Human Sequencing Studies Research Articles

Multiomic Analysis and CRISPR Perturbation Screens Identify Endothelial Cell Programs and Novel Therapeutic Targets for Coronary Artery Disease.

Endothelial cells (EC) are an important mediator of atherosclerosis and vascular disease. Their exposure to atherogenic risk factors such as hypertension and serum cholesterol leads to endothelial dysfunction and many disease-associated processes. Identifying which of these multiple EC functions is causally related to disease risk has been challenging. There is evidence from in vivo models and human sequencing studies that dysregulation of nitric oxide production directly affects risk of coronary artery disease. Human genetics can help prioritize the other EC functions with causal relationships because germline mutations are acquired at birth and serve as a randomized test of which pathways affect disease risk. Though several coronary artery disease risk variants have been linked to EC function, this process has been slow and laborious. Unbiased analyses of EC dysfunction using multiomic approaches promise to identify the causal genetic mechanisms responsible for vascular disease. Here, we review the data from genomic, epigenomic, and transcriptomic studies that prioritize EC-specific causal pathways. New methods that CRISPR (clustered regularly interspaced short palindromic repeats) perturbation technology with genomic, epigenomic, and transcriptomic analysis promise to speed up the characterization of disease-associated genetic variation. We summarize several recent studies in ECs which use high-throughput genetic perturbation to identify disease-relevant pathways and novel mechanisms of disease. These genetically validated pathways can accelerate the identification of drug targets for the prevention and treatment of atherosclerosis.

Emerging Role of Acquired Mutations and Clonal Hematopoiesis in Atherosclerosis - Beyond Conventional Cardiovascular Risk Factors.

Accumulating evidence suggests that conventional cardiovascular risk factors are incompletely predictive of cardiovascular disease, as a substantial risk remains even when these factors are apparently managed well. In this context, clonal hematopoiesis has emerged as a new and potent risk factor for atherosclerotic cardiovascular disease and other cardiometabolic conditions. Clonal hematopoiesis typically arises from somatic mutations that confer a competitive advantage to a mutant hematopoietic stem cell, leading to its clonal expansion in the stem cell population and its progeny of blood leukocytes. Human sequencing studies and experiments in mice suggest that clonal hematopoiesis, at least when driven by certain mutations, contributes to accelerated atherosclerosis development. However, the epidemiology, biology and clinical implications of this phenomenon remain incompletely understood. Here, we review the current understanding of the connection between clonal hematopoiesis and atherosclerosis, and highlight knowledge gaps in this area of research.

Pathway analysis identified a significant association between cell-cell adherens junctions-related genes and non-syndromic cleft lip/palate in 895 Asian case-parent trios

ObjectivesTo study the association between the seven cell-cell adherens junctions (AJs) -related genes (CDH1, CTNND1, CTNNA1, ESRP1, ESRP2, PLEKHA5, and PLEKHA7) and non-syndromic cleft lip with or without cleft palate (NSCL/P) in the Asian population. DesignThe study included 895 NSCL/P case-parent trios of Asian ethnicity drawn from an international consortium established for a genome-wide association study. We performed association analysis by applying the genotypic transmission disequilibrium test for each of the 144 single nucleotide polymorphisms (SNPs), the versatile gene-based association study for the combined effects of all SNPs, all in or around the seven target genes, and the versatile pathway-based approach for the combined effects of these seven target genes, consecutively. ResultsIn our analysis, we found neither a significant SNP-based nor gene-based association with NSCL/P for any of the 144 relevant SNPs or the seven target genes. However, novel evidence of a significant association (P = 6.00 × 10−6) with NSCL/P was observed in the combined effects of these seven target genes in the versatile pathway-based analysis. ConclusionsOur results were consistent with the findings of previously published human sequencing studies and reinforced the role of these cell-cell AJs-related genes in the pathogenesis of NSCL/P that may be replicated using data from other genome-wide association studies. This pathway approach better reflects our current understanding of the biological mechanisms underlying the aetiology of NSCL/P.

Tet1-mediated modulation of Alzheimer's disease.

