high serum baff levels in patients with myositis

Background and objectives B-cell activating factor of the TNF family (BAFF) plays a role in (auto)antibody production. Elevated serum levels (sBAFF) were found in patients with myositis. Associations of elevated sBAFF with a SNP in the 5´regulatory region of the BAFF genewere reported in patients with autoimmune diseases, together with TTTT haplotype of the four SNPs located upstream of the BAFF gene. TTTT was associated with myositis in Czech patients, independently from the HLA-DRB1*03 allele. Here we evaluate associations of sBAFF with particular SNPs, anti-Jo-1-autoantibodies, HLA-DRB1*03, ILD and disease activity in myositis. Materials and methods The SNPs (rs9514828:871C>T, rs9514827:-2841T>C, rs3759467:-2704T>C and rs1041569:-2701T >A) were analysed by direct DNA sequencing in 311 patients with myositis (age:58.0 ± 14.5(years), females = 74%) and 113 healthy controls (age:40.6 ± 14.3, females = 70%). Levels of sBAFF were measured by ELISA. Autoantibodies were detected by immunoprecipitation. Multivariable logistic regression model for presence of anti-Jo-1-autoantibodies and nonparametric tests for group comparisons and correlation analysis were used. Results Analysed patients cohort included 150 DM, 139 PM, 51% ILD and 24% anti-Jo-1-positive patients. HLA-DRB1*03 was present in 45% of patients and 16% of controls. Higher s-BAFF levels were detected in: patients compared to healthy controls (p < 0.0001), patients with anti-Jo-1-autoantibodies compared to autoantibody negative (p = 0.02), patients with early (duration≤ 6 month; n = 114) compared to late disease (p = 0.038) and were associated with creatine kinase (CK) levels (rho=0.36;p < 0.0001) and disease duration (rho=-0.2;p = 0.0007). Logistic regression model explaining 49% (McFadden R2=0.49) of the data variability included, besides ILD (OR = 27.24;p < 0.0001) and HLA-DRB1*03 (OR = 5.26;p = 0.0009), significant additive effect of the BAFF gene SNPs: -2841T allele (OR = 2.55;p = 0.028) and a -871TT genotype (OR = 7.75;p = 0.007). The alternative substitution of CK concentration (OR = 1.48;p = 0.175) with sBAFF (OR = 0.67; p = 0.037)lowered model stability (R2 = 0.38). However, the additive effect of -871T allele to sBAFF levels in anti-Jo-1-positive patients compared to autoantibody-negative (higher in -871TT:p = 0.023 and -871CT:p = 0.043) and the opposite association in -2841TT genotype (higher in -2841CC: p = 0.033 and -2841CT:p = 0.068)were seen. Conclusion In addition to ILD and HLA-DRB1*03 allele, SNPs of the promotor region of the BAFF gene, -871C >T and -2841T >C, increase the risk of having anti-Jo-1-autoantibodies and high sBAFF levels in patients with myositis. Acknowledgements MZČR-Institutional support of research organisation-00023728.

A2.33 Haplotype TTTT in the BAFF gene is associated with idiopathic inflammatory myopathies in HLA-DRB1*03 negative patients

