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Purpose: In acute myeloid leukemia (AML), somatic mutations (commonly missense, nonsense, and frameshift indels) in RUNX1 are associated with a dismal clinical outcome. Inherited RUNX1 mutations cause familial platelet disorder. As approximately 5%–10% of germline RUNX1 mutations are large exonic deletions, we hypothesized that such exonic RUNX1 aberrations may also be acquired during the development of AML. Experimental Design: Sixty patients with well-characterized AML were analyzed with multiplex ligation-dependent probe amplification (n = 60), microarray (n = 11), and/or whole-genome sequencing (n = 8). Results: In total, 25 (42% of the cohort) RUNX1-aberrant patients (defined by the presence of classical mutations and/or exonic deletions) were identified. Sixteen patients (27%) carried only exonic deletions, 5 (8%) carried classical mutations, and 4 (7%) carried both exonic deletions and mutations. No significant difference was observed between patients with classical RUNX1 mutations and RUNX1 exonic deletions in median overall survival (OS, 53.1 vs. 38.8 months, respectively, P = 0.63). When applying the European Leukemia Net (ELN) classification including the RUNX1-aberrant group, 20% of the patients initially stratified as intermediate-risk (5% of the whole cohort) were reassigned to the high-risk group, which improved the performance of ELN classification regarding OS between intermediate- and high-risk groups (18.9 vs. 9.6 months, P = 0.09). Conclusions: Somatic RUNX1 exonic deletions constitute a novel recurrent aberration in AML. Our findings have important clinical implications regarding AML classification, risk stratification, and treatment decision. Moreover, they argue in favor of further investigating such genomic aberrations not only in RUNX1 but also in other genes implicated in cancer biology and management.

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Skeletal muscle fiber composition and capillary density influence physical performance and whole-body metabolic properties. ~45% of the variance in fiber type is heritable, which motivated us to perform a genome-wide association study of skeletal muscle histology from 656 Swedish men. Four independent variants were associated (p − 8) with proportion of type IIx fibers or capillary-to-fiber ratio (C:F). The strongest signal localized to the rs115660502 variant, where the G-allele corresponded with increased C:F and reduced skeletal muscle expression of the proximal gene, RAB3 GTPase Activating Non-Catalytic Protein Subunit 2 (RAB3GAP2). The G-allele was less frequent in elite short-track sprinters and more frequent in endurance athletes than in matched non-athlete (population) controls; RAB3GAP2 expression was reduced by high-intensity intermittent training. RAB3GAP2 protein was not uniformly expressed in muscle tissue but localized to the endothelium and capillaries. Experimental reduction of RAB3GAP2 in human endothelial cells led to increased tube formation in vitro, to regulation of secreted factors promoting angiogenesis and T-cell activation, to reduced intracellular levels of von Willebrand factor (VWF) and, post-implantation, to increased endothelial cell density in vivo in mice. The amount of RAB3GAP2 in skeletal muscle was positively associated with exercise-induced release of VWF in vivo in humans. By regulating the release of protein factors (VWF, CD70, TNC, TNXB, MCP1, IGFBP3, COL1A1, TFPI2 and tPA), RAB3GAP2 influences fitness adaptation after exercise by improving muscle healing and promotion of capillary formation.

Background Programmed cell death 1 (PD-1) and its ligands PD-L1 and PD-L2, as well as Indoleamine 2,3-deoxygenase (IDO1) can be expressed both by tumor and microenvironmental cells and are crucial for tumor immune escape. We aimed to evaluate the role of PD-1, its ligands and IDO1 in a cohort of patients with primary diffuse large B-cell lymphoma of the CNS (PCNSL). Material and methods Tissue microarrays (TMAs) were constructed in 45 PCNSL cases. RNA extraction from whole tissue sections and RNA sequencing were successfully performed in 33 cases. Immunohistochemical stainings for PD-1, PD-L1/paired box protein 5 (PAX-5), PD-L2/PAX-5 and IDO1, and Epstein-Barr virus encoding RNA (EBER) in situ hybridization were analyzed. Results High proportions of PD-L1 and PD-L2 positive tumor cells were observed in 11% and 9% of cases, respectively. High proportions of PD-L1 and PD-L2 positive leukocytes were observed in 55% and 51% of cases, respectively. RNA sequencing revealed that gene expression of IDO1 was high in patients with high proportion of PD-L1 positive leukocytes (p = .01). Protein expression of IDO1 in leukocytes was detected in 14/45 cases, in 79% of these cases a high proportion of PD-L1 positive leukocytes was observed. Gene expression of IDO1 was high in EBER-positive cases (p = .0009) and protein expression of IDO1 was detected in five of six EBER-positive cases. Conclusion Our study shows a significant association between gene and protein expression of IDO1 and protein expression of PD-L1 in the tumor microenvironment of PCNSL, possibly of importance for prediction of response to immunotherapies. Title in Thesis: PD-L1 and IDO1 are important immunosuppressive molecules in primary diffuse large B-cell lymphoma of the CNS

Supplemental Digital Content is available in the text., Background: Coronary artery disease (CAD) is accelerated in subjects with type 2 diabetes mellitus (T2D). Methods: To test whether this reflects differential genetic influences on CAD risk in subjects with T2D, we performed a systematic assessment of genetic overlap between CAD and T2D in 66 643 subjects (27 708 with CAD and 24 259 with T2D). Variants showing apparent association with CAD in stratified analyses or evidence of interaction were evaluated in a further 117 787 subjects (16 694 with CAD and 11 537 with T2D). Results: None of the previously characterized CAD loci was found to have specific effects on CAD in T2D individuals, and a genome-wide interaction analysis found no new variants for CAD that could be considered T2D specific. When we considered the overall genetic correlations between CAD and its risk factors, we found no substantial differences in these relationships by T2D background. Conclusions: This study found no evidence that the genetic architecture of CAD differs in those with T2D compared with those without T2D.

