Heterogeneous effects on type 2 diabetes and cardiovascular outcomes of genetic variants and traits associated with fasting insulin.

Hyperinsulinemia is a complex and heterogeneous phenotype that characterizes molecular alterations that precede the development of type 2 diabetes (T2D). It results from a complex combination of molecular processes, including insulin secretion and insulin sensitivity, that differ between individuals. To better understand the physiology of hyperinsulinemia and ultimately T2D, we implemented a genetic approach grouping fasting insulin (FI)-associated genetic variants based on their molecular and phenotypic similarities. We identified seven distinctive genetic clusters representing different physiologic mechanisms leading to rising FI levels, ranging from clusters of variants with effects on increased FI, but without increased risk of T2D (non-diabetogenic hyperinsulinemia), to clusters of variants that increase FI and T2D risk with demonstrated strong effects on body fat distribution, liver, lipid, and inflammatory processes (diabetogenic hyperinsulinemia). We generated cluster-specific polygenic scores in 1,104,258 individuals from five multi-ancestry cohorts to show that the clusters differed in associations with cardiometabolic traits. Among clusters characterized by non-diabetogenic hyperinsulinemia, there was both increased and decreased risk of coronary artery disease despite the non-increased risk of T2D. Similarly, the clusters characterized by diabetogenic hyperinsulinemia were associated with an increased risk of T2D, yet had differing risks of cardiovascular conditions, including coronary artery disease, myocardial infarction, and stroke. The strongest cluster-T2D associations were observed with the same direction of effect in non-Hispanic Black, Hispanic, non-Hispanic White, and non-Hispanic East Asian populations. These genetic clusters provide important insights into granular metabolic processes underlying the physiology of hyperinsulinemia, notably highlighting specific processes that decouple increasing FI levels from T2D and cardiovascular risk. Our findings suggest that increasing FI levels are not invariably associated with adverse cardiometabolic outcomes.

The Genetic Discrimination Observatory: confronting novel issues in genetic discrimination.

Genetic discrimination (GD) is the differential or unfair profiling of an individual on the basis of genetic data. This article summarizes the actions of the Genetic Discrimination Observatory (GDO) in addressing GD and recent developments in GD since late 2020. It shows how GD can take many forms in today's rapidly evolving society.

Alternative Splicing of Opioid Receptor Genes Shows a Conserved Pattern for 6TM Receptor Variants.

The opioid receptor (OPR) family comprises the mu-, delta-, and kappa-opioid, and nociceptin receptors that belong to the superfamily of 7-transmembrane spanning G protein-coupled receptors (GPCRs). The mu-opioid receptor is the main target for clinically used opioid analgesics, and its biology has been extensively studied. The N-terminally truncated 6TM receptors isoform produced through alternative splicing of the OPRM1 gene displays unique signaling and analgesic properties, but it is unclear if other OPRs have the same ability. In this study, we have built a comprehensive map of alternative splicing events that produce 6TM receptor variants in all the OPRs and demonstrated their evolutionary conservation. We then obtained evidence for their translation through ribosomal footprint analysis. We discovered that N-terminally truncated 6TM GPCRs are rare in the human genome and OPRs are overrepresented in this group. Finally, we also observed a significant enrichment of 6TM GPCR genes among genes associated with pain, psychiatric disorders, and addiction. Understanding the biology of 6TM receptors and leveraging this knowledge for drug development should pave the way for novel therapies.

Alternative Splicing of the Delta-Opioid Receptor Gene Suggests Existence of New Functional Isoforms.

The delta-opioid receptor (DOPr) participates in mediating the effects of opioid analgesics. However, no selective agonists have entered clinical care despite potential to ameliorate many neurological and psychiatric disorders. In an effort to address the drug development challenges, the functional contribution of receptor isoforms created by alternative splicing of the three-exonic coding gene, OPRD1, has been overlooked. We report that the gene is transcriptionally more diverse than previously demonstrated, producing novel protein isoforms in humans and mice. We provide support for the functional relevance of splice variants through context-dependent expression profiling (tissues, disease model) and conservation of the transcriptional landscape in closely related vertebrates. The conserved alternative transcriptional events have two distinct patterns. First, cassette exon inclusions between exons 1 and 2 interrupt the reading frame, producing truncated receptor fragments comprising only the first transmembrane (TM) domain, despite the lack of exact exon orthologues between distant species. Second, a novel promoter and transcriptional start site upstream of exon 2 produces a transcript of an N-terminally truncated 6TM isoform. However, a fundamental difference in the exonic landscaping as well as translation and translation products poses limits for modelling the human DOPr receptor system in mice.

