B cells are important in the pathogenesis of primary Sjögren's syndrome (pSS). Patients positive for Sjögren's syndrome antigen A/Sjögren syndrome antigen B (SSA/SSB) autoantibodies are more prone to ...systemic disease manifestations and adverse outcomes. We aimed to determine the role of B cell composition, gene expression, and B cell receptor usage in pSS subgroups stratified for SSA/SSB antibodies.
Over 230,000 B cells were isolated from peripheral blood of patients with pSS (n = 6 SSA-, n = 8 SSA+ single positive and n = 10 SSA/SSB+ double positive) and four healthy controls and processed for single-cell RNA sequencing (scRNA-seq) and single-cell variable, diversity, and joining (VDJ) gene sequencing (scVDJ-seq).
We show that SSA/SSB+ patients present the highest and lowest proportion of naïve and memory B cells, respectively, and the highest up-regulation of interferon-induced genes across all B cell subtypes. Differential usage of IGHV showed that IGHV1-69 and IGHV4-30-4 were more often used in all pSS subgroups compared with controls. Memory B cells from SSA/SSB+ patients displayed a higher proportion of cells with unmutated VDJ transcripts compared with other pSS patient groups and controls, indicating altered somatic hypermutation processes. Comparison with previous studies revealed heterogeneous clonotype pools, with little overlap in CDR3 sequences. Joint analysis using scRNA-seq and scVDJ-seq data allowed unsupervised stratification of patients with pSS and identified novel parameters that correlated to disease manifestations and antibody status.
We describe heterogeneity and molecular characteristics in B cells from patients with pSS, providing clues to intrinsic differences in B cells that affect the phenotype and outcome and allowing stratification of patients with pSS at improved resolution.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Increasing evidence suggests an epigenetic contribution to the pathogenesis of autoimmune diseases, including primary Sjögren's Syndrome (pSS). The aim of this study was to investigate the role of ...DNA methylation in pSS by analysing multiple tissues from patients and controls.
Genome-wide DNA methylation profiles were generated using HumanMethylation450K BeadChips for whole blood, CD19+ B cells and minor salivary gland biopsies. Gene expression was analysed in CD19+ B cells by RNA-sequencing. Analysis of genetic regulatory effects on DNA methylation at known pSS risk loci was performed.
We identified prominent hypomethylation of interferon (IFN)-regulated genes in whole blood and CD19+ B cells, including at the genes MX1, IFI44L and PARP9, replicating previous reports in pSS, as well as identifying a large number of novel associations. Enrichment for genomic overlap with histone marks for enhancer and promoter regions was observed. We showed for the first time that hypomethylation of IFN-regulated genes in pSS B cells was associated with their increased expression. In minor salivary gland biopsies we observed hypomethylation of the IFN-induced gene OAS2. Pathway and disease analysis resulted in enrichment of antigen presentation, IFN signalling and lymphoproliferative disorders. Evidence for genetic control of methylation levels at known pSS risk loci was observed.
Our study highlights the role of epigenetic regulation of IFN-induced genes in pSS where replication is needed for novel findings. The association with altered gene expression suggests a functional mechanism for differentially methylated CpG sites in pSS aetiology.
Understanding the relationship between genetic variation and biological function on a genomic scale is expected to provide fundamental new insights into the biology, evolution and pathophysiology of ...humans and other species. The hope that single nucleotide polymorphisms (SNPs) will allow genes that underlie complex disease to be identified, together with progress in identifying large sets of SNPs, are the driving forces behind intense efforts to establish the technology for large-scale analysis of SNPs. New genotyping methods that are high throughput, accurate and cheap are urgently needed for gaining full access to the abundant genetic variation of organisms.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Acute lymphoblastic leukemia (ALL) is the most common malignancy in children. ALL arises from the malignant transformation of progenitor B- and T-cells in the bone marrow into leukemic cells, but the ...mechanisms underlying this transformation are not well understood. Recent technical advances and decreasing costs of methods for high-throughput DNA sequencing and SNP genotyping have stimulated systematic studies of the epigenetic changes in leukemic cells from pediatric ALL patients. The results emerging from these studies are increasing our understanding of the epigenetic component of leukemogenesis and have demonstrated the potential of DNA methylation as a biomarker for lineage and subtype classification, prognostication, and disease progression in ALL. In this review, we provide a concise examination of the epigenetic studies in ALL, with a focus on DNA methylation and mutations perturbing genes involved in chromatin modification, and discuss the future role of epigenetic analyses in research and clinical management of ALL.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Genome-wide association studies with SNP markers are expected to allow identification of genes that underlie complex disorders. Hundreds of thousands of SNP markers will be required for comprehensive ...genome-wide association studies. The development of microarray-based methods for SNP genotyping on this scale remains a demanding task, despite many recent advances in technology for the production of high-density microarrays. A key technical obstacle is the PCR amplification step, which is required to reduce the complexity of and gain sufficient sensitivity for genotyping SNPs in large, diploid genomes. The multiplexing level that can be achieved in PCR does not match that of current microarray-based methods, making PCR the limiting step in the assays. Highly multiplexed microarray systems for SNP genotyping have recently been developed by combining well-known reaction principles for DNA amplification and SNP genotyping in clever ways. These new methods offer the potential of genome-wide SNP mapping of genes involved in complex diseases in the foreseeable future, provided that issues related to selection of the optimal SNP markers, sample throughput and the cost of the assays can be addressed.