BackgroundIn recent years, nucleotide sequencing has become increasingly instrumental in both research and clinical settings. This has led to an explosive growth in sequencing data produced worldwide. As the amount of data increases, so does the need for automated solutions for data processing and analysis. The concept of workflows has gained favour in the bioinformatics community, but there is little in the scientific literature describing end-to-end automation systems. Arteria is an automation system that aims at providing a solution to the data-related operational challenges that face sequencing core facilities.FindingsArteria is built on existing open source technologies, with a modular design allowing for a community-driven effort to create plug-and-play micro-services. In this article we describe the system, elaborate on the underlying conceptual framework, and present an example implementation. Arteria can be reduced to 3 conceptual levels: orchestration (using an event-based model of automation), process (the steps involved in processing sequencing data, modelled as workflows), and execution (using a series of RESTful micro-services). This creates a system that is both flexible and scalable. Arteria-based systems have been successfully deployed at 3 sequencing core facilities. The Arteria Project code, written largely in Python, is available as open source software, and more information can be found at https://arteria-project.github.io/ .ConclusionsWe describe the Arteria system and the underlying conceptual framework, demonstrating how this model can be used to automate data handling and analysis in the context of a sequencing core facility.

The genetic architecture of common traits, including the number, frequency, and effect sizes of inherited variants that contribute to individual risk, has been long debated. Genome-wide association studies have identified scores of common variants associated with type 2 diabetes, but in aggregate, these explain only a fraction of the heritability of this disease. Here, to test the hypothesis that lower-frequency variants explain much of the remainder, the GoT2D and T2D-GENES consortia performed whole-genome sequencing in 2,657 European individuals with and without diabetes, and exome sequencing in 12,940 individuals from five ancestry groups. To increase statistical power, we expanded the sample size via genotyping and imputation in a further 111,548 subjects. Variants associated with type 2 diabetes after sequencing were overwhelmingly common and most fell within regions previously identified by genome-wide association studies. Comprehensive enumeration of sequence variation is necessary to identify functional alleles that provide important clues to disease pathophysiology, but large-scale sequencing does not support the idea that lower-frequency variants have a major role in predisposition to type 2 diabetes.

A wealth of biospecimen samples are stored in modern globally distributed biobanks. Biomedical researchers worldwide need to be able to combine the available resources to improve the power of large-scale studies. A prerequisite for this effort is to be able to search and access phenotypic, clinical and other information about samples that are currently stored at biobanks in an integrated manner. However, privacy issues together with heterogeneous information systems and the lack of agreed-upon vocabularies have made specimen searching across multiple biobanks extremely challenging. We describe three case studies where we have linked samples and sample descriptions in order to facilitate global searching of available samples for research. The use cases include the ENGAGE (European Network for Genetic and Genomic Epidemiology) consortium comprising at least 39 cohorts, the SUMMIT (surrogate markers for micro- and macro-vascular hard endpoints for innovative diabetes tools) consortium and a pilot for data integration between a Swedish clinical health registry and a biobank. We used the Sample avAILability (SAIL) method for data linking: first, created harmonised variables and then annotated and made searchable information on the number of specimens available in individual biobanks for various phenotypic categories. By operating on this categorised availability data we sidestep many obstacles related to privacy that arise when handling real values and show that harmonised and annotated records about data availability across disparate biomedical archives provide a key methodological advance in pre-analysis exchange of information between biobanks, that is, during the project planning phase. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

© The Author(s) 2017. Published by Oxford University Press. All rights reserved. Progranulin is a secreted protein with important functions in processes including immune and inflammatory response, metabolism and embryonic development. The present study aimed at identification of genetic factors determining progranulin concentrations. We conducted a genome-wide association meta-analysis for serum progranulin in three independent cohorts from Europe: Sorbs (N=848) and KORA (N=1628) from Germany and PPP-Botnia (N=335) from Finland (total N=2811). Single nucleotide polymorphisms (SNPs) associated with progranulin levels were replicated in two additional German cohorts: LIFE-Heart Study (Leipzig; N=967) and Metabolic Syndrome Berlin Potsdam (Berlin cohort; N=833). We measured mRNA expression of genes in peripheral blood mononuclear cells (PBMC) by micro-arrays and performed mRNA expression quantitative trait and expression-progranulin association studies to functionally substantiate identified loci. Finally, we conducted siRNA silencing experiments in vitro to validate potential candidate genes within the associated loci. Heritability of circulating progranulin levels was estimated at 31.8% and 26.1% in the Sorbs and LIFE-Heart cohort, respectively. SNPs at three loci reached study-wide significance (rs660240 in CELSR 2-PSRC1-MYBPHL-SORT1, rs4747197 in CDH23- PSAP and rs5848 in GRN) explaining 19.4%/15.0% of the variance and 61%/57% of total heritability in the Sorbs/LIFE-Heart Study. The strongest evidence for association was at rs660240 (P=5.75x10 -50 ), which was also associated with mRNA expression of PSRC1 in PBMC (P=1.51x10 -21 ). Psrc1 knockdown in murine preadipocytes led to a consecutive 30% reduction in progranulin secretion. In conclusion, the present meta-GWAS combined with mRNA expression identified three loci associated with progranulin and supports the role of PSRC1 in the regulation of progranulin secretion.

Diabetes is the leading cause of end stage renal disease. Despite evidence for a substantial heritability of diabetic kidney disease, efforts to identify genetic susceptibility variants have had limited success. We extended previous efforts in three dimensions, examining a more comprehensive set of genetic variants in larger numbers of subjects with type 1 diabetes characterized for a wider range of cross-sectional diabetic kidney disease phenotypes. In 2,843 subjects, we estimated that the heritability of diabetic kidney disease was 35% (p=6x10-3 7 ). Genome-wide association analysis and replication in 12,540 individuals identified no single variants reaching stringent levels of significance and, despite excellent power, provided little independent confirmation of previously published associated variants. Whole exome sequencing in 997 subjects failed to identify any large-effect coding alleles of lower frequency influencing the risk of diabetic kidney disease. However, sets of alleles increasing body mass index (p=2.2×10-5) and the risk of type 2 diabetes (p=6.1x10-4 11 ) were associated with the risk of diabetic kidney disease. We also found genome-wide genetic correlation between diabetic kidney disease and failure at smoking cessation (p=1.1×10-4 13 ). Pathway analysis implicated ascorbate and aldarate metabolism (p=9×10-6), and pentose and glucuronate interconversions (p=3×10-6 14 ) in pathogenesis of diabetic kidney disease. These data provide further evidence for the role of genetic factors influencing diabetic kidney disease in those with type 1 diabetes and highlight some key pathways that may be responsible. Altogether these results reveal important biology behind the major cause of kidney disease.