BigDataScript: a scripting language for data pipelines.

MOTIVATION: The analysis of large biological datasets often requires complex processing pipelines that run for a long time on large computational infrastructures. We designed and implemented a simple script-like programming language with a clean and minimalist syntax to develop and manage pipeline execution and provide robustness to various types of software and hardware failures as well as portability. RESULTS: We introduce the BigDataScript (BDS) programming language for data processing pipelines, which improves abstraction from hardware resources and assists with robustness. Hardware abstraction allows BDS pipelines to run without modification on a wide range of computer architectures, from a small laptop to multi-core servers, server farms, clusters and clouds. BDS achieves robustness by incorporating the concepts of absolute serialization and lazy processing, thus allowing pipelines to recover from errors. By abstracting pipeline concepts at programming language level, BDS simplifies implementation, execution and management of complex bioinformatics pipelines, resulting in reduced development and debugging cycles as well as cleaner code. AVAILABILITY AND IMPLEMENTATION: BigDataScript is available under open-source license at http://pcingola.github.io/BigDataScript.

Convex lens-induced nanoscale templating.

We demonstrate a new platform, convex lens-induced nanoscale templating (CLINT), for dynamic manipulation and trapping of single DNA molecules. In the CLINT technique, the curved surface of a convex lens is used to deform a flexible coverslip above a substrate containing embedded nanotopography, creating a nanoscale gap that can be adjusted during an experiment to confine molecules within the embedded nanostructures. Critically, CLINT has the capability of transforming a macroscale flow cell into a nanofluidic device without the need for permanent direct bonding, thus simplifying sample loading, providing greater accessibility of the surface for functionalization, and enabling dynamic manipulation of confinement during device operation. Moreover, as DNA molecules present in the gap are driven into the embedded topography from above, CLINT eliminates the need for the high pressures or electric fields required to load DNA into direct-bonded nanofluidic devices. To demonstrate the versatility of CLINT, we confine DNA to nanogroove and nanopit structures, demonstrating DNA nanochannel-based stretching, denaturation mapping, and partitioning/trapping of single molecules in multiple embedded cavities. In particular, using ionic strengths that are in line with typical biological buffers, we have successfully extended DNA in sub-30-nm nanochannels, achieving high stretching (90%) that is in good agreement with Odijk deflection theory, and we have mapped genomic features using denaturation analysis.

Rare genomic structural variants in complex disease: lessons from the replication of associations with obesity.

The limited ability of common variants to account for the genetic contribution to complex disease has prompted searches for rare variants of large effect, to partly explain the 'missing heritability'. Analyses of genome-wide genotyping data have identified genomic structural variants (GSVs) as a source of such rare causal variants. Recent studies have reported multiple GSV loci associated with risk of obesity. We attempted to replicate these associations by similar analysis of two familial-obesity case-control cohorts and a population cohort, and detected GSVs at 11 out of 18 loci, at frequencies similar to those previously reported. Based on their reported frequencies and effect sizes (OR≥25), we had sufficient statistical power to detect the large majority (80%) of genuine associations at these loci. However, only one obesity association was replicated. Deletion of a 220 kb region on chromosome 16p11.2 has a carrier population frequency of 2×10(-4) (95% confidence interval [9.6×10(-5)-3.1×10(-4)]); accounts overall for 0.5% [0.19%-0.82%] of severe childhood obesity cases (P = 3.8×10(-10); odds ratio = 25.0 [9.9-60.6]); and results in a mean body mass index (BMI) increase of 5.8 kg.m(-2) [1.8-10.3] in adults from the general population. We also attempted replication using BMI as a quantitative trait in our population cohort; associations with BMI at or near nominal significance were detected at two further loci near KIF2B and within FOXP2, but these did not survive correction for multiple testing. These findings emphasise several issues of importance when conducting rare GSV association, including the need for careful cohort selection and replication strategy, accurate GSV identification, and appropriate correction for multiple testing and/or control of false discovery rate. Moreover, they highlight the potential difficulty in replicating rare CNV associations across different populations. Nevertheless, we show that such studies are potentially valuable for the identification of variants making an appreciable contribution to complex disease.

Novel association approach for variable number tandem repeats (VNTRs) identifies DOCK5 as a susceptibility gene for severe obesity.

