Peter Novota

Interaction of HLA-DRB1*03 and smoking for the development of anti-Jo-1 antibodies in adult idiopathic inflammatory myopathies: a European-wide case study

Objectives HLA-DRB1*03 is strongly associated with anti-Jo-1-positive idiopathic inflammatory myopathies (IIM) and there is now increasing evidence that Jo-1 antigen is preferentially expressed in lung tissue. This study examined whether smoking was associated with the development of anti-Jo-1 antibodies in HLA-DRB1*03-positive IIM. Methods IIM cases were selected with concurrent information regarding HLA-DRB1 status, smoking history and anti-Jo-1 antibody status. DNA was genotyped at DRB1 using a commercial sequence-specific oligonucleotide kit. Anti-Jo-1 antibody status was established using a line blot assay or immunoprecipitation. Results 557 Caucasian IIM patients were recruited from Hungary (181), UK (99), Sweden (94) and Czech Republic (183). Smoking frequency was increased in anti-Jo-1-positive IIM cases, and reached statistical significance in Hungarian IIM (45% Jo-1-positive vs 17% Jo-1-negative, OR 3.94, 95% CI 1.53 to 9.89, p<0.0001). A strong association between HLA-DRB1*03 and anti-Jo-1 status was observed across all four cohorts (DRB1*03 frequency: 74% Jo-1-positive vs 35% Jo-1-negative, OR 5.55, 95% CI 3.42 to 9.14, p<0.0001). The frequency of HLA-DRB1*03 was increased in smokers. The frequency of anti-Jo-1 was increased in DRB1*03-positive smokers vs DRB1*03-negative non-smokers (42% vs 8%, OR 7.75, 95% CI 4.21 to 14.28, p<0.0001) and DRB1*03-positive non-smokers (42% vs 31%, p=0.08). In DRB1*03-negative patients, anti-Jo-1 status between smokers and non-smokers was not significantly different. No significant interaction was noted between smoking and DRB1*03 status using anti-Jo-1 as the outcome measure. Conclusion Smoking appears to be associated with an increased risk of possession of anti-Jo-1 in HLA-DRB1*03-positive IIM cases. The authors hypothesise that an interaction between HLA-DRB1*03 and smoking may prime the development of anti-Jo-1 antibodies.

HLA DRB1, DQB1 and insulin promoter VNTR polymorphisms: interactions and the association with adult‐onset diabetes mellitus in Czech patients

Both the human leucocyte antigen (HLA) DRB1 and the HLA DQB1 gene loci play a role in the development and progression of autoimmune diabetes mellitus (T1DM). Similarly, the insulin promoter variable number tandem repeats (INS‐VNTR) polymorphism is also involved in the pathogenesis of diabetes mellitus (DM). We studied the association between each of these polymorphisms and DM diagnosed in patients older than age 35 years. Furthermore, we analysed possible interactions between HLA DRB1/DQB1 and INS‐VNTR polymorphisms. Based on C‐peptide and GADA levels we were able to distinguish three types of diabetes: T1DM, latent autoimmune diabetes in adults (LADA) and T2DM. INS‐VNTR was genotyped indirectly by typing INS–23HphI A/T polymorphism. The genotype and allele frequencies of INS–23HphI did not differ between each of the diabetic groups and group of healthy subjects. We did, however, observe an association between the INS–23HphI alleles, genotypes and C‐peptide secretion in all diabetic patients: A allele frequency was 86.2% in the C‐peptide‐negative group vs. 65.4% in the C‐peptide‐positive group (Pcorr. < 0.005); AA genotype was found to be 72.4% in the C‐peptide‐negative group vs. 42.6% in the C‐peptide‐positive groups (Pcorr. < 0.01). The HLA genotyping revealed a significantly higher frequency of HLA DRB1*03 allele in both T1DM and LADA groups when compared to healthy subjects: T1DM (25.7%) vs. control group (10.15%), odds ratio (OR) = 3.06, P < 0.05; LADA (27.6%) vs. control (10.15%), OR = 3.37, P < 0.01. The simultaneous presence of both HLA DRB1*04 and INS–23HphI AA genotype was detected in 37.5% of the T1DM group compared to only 9.2% of the healthy individuals group (OR = 5.9, Pcorr. < 0.007). We summarize that in the Central Bohemian population of the Czech Republic, the INS–23HphI A allele appears to be associated with a decrease in pancreatic beta cell secretory activity. HLA genotyping points to at least a partial difference in mechanism, which leads to T1DM and LADA development as well as a more diverse genetic predisposition in juvenile‐ and adult‐onset diabetes. The simultaneous effect of HLA and INS‐VNTR alleles/genotypes predispose individuals to an increased risk of diabetes development.

