Misexpression of inactive genes in whole blood is associated with nearby rare structural variants.

Gene misexpression is the aberrant transcription of a gene in a context where it is usually inactive. Despite its known pathological consequences in specific rare diseases, we have a limited understanding of its wider prevalence and mechanisms in humans. To address this, we analyzed gene misexpression in 4,568 whole-blood bulk RNA sequencing samples from INTERVAL study blood donors. We found that while individual misexpression events occur rarely, in aggregate they were found in almost all samples and a third of inactive protein-coding genes. Using 2,821 paired whole-genome and RNA sequencing samples, we identified that misexpression events are enriched in cis for rare structural variants. We established putative mechanisms through which a subset of SVs lead to gene misexpression, including transcriptional readthrough, transcript fusions, and gene inversion. Overall, we develop misexpression as a type of transcriptomic outlier analysis and extend our understanding of the variety of mechanisms by which genetic variants can influence gene expression.

Deconvolution of bulk blood eQTL effects into immune cell subpopulations.

BACKGROUND: Expression quantitative trait loci (eQTL) studies are used to interpret the function of disease-associated genetic risk factors. To date, most eQTL analyses have been conducted in bulk tissues, such as whole blood and tissue biopsies, which are likely to mask the cell type-context of the eQTL regulatory effects. Although this context can be investigated by generating transcriptional profiles from purified cell subpopulations, current methods to do this are labor-intensive and expensive. We introduce a new method, Decon2, as a framework for estimating cell proportions using expression profiles from bulk blood samples (Decon-cell) followed by deconvolution of cell type eQTLs (Decon-eQTL). RESULTS: The estimated cell proportions from Decon-cell agree with experimental measurements across cohorts (R ≥ 0.77). Using Decon-cell, we could predict the proportions of 34 circulating cell types for 3194 samples from a population-based cohort. Next, we identified 16,362 whole-blood eQTLs and deconvoluted cell type interaction (CTi) eQTLs using the predicted cell proportions from Decon-cell. CTi eQTLs show excellent allelic directional concordance with eQTL (≥ 96-100%) and chromatin mark QTL (≥87-92%) studies that used either purified cell subpopulations or single-cell RNA-seq, outperforming the conventional interaction effect. CONCLUSIONS: Decon2 provides a method to detect cell type interaction effects from bulk blood eQTLs that is useful for pinpointing the most relevant cell type for a given complex disease. Decon2 is available as an R package and Java application (https://github.com/molgenis/systemsgenetics/tree/master/Decon2) and as a web tool (www.molgenis.org/deconvolution).

A comprehensive analysis of microProteins reveals their potentially widespread mechanism of transcriptional regulation.

Truncated transcription factor-like proteins called microProteins (miPs) can modulate transcription factor activities, thereby increasing transcriptional regulatory complexity. To understand their prevalence, evolution, and function, we predicted over 400 genes that encode putative miPs from Arabidopsis (Arabidopsis thaliana) using a bioinformatics pipeline and validated two novel miPs involved in flowering time and response to abiotic and biotic stress. We provide an evolutionary perspective for a class of miPs targeting homeodomain transcription factors in plants and metazoans. We identify domain loss as one mechanism of miP evolution and suggest the possible roles of miPs on the evolution of their target transcription factors. Overall, we reveal a prominent layer of transcriptional regulation by miPs, show pervasiveness of such proteins both within and across genomes, and provide a framework for studying their function and evolution.

KidneyNetwork: using kidney-derived gene expression data to predict and prioritize novel genes involved in kidney disease.