Human sequencing studies have identified roughly 30 risk loci associated Alzheimer's disease (AD). However, these loci only explain a portion of disease pathogenicity. Recent studies support a link between gene-environment interactions and AD, and published work from our lab indicates Tet1 mediates gene-environment interactions and Tet1 KO reduces stress response in mice [1]. Furthermore, DNA deep sequencing studies identified an enrichment of rare Tet2 variants associated with AD [2]. This study examines how knockout of the Tet1, a DNA 5-hydroxymethylcytosine (5hmC) regulator, influences AD-associated transcription and pathological outcomes in the 5xFAD AD mouse model. We measured cognitive/behavioral, 5hmC, and expression profiles of a Tet1 heterozygous knockout in the 5xFAD mouse model to examine the influences of Tet1 KO on AD pathogenicity. We show significant differences in behavior, cognition, 5hmC methylation and expression profiles of FAD/Tet1+/- mice relative to WT and FAD. In relation to FAD, FAD/Tet1+/- improved stress response in the Tail-Suspension Assay (p = 0.0438), and relative to WT mice, FAD/Tet1+/- mice display a reduction in their latency to mount the platform in the Morris water maze (p = 0.01). Analysis of 5hmC capture data revealed 2536 differentially hydroxymethylated regions (DhMRs) upregulated in FADTet1 and 410 DhMRs downregulated relative FAD (FDR > 0.05). Analysis of the expression data highlighted 132 differentially expressed genes (DEGs) in FAD/Tet1+/- relative to FAD (FDR > 0.05). Among the genes exclusive to the FAD/Tet1 mice were Nr4A1, Arc, and APLP1. Notably, Nr4A1 appeared in both the 5hmC and RNA-Seq data with increased levels of 5hmC and gene expression relative to FAD mice. Gene ontology analysis of RNA expression data identified significant changes in pathways involved in regulation of ion transmembrane transport, behavioral fear response, regulation of trans-synaptic signaling, and learning or memory. Taken together, these results suggest Tet1 modulates AD-associated gene regulation, expression and pathology.

The mutational constraint spectrum quantified from variation in 141,456 humans

Genetic variants that inactivate protein-coding genes are a powerful source of information about the phenotypic consequences of gene disruption: genes that are crucial for the function of an organism will be depleted of such variants in natural populations, whereas non-essential genes will tolerate their accumulation. However, predicted loss-of-function variants are enriched for annotation errors, and tend to be found at extremely low frequencies, so their analysis requires careful variant annotation and very large sample sizes1. Here we describe the aggregation of 125,748 exomes and 15,708 genomes from human sequencing studies into the Genome Aggregation Database (gnomAD). We identify 443,769 high-confidence predicted loss-of-function variants in this cohort after filtering for artefacts caused by sequencing and annotation errors. Using an improved model of human mutation rates, we classify human protein-coding genes along a spectrum that represents tolerance to inactivation, validate this classification using data from model organisms and engineered human cells, and show that it can be used to improve the power of gene discovery for both common and rare diseases.

Using human sequencing to guide craniofacial research.

A recent convergence of technological innovations has re-energized the ability to apply genetics to research in human craniofacial development. Next-generation exome and whole genome sequencing have significantly dropped in price, making it relatively trivial to sequence and analyze patients and families with congenital craniofacial anomalies. A concurrent revolution in genome editing with the use of the CRISPR-Cas9 system enables the rapid generation of animal models, including mouse, which can precisely recapitulate human variants. Here, we summarize the choices currently available to the research community. We illustrate this approach with the study of a family with a novel craniofacial syndrome with dominant inheritance pattern. The genomic analysis suggested a causal variant in AMOTL1 which we modeled in mice. We also made a novel deletion allele of Amotl1. Our results indicate that Amotl1 is not required in the mouse for survival to weaning. Mice carrying the variant identified in the human sequencing studies, however, do not survive to weaning in normal ratios. The cause of death is not understood for these mice complicating our conclusions about the pathogenicity in the index patient. Thus, we highlight some of the powerful opportunities and confounding factors confronting current craniofacial genetic research.

Sequencing studies in human genetics: design and interpretation

Next-generation sequencing is becoming the primary discovery tool in human genetics. There have been many clear successes in identifying genes that are responsible for Mendelian diseases, and sequencing approaches are now poised to identify the mutations that cause undiagnosed childhood genetic diseases and those that predispose individuals to more common complex diseases. There are, however, growing concerns that the complexity and magnitude of complete sequence data could lead to an explosion of weakly justified claims of association between genetic variants and disease. Here, we provide an overview of the basic workflow in next-generation sequencing studies and emphasize, where possible, measures and considerations that facilitate accurate inferences from human sequencing studies.