Publications by authors named "Johanna K Sandling"

44 Publications

Contribution of rare genetic variation to disease susceptibility in a large Scandinavian myositis cohort.

Arthritis Rheumatol 2021 Jul 19. Epub 2021 Jul 19.

Division of Rheumatology, Department of Medicine, Solna, Karolinska Institutet, and Karolinska University Hospital, Stockholm, Sweden.

Objective: Idiopathic inflammatory myopathies (IIM) are a heterogeneous group of complex autoimmune conditions characterized by inflammation in skeletal muscle and extra-muscular compartments and interferon system activation. Here we aimed to examine the contribution of genetic variation to disease susceptibility and to identify novel avenues for research in IIM.

Methods: Targeted DNA sequencing was used to mine coding and potentially regulatory single nucleotide variants from ~1900 immune-related genes in a Scandinavian case-control cohort of 454 IIM patients and 1,024 healthy controls. Gene-based aggregate testing, together with rare variant- and gene-level enrichment analyses, was implemented to explore genotype-phenotype relations.

Results: Gene-based aggregate tests of all variants, including rare variants, identified IFI35 as potential genetic risk locus for IIM, suggesting a genetic signature of type I interferon pathway activation. Functional annotation of the IFI35 locus highlighted a regulatory network linked to the skeletal muscle-specific gene PTGES3L, as a potential candidate for IIM pathogenesis. Aggregate genetic associations with AGER and PSMB8 in the Major Histocompatibility Complex locus were detected in the anti-synthetase syndrome subgroup, which additionally showed a less marked genetic signature of the type I interferon pathway. Enrichment analyses indicated a burden of synonymous and non-coding rare variants in IIM patients, suggesting increased disease predisposition associated with these classes of rare variants.

Conclusion: Our study suggests the contribution of rare genetic variation to disease susceptibility in IIM and specific patient subgroups, and pinpoints genetic associations consistent with previous findings by gene expression profiling. These features highlight genetic profiles potentially relevant to disease pathogenesis.
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http://dx.doi.org/10.1002/art.41929DOI Listing
July 2021

Variants in BANK1 are associated with lupus nephritis of European ancestry.

Genes Immun 2021 Jul 14;22(3):194-202. Epub 2021 Jun 14.

Department of Medical Sciences and Science for Life Laboratory, Uppsala University, Uppsala, Sweden.

The genetic background of lupus nephritis (LN) has not been completely elucidated. We performed a case-only study of 2886 SLE patients, including 947 (33%) with LN. Renal biopsies were available from 396 patients. The discovery cohort (Sweden, n = 1091) and replication cohort 1 (US, n = 962) were genotyped on the Immunochip and replication cohort 2 (Denmark/Norway, n = 833) on a custom array. Patients with LN, proliferative nephritis, or LN with end-stage renal disease were compared with SLE without nephritis. Six loci were associated with LN (p < 1 × 10, NFKBIA, CACNA1S, ITGA1, BANK1, OR2Y, and ACER3) in the discovery cohort. Variants in BANK1 showed the strongest association with LN in replication cohort 1 (p = 9.5 × 10) and proliferative nephritis in a meta-analysis of discovery and replication cohort 1. There was a weak association between BANK1 and LN in replication cohort 2 (p = 0.052), and in the meta-analysis of all three cohorts the association was strengthened (p = 2.2 × 10). DNA methylation data in 180 LN patients demonstrated methylation quantitative trait loci (meQTL) effects between a CpG site and BANK1 variants. To conclude, we describe genetic variations in BANK1 associated with LN and evidence for genetic regulation of DNA methylation within the BANK1 locus. This indicates a role for BANK1 in LN pathogenesis.
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http://dx.doi.org/10.1038/s41435-021-00142-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8277572PMC
July 2021

Interaction between the rs11889341(T) risk allele and smoking confers increased risk of myocardial infarction and nephritis in patients with systemic lupus erythematosus.

Ann Rheum Dis 2021 Mar 25. Epub 2021 Mar 25.

Department of Medical Sciences, Rheumatology and Science for Life Laboratory, Uppsala University, Uppsala, Sweden.

Objective: To investigate how genetics influence the risk of smoking-related systemic lupus erythematosus (SLE) manifestations.

Methods: Patients with SLE (n=776, n=836) were genotyped using the 200K Immunochip single nucleotide polymorphisms (SNP) Array (Illumina) and a custom array. Sixty SNPs with SLE association (p<5.0×10) were analysed. Signal transducer and activator of transcription 4 (STAT4) activation was assessed in stimulated peripheral blood mononuclear cells from healthy controls (n=45).

Results: In the discovery cohort, smoking was associated with myocardial infarction (MI) (OR 1.96 (95% CI 1.09 to 3.55)), with a greater effect in patients carrying any rs11889341 risk allele (OR 2.72 (95% CI 1.24 to 6.00)) or two risk alleles (OR 8.27 (95% CI 1.48 to 46.27)).Smokers carrying the risk allele also displayed an increased risk of nephritis (OR 1.47 (95% CI 1.06 to 2.03)). In the replication cohort, the high risk of MI in smokers carrying the risk allele and the association between the risk allele and nephritis in smokers were confirmed (OR 6.19 (95% CI 1.29 to 29.79) and 1.84 (95% CI 1.05 to 3.29), respectively).The interaction between smoking and the risk allele resulted in further increase in the risk of MI (OR 2.14 (95% CI 1.01 to 4.62)) and nephritis (OR 1.53 (95% CI 1.08 to 2.17)), with 54% (MI) and 34% (nephritis) of the risk attributable to the interaction. Levels of interleukin-12-induced phosphorylation of STAT4 in CD8+ T cells were higher in smokers than in non-smokers (mean geometric fluorescence intensity 1063 vs 565, p=0.0063).Lastly, the rs564799 risk allele displayed association with MI in both cohorts (OR 1.53 (95% CI 1.01 to 2.31) and 2.15 (95% CI 1.08 to 4.26), respectively).

Conclusions: Smoking in the presence of the risk gene variant appears to increase the risk of MI and nephritis in SLE. Our results also highlight the role of the IL12-STAT4 pathway in SLE-cardiovascular morbidity.
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http://dx.doi.org/10.1136/annrheumdis-2020-219727DOI Listing
March 2021

Function of multiple sclerosis-protective HLA class I alleles revealed by genome-wide protein-quantitative trait loci mapping of interferon signalling.

PLoS Genet 2020 10 26;16(10):e1009199. Epub 2020 Oct 26.

Rheumatology and Science for Life Laboratories, Department of Medical Sciences, Uppsala University, Uppsala, Sweden.

