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Identification of genes that play a crucial role in regulating cholesterol levels

Genome-wide association studies (GWAS), which were combined with RNA interference studies and microscope-based functional analyses, enabled researchers from the European Molecular Biology Laboratory and Heidelberg University Hospital to identify genes that play key roles in the regulation of the blood cholesterol level. The new method leads to a better understanding of the relationships between the lipid metabolism and the development of cardiovascular diseases.

Dr. Heiko Runz, Institute of Human Genetics at Universität Heidelberg. © University Hospital Heidelberg

High cholesterol levels in the blood are one of the main risk factors for cardiovascular disease. Cholesterol-containing lipid depositions build up in the artery walls and form plaques, leading to atherosclerosis. Plaques that rupture may cause the formation of a thrombus that will slow or stop blood flow, leading to myocardial infarction or stroke. Cholesterol bound to low-density lipoprotein (LDL cholesterol) – the packaging form through which cholesterol is transported from the liver to other organs and tissues of the body – is seen as the main culprit. The concept of disease-causing high blood cholesterol levels is associated with powerful economic interests: cholesterol-lowering statins have become the best-selling class of drug worldwide and a cholesterol-free diet is an important segment of the food and beverage industries that is intensely advertised.

However, the relationship between elevated blood cholesterol levels and cardiovascular diseases is unclear and highly controversial. Cholesterol is not only a vital component of cell membranes (of the brain in particular), the body also synthesises most of the required cholesterol itself. Around 90 percent of all cholesterol is located inside cells. The cells release and take up cholesterol to and from the bloodstream. Imbalances in relevant cellular mechanisms (i.e. the secretory pathways between the endoplasmic reticulum (ER), Golgi apparatus and plasma membrane) are thus an important contributor to variation in blood cholesterol levels and associated diseases. It has also been known for quite some time that alterations in blood cholesterol levels are highly heritable, but the genetic basis and molecular mechanisms leading to disease are still largely unknown.

Molecular Medicine Partnership Unit

Dr. Rainer Pepperkok, European Molecular Biology Laboratory, Heidelberg. © EMBL

In a large cooperative project (“Cell biology and disorders of cholesterol metabolism”) scientists and doctors from Heidelberg are investigating the factors that regulate the cellular cholesterol metabolism and associated diseases. The project team, which is led by Dr. Rainer Pepperkok from the European Molecular Biology Laboratory (EMBL) and Dr. Heiko Runz from the Institute of Human Genetics at the University of Heidelberg, is part of the Molecular Medicine Partnership Unit (MMPU), a joint initiative of the EMBL and Heidelberg University Hospital. 

Pepperkok, cell biologist and head of the Advanced Light Microscopy Core Facility at EMBL, is specifically focused on membrane trafficking in the secretory pathway from the ER to the Golgi apparatus. In order to study the proteins that are involved in the temporal and spatial organisation of ER-exit sites and the biogenesis of the Golgi complex, Pepperkok developed new approaches to study living cells, including ones involving GFP- (green fluorescent protein) labelled proteins and the systematic silencing of individual genes using RNA interference (RNAi). Human geneticist Runz is specifically focused on cell-based functional genomics, i.e. the combination of cell biology and genetics, with the goal of deriving information on gene function from genomic data. In a study, which combined the methodological approaches of the two laboratories, the MMPU team has been able to identify genes that are pivotal in regulating the cholesterol level in cells and blood. 

Genome-wide association studies, RNA interference and microcope-based functional analyses

Schematic of the steps involved in cellular membrane transport (COPII – coated vesicle protein II: involved in ER to Golgi transport; COPI – coated vesicle protein I: involved in Golgi to ER transport; VTC – vesicular-tubular cluster; TGN – trans-Golgi network). © Pepperkok, EMBL

Around 120 loci in the human genome have so far been identified to have an association with alterations in the lipid content and the blood cholesterol level in particular and to lead to an increased risk of developing cardiovascular diseases such as atherosclerosis and myocardial infarction. So-called genome-wide association studies (GWAS) were carried out to identify genes involved in the cellular cholesterol metabolism: the genomes of hundreds of thousands of individuals were screened for variations (e.g., in SNPs, single nucleotide polymorphisms) that occur in a population with the symptoms under investigation in comparison to a control group. All genes that were known to play a role in diseased lipid metabolism were also identified using GWAS. 

However, the function of most gene loci identified was unknown and could not be elucidated using the epidemiological genome analyses. Investigations on the phenotype level that enable the identification of candidate genes by disease-related gene expression analyses also had to be carried out. 

Identification of new cholesterol-regulating genes using high-resolution fluorescence microscopy. Top left: cholesterol uptake (orange) into cells; top right: free cholesterol (blue) in the cells; bottom left: localisation of a new cholesterol-regulating protein (green); bottom right: cell mass (grey) and cholesterol particles (white) taken up. © PLoS Genetics; P. Blattmann/EMBL; C. Schuberth/University Hospital Heidelberg.

The team led by Runz and Pepperkok investigated 133 genes within 56 of the 120 or so loci previously identified by GWAS as being linked with blood lipid levels and cardiovascular disease. The researchers used RNA interference (RNAi) to test the function of these genes by selectively decreasing their expression and measuring what changes this induced in cholesterol metabolism. The mRNAs of the candidate genes were captured with specific synthetic small RNA molecules. A high-resolution fluorescence microscopy platform, including image acquisition and automated analysis of cellular phenotypes, was subsequently used to find out whether cells in which the respective genes had been silenced showed defects in the uptake or deposition of labelled cholesterol (see figure). From this, the researchers were able to deduce which of the genes are most likely involved in increasing blood cholesterol levels and onset of the disease.

Referring to this new approach that enabled the identification and functional validation of genes involved in the regulation of cholesterol levels, Rainer Pepperkok comments: “This is the first large-scale RNA interference study carried out with genes previously identified by GWAS. It has the potential to identify from a vast number of candidate genes those genes that are involved in the regulation of cholesterol levels. We will be able to specifically focus on these genes in future studies.” Heiko Runz, who currently works at the Center for Human Genetic Research at the Massachusetts General Hospital in Boston, USA, emphasised the major medical importance of the study: “In principle, our approach can be applied to any disease that has a noticeable effect on cells. We have identified many new genes that affect the critical process of cellular cholesterol uptake and probably also play a key role in the development of coronary heart disease and myocardial infarction. Future studies will be carried out to obtain insights into which genes and DNA sequence variants are the most relevant in the disease process and whether the defects of these genes can be compensated with drugs.” 

The genes identified might thus contribute to a better understanding of mechanisms that lead to cardiovascular diseases and potentially also improve the prediction and diagnosis of such diseases, which are the primary cause of death in Western industrialised nations. 

Reference:
Peter Blattmann, Christian Schuberth, Rainer Pepperkok, Heiko Runz (2013): RNAi-based functional profiling of loci from blood lipid genome-wide association studies identifies genes with cholesterol-regulatory function. PLOS Genetics 9(2): e1003338.

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