Genetic variation can modulate gene expression, and thereby phenotypic variation and susceptibility to complex diseases such as type 2 diabetes (T2D). Here we harnessed the potential of DNA and RNA sequencing in human pancreatic islets from 89 deceased donors to identify genes of potential importance in the pathogenesis of T2D. We present a catalog of genetic variants regulating gene expression (eQTL) and exon use (sQTL), including many long noncoding RNAs, which are enriched in known T2D-associated loci. Of 35 eQTL genes, whose expression differed between normoglycemic and hyperglycemic individuals, siRNA of tetraspanin 33 (TSPAN33), 5'-nucleotidase, ecto (NT5E), transmembrane emp24 protein transport domain containing 6 (TMED6), and p21 protein activated kinase 7 (PAK7) in INS1 cells resulted in reduced glucose-stimulated insulin secretion. In addition, we provide a genome-wide catalog of allelic expression imbalance, which is also enriched in known T2D-associated loci. Notably, allelic imbalance in paternally expressed gene 3 (PEG3) was associated with its promoter methylation and T2D status. Finally, RNA editing events were less common in islets than previously suggested in other tissues. Taken together, this study provides new insights into the complexity of gene regulation in human pancreatic islets and better understanding of how genetic variation can influence glucose metabolism.

Although meta-analyses of genome-wide association studies have identified >60 single nucleotide polymorphisms (SNPs) associated with type 2 diabetes and/or glycemic traits, there is little information on whether these variants also affect α-cell function. The aim of the current study was to evaluate the effects of glycemia-associated genetic loci on islet function in vivo and in vitro. We studied 43 SNPs in 4,654 normoglycemic participants from the Finnish population-based Prevalence, Prediction, and Prevention of Diabetes-Botnia (PPP-Botnia) Study. Islet function was assessed, in vivo, by measuring insulin and glucagon concentrations during oral glucose tolerance test, and, in vitro, by measuring glucose-stimulated insulin and glucagon secretion from human pancreatic islets. Carriers of risk variants in BCL11A, HHEX, ZBED3, HNF1A, IGF1, and NOTCH2 showed elevated whereas those in CRY2, IGF2BP2, TSPAN8, and KCNJ11 showed decreased fasting and/or 2-h glucagon concentrations in vivo. Variants in BCL11A, TSPAN8, and NOTCH2 affected glucagon secretion both in vivo and in vitro. The MTNR1B variant was a clear outlier in the relationship analysis between insulin secretion and action, as well as between insulin, glucose, and glucagon. Many of the genetic variants shown to be associated with type 2 diabetes or glycemic traits also exert pleiotropic in vivo and in vitro effects on islet function.

Genome-wide association studies (GWAS) have identified more than 65 genetic loci associated with risk of type 2 diabetes. However, the contribution of distorted parental transmission of alleles to risk of type 2 diabetes has been mostly unexplored. Our goal was therefore to search for parent-of-origin effects (POE) among type 2 diabetes loci in families. Families from the Botnia study (n = 4,211, 1,083 families) were genotyped for 72 single-nucleotide polymorphisms (SNPs) associated with type 2 diabetes and assessed for POE on type 2 diabetes. The family-based Hungarian Transdanubian Biobank (HTB) (n = 1,463, >135 families) was used to replicate SNPs showing POE. Association of type 2 diabetes loci within families was also tested. Three loci showed nominal POE, including the previously reported variants in KCNQ1, for type 2 diabetes in families from Botnia (rs2237895: p POE = 0.037), which can be considered positive controls. The strongest POE was seen for rs7578597 SNP in the THADA gene, showing excess transmission of the maternal risk allele T to diabetic offspring (Botnia: p POE = 0.01; HTB p POE = 0.045). These data are consistent with previous evidence of allelic imbalance for expression in islets, suggesting that the THADA gene can be imprinted in a POE-specific fashion. Five CpG sites, including those flanking rs7578597, showed differential methylation between diabetic and non-diabetic donor islets. Taken together, the data emphasise the need for genetic studies to consider from which parent an offspring has inherited a susceptibility allele.

Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone with extrapancreatic effects beyond glycemic control. Here we demonstrate unexpected effects of GIP signaling in the vasculature. GIP induces the expression of the proatherogenic cytokine osteopontin (OPN) in mouse arteries via local release of endothelin-1 and activation of CREB. Infusion of GIP increases plasma OPN concentrations in healthy individuals. Plasma endothelin-1 and OPN concentrations are positively correlated in patients with critical limb ischemia. Fasting GIP concentrations are higher in individuals with a history of cardiovascular disease (myocardial infarction, stroke) when compared with control subjects. GIP receptor (GIPR) and OPN mRNA levels are higher in carotid endarterectomies from patients with symptoms (stroke, transient ischemic attacks, amaurosis fugax) than in asymptomatic patients, and expression associates with parameters that are characteristic of unstable and inflammatory plaques (increased lipid accumulation, macrophage infiltration, and reduced smooth muscle cell content). While GIPR expression is predominantly endothelial in healthy arteries from humans, mice, rats, and pigs, remarkable upregulation is observed in endothelial and smooth muscle cells upon culture conditions, yielding a “vascular disease–like” phenotype. Moreover, the common variant rs10423928 in the GIPR gene is associated with increased risk of stroke in patients with type 2 diabetes.

