Klementy Shchetynsky

Epigenome-wide association data implicate DNA methylation as an intermediary of genetic risk in rheumatoid arthritis

Epigenetic mechanisms integrate genetic and environmental causes of disease, but comprehensive genome-wide analyses of epigenetic modifications have not yet demonstrated robust association with common diseases. Using Illumina HumanMethylation450 arrays on 354 anti-citrullinated protein antibody–associated rheumatoid arthritis cases and 337 controls, we identified two clusters within the major histocompatibility complex (MHC) region whose differential methylation potentially mediates genetic risk for rheumatoid arthritis. To reduce confounding factors that have hampered previous epigenome-wide studies, we corrected for cellular heterogeneity by estimating and adjusting for cell-type proportions in our blood-derived DNA samples and used mediation analysis to filter out associations likely to be a consequence of disease. Four CpGs also showed an association between genotype and variance of methylation. The associations for both clusters replicated at least one CpG (P < 0.01), with the rest showing suggestive association, in monocyte cell fractions in an independent cohort of 12 cases and 12 controls. Thus, DNA methylation is a potential mediator of genetic risk.

High-Density Genetic Mapping Identifies New Susceptibility Variants in Sarcoidosis Phenotypes and Shows Genomic-driven Phenotypic Differences.

RATIONALE Sarcoidosis is a multisystem disease of unknown cause. Löfgrens syndrome (LS) is a characteristic subgroup of sarcoidosis that is associated with a good prognosis in sarcoidosis. However, little is known about its genetic architecture or its broader phenotype, non-LS sarcoidosis. OBJECTIVES To address the genetic architecture of sarcoidosis phenotypes, LS and non-LS. METHODS An association study in a white Swedish cohort of 384 LS, 664 non-LS, and 2,086 control subjects, totaling 3,134 subjects using a fine-mapping genotyping platform was conducted. Replication was performed in four independent cohorts, three of white European descent (Germany, n = 4,975; the Netherlands, n = 613; and Czech Republic, n = 521), and one of black African descent (United States, n = 1,657), totaling 7,766 subjects. MEASUREMENTS AND MAIN RESULTS A total of 727 LS-associated variants expanding throughout the extended major histocompatibility complex (MHC) region and 68 non-LS-associated variants located in the MHC class II region were identified and confirmed. A shared overlap between LS and non-LS defined by 17 variants located in the MHC class II region was found. Outside the MHC region, two LS-associated loci, in ADCY3 and between CSMD1 and MCPH1, were observed and replicated. CONCLUSIONS Comprehensive and integrative analyses of genetics, transcription, and pathway modeling on LS and non-LS indicates that these sarcoidosis phenotypes have different genetic susceptibility, genomic distributions, and cellular activities, suggesting distinct molecular mechanisms in pathways related to immune response with a common region.

The balance of expression of PTPN22 splice forms is significantly different in rheumatoid arthritis patients compared with controls

BackgroundThe R620W variant in protein tyrosine phosphatase non-receptor 22 (PTPN22) is associated with rheumatoid arthritis (RA). The PTPN22 gene has alternatively spliced transcripts and at least two of the splice forms have been confirmed to encode different PTPN22 (LYP) proteins, but detailed information regarding expression of these is lacking, especially with regard to autoimmune diseases.MethodsWe have investigated the mRNA expression of known PTPN22 splice forms with TaqMan real-time PCR in relation to ZNF592 as an endogenous reference in peripheral blood cells from three independent cohorts with RA patients (n = 139) and controls (n = 111) of Caucasian origin. Polymorphisms in the PTPN22 locus (25 SNPs) and phenotypic data (gender, disease activity, ACPA and RF status) were used for analysis. Additionally, we addressed possible effects of methotrexate treatment on PTPN22 expression.ResultsWe found consistent differences in the expression of the PTPN22 splice forms in unstimulated peripheral blood mononuclear cells between RA patients and normal controls. This difference was more pronounced when comparing the ratio of splice forms and was not affected by methotrexate treatment.ConclusionsOur data show that RA patients and healthy controls have a shift in balance of expression of splice forms derived from the PTPN22 gene. This balance seems not to be caused by treatment and may be of importance during immune response due to great structural differences in the encoded PTPN22 proteins.

