Macrothrombocytopenia (MTP) is a heterogeneous group of disorders characterized by enlarged and reduced numbers of circulating platelets, sometimes resulting in abnormal bleeding. In most MTP, this ...phenotype arises because of altered regulation of platelet formation from megakaryocytes (MKs). We report the identification of DIAPH1, which encodes the Rho-effector diaphanous-related formin 1 (DIAPH1), as a candidate gene for MTP using exome sequencing, ontological phenotyping, and similarity regression. We describe 2 unrelated pedigrees with MTP and sensorineural hearing loss that segregate with a DIAPH1 R1213* variant predicting partial truncation of the DIAPH1 diaphanous autoregulatory domain. The R1213* variant was linked to reduced proplatelet formation from cultured MKs, cell clustering, and abnormal cortical filamentous actin. Similarly, in platelets, there was increased filamentous actin and stable microtubules, indicating constitutive activation of DIAPH1. Overexpression of DIAPH1 R1213* in cells reproduced the cytoskeletal alterations found in platelets. Our description of a novel disorder of platelet formation and hearing loss extends the repertoire of DIAPH1-related disease and provides new insight into the autoregulation of DIAPH1 activity.
•A gain-of-function variant in DIAPH1 causes macrothrombocytopenia and hearing loss and extends the spectrum of DIAPH1-related disease.•Our findings of altered megakaryopoiesis and platelet cytoskeletal regulation highlight a critical role for DIAPH1 in platelet formation.
Linking non-coding genetic variants associated with the risk of diseases or disease-relevant traits to target genes is a crucial step to realize GWAS potential in the introduction of precision ...medicine. Here we set out to determine the mechanisms underpinning variant association with platelet quantitative traits using cell type-matched epigenomic data and promoter long-range interactions. We identify potential regulatory functions for 423 of 565 (75%) non-coding variants associated with platelet traits and we demonstrate, through ex vivo and proof of principle genome editing validation, that variants in super enhancers play an important role in controlling archetypical platelet functions.
Primary membranoproliferative GN, including complement 3 (C3) glomerulopathy, is a rare, untreatable kidney disease characterized by glomerular complement deposition. Complement gene mutations can ...cause familial C3 glomerulopathy, and studies have reported rare variants in complement genes in nonfamilial primary membranoproliferative GN.
We analyzed whole-genome sequence data from 165 primary membranoproliferative GN cases and 10,250 individuals without the condition (controls) as part of the National Institutes of Health Research BioResource-Rare Diseases Study. We examined copy number, rare, and common variants.
Our analysis included 146 primary membranoproliferative GN cases and 6442 controls who were unrelated and of European ancestry. We observed no significant enrichment of rare variants in candidate genes (genes encoding components of the complement alternative pathway and other genes associated with the related disease atypical hemolytic uremic syndrome; 6.8% in cases versus 5.9% in controls) or exome-wide. However, a significant common variant locus was identified at 6p21.32 (rs35406322) (
=3.29×10
; odds ratio OR, 1.93; 95% confidence interval 95% CI, 1.53 to 2.44), overlapping the HLA locus. Imputation of HLA types mapped this signal to a haplotype incorporating DQA1*05:01, DQB1*02:01, and DRB1*03:01 (
=1.21×10
; OR, 2.19; 95% CI, 1.66 to 2.89). This finding was replicated by analysis of HLA serotypes in 338 individuals with membranoproliferative GN and 15,614 individuals with nonimmune renal failure.
We found that HLA type, but not rare complement gene variation, is associated with primary membranoproliferative GN. These findings challenge the paradigm of complement gene mutations typically causing primary membranoproliferative GN and implicate an underlying autoimmune mechanism in most cases.
Bulk and single-cell DNA sequencing has enabled reconstructing clonal substructures of somatic tissues from frequency and cooccurrence patterns of somatic variants. However, approaches to ...characterize phenotypic variations between clones are not established. Here we present cardelino (https://github.com/single-cell-genetics/cardelino), a computational method for inferring the clonal tree configuration and the clone of origin of individual cells assayed using single-cell RNA-seq (scRNA-seq). Cardelino flexibly integrates information from imperfect clonal trees inferred based on bulk exome-seq data, and sparse variant alleles expressed in scRNA-seq data. We apply cardelino to a published cancer dataset and to newly generated matched scRNA-seq and exome-seq data from 32 human dermal fibroblast lines, identifying hundreds of differentially expressed genes between cells from different somatic clones. These genes are frequently enriched for cell cycle and proliferation pathways, indicating a role for cell division genes in somatic evolution in healthy skin.