Aims/hypothesis Human complex metabolic traits are in part regulated by genetic determinants. Here we applied exome sequencing to identify novel associations of coding polymorphisms at minor allele frequencies (MAFs) >1% with common metabolic phenotypes. Methods The study comprised three stages. We performed medium-depth (8×) whole exome sequencing in 1,000 cases with type 2 diabetes, BMI >27.5 kg/m2 and hypertension and in 1,000 controls (stage 1). We selected 16,192 polymorphisms nominally associated (p 1%. In stage 2 we identified 51 potential associations with one or more of eight metabolic phenotypes covered by 45 unique polymorphisms. In meta-analyses of stage 2 and stage 3 results, we demonstrated robust associations for coding polymorphisms in CD300LG (fasting HDL-cholesterol: MAF 3.5%, p = 8.5 × 10−14), COBLL1 (type 2 diabetes: MAF 12.5%, OR 0.88, p = 1.2 × 10−11) and MACF1 (type 2 diabetes: MAF 23.4%, OR 1.10, p = 8.2 × 10−10). Conclusions/interpretation We applied exome sequencing as a basis for finding genetic determinants of metabolic traits and show the existence of low-frequency and common coding polymorphisms with impact on common metabolic traits. Based on our study, coding polymorphisms with MAF above 1% do not seem to have particularly high effect sizes on the measured metabolic traits. Electronic supplementary material The online version of this article (doi:10.1007/s00125-012-2756-1) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Reduced insulin release has been linked to defect exocytosis in β-cells. However, whether expression of genes suggested to be involved in the exocytotic process (exocytotic genes) is altered in pancreatic islets from patients with type 2 diabetes (T2D), and correlate to insulin secretion, needs to be further investigated. Analysing expression levels of 23 exocytotic genes using microarray revealed reduced expression of five genes in human T2D islets (χ(2)=13.25; p

Group Authors : DIAGRAM Consortium MUTHER Consortium variants influencing fasting glycemic traits and insulin resistance Seuls les 100 premiers auteurs ont été conservés dans la publication. La liste complète des auteurs et des des affiliations est disponible sur la publication https://www.nature.com/articles/ng.2274.pdf p. 667-669.; International audience; Recent genome-wide association studies have described many loci implicated in type 2 diabetes (T2D) pathophysiology and beta-cell dysfunction but have contributed little to the understanding of the genetic basis of insulin resistance. We hypothesized that genes implicated in insulin resistance pathways might be uncovered by accounting for differences in body mass index (BMI) and potential interactions between BMI and genetic variants. We applied a joint meta-analysis approach to test associations with fasting insulin and glucose on a genome-wide scale. We present six previously unknown loci associated with fasting insulin at P < 5 x 10(-8) in combined discovery and follow-up analyses of 52 studies comprising up to 96,496 non-diabetic individuals. Risk variants were associated with higher triglyceride and lower high-density lipoprotein (HDL) cholesterol levels, suggesting a role for these loci in insulin resistance pathways. The discovery of these loci will aid further characterization of the role of insulin resistance in T2D pathophysiology.

ContextMitochondrial ATP production is important in the regulation of glucose-stimulated insulin secretion. Genetic factors may modulate the capacity of the β-cells to secrete insulin and thereby contribute to the risk of type 2 diabetes.ObjectiveThe aim of this study was to identify genetic loci in or adjacent to nuclear-encoded genes of the oxidative phosphorylation (OXPHOS) pathway that are associated with insulin secretion in vivo.Design and methodsTo find polymorphisms associated with glucose-stimulated insulin secretion, data from a genome-wide association study (GWAS) of 1467 non-diabetic individuals, including the Diabetes Genetic Initiative (DGI), was examined. A total of 413 single nucleotide polymorphisms with a minor allele frequency ≥0.05 located in or adjacent to 76 OXPHOS genes were included in the DGI GWAS. A more extensive population-based study of 4323 non-diabetics, the PPP-Botnia, was used as a replication cohort. Insulinogenic index during an oral glucose tolerance test was used as a surrogate marker of glucose-stimulated insulin secretion. Multivariate linear regression analyses were used to test genotype–phenotype associations.ResultsTwo common variants were identified in the DGI, where the major C-allele of rs606164, adjacent to NADH dehydrogenase (ubiquinone) 1 subunit C2 (NDUFC2), and the minor G-allele of rs1323070, adjacent to cytochrome c oxidase subunit VIIa polypeptide 2 (COX7A2), showed nominal associations with decreased glucose-stimulated insulin secretion (P=0.0009, respective P=0.003). These associations were replicated in PPP-Botnia (P=0.002 and P=0.05).ConclusionOur study shows that genetic variation near genes involved in OXPHOS may influence glucose-stimulated insulin secretion in vivo.

Object Family studies have suggested a role of genetic factors in susceptibility to aneurysmal subarachnoid hemorrhage (aSAH), but the underlying genetic risk factors remain poorly defined. There is an activation of the fibrinolytic system in aSAH, and fibrinolytic markers may be useful in predicting outcome. The authors investigate associations between putative functional variants in genes of importance for fibrinolysis and aSAH and/or outcome following aSAH. Methods One hundred eighty-three patients presenting with aSAH at a neurointensive care unit were consecutively recruited. Two healthy controls per case, matched for age, sex, and geographic region, were randomly recruited. Outcome was assessed after 1 year according to the extended Glasgow Outcome Scale. Single nucleotide polymorphisms (SNPs) in the tissue-type plasminogen activator (tPA), plasminogen activator inhibitor type 1 (PAI-1), thrombin activatable fibrinolysis inhibitor (TAFI), and factor XIII (FXIII) genes were investigated. Results Participants carrying the FXIII 34Leu allele showed an increased risk of aSAH. When adjusting for smoking and hypertension, 2 haplotypes, differing on either the FXIII Val34Leu or the Pro564Leu position, showed an association to aSAH. No significant association was observed for the tPA -7351 C > T, PAI-1 -675 4G > 5G, or TAFI Ala147Thr SNPs. No specific SNP or haplotype was associated with outcome after aSAH, whereas a weak association was observed for a tPA/PAI-1 genotype combination. Conclusions Polymorphisms in the FXIII gene showed association to aSAH. The finding of an increased risk of bleeding in FXIII 34Leu carriers is biologically plausible.