Interferons (IFNs) are cytokines that are central to the host defence against viruses and other microorganisms. If not properly regulated, IFNs may contribute to the pathogenesis of inflammatory autoimmune, or infectious diseases. To identify genetic polymorphisms regulating the IFN system we performed an unbiased genome-wide protein-quantitative trait loci (pQTL) mapping of cell-type specific type I and type II IFN receptor levels and their responses in immune cells from 303 healthy individuals. Seven genome-wide significant (p < 5.0E-8) pQTLs were identified. Two independent SNPs that tagged the multiple sclerosis (MS)-protective HLA class I alleles A*02/A*68 and B*44, respectively, were associated with increased levels of IFNAR2 in B and T cells, with the most prominent effect in IgD-CD27+ memory B cells. The increased IFNAR2 levels in B cells were replicated in cells from an independent set of healthy individuals and in MS patients. Despite increased IFNAR2 levels, B and T cells carrying the MS-protective alleles displayed a reduced response to type I IFN stimulation. Expression and methylation-QTL analysis demonstrated increased mRNA expression of the pseudogene HLA-J in B cells carrying the MS-protective class I alleles, possibly driven via methylation-dependent transcriptional regulation. Together these data suggest that the MS-protective effects of HLA class I alleles are unrelated to their antigen-presenting function, and propose a previously unappreciated function of type I IFN signalling in B and T cells in MS immune-pathogenesis.
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http://dx.doi.org/10.1371/journal.pgen.1009199DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7644105PMC
October 2020

Molecular pathways in patients with systemic lupus erythematosus revealed by gene-centred DNA sequencing.

Ann Rheum Dis 2021 01 9;80(1):109-117. Epub 2020 Oct 9.

Department of Biomedical and Clinical Sciences, Division of Inflammation and Infection, Linköping University, Linköping, Sweden.

Objectives: Systemic lupus erythematosus (SLE) is an autoimmune disease with extensive heterogeneity in disease presentation between patients, which is likely due to an underlying molecular diversity. Here, we aimed at elucidating the genetic aetiology of SLE from the immunity pathway level to the single variant level, and stratify patients with SLE into distinguishable molecular subgroups, which could inform treatment choices in SLE.

Methods: We undertook a pathway-centred approach, using sequencing of immunological pathway genes. Altogether 1832 candidate genes were analysed in 958 Swedish patients with SLE and 1026 healthy individuals. Aggregate and single variant association testing was performed, and we generated pathway polygenic risk scores (PRS).

Results: We identified two main independent pathways involved in SLE susceptibility: T lymphocyte differentiation and innate immunity, characterised by HLA and interferon, respectively. Pathway PRS defined pathways in individual patients, who on average were positive for seven pathways. We found that SLE organ damage was more pronounced in patients positive for the T or B cell receptor signalling pathways. Further, pathway PRS-based clustering allowed stratification of patients into four groups with different risk score profiles. Studying sets of genes with priors for involvement in SLE, we observed an aggregate common variant contribution to SLE at genes previously reported for monogenic SLE as well as at interferonopathy genes.

Conclusions: Our results show that pathway risk scores have the potential to stratify patients with SLE beyond clinical manifestations into molecular subsets, which may have implications for clinical follow-up and therapy selection.
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http://dx.doi.org/10.1136/annrheumdis-2020-218636DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7788061PMC
January 2021

Genetic and clinical basis for two distinct subtypes of primary Sjögren's syndrome.

Rheumatology (Oxford) 2021 02;60(2):837-848

Science for Life Laboratory, Department of Medical Biochemistry and Microbiology, Uppsala, Sweden.

Objectives: Clinical presentation of primary Sjögren's syndrome (pSS) varies considerably. A shortage of evidence-based objective markers hinders efficient drug development and most clinical trials have failed to reach primary endpoints.

Methods: We performed a multicentre study to identify patient subgroups based on clinical, immunological and genetic features. Targeted DNA sequencing of 1853 autoimmune-related loci was performed. After quality control, 918 patients with pSS, 1264 controls and 107 045 single nucleotide variants remained for analysis. Replication was performed in 177 patients with pSS and 7672 controls.

Results: We found strong signals of association with pSS in the HLA region. Principal component analysis of clinical data distinguished two patient subgroups defined by the presence of SSA/SSB antibodies. We observed an unprecedented high risk of pSS for an association in the HLA-DQA1 locus of odds ratio 6.10 (95% CI: 4.93, 7.54, P=2.2×10-62) in the SSA/SSB-positive subgroup, while absent in the antibody negative group. Three independent signals within the MHC were observed. The two most significant variants in MHC class I and II respectively, identified patients with a higher risk of hypergammaglobulinaemia, leukopenia, anaemia, purpura, major salivary gland swelling and lymphadenopathy. Replication confirmed the association with both MHC class I and II signals confined to SSA/SSB antibody positive pSS.

Conclusion: Two subgroups of patients with pSS with distinct clinical manifestations can be defined by the presence or absence of SSA/SSB antibodies and genetic markers in the HLA locus. These subgroups should be considered in clinical follow-up, drug development and trial outcomes, for the benefit of both subgroups.
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http://dx.doi.org/10.1093/rheumatology/keaa367DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7850528PMC
February 2021

Contributions of de novo variants to systemic lupus erythematosus.

Eur J Hum Genet 2021 Jan 28;29(1):184-193. Epub 2020 Jul 28.

Department of Medical Sciences, Molecular Medicine and Science for Life Laboratory, Uppsala University, 751 23, Uppsala, Sweden.

By performing whole-genome sequencing in a Swedish cohort of 71 parent-offspring trios, in which the child in each family is affected by systemic lupus erythematosus (SLE, OMIM 152700), we investigated the contribution of de novo variants to risk of SLE. We found de novo single nucleotide variants (SNVs) to be significantly enriched in gene promoters in SLE patients compared with healthy controls at a level corresponding to 26 de novo promoter SNVs more in each patient than expected. We identified 12 de novo SNVs in promoter regions of genes that have been previously implicated in SLE, or that have functions that could be of relevance to SLE. Furthermore, we detected three missense de novo SNVs, five de novo insertion-deletions, and three de novo structural variants with potential to affect the expression of genes that are relevant for SLE. Based on enrichment analysis, disease-affecting de novo SNVs are expected to occur in one-third of SLE patients. This study shows that de novo variants in promoters commonly contribute to the genetic risk of SLE. The fact that de novo SNVs in SLE were enriched to promoter regions highlights the importance of using whole-genome sequencing for identification of de novo variants.
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http://dx.doi.org/10.1038/s41431-020-0698-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7852530PMC
January 2021

High genetic risk score is associated with early disease onset, damage accrual and decreased survival in systemic lupus erythematosus.

Ann Rheum Dis 2020 03 11;79(3):363-369. Epub 2019 Dec 11.

Rheumatology and Science for Life Laboratory, Department of Medical Sciences, Uppsala University, Uppsala, Sweden.

Objectives: To investigate associations between a high genetic disease risk and disease severity in patients with systemic lupus erythematosus (SLE).

Methods: Patients with SLE (n=1001, discovery cohort and n=5524, replication cohort) and healthy controls (n=2802 and n=9859) were genotyped using a 200K Immunochip single nucleotide polymorphism array. A genetic risk score (GRS) was assigned to each individual based on 57 SLE risk loci.