Genetic testing in patients with suspected hereditary kidney disease may not reveal the genetic cause for the disorder as potentially pathogenic variants can reside in genes that are not yet known to be involved in kidney disease. We have developed KidneyNetwork, that utilizes tissue-specific expression to inform candidate gene prioritization specifically for kidney diseases. KidneyNetwork is a novel method constructed by integrating a kidney RNA-sequencing co-expression network of 878 samples with a multi-tissue network of 31,499 samples. It uses expression patterns and established gene-phenotype associations to predict which genes could be related to what (disease) phenotypes in an unbiased manner. We applied KidneyNetwork to rare variants in exome sequencing data from 13 kidney disease patients without a genetic diagnosis to prioritize candidate genes. KidneyNetwork can accurately predict kidney-specific gene functions and (kidney disease) phenotypes for disease-associated genes. The intersection of prioritized genes with genes carrying rare variants in a patient with kidney and liver cysts identified ALG6 as plausible candidate gene. We strengthen this plausibility by identifying ALG6 variants in several cystic kidney and liver disease cases without alternative genetic explanation. We present KidneyNetwork, a publicly available kidney-specific co-expression network with optimized gene-phenotype predictions for kidney disease phenotypes. We designed an easy-to-use online interface that allows clinicians and researchers to use gene expression and co-regulation data and gene-phenotype connections to accelerate advances in hereditary kidney disease diagnosis and research. TRANSLATIONAL STATEMENT: Genetic testing in patients with suspected hereditary kidney disease may not reveal the genetic cause for the patient's disorder. Potentially pathogenic variants can reside in genes not yet known to be involved in kidney disease, making it difficult to interpret the relevance of these variants. This reveals a clear need for methods to predict the phenotypic consequences of genetic variation in an unbiased manner. Here we describe KidneyNetwork, a tool that utilizes tissue-specific expression to predict kidney-specific gene functions. Applying KidneyNetwork to a group of undiagnosed cases identified ALG6 as a candidate gene in cystic kidney and liver disease. In summary, KidneyNetwork can aid the interpretation of genetic variants and can therefore be of value in translational nephrogenetics and help improve the diagnostic yield in kidney disease patients.

Brain expression quantitative trait locus and network analyses reveal downstream effects and putative drivers for brain-related diseases.

Identification of therapeutic targets from genome-wide association studies (GWAS) requires insights into downstream functional consequences. We harmonized 8,613 RNA-sequencing samples from 14 brain datasets to create the MetaBrain resource and performed cis- and trans-expression quantitative trait locus (eQTL) meta-analyses in multiple brain region- and ancestry-specific datasets (n ≤ 2,759). Many of the 16,169 cortex cis-eQTLs were tissue-dependent when compared with blood cis-eQTLs. We inferred brain cell types for 3,549 cis-eQTLs by interaction analysis. We prioritized 186 cis-eQTLs for 31 brain-related traits using Mendelian randomization and co-localization including 40 cis-eQTLs with an inferred cell type, such as a neuron-specific cis-eQTL (CYP24A1) for multiple sclerosis. We further describe 737 trans-eQTLs for 526 unique variants and 108 unique genes. We used brain-specific gene-co-regulation networks to link GWAS loci and prioritize additional genes for five central nervous system diseases. This study represents a valuable resource for post-GWAS research on central nervous system diseases.

Feasibility of predicting allele specific expression from DNA sequencing using machine learning.

Allele specific expression (ASE) concerns divergent expression quantity of alternative alleles and is measured by RNA sequencing. Multiple studies show that ASE plays a role in hereditary diseases by modulating penetrance or phenotype severity. However, genome diagnostics is based on DNA sequencing and therefore neglects gene expression regulation such as ASE. To take advantage of ASE in absence of RNA sequencing, it must be predicted using only DNA variation. We have constructed ASE models from BIOS (n = 3432) and GTEx (n = 369) that predict ASE using DNA features. These models are highly reproducible and comprise many different feature types, highlighting the complex regulation that underlies ASE. We applied the BIOS-trained model to population variants in three genes in which ASE plays a clinically relevant role: BRCA2, RET and NF1. This resulted in predicted ASE effects for 27 variants, of which 10 were known pathogenic variants. We demonstrated that ASE can be predicted from DNA features using machine learning. Future efforts may improve sensitivity and translate these models into a new type of genome diagnostic tool that prioritizes candidate pathogenic variants or regulators thereof for follow-up validation by RNA sequencing. All used code and machine learning models are available at GitHub and Zenodo.

Potential impact of celiac disease genetic risk factors on T cell receptor signaling in gluten-specific CD4+ T cells.

