Publications by authors named "Claes Wadelius"

75 Publications

Genome-wide association study of liver enzyme elevation in rheumatoid arthritis patients starting methotrexate.

Pharmacogenomics 2021 Oct 15;22(15):973-982. Epub 2021 Sep 15.

Department of Medical Sciences, Clinical Pharmacogenomics & Science for Life Laboratory, Uppsala University, SE-751 85, Uppsala, Sweden.

To identify novel genetic variants predisposing to elevation of Alanine aminotransferase (ALT) in rheumatoid arthritis (RA) patients after initiation of methotrexate (MTX) treatment. We performed genome-wide association studies in 198 RA patients starting MTX. Outcomes were maximum level of ALT and ALT >1.5-times the upper level of normal within the first 6 months of treatment. (rs72675408) was significantly associated with maximum level of ALT (p = 4.36 × 10). This variant is in linkage disequilibrium with rs72675451, which is associated with differential expression of and . We found an association between ALT elevation and genetic variants that may regulate the expression of and . encodes a janus kinase involved in the pathogenesis of RA.
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http://dx.doi.org/10.2217/pgs-2021-0064DOI Listing
October 2021

The Thioesterase as a Regulator of Lipid Metabolism in Type 2 Diabetes Detected in a Multi-Omics Study of Human Liver.

OMICS 2021 Oct 13;25(10):652-659. Epub 2021 Sep 13.

Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.

Type 2 diabetes (T2D) is characterized by pathophysiological alterations in lipid metabolism. One strategy to understand the molecular mechanisms behind these abnormalities is to identify -regulatory elements (CREs) located in chromatin-accessible regions of the genome that regulate key genes. In this study we integrated assay for transposase-accessible chromatin followed by sequencing (ATAC-seq) data, widely used to decode chromatin accessibility, with multi-omics data and publicly available CRE databases to identify candidate CREs associated with T2D for further experimental validations. We performed high-sensitive ATAC-seq in nine human liver samples from normal and T2D donors, and identified a set of differentially accessible regions (DARs). We identified seven DARs including a candidate enhancer for the gene that regulates the balance of acyl-CoA and free fatty acids (FFAs) in the cytoplasm. The relevance of regulation in T2D was supported by the analysis of transcriptomics and proteomics data in liver tissue. Long-chain acyl-CoA thioesterases (ACOTs) are a group of enzymes that hydrolyze acyl-CoA esters to FFAs and coenzyme A. ACOTs have been associated with regulation of triglyceride levels, fatty acid oxidation, mitochondrial function, and insulin signaling, linking their regulation to the pathogenesis of T2D. Our strategy integrating chromatin accessibility with DNA binding and other types of omics provides novel insights on the role of genetic regulation in T2D and is extendable to other complex multifactorial diseases.
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http://dx.doi.org/10.1089/omi.2021.0093DOI Listing
October 2021

Functional annotation of noncoding mutations in cancer.

Life Sci Alliance 2021 09 19;4(9). Epub 2021 Jul 19.

Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden

In a cancer genome, the noncoding sequence contains the vast majority of somatic mutations. While very few are expected to be cancer drivers, those affecting regulatory elements have the potential to have downstream effects on gene regulation that may contribute to cancer progression. To prioritize regulatory mutations, we screened somatic mutations in the Pan-Cancer Analysis of Whole Genomes cohort of 2,515 cancer genomes on individual bases to assess their potential regulatory roles in their respective cancer types. We found a highly significant enrichment of regulatory mutations associated with the deamination signature overlapping a CpG site in the CCAAT/Enhancer Binding Protein β recognition sites in many cancer types. Overall, 5,749 mutated regulatory elements were identified in 1,844 tumor samples from 39 cohorts containing 11,962 candidate regulatory mutations. Our analysis indicated 20 or more regulatory mutations in 5.5% of the samples, and an overall average of six per tumor. Several recurrent elements were identified, and major cancer-related pathways were significantly enriched for genes nearby the mutated regulatory elements. Our results provide a detailed view of the role of regulatory elements in cancer genomes.
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http://dx.doi.org/10.26508/lsa.201900523DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8321657PMC
September 2021

Polymorphisms rs55710213 and rs56334587 regulate SCD1 expression by modulating HNF4A binding.

Biochim Biophys Acta Gene Regul Mech 2021 08 22;1864(8):194724. Epub 2021 Jun 22.

Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden. Electronic address:

The stearoyl-CoA desaturase 1 (SCD1) gene at 10q24.31 encodes the rate limiting enzyme SCD1 that catalyzes the biosynthesis of monounsaturated fatty acids (MUFAs) from saturated fatty acids (SFAs). Dysregulated SCD1 activity has been observed in many human diseases including non-alcoholic fatty liver disease (NAFLD), obesity, hypertension, hyperlipidemia, metabolic syndrome and several types of cancer. HNF4A is a central regulator of glucose and lipid metabolism and previous studies suggested that it is deeply involved in regulating the SCD1 activity in the liver. However, the underlying mechanisms on whether and how SCD1 is regulated by HNF4A have not been explored in detail. In this study, we found that HNF4A regulates SCD1 expression by directly binding to the key regulatory regions in the SCD1 locus. Knocking down of HNF4A significantly downregulated the expression of SCD1. Variants rs55710213 and rs56334587 in intron 5 of SCD1 directly reside in a canonical HNF4A binding site. The GG haplotype of rs55710213 and rs56334587 is associated with decreased SCD1 activity by disrupting the binding of HNF4A, which further decreased the enhancer activity and SCD1 expression. In conclusion, our study demonstrated that SCD1 is directly regulated by HNF4A, which may be helpful in the understanding of the altered metabolic pathways in many diseases associated with dysregulated SCD1 or HNF4A or both.
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http://dx.doi.org/10.1016/j.bbagrm.2021.194724DOI Listing
August 2021

Multifaceted regulation of hepatic lipid metabolism by YY1.

