Systematic elucidation of genetic mechanisms underlying cholesterol uptake

Abstract

Genetic variation contributes greatly to LDL cholesterol (LDL-C) levels and coronary artery disease risk. By combined analysis of rare coding variants from the UK Biobank and genome-scale CRISPR-Cas9 knockout and activation screening, we have substantially improved the identification of genes whose disruption alters serum LDL-C levels. We identify 21 genes in which rare coding variants significantly alter LDL-C levels at least partially through altered LDL-C uptake. We use co-essentiality-based gene module analysis to show that dysfunction of the RAB10 vesicle transport pathway leads to hypercholesterolemia in humans and mice by impairing surface LDL receptor levels. Further, we demonstrate that loss of function ofOTX2leads to robust reduction in serum LDL-C levels in mice and humans by increasing cellular LDL-C uptake. Altogether, we present an integrated approach that improves our understanding of genetic regulators of LDL-C levels and provides a roadmap for further efforts to dissect complex human disease genetics.

Joint genotypic and phenotypic outcome modeling improves base editing variant effect quantification

“…The first library (LDL-C GWAS library) targets 583 variants associated with LDL-C levels from the UK Biobank GWAS cohort ( Methods , Supplementary Table 1 ). We included fine-mapped variants with posterior inclusion probability (PIP) > 0.25 from either the SUSIE or Polyfun fine-mapping pipelines 43,44 , and also variants with PIP > 0.1 within 250 kb of any of 490 genes found to significantly alter LDL-C uptake from recent CRISPR-Cas9 knockout screens 37,45 . We designed five tiled gRNAs for each variant allele that place the variant in positions shown to induce most efficient editing with ABE8e ( Fig.…”

“…1e ) 46 . Positive control gRNAs which ablate splice donor and acceptor consensus sites in six genes found to have significantly altered LDL-C uptake upon knockout 37 , and 100 non-targeting negative control gRNAs that tile 20 synthetic variants were included, for a total of 3,455 gRNAs.…”

“…We identified 54 variants that significantly alter LDL-C uptake (95% CI does not contain 0, Supplementary Table 4 ). These variants include intronic variants in well-known LDL-C uptake mediators whose knockout altered LDL-C uptake in a recent genome-scale CRISPR screen 37 such as ABCA1 , LDLR , and SCARB1 (Fig. 3e).…”

“…We have modeled the impacts of both common GWAS-associated and rare LDLR coding variants through base editing installation followed by cellular uptake of fluorescent LDL-C in HepG2 cells, which provides a scalable flow cytometric assay to measure a key contributing factor of serum LDL-C levels 37 given the majority of serum LDL-C is cleared in liver 38 . By applying our experimental-computational pipeline to this screen model, we identify LDL uptake-altering GWAS-associated variants and characterize their downstream impact on chromatin accessibility, transcription factor binding, and gene expression that leads to differential LDL uptake.…”

CRISPR screening in cardiovascular research

“…Similar readout and cell sorting strategy were employed, however, this study mainly focused on those genes adjacent to variants associated with serum LDL cholesterol from human Genome-Wide Association Study (GWAS) data, and CRISPRa screen was also employed as an orthogonal approach. In this way, they found that 21 genes with human genetic evidence may regulate LDL cholesterol uptake including RAB10 and OTX2 ( Hamilton et al, 2023 ). Another group performed similar CRISPR screen in HepG2 cells at whole genome scale to explore genes whose loss-of-function compromises LDL cholesterol uptake, and determined transgelin (encoded by TAGLN ) as a novel hit which acts by disrupting actin-dependent clathrin-mediated endocytosis of LDL ( Lucero et al, 2022 ).…”