Celiac disease is an auto-immune disease in which an immune response to dietary gluten leads to inflammation and subsequent atrophy of small intestinal villi, causing severe bowel discomfort and malabsorption of nutrients. The major instigating factor for the immune response in celiac disease is the activation of gluten-specific CD4+ T cells expressing T cell receptors that recognize gluten peptides presented in the context of HLA-DQ2 and DQ8. Here we provide an in-depth characterization of 28 gluten-specific T cell clones. We assess their transcriptional and epigenetic response to T cell receptor stimulation and link this to genetic factors associated with celiac disease. Gluten-specific T cells have a distinct transcriptional profile that mostly resembles that of Th1 cells but also express cytokines characteristic of other types of T-helper cells. This transcriptional response appears not to be regulated by changes in chromatin state, but rather by early upregulation of transcription factors and non-coding RNAs that likely orchestrate the subsequent activation of genes that play a role in immune pathways. Finally, integration of chromatin and transcription factor binding profiles suggest that genes activated by T cell receptor stimulation of gluten­specific T cells may be impacted by genetic variation at several genetic loci associated with celiac disease.

Epigenome-wide association meta-analysis of DNA methylation with coffee and tea consumption.

Coffee and tea are extensively consumed beverages worldwide which have received considerable attention regarding health. Intake of these beverages is consistently linked to, among others, reduced risk of diabetes and liver diseases; however, the mechanisms of action remain elusive. Epigenetics is suggested as a mechanism mediating the effects of dietary and lifestyle factors on disease onset. Here we report the results from epigenome-wide association studies (EWAS) on coffee and tea consumption in 15,789 participants of European and African-American ancestries from 15 cohorts. EWAS meta-analysis of coffee consumption reveals 11 CpGs surpassing the epigenome-wide significance threshold (P-value <1.1×10-7), which annotated to the AHRR, F2RL3, FLJ43663, HDAC4, GFI1 and PHGDH genes. Among them, cg14476101 is significantly associated with expression of the PHGDH and risk of fatty liver disease. Knockdown of PHGDH expression in liver cells shows a correlation with expression levels of genes associated with circulating lipids, suggesting a role of PHGDH in hepatic-lipid metabolism. EWAS meta-analysis on tea consumption reveals no significant association, only two CpGs annotated to CACNA1A and PRDM16 genes show suggestive association (P-value <5.0×10-6). These findings indicate that coffee-associated changes in DNA methylation levels may explain the mechanism of action of coffee consumption in conferring risk of diseases.

Common and rare variant association analyses in amyotrophic lateral sclerosis identify 15 risk loci with distinct genetic architectures and neuron-specific biology.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with a lifetime risk of one in 350 people and an unmet need for disease-modifying therapies. We conducted a cross-ancestry genome-wide association study (GWAS) including 29,612 patients with ALS and 122,656 controls, which identified 15 risk loci. When combined with 8,953 individuals with whole-genome sequencing (6,538 patients, 2,415 controls) and a large cortex-derived expression quantitative trait locus (eQTL) dataset (MetaBrain), analyses revealed locus-specific genetic architectures in which we prioritized genes either through rare variants, short tandem repeats or regulatory effects. ALS-associated risk loci were shared with multiple traits within the neurodegenerative spectrum but with distinct enrichment patterns across brain regions and cell types. Of the environmental and lifestyle risk factors obtained from the literature, Mendelian randomization analyses indicated a causal role for high cholesterol levels. The combination of all ALS-associated signals reveals a role for perturbations in vesicle-mediated transport and autophagy and provides evidence for cell-autonomous disease initiation in glutamatergic neurons.

Meta-analyses identify DNA methylation associated with kidney function and damage.

Chronic kidney disease is a major public health burden. Elevated urinary albumin-to-creatinine ratio is a measure of kidney damage, and used to diagnose and stage chronic kidney disease. To extend the knowledge on regulatory mechanisms related to kidney function and disease, we conducted a blood-based epigenome-wide association study for estimated glomerular filtration rate (n = 33,605) and urinary albumin-to-creatinine ratio (n = 15,068) and detected 69 and seven CpG sites where DNA methylation was associated with the respective trait. The majority of these findings showed directionally consistent associations with the respective clinical outcomes chronic kidney disease and moderately increased albuminuria. Associations of DNA methylation with kidney function, such as CpGs at JAZF1, PELI1 and CHD2 were validated in kidney tissue. Methylation at PHRF1, LDB2, CSRNP1 and IRF5 indicated causal effects on kidney function. Enrichment analyses revealed pathways related to hemostasis and blood cell migration for estimated glomerular filtration rate, and immune cell activation and response for urinary albumin-to-creatinineratio-associated CpGs.