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DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
STAT1 gain‐of‐function (GOF) variants lead to defective Th17 cell development and chronic mucocutaneous candidiasis (CMC), but frequently also to autoimmunity. Stimulation of cells with STAT1 ...inducing cytokines like interferons (IFN) result in hyperphosphorylation and delayed dephosphorylation of GOF STAT1. However, the mechanism how the delayed dephosphorylation exactly causes the increased expression of STAT1‐dependent genes, and how the intracellular signal transduction from cytokine receptors is affected, remains unknown. In this study we show that the circulating levels of IFN‐α were not persistently elevated in STAT1 GOF patients. Nevertheless, the expression of interferon signature genes was evident even in the patient with low or undetectable serum IFN‐α levels. Chromatin immunoprecipitation (ChIP) experiments revealed that the active chromatin mark trimethylation of lysine 4 of histone 3 (H3K4me3), was significantly enriched in areas associated with interferon‐stimulated genes in STAT1 GOF cells in comparison to cells from healthy donors. This suggests that the chromatin binding of GOF STAT1 variant promotes epigenetic changes compatible with higher gene expression and elevated reactivity to type I interferons, and possibly predisposes for interferon‐related autoimmunity. The results also suggest that epigenetic rewiring may be responsible for treatment failure of Janus kinase 1/2 (JAK1/2) inhibitors in certain patients.
Type I IFN activates STAT1 signaling pathway. In STAT1 GOF cells interferon‐stimulated gene (ISG) expression is higher and the active chromatin mark H3K4me3 is enriched in areas associated with ISGs. GOF STAT1 variant may promote epigenetic changes leading to elevated reactivity to type I IFN, and predispose for autoimmunity.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Systemic lupus erythematosus (SLE) is a genetically complex autoimmune disease characterized by loss of immune tolerance to nuclear and cell surface antigens. Previous genome-wide association studies ...(GWAS) had modest sample sizes, reducing their scope and reliability. Our study comprised 7,219 cases and 15,991 controls of European ancestry, constituting a new GWAS, a meta-analysis with a published GWAS and a replication study. We have mapped 43 susceptibility loci, including ten new associations. Assisted by dense genome coverage, imputation provided evidence for missense variants underpinning associations in eight genes. Other likely causal genes were established by examining associated alleles for cis-acting eQTL effects in a range of ex vivo immune cells. We found an over-representation (n = 16) of transcription factors among SLE susceptibility genes. This finding supports the view that aberrantly regulated gene expression networks in multiple cell types in both the innate and adaptive immune response contribute to the risk of developing SLE.
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IJS, NUK, SBMB, UL, UM, UPUK
Structural chromosomal rearrangements that can lead to in-frame gene-fusions are a leading source of information for diagnosis, risk stratification, and prognosis in pediatric acute lymphoblastic ...leukemia (ALL). Traditional methods such as karyotyping and FISH struggle to accurately identify and phase such large-scale chromosomal aberrations in ALL genomes. We therefore evaluated linked-read WGS for detecting chromosomal rearrangements in primary samples of from 12 patients diagnosed with ALL. We assessed the effect of input DNA quality on phased haplotype block size and the detectability of copy number aberrations and structural variants in the ALL genomes. We found that biobanked DNA isolated by standard column-based extraction methods was sufficient to detect chromosomal rearrangements even at low 10x sequencing coverage. Linked-read WGS enabled precise, allele-specific, digital karyotyping at a base-pair resolution for a wide range of structural variants including complex rearrangements and aneuploidy assessment. With use of haplotype information from the linked-reads, we also identified previously unknown structural variants, such as a compound heterozygous deletion of ERG in a patient with the DUX4-IGH fusion gene. We conclude that linked-read WGS allows detection of important pathogenic variants in ALL genomes at a resolution beyond that of traditional karyotyping and FISH.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Ticagrelor, a direct-acting P2Y12-receptor antagonist, is rapidly absorbed and partly metabolized to the major metabolite AR-C124910XX (ARC). To identify single-nucleotide polymorphisms (SNPs) ...associated with pharmacokinetics of ticagrelor and clinical outcomes, we performed a genome-wide association study (GWAS) in patients treated with ticagrelor in the PLATO trial.
A two-stage design was used for the GWAS with discovery (discovery phase: n = 1812) and replication cohorts (replication phase: n = 1941). The steady-state area under the curve (AUCss) values, estimated by the population pharmacokinetic (PK) models, were log transformed and analysed on a genome-wide scale using linear regression. SNPs were analysed against clinical events using Cox-regression in 4990 patients. An SNP (rs113681054) in SLCO1B1 was associated with levels of ticagrelor (P = 1.1 × 10(-6)) and ARC (P = 4.6 × 10(-13)). This SNP is in linkage disequilibrium with a functional variant (rs4149056) that results in decreased OATP1B1 transporter activity. Ticagrelor levels were also associated with two independent SNPs (rs62471956, P = 7.7 × 10(-15) and rs56324128, P = 9.7 × 10(-12)) in the CYP3A4 region. Further, ARC levels were associated with rs61361928 (P = 3.0 × 10(-14)) in UGT2B7. At all loci, the effects were small. None of the identified SNPs that affected ticagrelor PK were associated with the primary composite outcome (cardiovascular death myocardial infarction, and stroke), non-CABG-related bleeds or investigator-reported dyspnoea.
In patients with ACS, ticagrelor pharmacokinetics is influenced by three genetic loci (SLCO1B1, UGT2B7, and CYP3A4). However, the modest genetic effects on ticagrelor plasma levels did not translate into any detectable effect on efficacy or safety during ticagrelor treatment.
NCT00391872.
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