The interindividual variation in the functional response of platelets to activation by agonists is heritable. Genome-wide association studies (GWASs) of quantitative measures of platelet function ...have identified fewer than 20 distinctly associated variants, some with unknown mechanisms. Here, we report GWASs of pathway-specific functional responses to agonism by adenosine 5′-diphosphate, a glycoprotein VI–specific collagen mimetic, and thrombin receptor-agonist peptides, each specific to 1 of the G protein–coupled receptors PAR-1 and PAR-4, in subsets of 1562 individuals. We identified an association (P = 2.75 × 10−40) between a common intronic variant, rs10886430, in the G protein–coupled receptor kinase 5 gene (GRK5) and the sensitivity of platelets to activate through PAR-1. The variant resides in a megakaryocyte-specific enhancer that is bound by the transcription factors GATA1 and MEIS1. The minor allele (G) is associated with fewer GRK5 transcripts in platelets and the greater sensitivity of platelets to activate through PAR-1. We show that thrombin-mediated activation of human platelets causes binding of GRK5 to PAR-1 and that deletion of the mouse homolog Grk5 enhances thrombin-induced platelet activation sensitivity and increases platelet accumulation at the site of vascular injury. This corroborates evidence that the human G allele of rs10886430 is associated with a greater risk for cardiovascular disease. In summary, by combining the results of pathway-specific GWASs and expression quantitative trait locus studies in humans with the results from platelet function studies in Grk5−/− mice, we obtain evidence that GRK5 regulates the human platelet response to thrombin via the PAR-1 pathway.
•Platelet reactivity via the PAR-1 thrombin receptor is mediated by GRK5.•Platelet GRK5 is associated with thrombus formation in humans and mice.
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Technology utilizing human induced pluripotent stem cells (iPS cells) has enormous potential to provide improved cellular models of human disease. However, variable genetic and phenotypic ...characterization of many existing iPS cell lines limits their potential use for research and therapy. Here we describe the systematic generation, genotyping and phenotyping of 711 iPS cell lines derived from 301 healthy individuals by the Human Induced Pluripotent Stem Cells Initiative. Our study outlines the major sources of genetic and phenotypic variation in iPS cells and establishes their suitability as models of complex human traits and cancer. Through genome-wide profiling we find that 5-46% of the variation in different iPS cell phenotypes, including differentiation capacity and cellular morphology, arises from differences between individuals. Additionally, we assess the phenotypic consequences of genomic copy-number alterations that are repeatedly observed in iPS cells. In addition, we present a comprehensive map of common regulatory variants affecting the transcriptome of human pluripotent cells.
Characterizing the multifaceted contribution of genetic and epigenetic factors to disease phenotypes is a major challenge in human genetics and medicine. We carried out high-resolution genetic, ...epigenetic, and transcriptomic profiling in three major human immune cell types (CD14+ monocytes, CD16+ neutrophils, and naive CD4+ T cells) from up to 197 individuals. We assess, quantitatively, the relative contribution of cis-genetic and epigenetic factors to transcription and evaluate their impact as potential sources of confounding in epigenome-wide association studies. Further, we characterize highly coordinated genetic effects on gene expression, methylation, and histone variation through quantitative trait locus (QTL) mapping and allele-specific (AS) analyses. Finally, we demonstrate colocalization of molecular trait QTLs at 345 unique immune disease loci. This expansive, high-resolution atlas of multi-omics changes yields insights into cell-type-specific correlation between diverse genomic inputs, more generalizable correlations between these inputs, and defines molecular events that may underpin complex disease risk.