Association of MHC class I chain related gene-A microsatellite polymorphism with the susceptibility to T1DM and LADA in Czech adult patients.

The results in this study suggest that microsatellite polymorphism within the transmembrane region of MIC‐A gene is associated with genetic susceptibility to adult‐onset of type 1 diabetes mellitus (T1DM), MIC‐A5.1 allele, corrected P = 0.001, whereas it is not associated with latent autoimmune diabetes in adults (LADA) in Czech population. According to our findings, we can hypothesize that adult‐onset T1DM and LADA may have partly different immunogenetic aetiopathogenesis.

Autoimmune Diabetes Mellitus with Adult Onset and Type 1 Diabetes Mellitus in Children Have Different Genetic Predispositions

Abstract:  Type 1 diabetes with manifestation after 35 years of age is defined by CP <200 pmol/L and institution of insulin therapy within 6 months after diagnosis. Latent autoimmune diabetes mellitus in adults (LADA) manifesting after 35 years of age is defined by minimum 6 months after diagnosis without insulin therapy and C peptide (CP) >200 pmol/L and antiGAD > 50 ng/mL. We aimed to find a possible genetic discrimination among different types of autoimmune diabetes. To accomplish this goal, we analyzed DNA samples from 31 LADA patients, 75 patients with adult‐onset type 1 diabetes mellitus, 188 type 1 diabetic children, and 153 healthy adult individuals. We studied five genetic loci on chromosomes 6, 11, 4, and 14: HLA DRB1 and DQB1 alleles, major histocompatibility complex (MHC) class I–related gene‐A (MIC‐A) microsatellite polymorphism, interleukin (IL)‐18 single nucleotide polymorphism, the microsatellite polymorphism of nuclear factor kappa B gene (NF‐κB1), and the single nucleotide polymorphism of a gene for its inhibitor (NF‐κBIA). HLA‐DR3 was detected as the predisposition allele for LADA (OR = 4.94, P < 0.0001). Further we found a statistically significant increase of NF‐κBIA AA genotype (OR = 2.68, P < 0.01). On the other hand, DRB1*04, which is linked with DQB1*0302, was observed as a risk factor in patients with type 1 diabetes mellitus (T1DM) onset after 35 years of age (OR = 10.47, P < 0.0001 and OR = 9.49, P < 0.0001, respectively). There was also an association with MIC‐A5.1 (OR = 2.14, P < 0.01). Statistically significant difference was found in the distribution of IL‐18 promoter –607 (C/A) polymorphism between LADA and T1DM in adults (P < 0.01). We conclude that all subgroups of autoimmune diabetes have partly different immunogenetic predisposition.

The expression regulation of the HSPA1B gene in patients with myositis is not dependent on the presence of HLA-DRB1*03 risk allele

Background and objectives Genes within the major histocompatibility complex (MHC) region has a strong genetic relevance in autoimmunity development. The involvement of the class I and class II genes, as well as class III (non-Class I/II MHC) genes has been proposed. For the myositis development, the HLA-DRB1*03 is a known risk factor in Caucasian population. However, there are another genes appearing to be significant players in the ethiology of myositis located near the HLA-DRB1 locus. In the present study the authors have focused on one of these risk factors – the regulation of MHC-linked inducible HSP70 genes expression and their relation to the known immunogenetic risk factor located within the MHC (HLA-DRB1*03). Materials and methods The authors have investigated the gene specific HSP70 expression in 20 patients with dermatopolymyositis and 15 healthy people matching in age as control samples. Expression levels of the two inducible HSP70 genes (HSPA1A, HSPA1B) were analysed both in patients and controls. Both of the groups were additionally genotyped for HLA-DRB1 locus. Myositis-specific and associated autoantibodies were also identified in patients. Results The expression of both, the HSPA1A and HSPA1B genes was significantly upregulated (p<0.001; p<0.05) in patients suffering from myositis when compared to controls. The expression regulation of the HSPA1A was found to be associated with the presence of the HLA-DRB1*03 risk allele in patients. However, this was not observed for the HSPA1B gene. In contrast, the authors found a positive correlation between the expression regulation of the HSPA1B gene and the presence of disease specific autoantibodies in myositis patients. Additionally, positive correlation between the presence of disease specific autoantibodies and the HLA-DRB1*03 risk allele was found. None of these observations were found in healthy controls. Conclusions The results suggest that the two MHC-linked inducible genes are differentially expressed in dependence on the autoantibody or HLA risk allele presence. The differential gene expression regulation shows that the HSPA1B is an – on HLA-DRB1 – independent molecular marker for myositis development.

