Avanthi Raghavan

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.

CRISPR-Cas9 Targeting of PCSK9 in Human Hepatocytes In Vivo—Brief Report

Objective—Although early proof-of-concept studies of somatic in vivo genome editing of the mouse ortholog of proprotein convertase subtilisin/kexin type 9 (Pcsk9) in mice have established its therapeutic potential for the prevention of cardiovascular disease, the unique nature of genome-editing technology—permanent alteration of genomic DNA sequences—mandates that it be tested in vivo against human genes in normal human cells with human genomes to give reliable preclinical insights into the efficacy (on-target mutagenesis) and safety (lack of off-target mutagenesis) of genome-editing therapy before it can be used in patients. Approach and Results—We used a clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated (Cas) 9 genome-editing system to target the human PCSK9 gene in chimeric liver-humanized mice bearing human hepatocytes. We demonstrated high on-target mutagenesis (approaching 50%), greatly reduced blood levels of human PCSK9 protein, and minimal off-target mutagenesis. Conclusions—This work yields important information on the efficacy and safety of CRISPR-Cas9 therapy targeting the human PCSK9 gene in human hepatocytes in vivo, and it establishes humanized mice as a useful platform for the preclinical assessment of applications of somatic in vivo genome editing.

CRISPR-Cas9 Targeting of PCSK9 in Human Hepatocytes In Vivo

Objective—Although early proof-of-concept studies of somatic in vivo genome editing of the mouse ortholog of proprotein convertase subtilisin/kexin type 9 (Pcsk9) in mice have established its therapeutic potential for the prevention of cardiovascular disease, the unique nature of genome-editing technology—permanent alteration of genomic DNA sequences—mandates that it be tested in vivo against human genes in normal human cells with human genomes to give reliable preclinical insights into the efficacy (on-target mutagenesis) and safety (lack of off-target mutagenesis) of genome-editing therapy before it can be used in patients. Approach and Results—We used a clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated (Cas) 9 genome-editing system to target the human PCSK9 gene in chimeric liver-humanized mice bearing human hepatocytes. We demonstrated high on-target mutagenesis (approaching 50%), greatly reduced blood levels of human PCSK9 protein, and minimal off-target mutagenesis. Conclusions—This work yields important information on the efficacy and safety of CRISPR-Cas9 therapy targeting the human PCSK9 gene in human hepatocytes in vivo, and it establishes humanized mice as a useful platform for the preclinical assessment of applications of somatic in vivo genome editing.

Sex Differences in Select Non-communicable HIV-Associated Comorbidities: Exploring the Role of Systemic Immune Activation/Inflammation

Purpose of the ReviewThe goals of this review are to (1) explore HIV-associated cardiovascular disease (CVD), neurocognitive impairment, and non-AIDS-defining cancers (NADC) as heterogeneous model disease states fuelled in part by systemic immune activation/inflammation; (2) consider sex differences in the epidemiology of these diseases in both high-resource and lower-resource settings; and (3) examine biological and environmental factors which may contribute to heightened systemic immune activation/inflammation specifically among women living with HIV (WLHIV).Recent FindingsThe observation that WLHIV have higher levels of systemic immune activation/inflammation than men living with HIV (MLHIV) may be relevant to sex differences in select non-communicable HIV-associated comorbidities. Heightened systemic immune activation among WLHIV may be influenced by sex-specific responses to the virus and to immunomodulatory agents, as well as by behavioral choices/comorbid conditions and perturbations in the hypothalamic-pituitary-gonadal axis.SummaryAdditional research is needed to elucidate region-specific drivers of heightened systemic immune activation/inflammation among WLHIV and to determine whether WLHIV who present with one immune-mediated HIV-associated comorbidity (e.g., cognitive impairment) may be at increased risk for another (e.g., CVD, NADC). This kind of research would facilitate improved risk prediction for non-communicable HIV-associated comorbidities among WLHIV and the development of targeted immunomodulatory prevention strategies.

High-throughput Screening and CRISPR-Cas9 Modeling of Causal Lipid-associated Expression Quantitative Trait Locus Variants

Genome-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 Summary Genome-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.

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

In the version of this article originally published, minus signs were missing from the three β-values for BMI given in Table 1. The errors have been corrected in the HTML and PDF versions of the article.