Background and objectives B-cell activating factor of the TNF family (BAFF) plays a role in (auto)antibody production. Patients with idiopathic inflammatory myopathies (IIMs or myositis) have elevated levels of BAFF in the serum which associate with disease activity. We have previously found an association between TTTT haplotype from the four single nucleotide polymorphisms (SNPs) in the promoter region of BAFF gene (rs9514827, rs3759467, rs1041569 and rs9514828) with presence of myositis in our cohort of IIM patients. The HLA-DRB1*03 allele is a well-known genetic risk factor for myositis in Caucasians. In this study we have analysed the possible interference of HLA-DRB1*03 allele with the presence of the particular SNPs of BAFF gene in patients with myositis. Materials and methods The SNPs (rs9514827, rs3759467, rs1041569 and rs9514828) were analysed by direct DNA sequencing in a cohort of 311 patients with myositis (age 5–80 years, females 74%) and in 113 healthy controls (age 18–79 years, females 69%). The HLA-DRB1 genotyping with PCR using sequence-specific oligonucleotide and sequence based typing technique was available in the subgroups of 297 patients and 103 controls. The χ2 or Fisher tests for analysis of allelic, haplotype and genotype associations with Bonferroni’s correction were used. Results The four BAFF SNPs were in strong linkage disequilibrium and formed four common haplotypes (TTAC, CTAT, TCAC, TTTT) in both IIM patients and controls, compatible with already reported results. A significantly higher frequency of the TTTT haplotype was present in myositis patients compared to healthy controls (16% vs. 10%; OR = 1.65, 95% CI = 1.03–2.65; p < 0.05). There were no significant differences between patients and controls in the frequencies of alleles and genotypes. 162 IIM patients (55%) and 87 healthy controls (84%) were HLA-DRB1*03 negative and a significant association of the TTTT haplotype in IIM patients was confirmed by a higher frequency (19%) compared to controls (11.5%; OR = 1.79, 95% CI = 1.04–3.07; p < 0.05) within the DRB1*03 negative cohorts. The trend for lower frequency of -2701A allele and AA genotype (rs1041569) in myositis was also found within the HLA-DRB1*03 negative cohort, with IIM patients having a significantly lower proportion of AA genotype compared to controls (62% vs. 77%; OR = 0.48, CI = 0.27–0.87; p < 0.05). Conclusions An association of the TTTT haplotype with myositis and a lower frequency of -2701 A allele and AA genotype in patients with IIM are independent from the presence of HLA-DRB1*03 risk allele. Acknowledgements IGA-MZ CR NT/12438–4, IMI-funded project BeTheCure, 115142-2

AB0009 Association of MHC Haplotypes with Idiopathic Inflammatory Myopathy in Cohort of Czech Patients

Background Idiopathic inflammatory myopathies (IIM) belong to the group of systemic autoimmune diseases. IIM are characterized clinically by chronic muscle inflammation leading to a dysfunction and/or destruction of muscle cells. Like many other autoimmune diseases, IIM have its major genetic predisposition linked to the MHC genes. In this study, we have analyzed MHC haplotypes involving alleles of HLA class II genes (DQB1, DRB1) and 6 polymorphisms located within the HSP70 genes (HSPA1A, HSPA1B and HSPA1L). Frequencies of all polymorphisms and haplotypes were studied in a cohort of patients with IIM and compared with healthy controls. Objectives The aim of this study was to investigate the relation between MHC haplotypes and the risk of IIM development in a single center cohort. Methods We have analyzed 82 patients with dermatomyositis (DM) and 70 patients with polymyositis (PM) diagnosed according to Bohan and Peter criteria and compared them with 150 healthy controls. In total, six genetic polymorphisms located within the three HSP70 genes were analyzed by direct genomic DNA sequencing. Additionally, HLA-DRB1 and HLA-DQB1 loci were genotyped using the commercial LABType® SSO kit (One Lambda, USA). The statistical analysis was done using Fishers exact test with calculated p<0.05 considered as statistically significant. Results The most frequent MHC haplotype in controls, total IIM, as well as in DM and PM subgroups was found to be the HLA-DQB1*02-DRB1*03:01-T-C-C-G-C-INS haplotype. However, the difference in frequency between patients and controls reached statistical significance only for IIM overall (p<0.01; OR=1.90, CI (95%): 1.15, 3.13) and the DM subgroup (p<0.05; OR=1.82, CI (95%): 1.02, 3.23). There was not a statistically significant difference between the PM subgroup and controls. The HLA-DQB1*05–DRB1*16:01-T-A-G-A-G-DEL haplotype was found to have the strongest disease association in all patient subgroups. The difference was the highest in the IIM overall (p<0.0001; OR=32.18, CI (95%): 1.92, 540.60) and remained significant when the DM subgroup (p<0.01; OR=32.64, CI (95%): 1.87, 569.60) as well as the PM subgroup (p<0.001; OR=29.04, CI (95%): 1.62, 519.70) were analyzed separately. When comparing the MHC background of DM and PM, we have found another haplotype (HLA-DQB1*03-DRB1*11:01-T-A-G-A-G-DEL) differentially associated with these diagnoses. The difference in frequency of this haplotype between DM and PM reached statistical significance with p<0.01. Conclusions Our results show a strong association between distinct MHC haplotypes and IIM in general. Additionally, we have identified one MHC haplotype suggesting, that DM and PM may have partially different genetic background. HSP70 polymorphisms are located nearby the HLA-DRB1 gene. Therefore, combination of distinct HLA-DRB1 alleles and HSP70 polymorphisms can help in identification of higher genetic risk for development of myositis. Acknowledgements This study was supported by the Internal Grant Agency of the Ministry of Health in the Czech Republic [MZČR NT 13699], and Institutional support of MHCR VZ (00023728). Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.4166