Fisetin protects against cardiac cell death through reduction of ROS production and caspases activity.

Myocardial infarction (MI) is a leading cause of death worldwide. Reperfusion is considered as an optimal therapy following cardiac ischemia. However, the promotion of a rapid elevation of O2 levels in ischemic cells produces high amounts of reactive oxygen species (ROS) leading to myocardial tissue injury. This phenomenon is called ischemia reperfusion injury (IRI). We aimed at identifying new and effective compounds to treat MI and minimize IRI. We previously studied heart regeneration following myocardial injury in zebrafish and described each step of the regeneration process, from the day of injury until complete recovery, in terms of transcriptional responses. Here, we mined the data and performed a deep in silico analysis to identify drugs highly likely to induce cardiac regeneration. Fisetin was identified as the top candidate. We validated its effects in an in vitro model of MI/IRI in mammalian cardiac cells. Fisetin enhances viability of rat cardiomyocytes following hypoxia/starvation - reoxygenation. It inhibits apoptosis, decreases ROS generation and caspase activation and protects from DNA damage. Interestingly, fisetin also activates genes involved in cell proliferation. Fisetin is thus a highly promising candidate drug with clinical potential to protect from ischemic damage following MI and to overcome IRI.

Association of maternal prenatal smoking GFI1-locus and cardio-metabolic phenotypes in 18,212 adults.

BACKGROUND: DNA methylation at the GFI1-locus has been repeatedly associated with exposure to smoking from the foetal period onwards. We explored whether DNA methylation may be a mechanism that links exposure to maternal prenatal smoking with offspring's adult cardio-metabolic health. METHODS: We meta-analysed the association between DNA methylation at GFI1-locus with maternal prenatal smoking, adult own smoking, and cardio-metabolic phenotypes in 22 population-based studies from Europe, Australia, and USA (n = 18,212). DNA methylation at the GFI1-locus was measured in whole-blood. Multivariable regression models were fitted to examine its association with exposure to prenatal and own adult smoking. DNA methylation levels were analysed in relation to body mass index (BMI), waist circumference (WC), fasting glucose (FG), high-density lipoprotein cholesterol (HDL-C), triglycerides (TG), diastolic, and systolic blood pressure (BP). FINDINGS: Lower DNA methylation at three out of eight GFI1-CpGs was associated with exposure to maternal prenatal smoking, whereas, all eight CpGs were associated with adult own smoking. Lower DNA methylation at cg14179389, the strongest maternal prenatal smoking locus, was associated with increased WC and BP when adjusted for sex, age, and adult smoking with Bonferroni-corrected P < 0·012. In contrast, lower DNA methylation at cg09935388, the strongest adult own smoking locus, was associated with decreased BMI, WC, and BP (adjusted 1 × 10-7 < P < 0.01). Similarly, lower DNA methylation at cg12876356, cg18316974, cg09662411, and cg18146737 was associated with decreased BMI and WC (5 × 10-8 < P < 0.001). Lower DNA methylation at all the CpGs was consistently associated with higher TG levels. INTERPRETATION: Epigenetic changes at the GFI1 were linked to smoking exposure in-utero/in-adulthood and robustly associated with cardio-metabolic risk factors. FUND: European Union's Horizon 2020 research and innovation programme under grant agreement no. 633595 DynaHEALTH.

Identification of context-dependent expression quantitative trait loci in whole blood.

Genetic risk factors often localize to noncoding regions of the genome with unknown effects on disease etiology. Expression quantitative trait loci (eQTLs) help to explain the regulatory mechanisms underlying these genetic associations. Knowledge of the context that determines the nature and strength of eQTLs may help identify cell types relevant to pathophysiology and the regulatory networks underlying disease. Here we generated peripheral blood RNA-seq data from 2,116 unrelated individuals and systematically identified context-dependent eQTLs using a hypothesis-free strategy that does not require previous knowledge of the identity of the modifiers. Of the 23,060 significant cis-regulated genes (false discovery rate (FDR) ≤ 0.05), 2,743 (12%) showed context-dependent eQTL effects. The majority of these effects were influenced by cell type composition. A set of 145 cis-eQTLs depended on type I interferon signaling. Others were modulated by specific transcription factors binding to the eQTL SNPs.