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].

THU0065 Expression Profiling of Extracellular Serum Micrornas in Patients with Idiopathic Inflammatory Myopathy

Background Idiopathic inflammatory myopathies (IIMs) are chronic autoimmune disorders characterized by skeletal muscle weakness, inflammatory immune cells in muscle tissues and frequent presence of serum autoantibodies. Besides a genetic risk located within the MHC complex, also epigenetic regulations including changes in miRNAs expression profiles have been implicated recently in many autoimmune diseases. Extracellular miRNAs circulate in the bloodstream and such miRNAs are remarkably stable. It is hypothesized that circulating miRNA may be delivered to recipient cells and regulate translation of target genes. Objectives The aim of this study was to analyze the differences in expression profiles of circulating extracellular miRNAs between patients and controls with possible selection of relevant candidate miRNA molecules involved in disease progression. Methods Expression profile of 1,347 miRNA molecules was analyzed in 47 myositis patients and 16 healthy controls. Circulating miRNAs were prepared from serum using the Trizol® reagent. The expression of miRNAs was measured using the 44K high density microarray from Agilent Company. Results The miRNA expression profiles clearly separate between healthy controls and patients with myositis. In total, 87 miRNA molecules were found to be differentially expressed in myositis patients. Analysis of the associated biological pathways regulated by the detected miRNAs revealed that 44 (50%) differentially expressed miRNAs are predicted to regulate immune response, 38 (43%) miRNAs are related to muscle activity and 40 (46%) miRNAs were predicted to be involved in cell death. Conclusions A number of miRNAs differentially expressed in patients with IIMs were identified. These molecules are potentially involved in the etiopathogenesis of the disease since all are related to important immune mechanisms or muscle function. Acknowledgements This study was supported by the Internal Grant Agency of the Ministry of Health in the Czech Republic [MZČR NT 12452-4], Institutional support of MHCR VZ (00023728) and The Charles University Grant agency (No.621812). Disclosure of Interest None Declared

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

A3.29 Micro-RNA molecules are clearly involved in pathogenesis of idiopathic inflammatory myopathy

Background and Objectives MicroRNAs can regulate the gene expression and cellular functions such as cell cycle, differentiation, or apoptosis. Emerging evidence has demonstrated that miRNAs play a vital role also in the regulation of immunological functions, including the development of autoimmunity. Like in most of other autoimmune diseases, the etiopathogenesis of Idiopathic inflammatory myopathy (IIM) is also believed to be triggered by environmental factors in combination with susceptible genetic background. Beside a genetic risk located within the MHC complex, the role of epigenetic regulations including changes in miRNAs expression profiles in autoimmunity development have recently also been implicated. The aim of this study was to look for specific miRNA expression patterns in the serum, peripheral blood mononuclear cells (PBMCs) and muscle biopsies of patients suffering from idiopathic inflammatory myopathy. Materials and Methods RNA was isolated from serum, PBMCs and muscle biopsy samples of patients suffering from IIM using modified Trizol-chloroform method. The expression profile of miRNAs was determined by 60K high density microarray from Agilent Company. Results In total, 87 miRNAs were found to be differentially expressed at a significant level (p<0.05) in serum samples of patients with IIM. Under the same conditions for comparison, 14 miRNAs were differentially expressed in PBMCs (p<0.05) and 78 miRNAs in muscle biopsies (p<0.05). What more, the expression of 10 miRNA molecules have shown the same expression regulation among either serum and muscle biopsy, or PBMCs and muscle biopsy samples. Nine of them were upregulated in serum and muscle biopsy samples; one of them was upregulated in PBMCs and biopsy samples. Beside this concordance, we could see significant differences in expression between patients and controls in all of these 10 miRNA molecules (p<0.05). Conclusions This is the first study providing information about complete expression profile of miRNA molecules found in the serum, PBMCs and muscle biopsy samples of patients with IIM. We have found that a number of miRNAs were differentially expressed in diverse tissue samples indicating tissue specific expression of miRNAs. The “missregulated” miRNAs might potentially act as biomarkers of initiated systemic inflammatory response, or functional molecules related to muscle tissue injury, or molecules involved in regulation of immune response. Acknowledgement This study was supported by the Internal Grant Agency of the Ministry of Health in the Czech Republic [MZČR NT 12452-4], Institutional support of MHCR VZ (00023728) and The Charles University Grant Agency (No.621812).