Life Sci Alliance 2021 07 7;4(7). Epub 2021 Jun 7.

Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden

Recent studies suggested that dysregulated plays a pivotal role in many liver diseases. To obtain a detailed view of genes and pathways regulated by YY1 in the liver, we carried out RNA sequencing in HepG2 cells after YY1 knockdown. A rigid set of 2,081 differentially expressed genes was identified by comparing the YY1-knockdown samples (n = 8) with the control samples (n = 14). YY1 knockdown significantly decreased the expression of several key transcription factors and their coactivators in lipid metabolism. This is illustrated by YY1 regulating PPARA expression through binding to its promoter and enhancer regions. Our study further suggest that down-regulation of the key transcription factors together with YY1 knockdown significantly decreased the cooperation between YY1 and these transcription factors at various regulatory regions, which are important in regulating the expression of genes in hepatic lipid metabolism. This was supported by the finding that the expression of and , encoding key enzymes in lipogenesis, were regulated by the cooperation between YY1 and PPARA/RXRA complex over their promoters.
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http://dx.doi.org/10.26508/lsa.202000928DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8200296PMC
July 2021

Single nucleus transcriptomics data integration recapitulates the major cell types in human liver.

Hepatol Res 2021 Feb 28;51(2):233-238. Epub 2020 Nov 28.

Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.

Aim: The aim of this study was to explore the benefits of data integration from different platforms for single nucleus transcriptomics profiling to characterize cell populations in human liver.

Methods: We generated single-nucleus RNA sequencing data from Chromium 10X Genomics and Drop-seq for a human liver sample. We utilized state of the art bioinformatics tools to undertake a rigorous quality control and to integrate the data into a common space summarizing the gene expression variation from the respective platforms, while accounting for known and unknown confounding factors.

Results: Analysis of single nuclei transcriptomes from both 10X and Drop-seq allowed identification of the major liver cell types, while the integrated set obtained enough statistical power to separate a small population of inactive hepatic stellate cells that was not characterized in either of the platforms.

Conclusions: Integration of droplet-based single nucleus transcriptomics data enabled identification of a small cluster of inactive hepatic stellate cells that highlights the potential of our approach. We suggest single-nucleus RNA sequencing integrative approaches could be utilized to design larger and cost-effective studies.
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http://dx.doi.org/10.1111/hepr.13585DOI Listing
February 2021

Integration of whole-body [F]FDG PET/MRI with non-targeted metabolomics can provide new insights on tissue-specific insulin resistance in type 2 diabetes.

Sci Rep 2020 05 20;10(1):8343. Epub 2020 May 20.

Department of Surgical Sciences, section of Radiology, Uppsala University, Uppsala, Sweden.

Alteration of various metabolites has been linked to type 2 diabetes (T2D) and insulin resistance. However, identifying significant associations between metabolites and tissue-specific phenotypes requires a multi-omics approach. In a cohort of 42 subjects with different levels of glucose tolerance (normal, prediabetes and T2D) matched for age and body mass index, we calculated associations between parameters of whole-body positron emission tomography (PET)/magnetic resonance imaging (MRI) during hyperinsulinemic euglycemic clamp and non-targeted metabolomics profiling for subcutaneous adipose tissue (SAT) and plasma. Plasma metabolomics profiling revealed that hepatic fat content was positively associated with tyrosine, and negatively associated with lysoPC(P-16:0). Visceral adipose tissue (VAT) and SAT insulin sensitivity (K), were positively associated with several lysophospholipids, while the opposite applied to branched-chain amino acids. The adipose tissue metabolomics revealed a positive association between non-esterified fatty acids and, VAT and liver K. Bile acids and carnitines in adipose tissue were inversely associated with VAT K. Furthermore, we detected several metabolites that were significantly higher in T2D than normal/prediabetes. In this study we present novel associations between several metabolites from SAT and plasma with the fat fraction, volume and insulin sensitivity of various tissues throughout the body, demonstrating the benefit of an integrative multi-omics approach.
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http://dx.doi.org/10.1038/s41598-020-64524-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7239946PMC
May 2020

A Multi-Omics Approach to Liver Diseases: Integration of Single Nuclei Transcriptomics with Proteomics and HiCap Bulk Data in Human Liver.

OMICS 2020 04 16;24(4):180-194. Epub 2020 Mar 16.

Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.

