Using Genomics to Identify Novel Therapeutic Targets for Aortic Disease.

Aortic disease, including dissection, aneurysm, and rupture, carries significant morbidity and mortality and is a notable cause of sudden cardiac death. Much of our knowledge regarding the genetic basis of aortic disease has relied on the study of individuals with Mendelian aortopathies and, until recently, the genetic determinants of population-level variance in aortic phenotypes remained unclear. However, the application of machine learning methodologies to large imaging datasets has enabled researchers to rapidly define aortic traits and mine dozens of novel genetic associations for phenotypes such as aortic diameter and distensibility. In this review, we highlight the emerging potential of genomics for identifying causal genes and candidate drug targets for aortic disease. We describe how deep learning technologies have accelerated the pace of genetic discovery in this field. We then provide a blueprint for translating genetic associations to biological insights, reviewing techniques for locus and cell type prioritization, high-throughput functional screening, and disease modeling using cellular and animal models of aortic disease

Regulatory variants at KLF14 influence type 2 diabetes risk via a female-specific effect on adipocyte size and body composition

Individual risk of type 2 diabetes (T2D) is modified by perturbations to the mass, distribution and function of adipose tissue. To investigate the mechanisms underlying these associations, we explored the molecular, cellular and whole-body effects of T2D-associated alleles near KLF14. We show that KLF14 diabetes-risk alleles act in adipose tissue to reduce KLF14 expression and modulate, in trans, the expression of 385 genes. We demonstrate, in human cellular studies, that reduced KLF14 expression increases pre-adipocyte proliferation but disrupts lipogenesis, and in mice, that adipose tissue–specific deletion of Klf14 partially recapitulates the human phenotype of insulin resistance, dyslipidemia and T2D. We show that carriers of the KLF14 T2D risk allele shift body fat from gynoid stores to abdominal stores and display a marked increase in adipocyte cell size, and that these effects on fat distribution, and the T2D association, are female specific. The metabolic risk associated with variation at this imprinted locus depends on the sex both of the subject and of the parent from whom the risk allele derives

Mining the LIPG Allelic Spectrum Reveals the Contribution of Rare and Common Regulatory Variants to HDL Cholesterol

Genome-wide association studies (GWAS) have successfully identified loci associated with quantitative traits, such as blood lipids. Deep resequencing studies are being utilized to catalogue the allelic spectrum at GWAS loci. The goal of these studies is to identify causative variants and missing heritability, including heritability due to low frequency and rare alleles with large phenotypic impact. Whereas rare variant efforts have primarily focused on nonsynonymous coding variants, we hypothesized that noncoding variants in these loci are also functionally important. Using the HDL-C gene LIPG as an example, we explored the effect of regulatory variants identified through resequencing of subjects at HDL-C extremes on gene expression, protein levels, and phenotype. Resequencing a portion of the LIPG promoter and 5′ UTR in human subjects with extreme HDL-C, we identified several rare variants in individuals from both extremes. Luciferase reporter assays were used to measure the effect of these rare variants on LIPG expression. Variants conferring opposing effects on gene expression were enriched in opposite extremes of the phenotypic distribution. Minor alleles of a common regulatory haplotype and noncoding GWAS SNPs were associated with reduced plasma levels of the LIPG gene product endothelial lipase (EL), consistent with its role in HDL-C catabolism. Additionally, we found that a common nonfunctional coding variant associated with HDL-C (rs2000813) is in linkage disequilibrium with a 5′ UTR variant (rs34474737) that decreases LIPG promoter activity. We attribute the gene regulatory role of rs34474737 to the observed association of the coding variant with plasma EL levels and HDL-C. Taken together, the findings show that both rare and common noncoding regulatory variants are important contributors to the allelic spectrum in complex trait loci

Leveraging CRISPR/Cas Genome Editing Technology to Identify and Characterize Causal GWAS Variants for Blood Lipids

Genome-wide association studies (GWAS) have identified a number of novel genetic loci linked to serum cholesterol and triglyceride levels. The causal DNA variants at these loci and the mechanism by which they influence phenotype and disease risk remain largely unexplored. Expression quantitative trait locus (eQTL) analyses of patient liver and adipose biopsies indicate that many lipid-associated variants influence gene expression in a cis-regulatory manner. However, linkage disequilibrium (LD) among neighboring single nucleotide polymorphisms (SNPs) at a GWAS-implicated locus makes it challenging to pinpoint the actual variant underlying an association signal. Here we performed high-throughput identification of putative disease-causal loci through a functional reporter-based screen, the massively parallel reporter assay (MPRA). We then validated prioritized variants using a combination of genome edited stem cells, clustered regularly interspaced short palindromic repeats (CRISPR) interference, and in vivo genome edited humanized mouse models to establish rs2277862-CPNE1, rs10889356-ANGPTL3, and rs12740374-SORT1 as causal SNP gene sets. These results highlight a novel experimental framework to discover causal genes and variants contributing to complex human traits

