Marisa Wong Medina, PhD
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Alternative splicing as a regulatory mechanism

Genes are comprised of exons and introns. Exons are protein-coding portions of the gene and are separated by introns, regions that are removed by a process called splicing to convert pre-messenger RNA (pre-mRNA) into mature messenger RNA (mRNA). Alternative splicing of pre-mRNAs produces diversity in gene expression. Multiple final mRNAs can be derived from a single gene via exon skipping or retention, intron retention, use of cryptic splice sites, or exon shuffling. Once considered to be a relatively infrequent occurrence, we now know that essential every gene with more than one exon undergoes alternative splicing, often with functional consequences. Notably, 15-50 percent of disease-causing mutations, including those in genes linked to cardiovascular disease, disrupt normal pre-mRNA splicing.

Cellular cholesterol is obtained through both de novo synthesis, for which 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) is the rate limiting enzyme, as well as uptake from the plasma, which is primarily mediated by the low density lipoprotein receptor (LDLR). Both HMGCR and LDLR undergo functionally relevant alternative splicing. Skipping of HMGCR exon 13 generates an alternatively spliced transcript, HMGCR13(-),that has been linked to cardiovascular disease. For example, we have demonstrated that inter-individual variation in HMGCR alternative splicing is a marker, and likely a determinant, of LDL cholesterol response to statin. In addition, SNPs within HMGCR and LDLR that modulate alternative splicing have been associated with variation in plasma LDL cholesterol in multiple independent populations, as well as familial hypercholesterolemia. These findings implicate alternative splicing as a physiologically and clinically relevant regulator of cholesterol metabolism.

Using cellular and in vivo models, we have found that genes involved in the cholesterol synthesis and uptake pathways undergo orchestrated changes at the level of alternative splicing in response to changes in cellular sterol content. These changes appear to be mediated by sterol regulation of specific splicing factors that target these genes. Overall, our results suggest that alternative splicing is a novel and generalized regulatory mechanism responsible for maintaining intracellular cholesterol homeostasis. Identification of novel mechanisms that ultimately impact circulating levels of cholesterol may inform the development of new drug targets developed to lower plasma cholesterol and reduce heart disease risk.

 

Statin pharmacogenomics

Statins are the most commonly prescribed class of drugs used for the prevention and treatment of cardiovascular disease, and function primarily through their ability to lower LDL cholesterol. However, the magnitude of this effect varies widely among individuals, with nearly a third of individuals failing to meet their lipid lowering goals on treatment. In collaboration with CHORI Senior Scientist Ronald M. Krauss, MD, we are investigating molecular and genetic determinants of inter-individual variation in statin-induced changes in plasma cholesterol. Toward this goal we are using a combination of approaches including genetic association studies in clinical trials and population-based cohorts, transcriptomic analyses of cellular statin response in a repository of lymphoblastoid cell lines derived from the Cholesterol and Pharmacogenetics clinical trial, as well as systems biology based approaches to combine and integrate omics-level datasets from our cellular and clinical cohorts. The results of this research could yield significant improvement in the ability to identify those individuals most likely to achieve cardiovascular benefit from statin treatment, as well as identify new pharmacologic approaches for increasing statin efficacy.

 

Functional validation of candidate genes identified through genetic studies

Genetic association and genetic linkage studies are two commonly used methods to identify novel loci associated with variation in human phenotypes. Recently very large-scale genome-wide association studies (GWAS) have identified a number of novel loci associated with variation in plasma cholesterol and coronary artery disease. Few of these candidate genes have been validated using animal and cellular models, thus the relationship between many of the identified candidate SNPs and genes with regulation of cholesterol metabolism remain unknown. In addition, while GWAS have successfully discovered many new loci, a large proportion of the genetic variance in plasma LDL-cholesterol has not yet been identified. We have performed genetic linkage studies using a cohort of family-based lymphoblastoid cell lines to identify quantitative trait loci associated with cellular markers of cholesterol metabolism. Thus, another goal of the lab is to functionally validate candidate genes identified by genetic association and linkage studies using cellular models, with the overall objective of elucidating novel molecular mechanisms that impact cholesterol metabolism. Discovery of such mechanisms, and the role specific proteins and enzymes that function within these pathways, may lead to the development to new therapeutics used for the treatment and prevention of cardiovascular disease.

 

Revised: Tuesday, March 29, 2016 9:15 AM

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