The liver is the largest solid organ and a primary metabolic hub. In recent years, intact cell nuclei were used to perform single-nuclei RNA-seq (snRNA-seq) for tissues difficult to dissociate and for flash-frozen archived tissue samples to discover unknown and rare cell subpopulations. In this study, we performed snRNA-seq of a liver sample to identify subpopulations of cells based on nuclear transcriptomics. In 4282 single nuclei, we detected, on average, 1377 active genes and we identified seven major cell types. We integrated data from 94,286 distal interactions ( < 0.05) for 7682 promoters from a targeted chromosome conformation capture technique (HiCap) and mass spectrometry proteomics for the same liver sample. We observed a reasonable correlation between proteomics and bulk snRNA-seq ( = 0.47) using tissue-independent gene-specific protein abundancy estimation factors. We specifically looked at genes of medical importance. The gene is involved in the pharmacogenetics of fluoropyrimidine toxicity and some of its variants are analyzed for clinical purposes. We identified a new putative polymorphic regulatory element, which may contribute to variation in toxicity. Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and we investigated all known risk genes. We identified a complex regulatory landscape for the gene with 16 candidate enhancers. Three of them harbor somatic motif breaking and other mutations in HCC in the Pan Cancer Analysis of Whole Genomes dataset and are candidates to contribute to malignancy. Our results highlight the potential of a multi-omics approach in the study of human diseases.
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http://dx.doi.org/10.1089/omi.2019.0215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7185313PMC
April 2020

Genome-wide association study of angioedema induced by angiotensin-converting enzyme inhibitor and angiotensin receptor blocker treatment.

Pharmacogenomics J 2020 12 21;20(6):770-783. Epub 2020 Feb 21.

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

Angioedema in the mouth or upper airways is a feared adverse reaction to angiotensin-converting enzyme inhibitor (ACEi) and angiotensin receptor blocker (ARB) treatment, which is used for hypertension, heart failure and diabetes complications. This candidate gene and genome-wide association study aimed to identify genetic variants predisposing to angioedema induced by these drugs. The discovery cohort consisted of 173 cases and 4890 controls recruited in Sweden. In the candidate gene analysis, ETV6, BDKRB2, MME, and PRKCQ were nominally associated with angioedema (p < 0.05), but did not pass Bonferroni correction for multiple testing (p < 2.89 × 10). In the genome-wide analysis, intronic variants in the calcium-activated potassium channel subunit alpha-1 (KCNMA1) gene on chromosome 10 were significantly associated with angioedema (p < 5 × 10). Whilst the top KCNMA1 hit was not significant in the replication cohort (413 cases and 599 ACEi-exposed controls from the US and Northern Europe), a meta-analysis of the replication and discovery cohorts (in total 586 cases and 1944 ACEi-exposed controls) revealed that each variant allele increased the odds of experiencing angioedema 1.62 times (95% confidence interval 1.05-2.50, p = 0.030). Associated KCNMA1 variants are not known to be functional, but are in linkage disequilibrium with variants in transcription factor binding sites active in relevant tissues. In summary, our data suggest that common variation in KCNMA1 is associated with risk of angioedema induced by ACEi or ARB treatment. Future whole exome or genome sequencing studies will show whether rare variants in KCNMA1 or other genes contribute to the risk of ACEi- and ARB-induced angioedema.
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http://dx.doi.org/10.1038/s41397-020-0165-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7674154PMC
December 2020

Analyses of non-coding somatic drivers in 2,658 cancer whole genomes.

Nature 2020 02 5;578(7793):102-111. Epub 2020 Feb 5.

Department of Physiology and Biophysics, Weill Cornell Medicine, New York, NY, USA.

The discovery of drivers of cancer has traditionally focused on protein-coding genes. Here we present analyses of driver point mutations and structural variants in non-coding regions across 2,658 genomes from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). For point mutations, we developed a statistically rigorous strategy for combining significance levels from multiple methods of driver discovery that overcomes the limitations of individual methods. For structural variants, we present two methods of driver discovery, and identify regions that are significantly affected by recurrent breakpoints and recurrent somatic juxtapositions. Our analyses confirm previously reported drivers, raise doubts about others and identify novel candidates, including point mutations in the 5' region of TP53, in the 3' untranslated regions of NFKBIZ and TOB1, focal deletions in BRD4 and rearrangements in the loci of AKR1C genes. We show that although point mutations and structural variants that drive cancer are less frequent in non-coding genes and regulatory sequences than in protein-coding genes, additional examples of these drivers will be found as more cancer genomes become available.
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http://dx.doi.org/10.1038/s41586-020-1965-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054214PMC
February 2020

rs953413 Regulates Polyunsaturated Fatty Acid Metabolism by Modulating ELOVL2 Expression.

iScience 2020 Feb 9;23(2):100808. Epub 2020 Jan 9.

Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden. Electronic address:

Long-chain polyunsaturated fatty acids (LC-PUFAs) influence human health in several areas, including cardiovascular disease, diabetes, fatty liver disease, and cancer. ELOVL2 encodes one of the key enzymes in the in vivo synthesis of LC-PUFAs from their precursors. Variants near ELOVL2 have repeatedly been associated with levels of LC-PUFA-derived metabolites in genome-wide association studies (GWAS), but the mechanisms behind these observations remain poorly defined. In this study, we found that rs953413, located in the first intron of ELOVL2, lies within a functional FOXA and HNF4α cooperative binding site. The G allele of rs953413 increases binding of FOXA1/FOXA2 and HNF4α to an evolutionarily conserved enhancer element, conferring allele-specific upregulation of the rs953413-associated gene ELOVL2. The expression of ELOVL2 was significantly downregulated by both FOXA1 and HNF4α knockdown and CRISPR/Cas9-mediated direct mutation to the enhancer element. Our results suggest that rs953413 regulates LC-PUFAs metabolism by altering ELOVL2 expression through FOXA1/FOXA2 and HNF4α cooperation.
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http://dx.doi.org/10.1016/j.isci.2019.100808DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7033636PMC
February 2020

Author Correction: Intra- and inter-individual metabolic profiling highlights carnitine and lysophosphatidylcholine pathways as key molecular defects in type 2 diabetes.