Results: SLE was more prevalent in the high, compared with the low, GRS-quartile (OR 12.32 (9.53 to 15.71), p=7.9×10 and OR 7.48 (6.73 to 8.32), p=2.2×10 for the discovery and the replication cohorts, respectively). In the discovery cohort, patients in the high GRS-quartile had a 6-year earlier mean disease onset (HR 1.47 (1.22 to 1.75), p=4.3×10), displayed higher prevalence of damage accrual (OR 1.47 (1.06 to 2.04), p=2.0×10), renal disorder (OR 2.22 (1.50 to 3.27), p=5.9×10), anti-dsDNA (OR 1.83 (1.19 to 2.81), p=6.1×10), end-stage renal disease (ESRD) (OR 5.58 (1.50 to 20.79), p=1.0×10), proliferative nephritis (OR 2.42 (1.30 to 4.49), p=5.1×10), anti-cardiolipin-IgG (OR 1.89 (1.13 to 3.18), p=1.6×10), anti-β-glycoprotein-I-IgG (OR 2.29 (1.29 to 4.06), p=4.8×10) and positive lupus anticoagulant test (OR 2.12 (1.16 to 3.89), p=1.5×10) compared with patients in the low GRS-quartile. Survival analysis showed earlier onset of the first organ damage (HR 1.51 (1.04 to 2.25), p=3.7×10), first cardiovascular event (HR 1.65 (1.03 to 2.64), p=2.6×10), nephritis (HR 2.53 (1.72 to 3.71), p=9.6×10), ESRD (HR 6.78 (1.78 to 26.86), p=6.5×10) and decreased overall survival (HR 1.83 (1.02 to 3.30), p=4.3×10) in high to low quartile comparison.

Conclusions: A high GRS is associated with increased risk of organ damage, renal dysfunction and all-cause mortality. Our results indicate that genetic profiling may be useful for predicting outcomes in patients with SLE.
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http://dx.doi.org/10.1136/annrheumdis-2019-216227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7034364PMC
March 2020

Shared and Unique Patterns of DNA Methylation in Systemic Lupus Erythematosus and Primary Sjögren's Syndrome.

Front Immunol 2019 30;10:1686. Epub 2019 Jul 30.

Section of Rheumatology and Science for Life Laboratory, Department of Medical Sciences, Uppsala University, Uppsala, Sweden.

To perform a cross-comparative analysis of DNA methylation in patients with systemic lupus erythematosus (SLE), patients with primary Sjögren's syndrome (pSS), and healthy controls addressing the question of epigenetic sharing and aiming to detect disease-specific alterations. DNA extracted from peripheral blood from 347 cases with SLE, 100 cases with pSS, and 400 healthy controls were analyzed on the Human Methylation 450k array, targeting 485,000 CpG sites across the genome. A linear regression model including age, sex, and blood cell type distribution as covariates was fitted, and association -values were Bonferroni corrected. A random forest machine learning classifier was designed for prediction of disease status based on DNA methylation data. We established a combined set of 4,945 shared differentially methylated CpG sites (DMCs) in SLE and pSS compared to controls. In pSS, hypomethylation at type I interferon induced genes was mainly driven by patients who were positive for Ro/SSA and/or La/SSB autoantibodies. Analysis of differential methylation between SLE and pSS identified 2,244 DMCs with a majority of sites showing decreased methylation in SLE compared to pSS. The random forest classifier demonstrated good performance in discerning between disease status with an area under the curve (AUC) between 0.83 and 0.96. The majority of differential DNA methylation is shared between SLE and pSS, however, important quantitative differences exist. Our data highlight neutrophil dysregulation as a shared mechanism, emphasizing the role of neutrophils in the pathogenesis of systemic autoimmune diseases. The current study provides evidence for genes and molecular pathways driving common and disease-specific pathogenic mechanisms.
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http://dx.doi.org/10.3389/fimmu.2019.01686DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6688520PMC
October 2020

Genetic variations in A20 DUB domain provide a genetic link to citrullination and neutrophil extracellular traps in systemic lupus erythematosus.

Ann Rheum Dis 2019 10 12;78(10):1363-1370. Epub 2019 Jul 12.

Research and Early Development, Respiratory, Inflammation and Autoimmune, BioPharmaceuticals R&D, AstraZeneca R&D Gothenburg, Mölndal, Sweden

Objectives: Genetic variations in (A20) de-ubiquitinase (DUB) domain increase the risk of systemic lupus erythematosus (SLE) and rheumatoid arthritis. A20 is a negative regulator of NF-κB but the role of its DUB domain and related genetic variants remain unclear. We aimed to study the functional effects of A20 DUB-domain alterations in immune cells and understand its link to SLE pathogenesis.

Methods: CRISPR/Cas9 was used to generate human U937 monocytes with A20 DUB-inactivating knock-in (KI) mutation. Whole genome RNA-sequencing was used to identify differentially expressed genes between WT and KI cells. Functional studies were performed in A20 U937 cells and in immune cells from A20 mice and genotyped healthy individuals with A20 DUB polymorphism rs2230926. Neutrophil extracellular trap (NET) formation was addressed ex vivo in neutrophils from A20 mice and SLE-patients with rs2230926.

Results: Genetic disruption of A20 DUB domain in human and murine myeloid cells did not give rise to enhanced NF-κB signalling. Instead, cells with mutation or rs2230926 polymorphism presented an upregulated expression of , an enzyme regulating protein citrullination and NET formation, two key mechanisms in autoimmune pathology. A20 cells exhibited enhanced protein citrullination and extracellular trap formation, which could be suppressed by selective PAD4 inhibition. Moreover, SLE-patients with rs2230926 showed increased NETs and increased frequency of autoantibodies to citrullinated epitopes.

Conclusions: We propose that genetic alterations disrupting the A20 DUB domain mediate increased susceptibility to SLE through the upregulation of with resultant protein citrullination and extracellular trap formation.
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http://dx.doi.org/10.1136/annrheumdis-2019-215434DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6788882PMC
October 2019

Circulating Levels of Interferon Regulatory Factor-5 Associates With Subgroups of Systemic Lupus Erythematosus Patients.

Front Immunol 2019 17;10:1029. Epub 2019 May 17.

Division of Rheumatology, Department of Medicine Solna, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden.

Systemic Lupus Erythematosus (SLE) is a heterogeneous autoimmune disease, which currently lacks specific diagnostic biomarkers. The diversity within the patients obstructs clinical trials but may also reflect differences in underlying pathogenesis. Our objective was to obtain protein profiles to identify potential general biomarkers of SLE and to determine molecular subgroups within SLE for patient stratification. Plasma samples from a cross-sectional study of well-characterized SLE patients ( = 379) and matched population controls ( = 316) were analyzed by antibody suspension bead array targeting 281 proteins. To investigate the differences between SLE and controls, Mann-Whitney -test with Bonferroni correction, generalized linear modeling and receiver operating characteristics (ROC) analysis were performed. K-means clustering was used to identify molecular SLE subgroups. We identified Interferon regulating factor 5 (IRF5), solute carrier family 22 member 2 (SLC22A2) and S100 calcium binding protein A12 (S100A12) as the three proteins with the largest fold change between SLE patients and controls (SLE/Control = 1.4, 1.4, and 1.2 respectively). The lowest -values comparing SLE patients and controls were obtained for S100A12, Matrix metalloproteinase-1 (MMP1) and SLC22A2 (p = 3 × 10, 3 × 10, and 5 × 10 respectively). In a set of 15 potential biomarkers differentiating SLE patients and controls, two of the proteins were transcription factors, i.e., IRF5 and SAM pointed domain containing ETS transcription factor (SPDEF). IRF5 was up-regulated while SPDEF was found to be down-regulated in SLE patients. Unsupervised clustering of all investigated proteins identified three molecular subgroups among SLE patients, characterized by (1) high levels of rheumatoid factor-IgM, (2) low IRF5, and (3) high IRF5. IRF5 expressing microparticles were analyzed by flow cytometry in a subset of patients to confirm the presence of IRF5 in plasma and detection of extracellular IRF5 was further confirmed by immunoprecipitation-mass spectrometry (IP-MS). Interestingly IRF5, a known genetic risk factor for SLE, was detected extracellularly and suggested by unsupervised clustering analysis to differentiate between SLE subgroups. Our results imply a set of circulating molecules as markers of possible pathogenic importance in SLE. We believe that these findings could be of relevance for understanding the pathogenesis and diversity of SLE, as well as for selection of patients in clinical trials.
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http://dx.doi.org/10.3389/fimmu.2019.01029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6533644PMC
September 2020