Integration of known DNA, RNA and protein biomarkers provides prediction of anti-TNF response in rheumatoid arthritis: results from the COMBINE study.

OBJECTIVE: In rheumatoid arthritis (RA) several recent efforts have sought to discover means of predicting which patients would benefit from treatment. However, results have been discrepant with few successful replications. Our objective was to build a biobank with DNA, RNA and protein measurements to test the claim that the current state-of-the-art precision medicine will benefit RA patients. METHODS: We collected 451 blood samples from 61 healthy individuals and 185 RA patients initiating treatment, before treatment initiation and at a 3 month follow-up time. All samples were subjected to high-throughput RNA sequencing, DNA genotyping, extensive proteomics and flow cytometry measurements, as well as comprehensive clinical phenotyping. Literature review identified 2 proteins, 52 single-nucleotide polymorphisms (SNPs) and 72 gene-expression biomarkers that had previously been proposed as predictors of Tumor Necrosis Factor (TNF) inhibitor response (ΔDAS28-CRP), RESULTS: From these published TNFi biomarkers we found that 2 protein, 2 SNP and 8 mRNA biomarkers could be replicated in the 59 TNF initiating patients. Combining these replicated biomarkers into a single signature we found that we could explain 51% of the variation in ΔDAS28-CRP. This corresponds to a sensitivity of 0.73 and specificity of 0.78 for the prediction of three month ΔDAS28-CRP better than −1.2. CONCLUSIONS: The COMBINE biobank is currently the largest collection of multi-omics data from RA patients with high potential for discovery and replication. Taking advantage of this we surveyed the current state-of-the-art of drug-response stratification in RA, and identified a small set of previously published biomarkers available in peripheral blood which predicts clinical response to TNF blockade in this independent cohort.

Genetic variation and epigenetic modification of the prodynorphin gene in peripheral blood cells in alcoholism

&NA; Dynorphins are critically involved in the development, maintenance and relapse of alcoholism. Alcohol‐induced changes in the prodynorphin gene expression may be influenced by both gene polymorphisms and epigenetic modifications. The present study of human alcoholics aims to evaluate DNA methylation patterns in the prodynorphin gene (PDYN) promoter and to identify single nucleotide polymorphisms (SNPs) associated with alcohol dependence and with altered DNA methylation. Genomic DNA was isolated from peripheral blood cells of alcoholics and healthy controls, and DNA methylation was studied in the PDYN promoter by bisulfite pyrosequencing. In alcoholics, DNA methylation increased in three of the seven CpG sites investigated, as well as in the average of the seven CpG sites. Data stratification showed lower increase in DNA methylation levels in individuals reporting craving and with higher levels of alcohol consumption. Association with alcoholism was observed for rs2235751 and the presence of the minor allele G was associated with reduced DNA methylation at PDYN promoter in females and younger subjects. Genetic and epigenetic factors within PDYN are related to risk for alcoholism, providing further evidence of its involvement on ethanol effects. These results might be of relevance for developing new biomarkers to predict disease trajectories and therapeutic outcome. HighlightsAltered DNA methylation in prodynorphin gene promoter in human alcoholicsSelected SNP associated with alcoholism and altered DNA methylationRelevant for biomarkers prediction of disease trajectories and therapeutic outcome

Expression of alternatively spliced variants of MAP2K4 gene in rheumatoid arthritis