Sci Rep 2019 Nov 28;9(1):18158. Epub 2019 Nov 28.

Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41598-019-54423-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6881403PMC
November 2019

Detailed Functional Characterization of a Waist-Hip Ratio Locus in 7p15.2 Defines an Enhancer Controlling Adipocyte Differentiation.

iScience 2019 Oct 10;20:42-59. Epub 2019 Sep 10.

Department of Medicine, Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Medical Sciences and Science for Life Laboratory, Uppsala University, Uppsala, Sweden; Stanford Cardiovascular Institute, Stanford University, Stanford, CA 94305, USA; Stanford Diabetes Research Center, Stanford University, Stanford, CA 94305, USA. Electronic address:

We combined CAGE sequencing in human adipocytes during differentiation with data from genome-wide association studies to identify an enhancer in the SNX10 locus on chromosome 7, presumably involved in body fat distribution. Using reporter assays and CRISPR-Cas9 gene editing in human cell lines, we characterized the role of the enhancer in adipogenesis. The enhancer was active during adipogenesis and responded strongly to insulin and isoprenaline. The allele associated with increased waist-hip ratio in human genetic studies was associated with higher enhancer activity. Mutations of the enhancer resulted in less adipocyte differentiation. RNA sequencing of cells with disrupted enhancer showed reduced expression of established adipocyte markers, such as ADIPOQ and LPL, and identified CHI3L1 on chromosome 1 as a potential gene involved in adipocyte differentiation. In conclusion, we identified and characterized an enhancer in the SNX10 locus and outlined its plausible mechanisms of action and downstream targets.
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http://dx.doi.org/10.1016/j.isci.2019.09.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6817687PMC
October 2019

Contribution of genetics to visceral adiposity and its relation to cardiovascular and metabolic disease.

Nat Med 2019 09 9;25(9):1390-1395. Epub 2019 Sep 9.

Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.

Visceral adipose tissue (VAT)-fat stored around the internal organs-has been suggested as an independent risk factor for cardiovascular and metabolic disease, as well as all-cause, cardiovascular-specific and cancer-specific mortality. Yet, the contribution of genetics to VAT, as well as its disease-related effects, are largely unexplored due to the requirement for advanced imaging technologies to accurately measure VAT. Here, we develop sex-stratified, nonlinear prediction models (coefficient of determination = 0.76; typical 95% confidence interval (CI) = 0.74-0.78) for VAT mass using the UK Biobank cohort. We performed a genome-wide association study for predicted VAT mass and identified 102 novel visceral adiposity loci. Predicted VAT mass was associated with increased risk of hypertension, heart attack/angina, type 2 diabetes and hyperlipidemia, and Mendelian randomization analysis showed visceral fat to be a causal risk factor for all four diseases. In particular, a large difference in causal effect between the sexes was found for type 2 diabetes, with an odds ratio of 7.34 (95% CI = 4.48-12.0) in females and an odds ratio of 2.50 (95% CI = 1.98-3.14) in males. Our findings bolster the role of visceral adiposity as a potentially independent risk factor, in particular for type 2 diabetes in Caucasian females. Independent validation in other cohorts is necessary to determine whether the findings can translate to other ethnicities, or outside the UK.
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http://dx.doi.org/10.1038/s41591-019-0563-7DOI Listing
September 2019

Intra- and inter-individual metabolic profiling highlights carnitine and lysophosphatidylcholine pathways as key molecular defects in type 2 diabetes.

Sci Rep 2019 07 4;9(1):9653. Epub 2019 Jul 4.

Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.

Type 2 diabetes (T2D) mellitus is a complex metabolic disease commonly caused by insulin resistance in several tissues. We performed a matched two-dimensional metabolic screening in tissue samples from 43 multi-organ donors. The intra-individual analysis was assessed across five key metabolic tissues (serum, visceral adipose tissue, liver, pancreatic islets and skeletal muscle), and the inter-individual across three different groups reflecting T2D progression. We identified 92 metabolites differing significantly between non-diabetes and T2D subjects. In diabetes cases, carnitines were significantly higher in liver, while lysophosphatidylcholines were significantly lower in muscle and serum. We tracked the primary tissue of origin for multiple metabolites whose alterations were reflected in serum. An investigation of three major stages spanning from controls, to pre-diabetes and to overt T2D indicated that a subset of lysophosphatidylcholines was significantly lower in the muscle of pre-diabetes subjects. Moreover, glycodeoxycholic acid was significantly higher in liver of pre-diabetes subjects while additional increase in T2D was insignificant. We confirmed many previously reported findings and substantially expanded on them with altered markers for early and overt T2D. Overall, the analysis of this unique dataset can increase the understanding of the metabolic interplay between organs in the development of T2D.
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http://dx.doi.org/10.1038/s41598-019-45906-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6609645PMC
July 2019

Studies of liver tissue identify functional gene regulatory elements associated to gene expression, type 2 diabetes, and other metabolic diseases.