Whole-genome sequencing identifies complex contributions to genetic risk by variants in genes causing monogenic systemic lupus erythematosus.

Hum Genet 2019 Feb 1;138(2):141-150. Epub 2019 Feb 1.

Department of Medical Sciences, Molecular Medicine and Science for Life Laboratory, Uppsala University, 751 23, Uppsala, Sweden.

Systemic lupus erythematosus (SLE, OMIM 152700) is a systemic autoimmune disease with a complex etiology. The mode of inheritance of the genetic risk beyond familial SLE cases is currently unknown. Additionally, the contribution of heterozygous variants in genes known to cause monogenic SLE is not fully understood. Whole-genome sequencing of DNA samples from 71 Swedish patients with SLE and their healthy biological parents was performed to investigate the general genetic risk of SLE using known SLE GWAS risk loci identified using the ImmunoChip, variants in genes associated to monogenic SLE, and the mode of inheritance of SLE risk alleles in these families. A random forest model for predicting genetic risk for SLE showed that the SLE risk variants were mainly inherited from one of the parents. In the 71 patients, we detected a significant enrichment of ultra-rare ( ≤ 0.1%) missense and nonsense mutations in 22 genes known to cause monogenic forms of SLE. We identified one previously reported homozygous nonsense mutation in the C1QC (Complement C1q C Chain) gene, which explains the immunodeficiency and severe SLE phenotype of that patient. We also identified seven ultra-rare, coding heterozygous variants in five genes (C1S, DNASE1L3, DNASE1, IFIH1, and RNASEH2A) involved in monogenic SLE. Our findings indicate a complex contribution to the overall genetic risk of SLE by rare variants in genes associated with monogenic forms of SLE. The rare variants were inherited from the other parent than the one who passed on the more common risk variants leading to an increased genetic burden for SLE in the child. Higher frequency SLE risk variants are mostly passed from one of the parents to the offspring affected with SLE. In contrast, the other parent, in seven cases, contributed heterozygous rare variants in genes associated with monogenic forms of SLE, suggesting a larger impact of rare variants in SLE than hitherto reported.
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http://dx.doi.org/10.1007/s00439-018-01966-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6373277PMC
February 2019

A rare regulatory variant in the MEF2D gene affects gene regulation and splicing and is associated with a SLE sub-phenotype in Swedish cohorts.

Eur J Hum Genet 2019 03 20;27(3):432-441. Epub 2018 Nov 20.

Science for Life Laboratory, Department of Medical Biochemistry and Microbiology, Uppsala University, Box 582, SE-751 24, Uppsala, Sweden.

Systemic lupus erythematosus (SLE) is an autoimmune disorder with heterogeneous clinical presentation and complex etiology involving the interplay between genetic, epigenetic, environmental and hormonal factors. Many common SNPs identified by genome wide-association studies (GWAS) explain only a small part of the disease heritability suggesting the contribution from rare genetic variants, undetectable in GWAS, and complex epistatic interactions. Using targeted re-sequencing of coding and conserved regulatory regions within and around 215 candidate genes selected on the basis of their known role in autoimmunity and genes associated with canine immune-mediated diseases, we identified a rare regulatory variant rs200395694:G > T located in intron 4 of the MEF2D gene encoding the myocyte-specific enhancer factor 2D transcription factor and associated with SLE in Swedish cohorts (504 SLE patients and 839 healthy controls, p = 0.014, CI = 1.1-10). Fisher's exact test revealed an association between the genetic variant and a triad of disease manifestations including Raynaud, anti-U1-ribonucleoprotein (anti-RNP), and anti-Smith (anti-Sm) antibodies (p = 0.00037) among the patients. The DNA-binding activity of the allele was further studied by EMSA, reporter assays, and minigenes. The region has properties of an active cell-specific enhancer, differentially affected by the alleles of rs200395694:G > T. In addition, the risk allele exerts an inhibitory effect on the splicing of the alternative tissue-specific isoform, and thus may modify the target gene set regulated by this isoform. These findings emphasize the potential of dissecting traits of complex diseases and correlating them with rare risk alleles with strong biological effects.
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http://dx.doi.org/10.1038/s41431-018-0297-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6460566PMC
March 2019

Cytokine production by activated plasmacytoid dendritic cells and natural killer cells is suppressed by an IRAK4 inhibitor.

Arthritis Res Ther 2018 Oct 24;20(1):238. Epub 2018 Oct 24.

Department of Medical Sciences, Rheumatology, Science for Life Laboratory, Uppsala University, Rudbecklaboratoriet, Dag Hammarskjölds v 20, C11, 751 85, Uppsala, Sweden.

Background: In systemic lupus erythematosus (SLE), immune complexes (ICs) containing self-derived nucleic acids trigger the synthesis of proinflammatory cytokines by immune cells. We asked how an interleukin (IL)-1 receptor-associated kinase 4 small molecule inhibitor (IRAK4i) affects RNA-IC-induced cytokine production compared with hydroxychloroquine (HCQ).

Methods: Plasmacytoid dendritic cells (pDCs) and natural killer (NK) cells were isolated from peripheral blood mononuclear cells (PBMCs) of healthy individuals. PBMCs from SLE patients and healthy individuals were depleted of monocytes. Cells were stimulated with RNA-containing IC (RNA-IC) in the presence or absence of IRAK4i I92 or HCQ, and cytokines were measured by immunoassay or flow cytometry. Transcriptome sequencing was performed on RNA-IC-stimulated pDCs from healthy individuals to assess the effect of IRAK4i and HCQ.

Results: In healthy individuals, RNA-IC induced interferon (IFN)-α, tumor necrosis factor (TNF)-α, IL-6, IL-8, IFN-γ, macrophage inflammatory protein (MIP)1-α, and MIP1-β production in pDC and NK cell cocultures. IFN-α production was selective for pDCs, whereas both pDCs and NK cells produced TNF-α. IRAK4i reduced the pDC and NK cell-derived cytokine production by 74-95%. HCQ interfered with cytokine production in pDCs but not in NK cells. In monocyte-depleted PBMCs, IRAK4i blocked cytokine production more efficiently than HCQ. Following RNA-IC activation of pDCs, 975 differentially expressed genes were observed (false discovery rate (FDR) < 0.05), with many connected to cytokine pathways, cell regulation, and apoptosis. IRAK4i altered the expression of a larger number of RNA-IC-induced genes than did HCQ (492 versus 65 genes).