Abstract 70: <i>KLF14</i> is a Novel Regulator of Human Metabolism

Human genetic studies have identified DNA variants near the KLF14 gene to be strongly associated with HDL cholesterol levels, triglyceride levels, risk of type 2 diabetes mellitus, and risk of coronary artery disease. Furthermore, the same variants are associated with both the expression level of KLF14 ( cis eQTL) and the expression levels of ten other genes related to metabolic traits located on different chromosomes ( trans eQTL) in human adipose tissues, suggesting that KLF14 may be a master regulator of gene expression in adipose tissue and a key player in human metabolism. In order to evaluate this hypothesis , we have rapidly and efficiently generated both KLF14 knockout human adipocytes (differentiated from human pluripotent stem cells) and Klf14 knockout mice through the use of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems. We have analyzed the KLF14 knockout adipocytes and the Klf14 knockout mice for metabolic phenotypes at several different levels. At an organismic level, we found Klf14 knockout mice displayed a variety of metabolic phenotypes—increased levels of triglycerides (up 61%), free fatty acids (up 75%), free glycerol (up 43%), and cholesterol levels (up 12%) in the blood. At a tissue level, we found Klf14 knockout adipose showed a broad activation of genes related to lipid synthesis and downregulation of groups of genes involved in muscle function and development, suggesting a switch between muscle (energy usage) and adipose (energy storage) programs. We also confirmed in Klf14 knockout adipose tissue significant changes in several of the ten trans eQTL genes reported in human adipose tissue. At a cellular level, we found in vitro differentiated Klf14 knockout adipocytes had increased adipogenesis with larger lipid droplets and more triglyceride accumulation (increased &gt;50%) compared to wild-type adipocytes. Thus, using novel genome-editing techniques, we were able to rapidly evaluate KLF14 function in mouse and human model systems. Taken together, the obtained data establish that KLF14 is a causal regulator of human metabolism, acting at least in part by modulating adipocyte function. Further studies in other metabolically active tissues such as liver and muscle are underway. </jats:p

Abstract 27: Therapeutic Targeting of Human Lipid Genes With in vivo CRISPR-Cas9 Genome Editing

Individuals with naturally occurring loss-of-function PCSK9, APOC3, and ANGPTL3 mutations experience reduced blood low-density lipoprotein cholesterol (LDL-C) levels, reduced blood triglyceride levels, and/or protection against cardiovascular disease. We recently established that genome editing using a Streptococcus pyogenes clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system, delivered by adenovirus, can efficiently introduce loss-of-function mutations into the endogenous mouse Pcsk9 gene in vivo, resulting in near-complete knockout of Pcsk9 and a 35%-40% reduction in the blood cholesterol level. Thus, the in vivo genome-editing approach may have therapeutic potential for the prevention of cardiovascular disease in humans. To better evaluate the feasibility of the approach in humans, we are performing preclinical studies using a liver-humanized mouse model in which the endogenous mouse hepatocytes have been replaced with human-derived hepatocytes and in which the plasma lipoprotein profile better reflects human physiology, particularly with respect to LDL-C. One disadvantage of the S. pyogenes CRISPR-Cas9 system is that its Cas9 gene is too large to fit in adeno-associated virus (AAV) vectors, which offer significant therapeutic advantages over adenoviruses. We have adapted the smaller Staphylococcus aureus CRISPR-Cas9 system and demonstrated efficient genome editing of the human PCSK9, APOC3, and ANGPTL3 genes in vitro. Additionally, using a minimal liver-specific promoter and a novel human-hepatocyte-optimized AAV capsid protein, we are employing AAV vectors to knock out each of the three genes in both primary human hepatocytes and liver-humanized mice. This is allowing us to interrogate both the efficacy (lowering of LDL-C and/or triglycerides) and safety (degree of off-target mutagenesis) of genome editing in human hepatocytes in vitro and in vivo. These translational studies are providing critical information on the viability of an approach that could ultimately yield a one-shot, long-term therapy that permanently reduces a person’s blood LDL-C and triglyceride levels, thus serving as a “vaccination” against cardiovascular disease.</jats:p

High-Throughput Screening and CRISPR-Cas9 Modeling of Causal Lipid-Associated Expression Quantitative Trait Locus Variants