Backgroundand objectives MAP2K4 encodes a mitogen activated protein kinase kinase 4 (MKK4), important for optimal activation of JNK1-3 and p38 – the two members of the MAP kinase family previously implicated in several rheumatic diseases, including rheumatoid arthritis (RA). Recently, MAP2K4 locus has been identified as being in statistical interaction with the major risk factor for RA - HLA-DRB1 shared epitope (SE) - in RA patients with anticitrullinated protein antibody (ACPA). In this study, the authors describe a novel splice variant of MAP2K4 (V2), and investigate the differential expression profile and phenotype associations of MAP2K4 splice variants in RA patients and healthy individuals. Methods The authors performed a discovery study to detect novel splice variants of MAP2K4 in human peripheral blood cells, and obtained sequence data for variants of interest. The relative expression of MAP2K4 forms in peripheral blood was investigated for 44 RA patients and 44 controls of Caucasian ancestry and analysed against available genotypic and phenotypic data. Results The authors detected a novel ‘skipped exon’ type splice variant of MAP2K4 in our study material. The MAP2K4 splice forms and were differently expressed in peripheral blood material from 88 RA cases and controls. Additionally, within the group of RA patients, a correlation was observed between MAP2K4 variants expression and phonotypical data for ACPA, rheumatoid factor, and SE. Conclusion Our results show differential expression ratio of the canonical and alternatively spliced MAP2K4 mRNAs in RA patients compared to healthy controls. This data implies that MAP2K4 splicing and expression profile may be associated with RA pathogenesis and should be assessed as a potential biomarker in the future.

T cells are influenced by a long non-coding RNA in the autoimmune associated PTPN2 locus

Non-coding SNPs in the protein tyrosine phosphatase non-receptor type 2 (PTPN2) locus have been linked with several autoimmune diseases, including rheumatoid arthritis, type I diabetes, and inflammatory bowel disease. However, the functional consequences of these SNPs are poorly characterized. Herein, we show in blood cells that SNPs in the PTPN2 locus are highly correlated with DNA methylation levels at four CpG sites downstream of PTPN2 and expression levels of the long non-coding RNA (lncRNA) LINC01882 downstream of these CpG sites. We observed that LINC01882 is mainly expressed in T cells and that anti-CD3/CD28 activated naïve CD4+ T cells downregulate the expression of LINC01882. RNA sequencing analysis of LINC01882 knockdown in Jurkat T cells, using a combination of antisense oligonucleotides and RNA interference, revealed the upregulation of the transcription factor ZEB1 and kinase MAP2K4, both involved in IL-2 regulation. Overall, our data suggests the involvement of LINC01882 in T cell activation and hints towards an auxiliary role of these non-coding SNPs in autoimmunity associated with the PTPN2 locus.

Systematic approach demonstrates enrichment of multiple interactions between non-HLA risk variants and HLA-DRB1 risk alleles in rheumatoid arthritis

Objective In anti-citrullinated protein antibody positive rheumatoid arthritis (ACPA-positive RA), a particular subset of HLA-DRB1 alleles, called shared epitope (SE) alleles, is a highly influential genetic risk factor. Here, we investigated whether non-HLA single nucleotide polymorphisms (SNP), conferring low disease risk on their own, interact with SE alleles more frequently than expected by chance and if such genetic interactions influence the HLA-DRB1 SE effect concerning risk to ACPA-positive RA. Methods We computed the attributable proportion (AP) due to additive interaction at genome-wide level for two independent ACPA-positive RA cohorts: the Swedish epidemiological investigation of rheumatoid arthritis (EIRA) and the North American rheumatoid arthritis consortium (NARAC). Then, we tested for differences in the AP p value distributions observed for two groups of SNPs, non-associated and associated with disease. We also evaluated whether the SNPs in interaction with HLA-DRB1 were cis-eQTLs in the SE alleles context in peripheral blood mononuclear cells from patients with ACPA-positive RA (SE-eQTLs). Results We found a strong enrichment of significant interactions (AP p<0.05) between the HLA-DRB1 SE alleles and the group of SNPs associated with ACPA-positive RA in both cohorts (Kolmogorov-Smirnov test D=0.35 for EIRA and D=0.25 for NARAC, p<2.2e-16 for both). Interestingly, 564 out of 1492 SNPs in consistent interaction for both cohorts were significant SE-eQTLs. Finally, we observed that the effect size of HLA-DRB1 SE alleles for disease decreases from 5.2 to 2.5 after removal of the risk alleles of the two top interacting SNPs (rs2476601 and rs10739581). Conclusion Our data demonstrate that there are massive genetic interactions between the HLA-DRB1 SE alleles and non-HLA genetic variants in ACPA-positive RA.