Conclusions: The IRAK4i I92 exhibits a broader inhibitory effect than HCQ on proinflammatory pathways triggered by RNA-IC, suggesting IRAK4 inhibition as a therapeutic option in SLE.
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http://dx.doi.org/10.1186/s13075-018-1702-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6235225PMC
October 2018

Epigenetic alterations in primary Sjögren's syndrome - an overview.

Clin Immunol 2018 11 9;196:12-20. Epub 2018 Apr 9.

Department of Medical Sciences, Rheumatology and Science for Life Laboratory, Uppsala University, Uppsala, Sweden.

Primary Sjögren's syndrome (pSS) is a chronic autoimmune rheumatic disease characterized by inflammation of exocrine glands, mainly salivary and lacrimal glands. In addition, pSS may affect multiple other organs resulting in systemic manifestations. Although the precise etiology of pSS remains elusive, pSS is considered to be a multi-factorial disease, where underlying genetic predisposition, environmental factors and epigenetic mechanisms contribute to disease development. Epigenetic mechanisms, such as DNA methylation, histone modifications and non-coding RNAs, may constitute a dynamic link between genome, environment and phenotypic manifestation by their modulating effects on gene expression. A growing body of studies reporting altered epigenetic landscapes in pSS suggests that epigenetic mechanisms play a role in the pathogenesis of pSS, and the reversible nature of epigenetic modifications suggests therapeutic strategies targeting epigenetic dysregulation in pSS. This article reviews our current understanding of epigenetic mechanisms in pSS and discusses implications for novel diagnostic and therapeutic approaches.
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http://dx.doi.org/10.1016/j.clim.2018.04.004DOI Listing
November 2018

Novel gene variants associated with cardiovascular disease in systemic lupus erythematosus and rheumatoid arthritis.

Ann Rheum Dis 2018 07 7;77(7):1063-1069. Epub 2018 Mar 7.

Department of Medical Sciences, Science for Life Laboratory, Rheumatology, Uppsala University, Uppsala, Sweden.

Objectives: Patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) have increased risk of cardiovascular disease (CVD). We investigated whether single nucleotide polymorphisms (SNPs) at autoimmunity risk loci were associated with CVD in SLE and RA.

Methods: Patients with SLE (n=1045) were genotyped using the 200K Immunochip SNP array (Illumina). The allele frequency was compared between patients with and without different manifestations of CVD. Results were replicated in a second SLE cohort (n=1043) and in an RA cohort (n=824). We analysed publicly available genetic data from general population, performed electrophoretic mobility shift assays and measured cytokine levels and occurrence of antiphospholipid antibodies (aPLs).

Results: We identified two new putative risk loci associated with increased risk for CVD in two SLE populations, which remained after adjustment for traditional CVD risk factors. An risk allele, rs17581834(T) was associated with stroke/myocardial infarction (MI) in SLE (OR 2.3 (1.5 to 3.4), P=8.5×10) and RA (OR 2.8 (1.4 to 5.6), P=3.8×10), meta-analysis (OR 2.5 (2.0 to 2.9), P=3.5×10), but not in population controls. The risk allele affected protein binding, and SLE patients with the risk allele had increased levels of plasma-IL10 (P=0.004) and aPL (P=0.01). An risk allele, rs799454(G) was associated with stroke/transient ischaemic attack in SLE (OR 1.7 (1.3 to 2.2), P=2.5×10) but not in RA. The risk allele is an expression quantitative trait locus for four genes.

Conclusions: The risk allele was associated with stroke/MI in SLE and RA, but not in the general population, indicating that shared immune pathways may be involved in the CVD pathogenesis in inflammatory rheumatic diseases.
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http://dx.doi.org/10.1136/annrheumdis-2017-212614DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6029634PMC
July 2018

DNA methylation mapping identifies gene regulatory effects in patients with systemic lupus erythematosus.

Ann Rheum Dis 2018 05 1;77(5):736-743. Epub 2018 Feb 1.

Department of Medical Sciences, Molecular Medicine and Science for Life Laboratory, Uppsala University, Uppsala, Sweden.

Objectives: Systemic lupus erythematosus (SLE) is a chronic autoimmune condition with heterogeneous presentation and complex aetiology where DNA methylation changes are emerging as a contributing factor. In order to discover novel epigenetic associations and investigate their relationship to genetic risk for SLE, we analysed DNA methylation profiles in a large collection of patients with SLE and healthy individuals.

Methods: DNA extracted from blood from 548 patients with SLE and 587 healthy controls were analysed on the Illumina HumanMethylation 450 k BeadChip, which targets 485 000 CpG sites across the genome. Single nucleotide polymorphism (SNP) genotype data for 196 524 SNPs on the Illumina ImmunoChip from the same individuals were utilised for methylation quantitative trait loci (-meQTLs) analyses.

Results: We identified and replicated differentially methylated CpGs (DMCs) in SLE at 7245 CpG sites in the genome. The largest methylation differences were observed at type I interferon-regulated genes which exhibited decreased methylation in SLE. We mapped -meQTLs and identified genetic regulation of methylation levels at 466 of the DMCs in SLE. The meQTLs for DMCs in SLE were enriched for genetic association to SLE, and included seven SLE genome-wide association study (GWAS) loci: (CD45), , , , , and . In addition, we observed association between genotype and variance of methylation at 20 DMCs in SLE, including at the locus.

Conclusions: Our results suggest that several of the genetic risk variants for SLE may exert their influence on the phenotype through alteration of DNA methylation levels at regulatory regions of target genes.
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http://dx.doi.org/10.1136/annrheumdis-2017-212379DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5909746PMC
May 2018

Novel risk genes for systemic lupus erythematosus predicted by random forest classification.

Sci Rep 2017 07 24;7(1):6236. Epub 2017 Jul 24.

Department of Medical Sciences, Molecular Medicine and Science for Life Laboratory, Uppsala University, Uppsala, Sweden.

Genome-wide association studies have identified risk loci for SLE, but a large proportion of the genetic contribution to SLE still remains unexplained. To detect novel risk genes, and to predict an individual's SLE risk we designed a random forest classifier using SNP genotype data generated on the "Immunochip" from 1,160 patients with SLE and 2,711 controls. Using gene importance scores defined by the random forest classifier, we identified 15 potential novel risk genes for SLE. Of them 12 are associated with other autoimmune diseases than SLE, whereas three genes (ZNF804A, CDK1, and MANF) have not previously been associated with autoimmunity. Random forest classification also allowed prediction of patients at risk for lupus nephritis with an area under the curve of 0.94. By allele-specific gene expression analysis we detected cis-regulatory SNPs that affect the expression levels of six of the top 40 genes designed by the random forest analysis, indicating a regulatory role for the identified risk variants. The 40 top genes from the prediction were overrepresented for differential expression in B and T cells according to RNA-sequencing of samples from five healthy donors, with more frequent over-expression in B cells compared to T cells.
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http://dx.doi.org/10.1038/s41598-017-06516-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5524838PMC
July 2017

Transancestral mapping and genetic load in systemic lupus erythematosus.