Interrogation of the Atherosclerosis-Associated SORT1 (Sortilin 1) Locus With Primary Human Hepatocytes, Induced Pluripotent Stem Cell-Hepatocytes, and Locus-Humanized Mice

Objective— The noncoding single-nucleotide polymorphism rs12740374 has been hypothesized to be the causal variant responsible for liver-specific modulation of SORT1(sortilin 1) expression (ie, expression quantitative trait locus) and, by extension, the association of the SORT1 locus on human chromosome 1p13 with low-density lipoprotein cholesterol levels and coronary heart disease. The goals of this study were to compare 3 different hepatocyte models in demonstrating that the rs12740374 minor allele sequence is responsible for transcriptional activation of SORT1 expression. Approach and Results— We found that although primary human hepatocytes of varied rs12740374 genotypes strongly replicated the SORT1 expression quantitative trait locus observed previously in whole-liver samples, a population cohort of induced pluripotent stem cell–derived hepatocyte-like cells poorly replicated the expression quantitative trait locus. In primary human hepatocytes from multiple individuals heterozygous at rs12740374, we used CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats–associated 9) to specifically target the rs12740374 minor allele sequence ex vivo, resulting in a reproducible reduction in SORT1 expression. We generated a locus-humanized transgenic mouse with a bacterial artificial chromosome bearing the human SORT1 locus with the rs12740374 minor allele. In this mouse model, we used CRISPR-Cas9 to target the rs12740374 minor allele sequence in the liver in vivo, resulting in a substantial reduction of hepatic SORT1 expression. Conclusions— The rs12740374 minor allele sequence enhances SORT1 expression in hepatocytes. CRISPR-Cas9 can be used in primary human hepatocytes ex vivo and locus-humanized mice in vivo to interrogate the function of noncoding regulatory regions. Induced pluripotent stem cell–derived hepatocyte-like cells experience limitations that prevent faithful modelling of some hepatocyte expression quantitative trait loci.

CRISPR-Cas9 Targeting ofPCSK9in Human Hepatocytes In Vivo—Brief ReportSignificance

Objective—Although early proof-of-concept studies of somatic in vivo genome editing of the mouse ortholog of proprotein convertase subtilisin/kexin type 9 (Pcsk9) in mice have established its therapeutic potential for the prevention of cardiovascular disease, the unique nature of genome-editing technology—permanent alteration of genomic DNA sequences—mandates that it be tested in vivo against human genes in normal human cells with human genomes to give reliable preclinical insights into the efficacy (on-target mutagenesis) and safety (lack of off-target mutagenesis) of genome-editing therapy before it can be used in patients. Approach and Results—We used a clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated (Cas) 9 genome-editing system to target the human PCSK9 gene in chimeric liver-humanized mice bearing human hepatocytes. We demonstrated high on-target mutagenesis (approaching 50%), greatly reduced blood levels of human PCSK9 protein, and minimal off-target mutagenesis. Conclusions—This work yields important information on the efficacy and safety of CRISPR-Cas9 therapy targeting the human PCSK9 gene in human hepatocytes in vivo, and it establishes humanized mice as a useful platform for the preclinical assessment of applications of somatic in vivo genome editing.

CRISPR-Cas9 Targeting of PCSK9 in Human Hepatocytes In Vivo—Brief ReportSignificance

Objective—Although early proof-of-concept studies of somatic in vivo genome editing of the mouse ortholog of proprotein convertase subtilisin/kexin type 9 (Pcsk9) in mice have established its therapeutic potential for the prevention of cardiovascular disease, the unique nature of genome-editing technology—permanent alteration of genomic DNA sequences—mandates that it be tested in vivo against human genes in normal human cells with human genomes to give reliable preclinical insights into the efficacy (on-target mutagenesis) and safety (lack of off-target mutagenesis) of genome-editing therapy before it can be used in patients. Approach and Results—We used a clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated (Cas) 9 genome-editing system to target the human PCSK9 gene in chimeric liver-humanized mice bearing human hepatocytes. We demonstrated high on-target mutagenesis (approaching 50%), greatly reduced blood levels of human PCSK9 protein, and minimal off-target mutagenesis. Conclusions—This work yields important information on the efficacy and safety of CRISPR-Cas9 therapy targeting the human PCSK9 gene in human hepatocytes in vivo, and it establishes humanized mice as a useful platform for the preclinical assessment of applications of somatic in vivo genome editing.