The association between the HLA-DRB1 shared epitope alleles and the risk of rheumatoid arthritis is influenced by massive gene-gene interactions.

In anti-citrullinated protein antibody positive rheumatoid arthritis (ACPA-positive RA), a particular subset of HLA-DRB1 alleles, called shared epitope alleles (SE), is the highest genetic risk factor. Here, we aimed to investigate whether gene-gene interactions influence this HLA-DRB1 related major disease risk; specifically, we set out to test if non-HLA SNPs, conferring low diseases risk on their own, can modulate the HLA-DRB1 SE effect to develop ACPA-positive RA. To address this question, we computed the attributable proportion (AP) due to additive interaction at genome-wide level for two independent ACPA-positive RA cohorts: the Swedish EIRA and the North American NARAC. We found a strong enrichment of significant interactions (AP p-values<0.05) between the HLA-DRB1 SE alleles and a group of SNPs associated with ACPA-positive RA in both cohorts (Kolmogorov-Smirnov [KS] test D=0.35 for EIRA and D=0.25 for NARAC, p<2.2e-16 for both). Interestingly, 201 out of 1,492 SNPs in consistent interaction for both cohorts, were eQTLs in SE alleles context in PBMCs from ACPA-positive RA patients. Finally, we observed that the effect size of HLA-DRB1 SE alleles for disease decreases from 5.2 to 2.5 after discounting the risk alleles of the two top interacting SNPs (rs2476601 and rs10739581, AP FDR corrected p <0.05). Our data demonstrate that the association between the HLA-DRB1 SE alleles and the risk of ACPA-positive RA is modulated by massive genetic interactions with non-HLA genetic variants.

Discovery of new candidate genes for rheumatoid arthritis through integration of genetic association data with expression pathway analysis

BackgroundHere we integrate verified signals from previous genetic association studies with gene expression and pathway analysis for discovery of new candidate genes and signaling networks, relevant for rheumatoid arthritis (RA).MethodRNA-sequencing-(RNA-seq)-based expression analysis of 377 genes from previously verified RA-associated loci was performed in blood cells from 5 newly diagnosed, non-treated patients with RA, 7 patients with treated RA and 12 healthy controls. Differentially expressed genes sharing a similar expression pattern in treated and untreated RA sub-groups were selected for pathway analysis. A set of “connector” genes derived from pathway analysis was tested for differential expression in the initial discovery cohort and validated in blood cells from 73 patients with RA and in 35 healthy controls.ResultsThere were 11 qualifying genes selected for pathway analysis and these were grouped into two evidence-based functional networks, containing 29 and 27 additional connector molecules. The expression of genes, corresponding to connector molecules was then tested in the initial RNA-seq data. Differences in the expression of ERBB2, TP53 and THOP1 were similar in both treated and non-treated patients with RA and an additional nine genes were differentially expressed in at least one group of patients compared to healthy controls. The ERBB2, TP53. THOP1 expression profile was successfully replicated in RNA-seq data from peripheral blood mononuclear cells from healthy controls and non-treated patients with RA, in an independent collection of samples.ConclusionIntegration of RNA-seq data with findings from association studies, and consequent pathway analysis implicate new candidate genes, ERBB2, TP53 and THOP1 in the pathogenesis of RA.