Variable number tandem repeats (VNTRs) constitute a relatively under-examined class of genomic variants in the context of complex disease because of their sequence complexity and the challenges in assaying them. Recent large-scale genome-wide copy number variant mapping and association efforts have highlighted the need for improved methodology for association studies using these complex polymorphisms. Here we describe the in-depth investigation of a complex region on chromosome 8p21.2 encompassing the dedicator of cytokinesis 5 (DOCK5) gene. The region includes two VNTRs of complex sequence composition which flank a common 3975 bp deletion, all three of which were genotyped by polymerase chain reaction and fragment analysis in a total of 2744 subjects. We have developed a novel VNTR association method named VNTRtest, suitable for association analysis of multi-allelic loci with binary and quantitative outcomes, and have used this approach to show significant association of the DOCK5 VNTRs with childhood and adult severe obesity (P(empirical)= 8.9 × 10(-8) and P= 3.1 × 10(-3), respectively) which we estimate explains ~0.8% of the phenotypic variance. We also identified an independent association between the 3975 base pair (bp) deletion and obesity, explaining a further 0.46% of the variance (P(combined)= 1.6 × 10(-3)). Evidence for association between DOCK5 transcript levels and the 3975 bp deletion (P= 0.027) and both VNTRs (P(empirical)= 0.015) was also identified in adipose tissue from a Swedish family sample, providing support for a functional effect of the DOCK5 deletion and VNTRs. These findings highlight the potential role of DOCK5 in human obesity and illustrate a novel approach for analysis of the contribution of VNTRs to disease susceptibility through association studies.

Analysis of beta-cell gene expression reveals inflammatory signaling and evidence of dedifferentiation following human islet isolation and culture.

The stresses encountered during islet isolation and culture may have deleterious effects on beta-cell physiology. However, the biological response of human islet cells to isolation remains poorly characterized. A better understanding of the network of signaling pathways induced by islet isolation and culturing may lead to strategies aimed at improving islet graft survival and function. Laser capture microdissection (LCM) was used to extract beta-cell RNA from 1) intact pancreatic islets, 2) freshly isolated islets, 3) islets cultured for 3 days, and changes in gene expression were examined by microarray analysis. We identified a strong inflammatory response induced by islet isolation that continues during in-vitro culture manifested by upregulation of several cytokines and cytokine-receptors. The most highly upregulated gene, interleukin-8 (IL-8), was induced by 3.6-fold following islet isolation and 56-fold after 3 days in culture. Immunofluorescence studies showed that the majority of IL-8 was produced by beta-cells themselves. We also observed that several pancreas-specific transcription factors were down-regulated in cultured islets. Concordantly, several pancreatic progenitor cell-specific transcription factors like SOX4, SOX9, and ID2 were upregulated in cultured islets, suggesting progressive transformation of mature beta-cell phenotype toward an immature endocrine cell phenotype. Our findings suggest islet isolation and culture induces an inflammatory response and loss of the mature endocrine cell phenotype. A better understanding of the signals required to maintain a mature beta-cell phenotype may help improve the efficacy of islet transplantation.

Analysis of early C2C12 myogenesis identifies stably and differentially expressed transcriptional regulators whose knock-down inhibits myoblast differentiation.

Myogenesis is a tightly controlled process involving the transcriptional activation and repression of thousands of genes. Although many components of the transcriptional network regulating the later phases of myogenesis have been identified, relatively few studies have described the transcriptional landscape during the first 24 h, when myoblasts commit to differentiate. Through dense temporal profiling of differentiating C2C12 myoblasts, we identify 193 transcriptional regulators (TRs) whose expression is significantly altered within the first 24 h of myogenesis. A high-content shRNA screen of 77 TRs involving 427 stable lines identified 42 genes whose knockdown significantly inhibits differentiation of C2C12 myoblasts. Of the TRs that were differentially expressed within the first 24 h, over half inhibited differentiation when knocked down, including known regulators of myogenesis (Myod1, Myog, and Myf5), as well as 19 TRs not previously associated with this process. Surprisingly, a similar proportion (55%) of shRNAs targeting TRs whose expression did not change also inhibited C2C12 myogenesis. We further show that a subset of these TRs inhibits myogenesis by downregulating expression of known regulatory and structural proteins. Our findings clearly illustrate that several TRs critical for C2C12 myogenesis are not differentially regulated, suggesting that approaches that focus functional studies on differentially-expressed transcripts will fail to provide a comprehensive view of this complex process.

The living microarray: a high-throughput platform for measuring transcription dynamics in single cells.