Summary. Background: Plasma fibrinogen level and fibrin clot structure are heritable traits that may be of importance in the pathogenesis of ischemic stroke. Objectives: To investigate associations between variation in the fibrinogen gamma (FGG), alpha (FGA) and beta (FGB) genes, fibrinogen level, and ischemic stroke. Methods: The Sahlgrenska Academy Study on Ischemic Stroke comprises 600 cases and 600 matched population controls. Stroke subtypes were defined according to TOAST criteria. Plasma fibrinogen level was measured by an automated clot-rate assay. Eight tagging single nucleotide polymorphisms (SNPs) were selected to capture genetic variation in the FGA, FGG, and FGB genes. Results: Plasma fibrinogen was independently associated with overall ischemic stroke and all subtypes, both in the acute stage (P

Using a genome-wide screen of 9.6 million genetic variants achieved through 1000 Genomes Project imputation in 62,166 samples,we identify association to lipid traits in 93 loci, including 79 previously identified loci with new lead SNPs and 10 new loci, 15 loci with a low-frequency lead SNP and 10 loci with a missense lead SNP, and 2 loci with an accumulation of rare variants. In six loci,SNPs with established function in lipid genetics (CELSR2, GCKR, LIPC and APOE) or candidate missense mutations with predicted damaging function (CD300LG and TM6SF2) explained the locus associations. The low-frequency variants increased the proportion of variance explained, particularly for low-density lipoprotein cholesterol and total cholesterol. Altogether, our results highlight the impact of low-frequency variants in complex traits and show that imputation offers a cost-effective alternative to resequencing. Refereed/Peer-reviewed

Observational studies have reported different effects of adiposity on cardiovascular risk factors across age and sex. Since cardiovascular risk factors are enriched in obese individuals, it has not been easy to dissect the effects of adiposity from those of other risk factors. We used a Mendelian randomization approach, applying a set of 32 genetic markers to estimate the causal effect of adiposity on blood pressure, glycemic indices, circulating lipid levels, and markers of inflammation and liver disease in up to 67,553 individuals. All analyses were stratified by age (cutoff 55 years of age) and sex. The genetic score was associated with BMI in both nonstratified analysis (P = 2.8 × 10−107) and stratified analyses (all P < 3.3 × 10−30). We found evidence of a causal effect of adiposity on blood pressure, fasting levels of insulin, C-reactive protein, interleukin-6, HDL cholesterol, and triglycerides in a nonstratified analysis and in the

BackgroundThere is a need to develop and validate surrogate markers of cardiovascular disease (CVD) in subjects with diabetes. The macrovascular changes associated with diabetes include aggravated atherosclerosis, increased arterial stiffness and endothelial dysfunction. The aim of this study was to determine which of these factors is most strongly associated with clinically manifest cardiovascular events. MethodsVascular changes were measured in a cohort of 458 subjects with type 2 diabetes (T2D) and CVD (myocardial infarction, stroke or lower extremity arterial disease), 527 subjects with T2D but without clinically manifest CVD and 515 subjects without T2D and with or without CVD. ResultsCarotid intima-media thickness (IMT) and ankle-brachial pressure index were independently associated with the presence of CVD in subjects with T2D, whereas pulse wave velocity and endothelial function provided limited independent additive information. Measurement of IMT in the carotid bulb provided better discrimination of the presence of CVD in subjects with T2D than measurement of IMT in the common carotid artery. The factors most significantly associated with increased carotid IMT in T2D were age, disease duration, systolic blood pressure, impaired renal function and increased arterial stiffness, whereas there were no or weak independent associations with metabolic factors and endothelial dysfunction. ConclusionsMeasures of atherosclerotic burden are associated with clinically manifest CVD in subjects with T2D. In addition, vascular changes that are not directly related to known metabolic risk factors are important in the development of both atherosclerosis and CVD in T2D. A better understanding of the mechanisms involved is crucial for enabling better identification of CVD risk in T2D. (Less)

Reference panels from the 1000 Genomes (1000G) Project Consortium provide near complete coverage of common and low-frequency genetic variation with minor allele frequency ≥0.5% across European ancestry populations. Within the European Network for Genetic and Genomic Epidemiology (ENGAGE) Consortium, we have undertaken the first large-scale meta-analysis of genome-wide association studies (GWAS), supplemented by 1000G imputation, for four quantitative glycaemic and obesity-related traits, in up to 87,048 individuals of European ancestry. We identified two loci for body mass index (BMI) at genome-wide significance, and two for fasting glucose (FG), none of which has been previously reported in larger meta-analysis efforts to combine GWAS of European ancestry. Through conditional analysis, we also detected multiple distinct signals of association mapping to established loci for waist-hip ratio adjusted for BMI (RSPO3) and FG (GCK and G6PC2). The index variant for one association signal at the G6PC2 locus is a low-frequency coding allele, H177Y, which has recently been demonstrated to have a functional role in glucose regulation. Fine-mapping analyses revealed that the non-coding variants most likely to drive association signals at established and novel loci were enriched for overlap with enhancer elements, which for FG mapped to promoter and transcription factor binding sites in pancreatic islets, in particular. Our study demonstrates that 1000G imputation and genetic fine-mapping of common and low-frequency variant association signals at GWAS loci, integrated with genomic annotation in relevant tissues, can provide insight into the functional and regulatory mechanisms through which their effects on glycaemic and obesity-related traits are mediated., Author Summary Human genetic studies have demonstrated that quantitative human anthropometric and metabolic traits, including body mass index, waist-hip ratio, and plasma concentrations of glucose and insulin, are highly heritable, and are established risk factors for type 2 diabetes and cardiovascular diseases. Although many regions of the genome have been associated with these traits, the specific genes responsible have not yet been identified. By making use of advanced statistical “imputation” techniques applied to more than 87,000 individuals of European ancestry, and publicly available “reference panels” of more than 37 million genetic variants, we have been able to identify novel regions of the genome associated with these glycaemic and obesity-related traits and localise genes within these regions that are most likely to be causal. This improved understanding of the biological mechanisms underlying glycaemic and obesity-related traits is extremely important because it may advance drug development for downstream disease endpoints, ultimately leading to public health benefits.