A Framework for Discovering, Designing, and Testing MicroProteins to Regulate Synthetic Transcriptional Modules.

Transcription factors often form protein complexes and give rise to intricate transcriptional networks. The regulation of transcription factor multimerization plays a key role in the fine-tuning of the underlying transcriptional pathways and can be exploited to modulate synthetic transcriptional modules. A novel regulation of protein complex formation is emerging: microProteins-truncated transcription factors-engage in protein-protein interactions with transcriptional complexes and modulate their transcriptional activity. Here, we outline a strategy for the discovery, design, and test of putative miPs to fine-tune the activity of transcription factors regulating synthetic or natural transcriptional circuits.

TMEM258 Is a Component of the Oligosaccharyltransferase Complex Controlling ER Stress and Intestinal Inflammation.

Significant insights into disease pathogenesis have been gleaned from population-level genetic studies; however, many loci associated with complex genetic disease contain numerous genes, and phenotypic associations cannot be assigned unequivocally. In particular, a gene-dense locus on chromosome 11 (61.5-61.65 Mb) has been associated with inflammatory bowel disease, rheumatoid arthritis, and coronary artery disease. Here, we identify TMEM258 within this locus as a central regulator of intestinal inflammation. Strikingly, Tmem258 haploinsufficient mice exhibit severe intestinal inflammation in a model of colitis. At the mechanistic level, we demonstrate that TMEM258 is a required component of the oligosaccharyltransferase complex and is essential for N-linked protein glycosylation. Consequently, homozygous deficiency of Tmem258 in colonic organoids results in unresolved endoplasmic reticulum (ER) stress culminating in apoptosis. Collectively, our results demonstrate that TMEM258 is a central mediator of ER quality control and intestinal homeostasis.

Analysis of the dynamic co-expression network of heart regeneration in the zebrafish.

The zebrafish has the capacity to regenerate its heart after severe injury. While the function of a few genes during this process has been studied, we are far from fully understanding how genes interact to coordinate heart regeneration. To enable systematic insights into this phenomenon, we generated and integrated a dynamic co-expression network of heart regeneration in the zebrafish and linked systems-level properties to the underlying molecular events. Across multiple post-injury time points, the network displays topological attributes of biological relevance. We show that regeneration steps are mediated by modules of transcriptionally coordinated genes, and by genes acting as network hubs. We also established direct associations between hubs and validated drivers of heart regeneration with murine and human orthologs. The resulting models and interactive analysis tools are available at http://infused.vital-it.ch. Using a worked example, we demonstrate the usefulness of this unique open resource for hypothesis generation and in silico screening for genes involved in heart regeneration.

microProtein Prediction Program (miP3): A Software for Predicting microProteins and Their Target Transcription Factors.

An emerging concept in transcriptional regulation is that a class of truncated transcription factors (TFs), called microProteins (miPs), engages in protein-protein interactions with TF complexes and provides feedback controls. A handful of miP examples have been described in the literature but the extent of their prevalence is unclear. Here we present an algorithm that predicts miPs and their target TFs from a sequenced genome. The algorithm is called miP prediction program (miP3), which is implemented in Python. The software will help shed light on the prevalence, biological roles, and evolution of miPs. Moreover, miP3 can be used to predict other types of miP-like proteins that may have evolved from other functional classes such as kinases and receptors. The program is freely available and can be applied to any sequenced genome.

A gene mapping bottleneck in the translational route from zebrafish to human.

Among a diversity of animal models of disease, the zebrafish is a promising model organism for enabling novel translational biomedical research. To fully achieve the latter, a key requirement is to match molecular readouts measured in zebrafish with information relevant to health and disease in humans. A fundamental step in this direction is to accurately map gene sequences from zebrafish to humans. Despite significant progress in genome annotation, this remains an intricate and time-consuming challenge. Here we discuss major obstacles that we had to overcome to systematically map genes from zebrafish to human. We identified important disparities, as well as partial agreements, between five public zebrafish-to-human homology resources. There is still a need for standardized, comprehensive genomic mappings between zebrafish and humans. Without this, efforts to use zebrafish as a powerful translational research tool will be stalled.