Hum Genomics 2019 04 29;13(1):20. Epub 2019 Apr 29.

Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.

Background: Genome-wide association studies (GWAS) of diseases and traits have found associations to gene regions but not the functional SNP or the gene mediating the effect. Difference in gene regulatory signals can be detected using chromatin immunoprecipitation and next-gen sequencing (ChIP-seq) of transcription factors or histone modifications by aligning reads to known polymorphisms in individual genomes. The aim was to identify such regulatory elements in the human liver to understand the genetics behind type 2 diabetes and metabolic diseases.

Methods: The genome of liver tissue was sequenced using 10X Genomics technology to call polymorphic positions. Using ChIP-seq for two histone modifications, H3K4me3 and H3K27ac, and the transcription factor CTCF, and our established bioinformatics pipeline, we detected sites with significant difference in signal between the alleles.

Results: We detected 2329 allele-specific SNPs (AS-SNPs) including 25 associated to GWAS SNPs linked to liver biology, e.g., 4 AS-SNPs at two type 2 diabetes loci. Two hundred ninety-two AS-SNPs were associated to liver gene expression in GTEx, and 134 AS-SNPs were located on 166 candidate functional motifs and most of them in EGR1-binding sites.

Conclusions: This study provides a valuable collection of candidate liver regulatory elements for further experimental validation.
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http://dx.doi.org/10.1186/s40246-019-0204-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6489362PMC
April 2019

Allele specific chromatin signals, 3D interactions, and motif predictions for immune and B cell related diseases.

Sci Rep 2019 02 25;9(1):2695. Epub 2019 Feb 25.

Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.

Several Genome Wide Association Studies (GWAS) have reported variants associated to immune diseases. However, the identified variants are rarely the drivers of the associations and the molecular mechanisms behind the genetic contributions remain poorly understood. ChIP-seq data for TFs and histone modifications provide snapshots of protein-DNA interactions allowing the identification of heterozygous SNPs showing significant allele specific signals (AS-SNPs). AS-SNPs can change a TF binding site resulting in altered gene regulation and are primary candidates to explain associations observed in GWAS and expression studies. We identified 17,293 unique AS-SNPs across 7 lymphoblastoid cell lines. In this set of cell lines we interrogated 85% of common genetic variants in the population for potential regulatory effect and we identified 237 AS-SNPs associated to immune GWAS traits and 714 to gene expression in B cells. To elucidate possible regulatory mechanisms we integrated long-range 3D interactions data to identify putative target genes and motif predictions to identify TFs whose binding may be affected by AS-SNPs yielding a collection of 173 AS-SNPs associated to gene expression and 60 to B cell related traits. We present a systems strategy to find functional gene regulatory variants, the TFs that bind differentially between alleles and novel strategies to detect the regulated genes.
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http://dx.doi.org/10.1038/s41598-019-39633-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6389883PMC
February 2019

Pandemrix-induced narcolepsy is associated with genes related to immunity and neuronal survival.

EBioMedicine 2019 Feb 30;40:595-604. Epub 2019 Jan 30.

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

Background: The incidence of narcolepsy rose sharply after the swine influenza A (H1N1) vaccination campaign with Pandemrix. Narcolepsy is an immune-related disorder with excessive daytime sleepiness. The most frequent form is strongly associated with HLA-DQB1*06:02, but only a minority of carriers develop narcolepsy. We aimed to identify genetic markers that predispose to Pandemrix-induced narcolepsy.

Methods: We tested for genome-wide and candidate gene associations in 42 narcolepsy cases and 4981 controls. Genotyping was performed on Illumina arrays, HLA alleles were imputed using SNP2HLA, and single nucleotide polymorphisms were imputed using the haplotype reference consortium panel. The genome-wide significance threshold was p < 5 × 10, and the nominal threshold was p < 0.05. Results were replicated in 32 cases and 7125 controls. Chromatin data was used for functional annotation.

Findings: Carrying HLA-DQB1*06:02 was significantly associated with narcolepsy, odds ratio (OR) 39.4 [95% confidence interval (CI) 11.3, 137], p = 7.9 × 10. After adjustment for HLA, GDNF-AS1 (rs62360233) was significantly associated, OR = 8.7 [95% CI 4.2, 17.5], p = 2.6 × 10, and this was replicated, OR = 3.4 [95% CI 1.2-9.6], p = 0.022. Functional analysis revealed variants in high LD with rs62360233 that might explain the detected association. The candidate immune-gene locus TRAJ (rs1154155) was nominally associated in both the discovery and replication cohorts, meta-analysis OR = 2.0 [95% CI 1.4, 2.8], p = 0.0002.

Interpretation: We found a novel association between Pandemrix-induced narcolepsy and the non-coding RNA gene GDNF-AS1, which has been shown to regulate expression of the essential neurotrophic factor GDNF. Changes in regulation of GDNF have been associated with neurodegenerative diseases. This finding may increase the understanding of disease mechanisms underlying narcolepsy. Associations between Pandemrix-induced narcolepsy and immune-related genes were replicated.
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http://dx.doi.org/10.1016/j.ebiom.2019.01.041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6413474PMC
February 2019

Genetic variants associated with angiotensin-converting enzyme inhibitor-induced cough: a genome-wide association study in a Swedish population.