Chemerin is an adipokine proposed to link obesity and chronic inflammation of adipose tissue. Genetic factors determining chemerin release from adipose tissue are yet unknown. We conducted a meta-analysis of genome-wide association studies (GWAS) for serum chemerin in three independent cohorts from Europe: Sorbs and KORA from Germany and PPP-Botnia from Finland (total N = 2,791). In addition, we measured mRNA expression of genes within the associated loci in peripheral mononuclear cells by micro-arrays, and within adipose tissue by quantitative RT-PCR and performed mRNA expression quantitative trait and expression-chemerin association studies to functionally substantiate our loci. Heritability estimate of circulating chemerin levels was 16.2% in the Sorbs cohort. Thirty single nucleotide polymorphisms (SNPs) at chromosome 7 within the retinoic acid receptor responder 2 (RARRES2)/Leucine Rich Repeat Containing (LRRC61) locus reached genome-wide significance (p, Author Summary Chemerin is an adipokine proposed to link obesity and chronic inflammation of adipose tissue. In the present study we show that circulating chemerin is a heritable trait. In a meta-analysis of genome-wide association studies (GWAS) of 2,791 individuals from Germany and Finland, we identified common genetic variants which associate with serum chemerin levels. The variants map within the retinoic acid receptor responder 2 (RARRES2)/Leucine Rich Repeat Containing (LRRC61) at chromosome 7. To better understand the potential functionality of the identified variants, we also provide insights into the mRNA expression of RARRES2 (encoding chemerin) in blood and adipose tissue. Our results highlight the role and function of genetic variation in the RARRES2 locus in the regulation of circulating chemerin concentrations.

Exome sequencing studies in complex diseases are challenged by the allelic heterogeneity, large number and modest effect sizes of associated variants on disease risk and the presence of large numbers of neutral variants, even in phenotypically relevant genes. Isolated populations with recent bottlenecks offer advantages for studying rare variants in complex diseases as they have deleterious variants that are present at higher frequencies as well as a substantial reduction in rare neutral variation. To explore the potential of the Finnish founder population for studying low-frequency (0.5–5%) variants in complex diseases, we compared exome sequence data on 3,000 Finns to the same number of non-Finnish Europeans and discovered that, despite having fewer variable sites overall, the average Finn has more low-frequency loss-of-function variants and complete gene knockouts. We then used several well-characterized Finnish population cohorts to study the phenotypic effects of 83 enriched loss-of-function variants across 60 phenotypes in 36,262 Finns. Using a deep set of quantitative traits collected on these cohorts, we show 5 associations (p, National Institutes of Health (U.S.) (NIH RC2 HL-102925)

The X chromosome (chrX) represents one potential source for the “missing heritability” for complex phenotypes, which thus far has remained underanalyzed in genome-wide association studies (GWAS). Here we demonstrate the benefits of including chrX in GWAS by assessing the contribution of 404,862 chrX SNPs to levels of twelve commonly studied cardiometabolic and anthropometric traits in 19,697 Finnish and Swedish individuals with replication data on 5,032 additional Finns. By using a linear mixed model, we estimate that on average 2.6% of the additive genetic variance in these twelve traits is attributable to chrX, this being in proportion to the number of SNPs in the chromosome. In a chrX-wide association analysis, we identify three novel loci: two for height (rs182838724 near FGF16/ATRX/MAGT1, joint P-value = 2.71×10−9, and rs1751138 near ITM2A, P-value = 3.03×10−10) and one for fasting insulin (rs139163435 in Xq23, P-value = 5.18×10−9). Further, we find that effect sizes for variants near ITM2A, a gene implicated in cartilage development, show evidence for a lack of dosage compensation. This observation is further supported by a sex-difference in ITM2A expression in whole blood (P-value = 0.00251), and is also in agreement with a previous report showing ITM2A escapes from X chromosome inactivation (XCI) in the majority of women. Hence, our results show one of the first links between phenotypic variation in a population sample and an XCI-escaping locus and pinpoint ITM2A as a potential contributor to the sexual dimorphism in height. In conclusion, our study provides a clear motivation for including chrX in large-scale genetic studies of complex diseases and traits., Author Summary The X chromosome (chrX) analyses have often been neglected in large-scale genome-wide association studies. Given that chrX contains a considerable proportion of DNA, we wanted to examine how the variation in the chromosome contributes to commonly studied phenotypes. To this end, we studied the associations of over 400,000 chrX variants with twelve complex phenotypes, such as height, in almost 25,000 Northern European individuals. Demonstrating the value of assessing chrX associations, we found that as a whole the variation in the chromosome influences the levels of many of these phenotypes and further identified three new genomic regions where the variants associate with height or fasting insulin levels. In one of these three associated regions, the region near ITM2A, we observed that there is a sex difference in the genetic effects on height in a manner consistent with a lack of dosage compensation in this locus. Further supporting this observation, ITM2A has been shown to be among those chrX genes where the X chromosome inactivation is incomplete. Identifying phenotype associations in regions like this where chrX allele dosages are not balanced between men and women can be particularly valuable in helping us to understand why some characteristics differ between sexes.