BACKGROUND: Current methods of measuring transcription in high-throughput have led to significant improvements in our knowledge of transcriptional regulation and Systems Biology. However, endpoint measurements obtained from methods that pool populations of cells are not amenable to studying time-dependent processes that show cell heterogeneity. RESULTS: Here we describe a high-throughput platform for measuring transcriptional changes in real time in single mammalian cells. By using reverse transfection microarrays we are able to transfect fluorescent reporter plasmids into 600 independent clusters of cells plated on a single microscope slide and image these clusters every 20 minutes. We use a fast-maturing, destabilized and nuclear-localized reporter that is suitable for automated segmentation to accurately measure promoter activity in single cells. We tested this platform with synthetic drug-inducible promoters that showed robust induction over 24 hours. Automated segmentation and tracking of over 11 million cell images during this period revealed that cells display substantial heterogeneity in their responses to the applied treatment, including a large proportion of transfected cells that do not respond at all. CONCLUSIONS: The results from our single-cell analysis suggest that methods that measure average cellular responses, such as DNA microarrays, RT-PCR and chromatin immunoprecipitation, characterize a response skewed by a subset of cells in the population. Our method is scalable and readily adaptable to studying complex systems, including cell proliferation, differentiation and apoptosis.

cnvHap: an integrative population and haplotype-based multiplatform model of SNPs and CNVs.

Although genome-wide association studies have uncovered single-nucleotide polymorphisms (SNPs) associated with complex disease, these variants account for a small portion of heritability. Some contribution to this 'missing heritability' may come from copy-number variants (CNVs), in particular rare CNVs; but assessment of this contribution remains challenging because of the difficulty in accurately genotyping CNVs, particularly small variants. We report a population-based approach for the identification of CNVs that integrates data from multiple samples and platforms. Our algorithm, cnvHap, jointly learns a chromosome-wide haplotype model of CNVs and cluster-based models of allele intensity at each probe. Using data for 50 French individuals assayed on four separate platforms, we found that cnvHap correctly detected at least 14% more deleted and 50% more amplified genotypes than PennCNV or QuantiSNP, with an 82% and 115% improvement for aberrations containing <10 probes. Combining data from multiple platforms additionally improved sensitivity.

Identification of novel chromosomal regions associated with airway hyperresponsiveness in recombinant congenic strains of mice.

Airway responsiveness is the ability of the airways to respond to bronchoconstricting stimuli by reducing their diameter. Airway hyperresponsiveness has been associated with asthma susceptibility in both humans and murine models, and it has been shown to be a complex and heritable trait. In particular, the A/J mouse strain is known to have hyperresponsive airways, while the C57BL/6 strain is known to be relatively refractory to bronchoconstricting stimuli. We analyzed recombinant congenic strains (RCS) of mice generated from these hyper- and hyporesponsive parental strains to identify genetic loci underlying the trait of airway responsiveness in response to methacholine as assessed by whole-body plethysmography. Our screen identified 16 chromosomal regions significantly associated with airway hyperresponsiveness (genome-wide P 73 cM), chromosome 7 (>63 cM), chromosome 8 (52-67 cM), chromosome 10 (3-7 cM and >68 cM), and chromosome 12 (25-38 cM and >52 cM). Our data identify several likely candidate genes from the 16 regions, including Ddr2, Hc, Fbn1, Flt3, Utrn, Enpp2, and Tsc.

Genomewide association study of a rapid progression cohort identifies new susceptibility alleles for AIDS (ANRS Genomewide Association Study 03).

BACKGROUND: Previous genomewide association studies (GWASs) of AIDS have targeted end points based on the control of viral load and disease nonprogression. The discovery of genetic factors that predispose individuals to rapid progression to AIDS should also reveal new insights into the molecular etiology of the pathology. METHODS: We undertook a case-control GWAS of a unique cohort of 85 human immunodeficiency virus type 1 (HIV-1)-infected patients who experienced rapid disease progression, using Illumina HumanHap300 BeadChips. The case group was compared with a control group of 1352 individuals for the 291,119 autosomal single-nucleotide polymorphisms (SNPs) passing the quality control tests, using the false-discovery rate (FDR) statistical method for multitest correction. RESULTS: Novel associations with rapid progression (FDR, < or = 25%) were identified for PRMT6 (P = 6.1 x 10(-7); odds ratio [OR], 0.24), SOX5 (P = 1.8 x 10(-6); OR, 0.45), RXRG (P = 3.9 x 10(-6); OR, 3.29), and TGFBRAP1 (P = 7 x 10(-6); OR, 0.34). The haplotype analysis identified exonic and promoter SNPs potentially important for PRMT6 and TGFBRAP1 function. CONCLUSIONS: The statistical and biological relevance of these associations and their high ORs underscore the power of extreme phenotypes for GWASs, even with a modest sample size. These genetic results emphasize the role of the transforming growth factor beta pathway in the pathogenesis of HIV-1 disease. Finally, the wealth of information provided by this study should help unravel new diagnostic and therapeutic targets.