Pharmacogenomics 2017 Feb 13;18(3):201-213. Epub 2017 Jan 13.

Department of Medical Sciences, Clinical Pharmacology & Science for Life Laboratory, Uppsala University, Uppsala, Sweden.

Aim: We conducted a genome-wide association study on angiotensin-converting enzyme inhibitor-induced cough and used our dataset to replicate candidate genes identified in previous studies.

Patients & Methods: A total of 124 patients and 1345 treated controls were genotyped using Illumina arrays. The genome-wide significance level was set to p < 5 × 10.

Results: We identified nearly genome-wide significant associations in CLASP1, PDE11A, KCNMB2, TGFA, SLC38A6 and MMP16. The strongest association was with rs62151109 in CLASP1 (odds ratio: 3.97; p = 9.44 × 10). All top hits except two were located in intronic or noncoding DNA regions. None of the candidate genes were significantly associated in our study.

Conclusion: Angiotensin-converting enzyme inhibitor-induced cough is potentially associated with genes that are independent of bradykinin pathways.
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http://dx.doi.org/10.2217/pgs-2016-0184DOI Listing
February 2017

PATZ1 down-regulates FADS1 by binding to rs174557 and is opposed by SP1/SREBP1c.

Nucleic Acids Res 2017 03;45(5):2408-2422

Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala 75237, Sweden.

The FADS1 and FADS2 genes in the FADS cluster encode the rate-limiting enzymes in the synthesis of long-chain polyunsaturated fatty acids (LC-PUFAs). Genetic variation in this region has been associated with a large number of diseases and traits many of them correlated to differences in metabolism of PUFAs. However, the causative variants leading to these associations have not been identified. Here we find that the multiallelic rs174557 located in an AluYe5 element in intron 1 of FADS1 is functional and lies within a PATZ1 binding site. The derived allele of rs174557, which is the common variant in most populations, diminishes binding of PATZ1, a transcription factor conferring allele-specific downregulation of FADS1. The PATZ1 binding site overlaps with a SP1 site. The competitive binding between the suppressive PATZ1 and the activating complex of SP1 and SREBP1c determines the enhancer activity of this region, which regulates expression of FADS1.
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http://dx.doi.org/10.1093/nar/gkw1186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5389558PMC
March 2017

Maps of context-dependent putative regulatory regions and genomic signal interactions.

Nucleic Acids Res 2016 Nov 12;44(19):9110-9120. Epub 2016 Sep 12.

Department of Cell and Molecular Biology, Uppsala University, Uppsala SE-751-24, Sweden

Gene transcription is regulated mainly by transcription factors (TFs). ENCODE and Roadmap Epigenomics provide global binding profiles of TFs, which can be used to identify regulatory regions. To this end we implemented a method to systematically construct cell-type and species-specific maps of regulatory regions and TF-TF interactions. We illustrated the approach by developing maps for five human cell-lines and two other species. We detected ∼144k putative regulatory regions among the human cell-lines, with the majority of them being ∼300 bp. We found ∼20k putative regulatory elements in the ENCODE heterochromatic domains suggesting a large regulatory potential in the regions presumed transcriptionally silent. Among the most significant TF interactions identified in the heterochromatic regions were CTCF and the cohesin complex, which is in agreement with previous reports. Finally, we investigated the enrichment of the obtained putative regulatory regions in the 3D chromatin domains. More than 90% of the regions were discovered in the 3D contacting domains. We found a significant enrichment of GWAS SNPs in the putative regulatory regions. These significant enrichments provide evidence that the regulatory regions play a crucial role in the genomic structural stability. Additionally, we generated maps of putative regulatory regions for prostate and colorectal cancer human cell-lines.
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http://dx.doi.org/10.1093/nar/gkw800DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5100580PMC
November 2016

Genetic prevention of hepatitis C virus-induced liver fibrosis by allele-specific downregulation of MERTK.

Hepatol Res 2017 Jul 29;47(8):826-830. Epub 2016 Sep 29.

Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.

Aim: Infection by hepatitis C virus (HCV) can result in the development of liver fibrosis and may eventually progress into cirrhosis and hepatocellular carcinoma. However, the molecular mechanisms for this process are not fully known. Several genome-wide association studies have been carried out to pinpoint causative variants in HCV-infected patient cohorts, but these variants are usually not the functional ones. The aim of this study was to identify the regulatory single nucleotide polymorphism associated with the risk of HCV-induced liver fibrosis and elucidate its molecular mechanism.

Methods: We utilized a bioinformatics approach to identify a non-coding regulatory variant, located in an intron of the MERTK gene, based on differential transcription factor binding between the alleles. We validated the results using expression reporter assays and electrophoresis mobility shift assays.

Results: Chromatin immunoprecipitation sequencing indicated that transcription factor(s) bind stronger to the A allele of rs6726639. Electrophoresis mobility shift assays supported these findings and suggested that the transcription factor is interferon regulatory factor 1 (IRF1). Luciferase report assays showed lower enhancer activity from the A allele and that IRF1 may act as a repressor.

Conclusions: Treatment of hepatitis C with interferon-α results in increased IRF1 levels and our data suggest that this leads to an allele-specific downregulation of MERTK mediated by an allelic effect on the regulatory element containing the functional rs6726639. This variant also shows the hallmarks for being the driver of the genome-wide association studies for reduced risk of liver fibrosis and non-alcoholic fatty liver disease at MERTK.
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http://dx.doi.org/10.1111/hepr.12810DOI Listing
July 2017

Genetic determinants of warfarin maintenance dose and time in therapeutic treatment range: a RE-LY genomics substudy.