FRI0520 Association Study of the BAFF Genetic Variations in Two Independent Cohorts with Idiopathic Inflammatory Myopathies

Background B-cell activating factor of the TNF family (BAFF) plays a role in (auto)antibody production. Patients with idiopathic inflammatory myopathies (IIMs or myositis) have elevated levels of BAFF in the serum (sBAFF) which associate with disease activity. Previously an association was shown between rs9514828 (-871 C/T) SNP (single nucleotide polymorphism) in the promoter region of the BAFF gene and idiopathic thrombocytopenic purpura and with phenotypic diversity in autoimmune rheumatic diseases but with inconsistent results. An association between the haplotype from the four SNPs located upstream of the BAFF gene and presence of myositis was found in one cohort of Czech patients (CZE). Objectives To replicate our findings in an independent cohort of myositis patients from Sweden (SWE) and to study correlations between particular SNPs and serum levels of BAFF. Methods Two cohorts of patients with myositis (CZE: n=311, age (years) 54.6, females 74% and SWE: n=307, age 54.1, females 64%) and healthy controls (CZE: n=103, age 42, females 69% and SWE: n=325, age 52.0, females 74%) were included in the study. SNPs (rs9514827, rs3759467, rs1041569 and rs9514828) were analysed by direct DNA sequencing and levels of sBAFF were measured by ELISA. The χ2 or Fisher tests for analysis of allelic, haplotype and genotype associations and nonparametric tests for group comparisons with Bonferronis correction were used. Results The four SNPs were in strong linkage disequilibrium and formed four common haplotypes (TTAC, CTAT, TCAC, TTTT) in both patients and controls, compatible to results reported from other populations. A significantly higher frequency of the TTTT haplotype was present in the CZE myositis patients compared to healthy controls (16% vs. 10%; OR=1.73, 95%CI=1.05-2.85, p<0.02), but not in the SWE cohort (patients 13% vs. controls 17%, p=0.07). CZE and SWE myositis patients did not differ significantly in allelic, genotype or haplotype frequencies, but a difference between the control groups was observed. SWE controls had higher frequencies of the rs1041569 T-allele and rs3759467 TC-genotype compared to CZE HC (19% vs 12% and 33% vs 19% respectively; p<0.05 for both). No associations were found between alleles or genotypes and serum levels of BAFF for any of the studied SNPs. Conclusions We found different patterns of associations between BAFF genetic polymorphism and myositis in two European populations. Our initial finding in Czech population could not be replicated in an independent cohort from Sweden. The difference between the control groups requires further analyses. Acknowledgements IGA -MZ Czech Republic NT/12438-4, ESF in the framework of the EuMyoNet, Krystufkova O. and Svitalkova T. contributed equally Disclosure of Interest O. Krystufkova Grant/research support: IGA -MZ Czech Republic NT/12438-4, ESF in the framework of the EuMyoNet, T. Svitalkova: None declared, H. Hulejova: None declared, M. Svetla: None declared, L. Plestilova: None declared, O. Pecha: None declared, H. Mann: None declared, A. Notarnicola: None declared, P. Venalis: None declared, L. Padyukov: None declared, P. Novota: None declared, I. Lundberg: None declared, J. Vencovsky: None declared DOI 10.1136/annrheumdis-2014-eular.4065

The expression profile of miR-23b is not altered in peripheral blood mononuclear cells of patients with idiopathic inflammatory myopathies.

Idiopathic inflammatory myopathies (IIM) belong to a group of autoimmune disorders, primarily characterized by chronic inflammation of human skeletal muscle tissue. The etiology of these diseases is unknown, however, genetic predisposition plays a significant role in disease onset. Beside the known genetic risk located in the MHC complex, the epigenetic modifications including changes in miRNAs expression profiles have been recently implicated recently in many autoimmune diseases. Micro RNA molecules are involved in many physiological processes, including the regulation of the immune response. In our study we have focused on the miR-23b, as it represents a novel promising autoimmunity regulator molecule. Downregulation of miR-23b was recently described in patients with rheumatoid arthritis and systemic lupus erythematosus. We have measured the expression miR-23b peripheral blood mononuclear cells of patients with dermatomyositis and polymyositis. No meaningful difference was found in comparison with healthy controls.