Nat Commun 2017 07 17;8:16021. Epub 2017 Jul 17.

Division of Rheumatology, Department of Medicine (DIMED), University of Padua, Padua 35122, Italy.

Systemic lupus erythematosus (SLE) is an autoimmune disease with marked gender and ethnic disparities. We report a large transancestral association study of SLE using Immunochip genotype data from 27,574 individuals of European (EA), African (AA) and Hispanic Amerindian (HA) ancestry. We identify 58 distinct non-HLA regions in EA, 9 in AA and 16 in HA (∼50% of these regions have multiple independent associations); these include 24 novel SLE regions (P<5 × 10), refined association signals in established regions, extended associations to additional ancestries, and a disentangled complex HLA multigenic effect. The risk allele count (genetic load) exhibits an accelerating pattern of SLE risk, leading us to posit a cumulative hit hypothesis for autoimmune disease. Comparing results across the three ancestries identifies both ancestry-dependent and ancestry-independent contributions to SLE risk. Our results are consistent with the unique and complex histories of the populations sampled, and collectively help clarify the genetic architecture and ethnic disparities in SLE.
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http://dx.doi.org/10.1038/ncomms16021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5520018PMC
July 2017

Epigenetic Patterns in Blood Associated With Lipid Traits Predict Incident Coronary Heart Disease Events and Are Enriched for Results From Genome-Wide Association Studies.

Circ Cardiovasc Genet 2017 Jan;10(1)

From the Department of Medical Sciences, Molecular Epidemiology and Science for Life Laboratory (Å.K.H., S.G., E.I.) and Department of Medical Sciences, Molecular Medicine and Science for Life Laboratory (J.K.S.), Uppsala University, Sweden; Cardiovascular Medicine unit, Department of Medicine Solna, Karolinska Institute, Stockholm, Sweden (Å.K.H.) Framingham Heart Study, MA (M.M.M., R.J., C.Y., C.L., T.H., S.D., L.A.C., D.L.); Department of Biostatistics (C.L., L.A.C., S.D.), Boston University, MA; Boston University, MA (M.M.M.); Department of Cardiology, Boston Children's Hospital, MA (M.M.M.); Population Sciences Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD (M.M.M., R.J., C.Y., C.L., T.H., D.L.); Centre for Cognitive Ageing and Cognitive Epidemiology (R.E.M., J.M.S., I.J.D.), Medical Genetics Section, Centre for Genomics and Experimental Medicine, Institute of Genetics and Molecular Medicine (R.E.M.), Alzheimer Scotland Dementia Research Centre (J.M.S.), and Department of Psychology (I.J.D.), University of Edinburgh, United Kingdom; Queensland Brain Institute, The University of Queensland, Brisbane, Australia (R.E.M., A.F.M., S.S.); Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland, Australia (S.S., A.F.M.); Hebrew Senior Life, Harvard Medical School, Boston, MA (R.J.); Department of Epidemiology, School of Public Health (M.R.I.) and Department of Biostatistics, Section on Statistical Genetics (D.Z.), University of Alabama at Birmingham; Department of Biostatistics, Harvard School of Public Health, Boston, MA (L. Liang); William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, United Kingdom (P.D.); Princess Al-Jawhara Al-Brahim Centre of Excellence in Research of Hereditary Disorders (PACER-HD), King Abdulaziz University, Jeddah, Saudi Arabia (P.D.); Department of Twin Research and Genetic Epidemiology, King's College London, United Kingdom (T.D.S.); Deparment of Medical Sciences, Cardiovascular Epidemiology, Uppsala University Hospital, Sweden (J.S., L.L.); Children's Hospital Oakland Research Institute, CA (R.M.K.); College of Public Health, University of Kentucky, Lexington (D.K.A.); and Department of Medicine, Division of Cardiovascular Medicine, Stanford University School of Medicine, CA (E.I.).

Background: Genome-wide association studies have identified loci influencing circulating lipid concentrations in humans; further information on novel contributing genes, pathways, and biology may be gained through studies of epigenetic modifications.

Methods And Results: To identify epigenetic changes associated with lipid concentrations, we assayed genome-wide DNA methylation at cytosine-guanine dinucleotides (CpGs) in whole blood from 2306 individuals from 2 population-based cohorts, with replication of findings in 2025 additional individuals. We identified 193 CpGs associated with lipid levels in the discovery stage (<1.08E-07) and replicated 33 (at Bonferroni-corrected <0.05), including 25 novel CpGs not previously associated with lipids. Genes at lipid-associated CpGs were enriched in lipid and amino acid metabolism processes. A differentially methylated locus associated with triglycerides and high-density lipoprotein cholesterol (HDL-C; cg27243685; =8.1E-26 and 9.3E-19) was associated with -expression of a reverse cholesterol transporter (=7.2E-28) and incident cardiovascular disease events (hazard ratio per SD increment, 1.38; 95% confidence interval, 1.15-1.66; =0.0007). We found significant -methylation quantitative trait loci at 64% of the 193 CpGs with an enrichment of signals from genome-wide association studies of lipid levels (=0.004, =0.008 and =0.00003) and coronary heart disease (=0.0007). For example, genome-wide significant variants associated with low-density lipoprotein cholesterol and coronary heart disease at were -methylation quantitative trait loci for a low-density lipoprotein cholesterol-related differentially methylated locus.

Conclusions: We report novel associations of DNA methylation with lipid levels, describe epigenetic mechanisms related to previous genome-wide association studies discoveries, and provide evidence implicating epigenetic regulation of reverse cholesterol transport in blood in relation to occurrence of cardiovascular disease events.
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http://dx.doi.org/10.1161/CIRCGENETICS.116.001487DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5331877PMC
January 2017

Epigenome-wide association study of body mass index, and the adverse outcomes of adiposity.

Nature 2017 01 21;541(7635):81-86. Epub 2016 Dec 21.

German Center for Diabetes Research (DZD), München-Neuherberg, Germany.

Approximately 1.5 billion people worldwide are overweight or affected by obesity, and are at risk of developing type 2 diabetes, cardiovascular disease and related metabolic and inflammatory disturbances. Although the mechanisms linking adiposity to associated clinical conditions are poorly understood, recent studies suggest that adiposity may influence DNA methylation, a key regulator of gene expression and molecular phenotype. Here we use epigenome-wide association to show that body mass index (BMI; a key measure of adiposity) is associated with widespread changes in DNA methylation (187 genetic loci with P < 1 × 10, range P = 9.2 × 10 to 6.0 × 10; n = 10,261 samples). Genetic association analyses demonstrate that the alterations in DNA methylation are predominantly the consequence of adiposity, rather than the cause. We find that methylation loci are enriched for functional genomic features in multiple tissues (P < 0.05), and show that sentinel methylation markers identify gene expression signatures at 38 loci (P < 9.0 × 10, range P = 5.5 × 10 to 6.1 × 10, n = 1,785 samples). The methylation loci identify genes involved in lipid and lipoprotein metabolism, substrate transport and inflammatory pathways. Finally, we show that the disturbances in DNA methylation predict future development of type 2 diabetes (relative risk per 1 standard deviation increase in methylation risk score: 2.3 (2.07-2.56); P = 1.1 × 10). Our results provide new insights into the biologic pathways influenced by adiposity, and may enable development of new strategies for prediction and prevention of type 2 diabetes and other adverse clinical consequences of obesity.
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http://dx.doi.org/10.1038/nature20784DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5570525PMC
January 2017

Higher Nevus Count Exhibits a Distinct DNA Methylation Signature in Healthy Human Skin: Implications for Melanoma.