Genetic and epigenetic mechanisms may interact and together affect biological processes and disease development. However, most previous studies have investigated genetic and epigenetic mechanisms independently, and studies examining their interactions throughout the human genome are lacking. To identify genetic loci that interact with the epigenome, we performed the first genome-wide DNA methylation quantitative trait locus (mQTL) analysis in human pancreatic islets. We related 574,553 single nucleotide polymorphisms (SNPs) with genome-wide DNA methylation data of 468,787 CpG sites targeting 99% of RefSeq genes in islets from 89 donors. We identified 67,438 SNP-CpG pairs in cis, corresponding to 36,783 SNPs (6.4% of tested SNPs) and 11,735 CpG sites (2.5% of tested CpGs), and 2,562 significant SNP-CpG pairs in trans, corresponding to 1,465 SNPs (0.3% of tested SNPs) and 383 CpG sites (0.08% of tested CpGs), showing significant associations after correction for multiple testing. These include reported diabetes loci, e.g. ADCY5, KCNJ11, HLA-DQA1, INS, PDX1 and GRB10. CpGs of significant cis-mQTLs were overrepresented in the gene body and outside of CpG islands. Follow-up analyses further identified mQTLs associated with gene expression and insulin secretion in human islets. Causal inference test (CIT) identified SNP-CpG pairs where DNA methylation in human islets is the potential mediator of the genetic association with gene expression or insulin secretion. Functional analyses further demonstrated that identified candidate genes (GPX7, GSTT1 and SNX19) directly affect key biological processes such as proliferation and apoptosis in pancreatic β-cells. Finally, we found direct correlations between DNA methylation of 22,773 (4.9%) CpGs with mRNA expression of 4,876 genes, where 90% of the correlations were negative when CpGs were located in the region surrounding transcription start site. Our study demonstrates for the first time how genome-wide genetic and epigenetic variation interacts to influence gene expression, islet function and potential diabetes risk in humans., Author Summary Inter-individual variation in genetics and epigenetics affects biological processes and disease susceptibility. However, most studies have investigated genetic and epigenetic mechanisms independently and to uncover novel mechanisms affecting disease susceptibility there is a highlighted need to study interactions between these factors on a genome-wide scale. To identify novel loci affecting islet function and potentially diabetes, we performed the first genome-wide methylation quantitative trait locus (mQTL) analysis in human pancreatic islets including DNA methylation of 468,787 CpG sites located throughout the genome. Our results showed that DNA methylation of 11,735 CpGs in 4,504 unique genes is regulated by genetic factors located in cis (67,438 SNP-CpG pairs). Furthermore, significant mQTLs cover previously reported diabetes loci including KCNJ11, INS, HLA, PDX1 and GRB10. We also found mQTLs associated with gene expression and insulin secretion in human islets. By performing causality inference tests (CIT), we identified CpGs where DNA methylation potentially mediates the genetic impact on gene expression and insulin secretion. Our functional follow-up experiments further demonstrated that identified mQTLs/genes (GPX7, GSTT1 and SNX19) directly affect pancreatic β-cell function. Together, our study provides a detailed map of genome-wide associations between genetic and epigenetic variation, which affect gene expression and insulin secretion in human pancreatic islets.

BACKGROUND: The incidence of hematologic cancers increases with age. These cancers are associated with recurrent somatic mutations in specific genes. We hypothesized that such mutations would be detectable in the blood of some persons who are not known to have hematologic disorders. METHODS: We analyzed whole-exome sequencing data from DNA in the peripheral-blood cells of 17,182 persons who were unselected for hematologic phenotypes. We looked for somatic mutations by identifying previously characterized single-nucleotide variants and small insertions or deletions in 160 genes that are recurrently mutated in hematologic cancers. The presence of mutations was analyzed for an association with hematologic phenotypes, survival, and cardiovascular events. RESULTS: Detectable somatic mutations were rare in persons younger than 40 years of age but rose appreciably in frequency with age. Among persons 70 to 79 years of age, 80 to 89 years of age, and 90 to 108 years of age, these clonal mutations were observed in 9.5% (219 of 2300 persons), 11.7% (37 of 317), and 18.4% (19 of 103), respectively. The majority of the variants occurred in three genes: DNMT3A, TET2, and ASXL1. The presence of a somatic mutation was associated with an increase in the risk of hematologic cancer (hazard ratio, 11.1; 95% confidence interval [CI], 3.9 to 32.6), an increase in all-cause mortality (hazard ratio, 1.4; 95% CI, 1.1 to 1.8), and increases in the risks of incident coronary heart disease (hazard ratio, 2.0; 95% CI, 1.2 to 3.4) and ischemic stroke (hazard ratio, 2.6; 95% CI, 1.4 to 4.8). CONCLUSIONS: Age-related clonal hematopoiesis is a common condition that is associated with increases in the risk of hematologic cancer and in all-cause mortality, with the latter possibly due to an increased risk of cardiovascular disease. (Funded by the National Institutes of Health and others.).

Using an integrative approach in which genetic variation, gene expression, and clinical phenotypes are assessed in relevant tissues may help functionally characterize the contribution of genetics to disease susceptibility. We sought to identify genetic variation influencing skeletal muscle gene expression (expression quantitative trait loci [eQTLs]) as well as expression associated with measures of insulin sensitivity. We investigated associations of 3,799,401 genetic variants in expression of >7,000 genes from three cohorts (n = 104). We identified 287 genes with cis-acting eQTLs (false discovery rate [FDR] Language of Original Document: English

Finnish samples have been extensively utilized in studying single-gene disorders, where the founder effect has clearly aided in discovery, and more recently in genome-wide association studies of complex traits, where the founder effect has had less obvious impacts. As the field starts to explore rare variants' contribution to polygenic traits, it is of great importance to characterize and confirm the Finnish founder effect in sequencing data and to assess its implications for rare-variant association studies. Here, we employ forward simulation, guided by empirical deep resequencing data, to model the genetic architecture of quantitative polygenic traits in both the general European and the Finnish populations simultaneously. We demonstrate that power of rare-variant association tests is higher in the Finnish population, especially when variants' phenotypic effects are tightly coupled with fitness effects and therefore reflect a greater contribution of rarer variants. SKAT-O, variable-threshold tests, and single-variant tests are more powerful than other rare-variant methods in the Finnish population across a range of genetic models. We also compare the relative power and efficiency of exome array genotyping to those of high-coverage exome sequencing. At a fixed cost, less expensive genotyping strategies have far greater power than sequencing; in a fixed number of samples, however, genotyping arrays miss a substantial portion of genetic signals detected in sequencing, even in the Finnish founder population. As genetic studies probe sequence variation at greater depth in more diverse populations, our simulation approach provides a framework for evaluating various study designs for gene discovery.