Pharmacogenomics 2016 08 4;17(13):1425-39. Epub 2016 Aug 4.

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

Aims: We investigated associations between genetic variation in candidate genes and on a genome-wide scale with warfarin maintenance dose, time in therapeutic range (TTR), and risk of major bleeding.

Materials & Methods: In total, 982 warfarin-treated patients from the RE-LY trial were studied.

Results: After adjusting for SNPs in VKORC1 and CYP2C9, SNPs in DDHD1 (rs17126068) and NEDD4 (rs2288344) were associated with dose. Adding these SNPs and CYP4F2 (rs2108622) to a base model increased R(2) by 2.9%. An SNP in ASPH (rs4379440) was associated with TTR (-6.8% per minor allele). VKORC1 was associated with time less than INR 2.0. VKORC1 and CYP2C9 were associated with time more than INR 3.0, but not with major bleeding.

Conclusions: We identified two novel genes associated with warfarin maintenance dose and one gene associated with TTR. These genes need to be replicated in an independent cohort.
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http://dx.doi.org/10.2217/pgs-2016-0061DOI Listing
August 2016

A Significant Regulatory Mutation Burden at a High-Affinity Position of the CTCF Motif in Gastrointestinal Cancers.

Hum Mutat 2016 09 2;37(9):904-13. Epub 2016 Jun 2.

Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, SE-751 08, Sweden.

Somatic mutations drive cancer and there are established ways to study those in coding sequences. It has been shown that some regulatory mutations are over-represented in cancer. We develop a new strategy to find putative regulatory mutations based on experimentally established motifs for transcription factors (TFs). In total, we find 1,552 candidate regulatory mutations predicted to significantly reduce binding affinity of many TFs in hepatocellular carcinoma and affecting binding of CTCF also in esophagus, gastric, and pancreatic cancers. Near mutated motifs, there is a significant enrichment of (1) genes mutated in cancer, (2) tumor-suppressor genes, (3) genes in KEGG cancer pathways, and (4) sets of genes previously associated to cancer. Experimental and functional validations support the findings. The strategy can be applied to identify regulatory mutations in any cell type with established TF motifs and will aid identifications of genes contributing to cancer.
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http://dx.doi.org/10.1002/humu.23014DOI Listing
September 2016

Allele-specific transcription factor binding in liver and cervix cells unveils many likely drivers of GWAS signals.

Genomics 2016 06 26;107(6):248-54. Epub 2016 Apr 26.

Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden. Electronic address:

Genome-wide association studies (GWAS) point to regions with associated genetic variants but rarely to a specific gene and therefore detailed knowledge regarding the genes contributing to complex traits and diseases remains elusive. The functional role of GWAS-SNPs is also affected by linkage disequilibrium with many variants on the same haplotype and sometimes in the same regulatory element almost equally likely to mediate the effect. Using ChIP-seq data on many transcription factors, we pinpointed genetic variants in HepG2 and HeLa-S3 cell lines which show a genome-wide significant difference in binding between alleles. We identified a collection of 3713 candidate functional regulatory variants many of which are likely drivers of GWAS signals or genetic difference in expression. A recent study investigated many variants before finding the functional ones at the GALNT2 locus, which we found in our genome-wide screen in HepG2. This illustrates the efficiency of our approach.
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http://dx.doi.org/10.1016/j.ygeno.2016.04.006DOI Listing
June 2016

Allele-specific transcription factor binding to common and rare variants associated with disease and gene expression.

Hum Genet 2016 May 18;135(5):485-497. Epub 2016 Mar 18.

Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.

Genome-wide association studies (GWAS) have identified a large number of disease-associated SNPs, but in few cases the functional variant and the gene it controls have been identified. To systematically identify candidate regulatory variants, we sequenced ENCODE cell lines and used public ChIP-seq data to look for transcription factors binding preferentially to one allele. We found 9962 candidate regulatory SNPs, of which 16 % were rare and showed evidence of larger functional effect than common ones. Functionally rare variants may explain divergent GWAS results between populations and are candidates for a partial explanation of the missing heritability. The majority of allele-specific variants (96 %) were specific to a cell type. Furthermore, by examining GWAS loci we found >400 allele-specific candidate SNPs, 141 of which were highly relevant in our cell types. Functionally validated SNPs support identification of an SNP in SYNGR1 which may expose to the risk of rheumatoid arthritis and primary biliary cirrhosis, as well as an SNP in the last intron of COG6 exposing to the risk of psoriasis. We propose that by repeating the ChIP-seq experiments of 20 selected transcription factors in three to ten people, the most common polymorphisms can be interrogated for allele-specific binding. Our strategy may help to remove the current bottleneck in functional annotation of the genome.
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http://dx.doi.org/10.1007/s00439-016-1654-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4835527PMC
May 2016

Novel regulatory variant detected on the VKORC1 haplotype that is associated with warfarin dose.

Pharmacogenomics 2016 08 5;17(12):1305-14. Epub 2016 Feb 5.

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

Aim: Warfarin dose requirement is associated with VKORC1 rs9923231, and we studied whether it is a functional variant.