J Invest Dermatol 2017 04 18;137(4):910-920. Epub 2016 Dec 18.

Department of Twin Research and Genetic Epidemiology, King's College London, London, UK.

High nevus count is the strongest risk factor for melanoma, and although gene variants have been discovered for both traits, epigenetic variation is unexplored. We investigated 322 healthy human skin DNA methylomes associated with total body nevi count, incorporating genetic and transcriptomic variation. DNA methylation changes were identified at genes involved in melanocyte biology, such as RAF1 (P = 1.2 × 10) and CTC1 (region: P = 6.3 × 10), and other genes including ARRDC1 (P = 3.1 × 10). A subset exhibited coordinated methylation and transcription changes within the same biopsy. The total analysis was also enriched for melanoma-associated DNA methylation variation (P = 6.33 × 10). In addition, we show that skin DNA methylation is associated in cis with known genome-wide association study single nucleotide polymorphisms for nevus count, at PLA2G6 (P = 1.7 × 10) and NID1 (P = 6.4 × 10), as well as melanoma risk, including in or near MC1R, MX2, and TERT/CLPTM1L (P < 1 × 10). Our analysis using a uniquely large dataset comprising healthy skin DNA methylomes identified known and additional regulatory loci and pathways in nevi and melanoma biology. This integrative study improves our understanding of predisposition to nevi and their potential contribution to melanoma pathogenesis.
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http://dx.doi.org/10.1016/j.jid.2016.11.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5754330PMC
April 2017

Immunoseq: the identification of functionally relevant variants through targeted capture and sequencing of active regulatory regions in human immune cells.

BMC Med Genomics 2016 09 13;9(1):59. Epub 2016 Sep 13.

Department of Human Genetics, McGill University, Montréal, Quebec, Canada.

Background: The observation that the genetic variants identified in genome-wide association studies (GWAS) frequently lie in non-coding regions of the genome that contain cis-regulatory elements suggests that altered gene expression underlies the development of many complex traits. In order to efficiently make a comprehensive assessment of the impact of non-coding genetic variation in immune related diseases we emulated the whole-exome sequencing paradigm and developed a custom capture panel for the known DNase I hypersensitive site (DHS) in immune cells - "Immunoseq".

Results: We performed Immunoseq in 30 healthy individuals where we had existing transcriptome data from T cells. We identified a large number of novel non-coding variants in these samples. Relying on allele specific expression measurements, we also showed that our selected capture regions are enriched for functional variants that have an impact on differential allelic gene expression. The results from a replication set with 180 samples confirmed our observations.

Conclusions: We show that Immunoseq is a powerful approach to detect novel rare variants in regulatory regions. We also demonstrate that these novel variants have a potential functional role in immune cells.
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http://dx.doi.org/10.1186/s12920-016-0220-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5022205PMC
September 2016

Epigenome-wide DNA methylation patterns associated with fatigue in primary Sjögren's syndrome.

Rheumatology (Oxford) 2016 06 10;55(6):1074-82. Epub 2016 Mar 10.

Clinical Immunology Unit, Department of Internal Medicine, Stavanger University Hospital, Stavanger, Norway.

Objective: Chronic fatigue is a common, disabling and poorly understood phenomenon. Recent studies indicate that epigenetic mechanisms may be involved in the expression of fatigue, a prominent feature of primary SS (pSS). The aim of this study was to investigate whether DNA methylation profiles of whole blood are associated with fatigue in patients with pSS.

Methods: Forty-eight pSS patients with high (n = 24) or low (n = 24) fatigue as measured by a visual analogue scale were included. Genome-wide DNA methylation was investigated using the Illumina HumanMethylation450 BeadChip array. After quality control, a total of 383 358 Cytosine-phosphate-Guanine (CpG) sites remained for further analysis. Age, sex and differential cell count estimates were included as covariates in the association model. A false discovery rate-corrected P < 0.05 was considered significant, and a cut-off of 3% average difference in methylation levels between high- and low-fatigue patients was applied.

Results: A total of 251 differentially methylated CpG sites were associated with fatigue. The CpG site with the most pronounced hypomethylation in pSS high fatigue annotated to the SBF2-antisense RNA1 gene. The most distinct hypermethylation was observed at a CpG site annotated to the lymphotoxin alpha gene. Functional pathway analysis of genes with differently methylated CpG sites in subjects with high vs low fatigue revealed enrichment in several pathways associated with innate and adaptive immunity.

Conclusion: Some genes involved in regulation of the immune system and in inflammation are differently methylated in pSS patients with high vs low fatigue. These findings point to functional networks that may underlie fatigue. Epigenetic changes could constitute a fatigue-regulating mechanism in pSS.
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http://dx.doi.org/10.1093/rheumatology/kew008DOI Listing
June 2016

Genome-wide DNA methylation analysis in multiple tissues in primary Sjögren's syndrome reveals regulatory effects at interferon-induced genes.

Ann Rheum Dis 2016 Nov 8;75(11):2029-2036. Epub 2016 Feb 8.

Rheumatology and Science for Life Laboratory, Department of Medical Sciences, Uppsala University, Uppsala, Sweden.

Objectives: 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.

Methods: 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.

Results: 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.

Conclusions: 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.
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http://dx.doi.org/10.1136/annrheumdis-2015-208659DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5099203PMC
November 2016

Epigenome-wide association study (EWAS) of BMI, BMI change and waist circumference in African American adults identifies multiple replicated loci.

Hum Mol Genet 2015 Aug 1;24(15):4464-79. Epub 2015 May 1.

Human Genetics Center, School of Public Health, University of Texas Health Sciences Center at Houston, Houston, TX 77030, USA, Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX 77030, USA.

Obesity is an important component of the pathophysiology of chronic diseases. Identifying epigenetic modifications associated with elevated adiposity, including DNA methylation variation, may point to genomic pathways that are dysregulated in numerous conditions. The Illumina 450K Bead Chip array was used to assay DNA methylation in leukocyte DNA obtained from 2097 African American adults in the Atherosclerosis Risk in Communities (ARIC) study. Mixed-effects regression models were used to test the association of methylation beta value with concurrent body mass index (BMI) and waist circumference (WC), and BMI change, adjusting for batch effects and potential confounders. Replication using whole-blood DNA from 2377 White adults in the Framingham Heart Study and CD4+ T cell DNA from 991 Whites in the Genetics of Lipid Lowering Drugs and Diet Network Study was followed by testing using adipose tissue DNA from 648 women in the Multiple Tissue Human Expression Resource cohort. Seventy-six BMI-related probes, 164 WC-related probes and 8 BMI change-related probes passed the threshold for significance in ARIC (P < 1 × 10(-7); Bonferroni), including probes in the recently reported HIF3A, CPT1A and ABCG1 regions. Replication using blood DNA was achieved for 37 BMI probes and 1 additional WC probe. Sixteen of these also replicated in adipose tissue, including 15 novel methylation findings near genes involved in lipid metabolism, immune response/cytokine signaling and other diverse pathways, including LGALS3BP, KDM2B, PBX1 and BBS2, among others. Adiposity traits are associated with DNA methylation at numerous CpG sites that replicate across studies despite variation in tissue type, ethnicity and analytic approaches.
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http://dx.doi.org/10.1093/hmg/ddv161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4492394PMC
August 2015

DNA methylation of lipid-related genes affects blood lipid levels.

