Article

RNAi–Based Functional Profiling of Loci from Blood Lipid Genome-Wide Association Studies Identifies Genes with Cholesterol-Regulatory Function

University of Oxford, United Kingdom
PLoS Genetics (Impact Factor: 7.53). 02/2013; 9(2):e1003338. DOI: 10.1371/journal.pgen.1003338
Source: PubMed

ABSTRACT

Genome-wide association studies (GWAS) are powerful tools to unravel genomic loci associated with common traits and complex human disease. However, GWAS only rarely reveal information on the exact genetic elements and pathogenic events underlying an association. In order to extract functional information from genomic data, strategies for systematic follow-up studies on a phenotypic level are required. Here we address these limitations by applying RNA interference (RNAi) to analyze 133 candidate genes within 56 loci identified by GWAS as associated with blood lipid levels, coronary artery disease, and/or myocardial infarction for a function in regulating cholesterol levels in cells. Knockdown of a surprisingly high number (41%) of trait-associated genes affected low-density lipoprotein (LDL) internalization and/or cellular levels of free cholesterol. Our data further show that individual GWAS loci may contain more than one gene with cholesterol-regulatory functions. Using a set of secondary assays we demonstrate for a number of genes without previously known lipid-regulatory roles (e.g. CXCL12, FAM174A, PAFAH1B1, SEZ6L, TBL2, WDR12) that knockdown correlates with altered LDL-receptor levels and/or that overexpression as GFP-tagged fusion proteins inversely modifies cellular cholesterol levels. By providing strong evidence for disease-relevant functions of lipid trait-associated genes, our study demonstrates that quantitative, cell-based RNAi is a scalable strategy for a systematic, unbiased detection of functional effectors within GWAS loci.

Full-text preview

Available from: plosone.org
  • Source
    • "Interestingly, WDR12 was also detected in the lighter fractions of the gradient, suggesting that WDR12 may be loosely associated with the PeBOW complex or may be involved in other cellular processes (Figure 3f). Recently, human WDR12 has been identified as a cholesterol-regulatory gene, although molecular mechanism of the regulation is unknown (Blattmann et al., 2013). Reduced global translation activities and reduced synthesis of mature 25S rRNA Since PES deficiency resulted in immediate growth arrest, and PES proteins cofractionated with ribosomes, we examined cellular protein translation activity, using 35 S-labeled methionine, on WT (wild-type) and AtPES RNAi seedlings (#28 and #38; Figure 4a). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Pescadillo (PES) is involved in diverse cellular processes such as embryonic development, ribosomal biogenesis, cell proliferation, and gene transcription in yeast and metazoans. In this study, we characterized cellular functions of plant PES in Nicotiana benthamiana, Arabidopsis, and tobacco BY-2 cells. A GFP fusion protein of PES is predominantly localized in the nucleolus, where its localization requires the N-terminal domain of PES. Silencing of plant PES led to growth arrest and acute cell death. PES interacts with plant homologs of BOP1 and WDR12 in the nucleolus, which are also nucleolar proteins involved in ribosome biogenesis of yeast and mammals. PES, BOP1, and WDR12 cofractionated with ribosome subunits. Depletion of any of these proteins led to defective biogenesis of the 60S ribosome large subunits and disruption of nucleolar morphology. PES-deficient plant cells also exhibited delayed maturation of 25S ribosomal RNA and suppressed global translation. During mitosis in tobacco BY-2 cells, PES is associated with the mitotic microtubules, including spindles and phragmoplasts, and PES deficiency disrupted spindle organization and chromosome arrangement. Collectively, these results suggest that plant PES has an essential role in cell growth and survival through its regulation of ribosome biogenesis and mitotic progression. This article is protected by copyright. All rights reserved.
    Preview · Article · Aug 2013 · The Plant Journal
  • [Show abstract] [Hide abstract]
    ABSTRACT: Hyperlipidemia is an important risk factor for cardiovascular disease globally, but there is still much mystery surrounding the topic of lipid regulation. Many studies have attempted to assess the underlying genetic basis of low-density lipoprotein (LDL) metabolism. Recently, multiple genome-wide association studies identified genes that strongly associate with plasma lipid concentration and cardiovascular disease. Compelling evidence linking the SORT1 gene to both LDL cholesterol (LDL-C) levels and the risk of coronary artery disease emerged from the data, prompting the search for the molecules and mechanisms responsible for this association. Three recent studies explored this relation through sortilin, the gene product of SORT1, and an intracellular trafficking molecule. Careful, hypothesis-driven experimental designs elucidated the potential mechanisms of sortilin's role in LDL-C metabolism. However, each study's conclusions differed in the details of SORT1's association to LDL-C and the subcellular mechanisms at work. Nevertheless, these 3 studies demonstrate how a complex disease such as hyperlipidemia can be evaluated from the scope of the genome down through the level of cellular regulation. Their findings serve as a platform for further study of LDL-C metabolism and hyperlipidemia while also providing lessons on how to better study other complex diseases.
    No preview · Article · Mar 2014 · Cardiology in review
  • [Show abstract] [Hide abstract]
    ABSTRACT: This years Deuel Conference on Lipids focused on the seminal contribution of human genetics to lipid biology and disease. Named in honor of Harry J Deuel Jr, famous single author of the three-volume textbook The Lipids (Interscience Publishers Inc., NY, USA) and established in 1955, the Deuel Conference is considered the longest-standing annual meeting on lipid metabolism in the USA. The 2013 meeting took place at the Silverado Resort of Napa Valley (CA, USA) and was characterized by first-rank scientific contributions and discussions in a friendly and informal setting. Four scientific sessions over 2 and a half days, a poster session and generous breaks allowed for stimulating scientific, as well as social interactions of scientists from academia and industry alike.
    No preview · Article · Jun 2013 · Clinical Lipidology
Show more