Modulation of the allergic asthma transcriptome following resiquimod treatment.

Resiquimod is a compound belonging to the imidazoquinoline family of compounds known to signal through Toll-like receptor 7. Resiquimod treatment has been demonstrated to inhibit the development of allergen induced asthma in experimental models. The aim of the present study was to elucidate the molecular processes that were altered following resiquimod treatment and allergen challenge in a mouse model of allergic asthma. Employing microarray analysis, we have characterized the "asthmatic" transcriptome of the lungs of A/J and C57BL/6 mice and determined that it includes genes involved in the control of cell cycle progression, the complement and coagulation cascades, and chemokine signaling. Our results demonstrated that resiquimod treatment resulted in the normalization of the expression of genes involved with airway remodeling, and generally, chemokine signaling. Resiquimod treatment also altered the expression of cell adhesion molecules, and molecules involved in natural killer (NK) cell-mediated cytotoxicity. Furthermore, we have demonstrated that systemic resiquimod administration resulted in the recruitment of NK cells to the lungs and livers of the mice, although no causal relationship between NK cell recruitment and treatment efficacy was found. Overall, our findings identified several genes, important in the development of asthma pathology, that were normalized following resiquimod treatment, thus improving our understanding of the molecular consequences of resiquimod treatment in the lung milieu. The recruitment of NK cells to the lungs may also have application in the treatment of virally induced asthma exacerbations.

TFCat: the curated catalog of mouse and human transcription factors.

Unravelling regulatory programs governed by transcription factors (TFs) is fundamental to understanding biological systems. TFCat is a catalog of mouse and human TFs based on a reliable core collection of annotations obtained by expert review of the scientific literature. The collection, including proven and homology-based candidate TFs, is annotated within a function-based taxonomy and DNA-binding proteins are organized within a classification system. All data and user-feedback mechanisms are available at the TFCat portal (http://www.tfcat.ca).

Genomewide association study of an AIDS-nonprogression cohort emphasizes the role played by HLA genes (ANRS Genomewide Association Study 02).

To elucidate the genetic factors predisposing to AIDS progression, we analyzed a unique cohort of 275 human immunodeficiency virus (HIV) type 1-seropositive nonprogressor patients in relation to a control group of 1352 seronegative individuals in a genomewide association study (GWAS). The strongest association was obtained for HCP5 rs2395029 (P=6.79x10(-10); odds ratio, 3.47) and was possibly linked to an effect of sex. Interestingly, this single-nucleotide polymorphism (SNP) was in high linkage disequilibrium with HLA-B, MICB, TNF, and several other HLA locus SNPs and haplotypes. A meta-analysis of our genomic data combined with data from the previously conducted Euro-CHAVI (Center for HIV/AIDS Vaccine Immunology) GWAS confirmed the HCP5 signal (P=3.02x10(-19)) and identified several new associations, all of them involving HLA genes: MICB, TNF, RDBP, BAT1-5, PSORS1C1, and HLA-C. Finally, stratification by HCP5 rs2395029 genotypes emphasized an independent role for ZNRD1, also in the HLA locus, and this finding was confirmed by experimental data. The present study, the first GWAS of HIV-1 nonprogressors, underscores the potential for some HLA genes to control disease progression soon after infection.

Genome-wide association study for early-onset and morbid adult obesity identifies three new risk loci in European populations.

We analyzed genome-wide association data from 1,380 Europeans with early-onset and morbid adult obesity and 1,416 age-matched normal-weight controls. Thirty-eight markers showing strong association were further evaluated in 14,186 European subjects. In addition to FTO and MC4R, we detected significant association of obesity with three new risk loci in NPC1 (endosomal/lysosomal Niemann-Pick C1 gene, P = 2.9 x 10(-7)), near MAF (encoding the transcription factor c-MAF, P = 3.8 x 10(-13)) and near PTER (phosphotriesterase-related gene, P = 2.1 x 10(-7)).