A7.7 Different Genetic Background of Dermatomyositis and Polymyositis in a Single Centre Cohort

Background and Objectives The idiopathic inflammatory myopathies (IIM) are systemic connective tissue diseases in which autoimmune pathology is responsible for promotion of chronic muscle inflammation and weakness. As in many other autoimmune diseases, the development of IIM is also associated with genes of HLA complex. The aim of this study was to determine the basic relation between alleles of HLA genes and IIM. Materials and Methods We have performed low to high resolution genotyping to characterise the allelic profiles of HLA-DRB1, -DQB1 and -DQA1 loci in a large group of single centre cohort of patients suffering from IIM (n = 269). The genomic DNA was prepared by standard DNA extraction methods and the HLA typing was done using the commercial LABType® SSO kit (One Lambda, USA). Statistical evaluation of results was done with chi-2 test and Fisher exact test. Autoantibody profiles were analysed with radioactive immunoprecipitation. Results The frequencies of HLA-DRB1*03:01 and -DRB1*16:01 alleles were increased in IIM patients and the difference reached statistical significance when compared to healthy controls (P < 0.01 for DRB1*03:01; P < 0.05 for DRB1*16:01). Different alleles were associated with dermatomyositis (DM) or polymyositis (PM). The frequency of DRB1*16:01 was significantly higher in DM patients (P < 0.01), whereas the frequency of DRB1*03:01 was higher in patients suffering from PM (P < 0.01), when compared to controls. Presence of HLA-DRB1*03:01 allele was associated with anti-Jo-1, anti-Ro52, or anti-Pm-Scl positivity in all IIM patients (P < 0.05). Interestingly, the DRB1*03:01 allele was also associated with the negativity for anti-p155/140 in our patients (P < 0.01). The DRB1*16:01 allele was associated with negativity of all studied autoantibodies, particularly in subgroup of DM patients (P < 0.05). Conclusions This study identifies different genetic background between patients with dermatomyositis and polymyositis in a homogenous population of patients from a single centre. Acknowledgement This work is supported by Internal Grant Agency of Ministry of Health of the Czech Republic NT/12438–4 and NT/13699.

A7.24 The Pentanucleotide Insertion in HSPA1B Gene is Associated with Idiopathic Inflammatory Myopathy

Background and Objectives The HSPA1B gene is one of the three HSP70 genes located within the Major Histocompatibility Complex (MHC) on chromosome 6p21. The HSP70 molecules in their intracellular form have antiapoptotic function and are responsible for stabilisation of protein structure; in their extracellular form, they act as mediators of immune response. The extracellular HSPs are part of the innate and adaptive immune response and are involved in the process of antigen presentation. The aim of our study was to find out if an association between polymorphisms of MHC located HSP70 genes and subgroups of idiopathic inflammatory myopathy exists. Materials and Methods We have analysed 177 patients suffering from idiopathic inflammatory myopathy (82 patients with dermatomyositis – DM, 71 patients with polymyositis – PM, 22 patients with cancer associated myositis, 2 patients with inclusion body myositis) and 59 healthy controls. In total, six genetic polymorphisms located within the three HSP70 genes were analysed by direct genomic DNA sequencing. The statistical analysis was done using Fisher’s exact test with calculated p < 0.05 considered as statistically significant. Results and Conclusions The frequency of the “INS” allele of the pentanucleotide insertion polymorphism in HSPA1B (Rs9281590) was increased in patients suffering from myositis (43.79%) in comparison with controls (32.20%; p < 0.05). The Odds Ratio calculated for this polymorphism was 1.64 (CI95% 1.056; 2.545). Its increased frequency was predominantly found in DM patients (p < 0.05); the allele distribution in PM patients did not significantly differ from controls. Presence of INS allele was strongly related to the well described HLA associated risk, the HLA-DRB1*03 allele (p < 0.001), found mostly in PM patients. INS allele is independent on other myositis associated HLA allele, HLA-DRB1*16, found increased in the population of our DM patients. Other polymorphisms analysed in this study did not show any relation to the myositis. Our findings support the hypothesis, that DM and PM have partially different genetic background. Acknowledgement This work is supported by Internal Grant Agency of Ministry of Health of the Czech Republic NT/13699.