Contains fulltext : 128630.pdf (Publisher’s version ) (Open Access) Smoking influences body weight such that smokers weigh less than non-smokers and smoking cessation often leads to weight increase. The relationship between body weight and smoking is partly explained by the effect of nicotine on appetite and metabolism. However, the brain reward system is involved in the control of the intake of both food and tobacco. We evaluated the effect of single-nucleotide polymorphisms (SNPs) affecting body mass index (BMI) on smoking behavior, and tested the 32 SNPs identified in a meta-analysis for association with two smoking phenotypes, smoking initiation (SI) and the number of cigarettes smoked per day (CPD) in an Icelandic sample (N=34,216 smokers). Combined according to their effect on BMI, the SNPs correlate with both SI (r=0.019, P=0.00054) and CPD (r=0.032, P=8.0 x 10(-7)). These findings replicate in a second large data set (N=127,274, thereof 76,242 smokers) for both SI (P=1.2 x 10(-5)) and CPD (P=9.3 x 10(-5)). Notably, the variant most strongly associated with BMI (rs1558902-A in FTO) did not associate with smoking behavior. The association with smoking behavior is not due to the effect of the SNPs on BMI. Our results strongly point to a common biological basis of the regulation of our appetite for tobacco and food, and thus the vulnerability to nicotine addiction and obesity.

Diabetic kidney disease, or diabetic nephropathy (DN), is a major complication of diabetes and the leading cause of end-stage renal disease (ESRD) that requires dialysis treatment or kidney transplantation. In addition to the decrease in the quality of life, DN accounts for a large proportion of the excess mortality associated with type 1 diabetes (T1D). Whereas the degree of glycemia plays a pivotal role in DN, a subset of individuals with poorly controlled T1D do not develop DN. Furthermore, strong familial aggregation supports genetic susceptibility to DN. However, the genes and the molecular mechanisms behind the disease remain poorly understood, and current therapeutic strategies rarely result in reversal of DN. In the GEnetics of Nephropathy: an International Effort (GENIE) consortium, we have undertaken a meta-analysis of genome-wide association studies (GWAS) of T1D DN comprising ∼2.4 million single nucleotide polymorphisms (SNPs) imputed in 6,691 individuals. After additional genotyping of 41 top ranked SNPs representing 24 independent signals in 5,873 individuals, combined meta-analysis revealed association of two SNPs with ESRD: rs7583877 in the AFF3 gene (P = 1.2×10−8) and an intergenic SNP on chromosome 15q26 between the genes RGMA and MCTP2, rs12437854 (P = 2.0×10−9). Functional data suggest that AFF3 influences renal tubule fibrosis via the transforming growth factor-beta (TGF-β1) pathway. The strongest association with DN as a primary phenotype was seen for an intronic SNP in the ERBB4 gene (rs7588550, P = 2.1×10−7), a gene with type 2 diabetes DN differential expression and in the same intron as a variant with cis-eQTL expression of ERBB4. All these detected associations represent new signals in the pathogenesis of DN., Author Summary The global prevalence of diabetes has reached epidemic proportions, constituting a major health care problem worldwide. Diabetic kidney disease, or diabetic nephropathy (DN)—the major long term microvascular complication of diabetes—is associated with excess mortality among patients with type 1 diabetes. Even though DN has been shown to cluster in families, the underlying genetic and molecular pathways remain poorly defined. We have undertaken the largest genome-wide association study and meta-analysis to date on DN and on its most severe form of kidney disease, end-stage renal disease (ESRD). We identified new loci significantly associated with diabetic ESRD: AFF3 and an intergenic locus on chromosome 15q26 residing between RGMA and MCTP2. Our functional analyses suggest that AFF3 influences renal tubule fibrosis, a pathological hallmark of severe DN. Another locus in ERBB4 was suggestively associated with DN and resides in the same intronic region as a variant affecting the expression of ERBB4. Subsequent pathway analysis of the genes co-expressed with ERBB4 indicated involvement of fibrosis.

The majority of genetic risk variants for type 2 diabetes (T2D) affect insulin secretion, but the mechanisms through which they influence pancreatic islet function remain largely unknown. We functionally characterized human islets to determine secretory, biophysical, and ultrastructural features in relation to genetic risk profiles in diabetic and nondiabetic donors. Islets from donors with T2D exhibited impaired insulin secretion, which was more pronounced in lean than obese diabetic donors. We assessed the impact of 14 disease susceptibility variants on measures of glucose sensing, exocytosis, and structure. Variants near TCF7L2 and ADRA2A were associated with reduced glucose-induced insulin secretion, whereas susceptibility variants near ADRA2A, KCNJ11, KCNQ1, and TCF7L2 were associated with reduced depolarization-evoked insulin exocytosis. KCNQ1, ADRA2A, KCNJ11, HHEX/IDE, and SLC2A2 variants affected granule docking. We combined our results to create a novel genetic risk score for β-cell dysfunction that includes aberrant granule docking, decreased Ca2+ sensitivity of exocytosis, and reduced insulin release. Individuals with a high risk score displayed an impaired response to intravenous glucose and deteriorating insulin secretion over time. Our results underscore the importance of defects in β-cell exocytosis in T2D and demonstrate the potential of cellular phenotypic characterization in the elucidation of complex genetic disorders.

Context: It has been shown that mitochondrial DNA (mtDNA) content is associated with type 2 diabetes (T2D) and related traits. However, empirical data, often based on small samples, did not confirm this observation in all studies. Therefore, the role of mtDNA content in T2D remains elusive. Objective: In this study, we assessed the heritability of mtDNA content in buccal cells and analyzed the association of mtDNA content in blood with prevalent and incident T2D. Design and Setting: mtDNA content from cells from buccal and blood samples was assessed using a real-time PCR-based assay. Heritability of mtDNA content was estimated in 391 twins from the Netherlands Twin Register. The association with prevalent T2D was tested in a case control study from The Netherlands (n = 329). Incident T2D was analyzed using prospective samples from Finland (n = 444) and The Netherlands (n = 238). Main Outcome Measures: We measured the heritability of mtDNA content and the association of mtDNA content in blood with prevalent and incident T2D. Results: A heritability of mtDNA content of 35% (19–48%) was estimated in the twin families. We did not observe evidence of an association between mtDNA content and prevalent or incident T2D and related traits. Furthermore, we observed a decline in mtDNA content with increasing age that was male specific (P = 0.001). Conclusion: In this study, we show that mtDNA content has a heritability of 35% in Dutch twins. There is no association between mtDNA content in blood and prevalent or incident T2D and related traits in our study samples.