AbstractGenome-wide association studies have identified a number of novel genetic loci linked to serum cholesterol and triglyceride levels. The causal DNA variants at these loci and the mechanisms by which they influence phenotype and disease risk remain largely unexplored. Expression quantitative trait locus analyses of patient liver and fat biopsies indicate that many lipid-associated variants influence gene expression in a cis-regulatory manner. However, linkage disequilibrium among neighboring SNPs at a genome-wide association study-implicated locus makes it challenging to pinpoint the actual variant underlying an association signal. We used a methodological framework for causal variant discovery that involves high-throughput identification of putative disease-causal loci through a functional reporter-based screen, the massively parallel reporter assay, followed by validation of prioritized variants in genome-edited human pluripotent stem cell models generated with CRISPR-Cas9. We complemented the stem cell models with CRISPR interference experiments in vitro and in knock-in mice in vivo. We provide validation for two high-priority SNPs, rs2277862 and rs10889356, being causal for lipid-associated expression quantitative trait loci. We also highlight the challenges inherent in modeling common genetic variation with these experimental approaches.Author SummaryGenome-wide association studies have identified numerous loci linked to a variety of clinical phenotypes. It remains a challenge to identify and validate the causal DNA variants in these loci. We describe the use of a high-throughput technique called the massively parallel reporter assay to analyze thousands of candidate causal DNA variants for their potential effects on gene expression. We use a combination of genome editing in human pluripotent stem cells, “CRISPR interference” experiments in other cultured human cell lines, and genetically modified mice to analyze the two highest-priority candidate DNA variants to emerge from the massively parallel reporter assay, and we confirm the relevance of the variants to nearby gene expression. These findings highlight a methodological framework with which to identify and functionally validate causal DNA variants.</jats:sec

The Longin Domain Regulates the Steady-State Dynamics of Sec22 in Plasmodium falciparum▿

The specificity of vesicle-mediated transport is largely regulated by the membrane-specific distribution of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. However, the signals and machineries involved in SNARE protein targeting to the respective intracellular locations are not fully understood. We have identified a Sec22 ortholog in Plasmodium falciparum (PfSec22) that contains an atypical insertion of the Plasmodium export element within the N-terminal longin domain. This Sec22 protein partially associates with membrane structures in the parasitized erythrocytes when expressed under the control of the endogenous promoter element. Our studies indicate that the atypical longin domain contains signals that are required for both endoplasmic reticulum (ER)/Golgi apparatus recycling of PfSec22 and partial export beyond the ER/Golgi apparatus interface. ER exit of PfSec22 is regulated by motifs within the α3 segment of the longin domain, whereas the recycling and export signals require residues within the N-terminal hydrophobic segment. Our data suggest that the longin domain of PfSec22 exhibits major differences from the yeast and mammalian orthologs, perhaps indicative of a novel mechanism for Sec22 trafficking in malaria parasites

Abstract 97: Genotype to Phenotype: Function of Common Noncoding and Rare Coding Variants In <i>ANGPTL3</i>

Human genetics studies have demonstrated a strong link between ANGPTL3 , which encodes lipoprotein lipase inhibitor Angiopoietin-like 3, and blood lipid phenotypes. Rare nonsense ANGPTL3 mutations were identified in patients with familial combined hypolipidemia, while common variants at the ANGPTL3 locus have been found by genome-wide association studies (GWASs) to associate with lower triglycerides (TGs) and low-density lipoprotein cholesterol. In light of the seemingly favorable clinical consequences of ANGPTL3 deficiency, we established an experimental framework to identify (1) causal common variants that regulate ANGPTL3 expression and (2) rare missense mutations that disrupt ANGPTL3 function. Using massively parallel reporter assays, we profiled the regulatory activity of all the common variants linked ( r 2 ≥ 0.5) to the lead GWAS SNP in the ANGPTL3 locus and found that rs10889356 demonstrated significant allele-specific enhancer activity. To validate this finding, we used CRISPR-Cas9 to alter the SNP in a human pluripotent stem cell line. When differentiated into hepatocytes, altered cells displayed a 67% increase in ANGPTL3 expression ( n = 4 wild-type and 4 mutant clones, P = 0.007). CRISPR interference using each of three guide RNAs targeting the SNP in HepG2 cells also substantially increased ANGPTL3 expression. These findings support rs10889356- ANGPTL3 as a causal SNP-gene set. Next, we examined the coding regions of ANGPTL3 in 20,000 sequenced individuals and sought to experimentally define rare missense variants using a mouse model. We used CRISPR-Cas9 to generate Angptl3 knockout mice, which exhibited decreased TG (61%, P &lt; 0.001) and decreased cholesterol (31%, P &lt; 0.002). We reconstituted the knockout mice to normal expression levels with adenoviruses expressing either wild-type ANGPTL3 or missense variant ANGPTL3 . So far we have assessed 28 rare missense variants computationally predicted to be deleterious, of which only 10—D42N, K58E, S117P, P264S, Q286H, L315S, L360Q, T383I, T383S, and Y417C—were validated as loss-of-function (conferring &lt;25% of wild-type activity as assessed by changes in both TG and cholesterol levels), underscoring the need for functional characterization of variants of uncertain significance. </jats:p