Materials & Methods: We selected variants in linkage disequilibrium with rs9923231 that bind transcription factors in an allele-specific way. Representative haplotypes were cloned or constructed, nuclear protein binding and transcriptional activity were evaluated.

Results: rs56314408C>T and rs2032915C>T were detected in a liver enhancer in linkage disequilibrium with rs9923231. The rs56314408-rs2032915 C-C haplotype preferentially bound nuclear proteins and had higher transcriptional activity than T-T and the African-specific T-C. A motif for TFAP2A/C was disrupted by rs56314408T. No difference in transcriptional activity was detected for rs9923231G>A.

Conclusion: Our results supported an activating role for rs56314408C, while rs9923231G>A had no evidence of being functional.
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http://dx.doi.org/10.2217/pgs-2015-0013DOI Listing
August 2016

Looking beyond GWAS: allele-specific transcription factor binding drives the association of GALNT2 to HDL-C plasma levels.

Lipids Health Dis 2016 Jan 27;15:18. Epub 2016 Jan 27.

Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.

Background: Plasma levels of high-density lipoprotein cholesterol (HDL-C) have been associated to cardiovascular disease. The high heritability of HDL-C plasma levels has been an incentive for several genome wide association studies (GWASs) which identified, among others, variants in the first intron of the GALNT2 gene strongly associated to HDL-C levels. However, the lead GWAS SNP associated to HDL-C levels in this genomic region, rs4846914, is located outside of transcription factor (TF) binding sites defined by chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) experiments in the ENCODE project and is therefore unlikely to be functional. In this study we apply a bioinformatics approach which rely on the premise that ChIP-seq reads can identify allele specific binding of a TF at cell specific regulatory elements harboring allele specific SNPs (AS-SNPs). EMSA and luciferase assays were used to validate the allele specific binding and to test the enhancer activity of the regulatory element harboring the AS-SNP rs4846913 as well as the neighboring rs2144300 which are in high LD with rs4846914.

Findings: Using luciferase assays we found that rs4846913 and the neighboring rs2144300 displayed allele specific enhancer activity. We propose that an inhibitor binds preferentially to the rs4846913-C allele with an inhibitory boost from the synergistic binding of other TFs at the neighboring SNP rs2144300. These events influence the transcription level of GALNT2.

Conclusions: The results suggest that rs4846913 and rs2144300 drive the association to HDL-C plasma levels through an inhibitory regulation of GALNT2 rather than the reported lead GWAS SNP rs4846914.
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http://dx.doi.org/10.1186/s12944-016-0183-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4728761PMC
January 2016

lobChIP: from cells to sequencing ready ChIP libraries in a single day.

Epigenetics Chromatin 2015 21;8:25. Epub 2015 Jul 21.

Science for Life Laboratory, Department of Immunology, Genetics and Pathology, BMC, Uppsala University, Box 815, 75108 Uppsala, Sweden.

Background: ChIP-seq is the method of choice for genome-wide studies of protein-DNA interactions. We describe a new method for ChIP-seq sample preparation, termed lobChIP, where the library reactions are performed on cross-linked ChIP fragments captured on beads.

Results: The lobChIP method was found both to reduce time and cost and to simplify the processing of many samples in parallel. lobChIP has an early incorporation of barcoded sequencing adaptors that minimizes the risk of sample cross-contamination and can lead to reduced amount of adaptor dimers in the sequencing libraries, while allowing for direct decross-linking and amplification of the sample.

Conclusions: With results for histone modifications and transcription factors, we show that lobChIP performs equal to or better than standard protocols and that it makes it possible to go from cells to sequencing ready libraries within a single day.
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http://dx.doi.org/10.1186/s13072-015-0017-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4507313PMC
July 2015

Different distribution of histone modifications in genes with unidirectional and bidirectional transcription and a role of CTCF and cohesin in directing transcription.

BMC Genomics 2015 Apr 15;16:300. Epub 2015 Apr 15.

Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, SE-751 08, Sweden.

Background: Several post-translational histone modifications are mainly found in gene promoters and are associated with the promoter activity. It has been hypothesized that histone modifications regulate the transcription, as opposed to the traditional view with transcription factors as the key regulators. Promoters of most active genes do not only initiate transcription of the coding sequence, but also a substantial amount of transcription of the antisense strand upstream of the transcription start site (TSS). This promoter feature has generally not been considered in previous studies of histone modifications and transcription factor binding.

Results: We annotated protein-coding genes as bi- or unidirectional depending on their mode of transcription and compared histone modifications and transcription factor occurrences between them. We found that H3K4me3, H3K9ac, and H3K27ac were significantly more enriched upstream of the TSS in bidirectional genes compared with the unidirectional ones. In contrast, the downstream histone modification signals were similar, suggesting that the upstream histone modifications might be a consequence of transcription rather than a cause. Notably, we found well-positioned CTCF and RAD21 peaks approximately 60-80 bp upstream of the TSS in the unidirectional genes. The peak heights were related to the amount of antisense transcription and we hypothesized that CTCF and cohesin act as a barrier against antisense transcription.

Conclusions: Our results provide insights into the distribution of histone modifications at promoters and suggest a novel role of CTCF and cohesin as regulators of transcriptional direction.
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http://dx.doi.org/10.1186/s12864-015-1485-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4446127PMC
April 2015
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