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•Genome, transcriptome, and epigenome reference panel in three human immune cell types•Identified 4,418 genes associated with epigenetic changes independent of genetics•Described genome-epigenome coordination defining cell-type-specific regulatory events•Functionally mapped disease mechanisms at 345 unique autoimmune disease loci
As part of the IHEC consortium, this study integrates genetic, epigenetic, and transcriptomic profiling in three immune cell types from nearly 200 people to characterize the distinct and cooperative contributions of diverse genomic inputs to transcriptional variation. Explore the Cell Press IHEC web portal at http://www.cell.com/consortium/IHEC.
Purpose
With growing evidence that rare single gene disorders present in the neonatal period, there is a need for rapid, systematic, and comprehensive genomic diagnoses in ICUs to assist acute and ...long-term clinical decisions. This study aimed to identify genetic conditions in neonatal (NICU) and paediatric (PICU) intensive care populations.
Methods
We performed trio whole genome sequence (WGS) analysis on a prospective cohort of families recruited in NICU and PICU at a single site in the UK. We developed a research pipeline in collaboration with the National Health Service to deliver validated pertinent pathogenic findings within 2–3 weeks of recruitment.
Results
A total of 195 families had whole genome analysis performed (567 samples) and 21% received a molecular diagnosis for the underlying genetic condition in the child. The phenotypic description of the child was a poor predictor of the gene identified in 90% of cases, arguing for gene agnostic testing in NICU/PICU. The diagnosis affected clinical management in more than 65% of cases (83% in neonates) including modification of treatments and care pathways and/or informing palliative care decisions. A 2–3 week turnaround was sufficient to impact most clinical decision-making.
Conclusions
The use of WGS in intensively ill children is acceptable and trio analysis facilitates diagnoses. A gene agnostic approach was effective in identifying an underlying genetic condition, with phenotypes and symptomatology being primarily used for data interpretation rather than gene selection. WGS analysis has the potential to be a first-line diagnostic tool for a subset of intensively ill children.
Approximately 2.4% of the human mitochondrial DNA (mtDNA) genome exhibits common homoplasmic genetic variation. We analyzed 12,975 whole-genome sequences to show that 45.1% of individuals from 1526 ...mother-offspring pairs harbor a mixed population of mtDNA (heteroplasmy), but the propensity for maternal transmission differs across the mitochondrial genome. Over one generation, we observed selection both for and against variants in specific genomic regions; known variants were more likely to be transmitted than previously unknown variants. However, new heteroplasmies were more likely to match the nuclear genetic ancestry as opposed to the ancestry of the mitochondrial genome on which the mutations occurred, validating our findings in 40,325 individuals. Thus, human mtDNA at the population level is shaped by selective forces within the female germ line under nuclear genetic control, which ensures consistency between the two independent genetic lineages.
The genetic cause of primary immunodeficiency disease (PID) carries prognostic information.
We conducted a whole-genome sequencing study assessing a large proportion of the NIHR BioResource–Rare ...Diseases cohort.
In the predominantly European study population of principally sporadic unrelated PID cases (n = 846), a novel Bayesian method identified nuclear factor κB subunit 1 (NFKB1) as one of the genes most strongly associated with PID, and the association was explained by 16 novel heterozygous truncating, missense, and gene deletion variants. This accounted for 4% of common variable immunodeficiency (CVID) cases (n = 390) in the cohort. Amino acid substitutions predicted to be pathogenic were assessed by means of analysis of structural protein data. Immunophenotyping, immunoblotting, and ex vivo stimulation of lymphocytes determined the functional effects of these variants. Detailed clinical and pedigree information was collected for genotype-phenotype cosegregation analyses.
Both sporadic and familial cases demonstrated evidence of the noninfective complications of CVID, including massive lymphadenopathy (24%), unexplained splenomegaly (48%), and autoimmune disease (48%), features prior studies correlated with worse clinical prognosis. Although partial penetrance of clinical symptoms was noted in certain pedigrees, all carriers have a deficiency in B-lymphocyte differentiation. Detailed assessment of B-lymphocyte numbers, phenotype, and function identifies the presence of an increased CD21low B-cell population. Combined with identification of the disease-causing variant, this distinguishes between healthy subjects, asymptomatic carriers, and clinically affected cases.
We show that heterozygous loss-of-function variants in NFKB1 are the most common known monogenic cause of CVID, which results in a temporally progressive defect in the formation of immunoglobulin-producing B cells.
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