Circ Cardiovasc Genet 2015 Apr 12;8(2):334-42. Epub 2015 Jan 12.

Background: Epigenetic mechanisms might be involved in the regulation of interindividual lipid level variability and thus may contribute to the cardiovascular risk profile. The aim of this study was to investigate the association between genome-wide DNA methylation and blood lipid levels high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, and total cholesterol. Observed DNA methylation changes were also further analyzed to examine their relationship with previous hospitalized myocardial infarction.

Methods And Results: Genome-wide DNA methylation patterns were determined in whole blood samples of 1776 subjects of the Cooperative Health Research in the Region of Augsburg F4 cohort using the Infinium HumanMethylation450 BeadChip (Illumina). Ten novel lipid-related CpG sites annotated to various genes including ABCG1, MIR33B/SREBF1, and TNIP1 were identified. CpG cg06500161, located in ABCG1, was associated in opposite directions with both high-density lipoprotein cholesterol (β coefficient=-0.049; P=8.26E-17) and triglyceride levels (β=0.070; P=1.21E-27). Eight associations were confirmed by replication in the Cooperative Health Research in the Region of Augsburg F3 study (n=499) and in the Invecchiare in Chianti, Aging in the Chianti Area study (n=472). Associations between triglyceride levels and SREBF1 and ABCG1 were also found in adipose tissue of the Multiple Tissue Human Expression Resource cohort (n=634). Expression analysis revealed an association between ABCG1 methylation and lipid levels that might be partly mediated by ABCG1 expression. DNA methylation of ABCG1 might also play a role in previous hospitalized myocardial infarction (odds ratio, 1.15; 95% confidence interval=1.06-1.25).

Conclusions: Epigenetic modifications of the newly identified loci might regulate disturbed blood lipid levels and thus contribute to the development of complex lipid-related diseases.
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http://dx.doi.org/10.1161/CIRCGENETICS.114.000804DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5012424PMC
April 2015

Meta-analysis in more than 17,900 cases of ischemic stroke reveals a novel association at 12q24.12.

Neurology 2014 Aug 16;83(8):678-85. Epub 2014 Jul 16.

Objectives: To perform a genome-wide association study (GWAS) using the Immunochip array in 3,420 cases of ischemic stroke and 6,821 controls, followed by a meta-analysis with data from more than 14,000 additional ischemic stroke cases.

Methods: Using the Immunochip, we genotyped 3,420 ischemic stroke cases and 6,821 controls. After imputation we meta-analyzed the results with imputed GWAS data from 3,548 cases and 5,972 controls recruited from the ischemic stroke WTCCC2 study, and with summary statistics from a further 8,480 cases and 56,032 controls in the METASTROKE consortium. A final in silico "look-up" of 2 single nucleotide polymorphisms in 2,522 cases and 1,899 controls was performed. Associations were also examined in 1,088 cases with intracerebral hemorrhage and 1,102 controls.

Results: In an overall analysis of 17,970 cases of ischemic stroke and 70,764 controls, we identified a novel association on chromosome 12q24 (rs10744777, odds ratio [OR] 1.10 [1.07-1.13], p = 7.12 × 10(-11)) with ischemic stroke. The association was with all ischemic stroke rather than an individual stroke subtype, with similar effect sizes seen in different stroke subtypes. There was no association with intracerebral hemorrhage (OR 1.03 [0.90-1.17], p = 0.695).

Conclusion: Our results show, for the first time, a genetic risk locus associated with ischemic stroke as a whole, rather than in a subtype-specific manner. This finding was not associated with intracerebral hemorrhage.
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http://dx.doi.org/10.1212/WNL.0000000000000707DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4150131PMC
August 2014

DNA methylation and body-mass index: a genome-wide analysis.

Lancet 2014 Jun 13;383(9933):1990-8. Epub 2014 Mar 13.

Department of Cardiovascular Sciences, University of Leicester, Leicester, UK; National Institute for Health Research Leicester Cardiovascular Biomedical Research Unit, Glenfield Hospital, Leicester, UK. Electronic address:

Background: Obesity is a major health problem that is determined by interactions between lifestyle and environmental and genetic factors. Although associations between several genetic variants and body-mass index (BMI) have been identified, little is known about epigenetic changes related to BMI. We undertook a genome-wide analysis of methylation at CpG sites in relation to BMI.

Methods: 479 individuals of European origin recruited by the Cardiogenics Consortium formed our discovery cohort. We typed their whole-blood DNA with the Infinium HumanMethylation450 array. After quality control, methylation levels were tested for association with BMI. Methylation sites showing an association with BMI at a false discovery rate q value of 0·05 or less were taken forward for replication in a cohort of 339 unrelated white patients of northern European origin from the MARTHA cohort. Sites that remained significant in this primary replication cohort were tested in a second replication cohort of 1789 white patients of European origin from the KORA cohort. We examined whether methylation levels at identified sites also showed an association with BMI in DNA from adipose tissue (n=635) and skin (n=395) obtained from white female individuals participating in the MuTHER study. Finally, we examined the association of methylation at BMI-associated sites with genetic variants and with gene expression.

Findings: 20 individuals from the discovery cohort were excluded from analyses after quality-control checks, leaving 459 participants. After adjustment for covariates, we identified an association (q value ≤0·05) between methylation at five probes across three different genes and BMI. The associations with three of these probes--cg22891070, cg27146050, and cg16672562, all of which are in intron 1 of HIF3A--were confirmed in both the primary and second replication cohorts. For every 0·1 increase in methylation β value at cg22891070, BMI was 3·6% (95% CI 2·4-4·9) higher in the discovery cohort, 2·7% (1·2-4·2) higher in the primary replication cohort, and 0·8% (0·2-1·4) higher in the second replication cohort. For the MuTHER cohort, methylation at cg22891070 was associated with BMI in adipose tissue (p=1·72 × 10(-5)) but not in skin (p=0·882). We observed a significant inverse correlation (p=0·005) between methylation at cg22891070 and expression of one HIF3A gene-expression probe in adipose tissue. Two single nucleotide polymorphisms--rs8102595 and rs3826795--had independent associations with methylation at cg22891070 in all cohorts. However, these single nucleotide polymorphisms were not significantly associated with BMI.

Interpretation: Increased BMI in adults of European origin is associated with increased methylation at the HIF3A locus in blood cells and in adipose tissue. Our findings suggest that perturbation of hypoxia inducible transcription factor pathways could have an important role in the response to increased weight in people.

Funding: The European Commission, National Institute for Health Research, British Heart Foundation, and Wellcome Trust.
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http://dx.doi.org/10.1016/S0140-6736(13)62674-4DOI Listing
June 2014
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