Type 2 diabetes mellitus (T2DM) and coronary artery disease (CAD) are leading causes of morbidity and mortality worldwide. Development of new and more effective approaches to prevention and treatment requires improved understanding of disease mechanisms. Genetic mapping in humans offers an approach to identify novel genes and DNA variants underlying the inherited contribution to disease susceptibility. Recently, we and others have used genome-wide association studies (GWAS) to identify novel genetic loci with strong association with blood lipid levels, CAD, and T2DM, along with a variety of related metabolic traits. Among the most intriguing of these loci is one on chromosome 7q32 that is just upstream of the KLF14 gene, which encodes a putative transcription factor. Although little is known of this gene's biology, single nucleotide polymorphisms (SNPs) in the locus are associated with high-density lipoprotein cholesterol (HDL-C), triglycerides, CAD, and T2DM. Expression quantitative trait locus (eQTL) studies in human adipose tissue have linked these same SNPs to the expression of ten genes that themselves harbor SNPs linked to a host of metabolic traits, including body mass index (BMI), fasting insulin levels, and fasting glucose levels. Based on these data, we hypothesize that KLF14 is a master regulator of the expression of a host of metabolic genes that together influence hyperglycemia, insulin resistance, obesity, blood lipid levels, and CAD-in other words, many of the cardinal features of the metabolic syndrome. We seek to test this hypothesis and better characterize the KLF14 molecular pathway. Building on our preliminary studies, we propose to use genome editing with cutting-edge clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems to knock out the KLF14 gene in human pluripotent stem cells, knock out the Klf14 gene in either the whole body or conditionally in metabolic tissues in mouse models, and knock in a FLAG tag into the endogenous KLF14 protein in mice with which to perform physiological ChIP experiments. We will use differentiated cells, primary tissues, and whole animals to comprehensively study the effects of KLF14 on gene expression and metabolic phenotypes. In doing this, we seek to establish a rapid and efficient multi-species approach with which to study the effects of metabolic genes discovered by gene mapping experiments.

Public Health Relevance

To develop new therapies to prevent diabetes and coronary artery disease, which are leading causes of morbidity and mortality worldwide, we and others have performed genetic studies to identify novel genes involved in these diseases. One such gene, KLF14, is associated with both diabetes and coronary artery disease as well as dyslipidemia, insulin resistance, and other features of the metabolic syndrome. Our goal is to use both human and mouse model systems to better understand the function of KLF14 and how it influences these diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK099571-03
Application #
9270020
Study Section
Cellular Aspects of Diabetes and Obesity Study Section (CADO)
Program Officer
Haft, Carol R
Project Start
2016-02-01
Project End
2019-01-31
Budget Start
2017-02-01
Budget End
2018-01-31
Support Year
3
Fiscal Year
2017
Total Cost
$326,025
Indirect Cost
$123,525
Name
University of Pennsylvania
Department
Internal Medicine/Medicine
Type
Domestic Higher Education
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Small, Kerrin S; Todor?evi?, Marijana; Civelek, Mete et al. (2018) Regulatory variants at KLF14 influence type 2 diabetes risk via a female-specific effect on adipocyte size and body composition. Nat Genet 50:572-580
Wang, Xiao; Raghavan, Avanthi; Peters, Derek T et al. (2018) Interrogation of the Atherosclerosis-Associated SORT1 (Sortilin 1) Locus With Primary Human Hepatocytes, Induced Pluripotent Stem Cell-Hepatocytes, and Locus-Humanized Mice. Arterioscler Thromb Vasc Biol 38:76-82
Musunuru, Kiran (2017) Genome Editing: The Recent History and Perspective in Cardiovascular Diseases. J Am Coll Cardiol 70:2808-2821
Chadwick, Alexandra C; Wang, Xiao; Musunuru, Kiran (2017) In Vivo Base Editing of PCSK9 (Proprotein Convertase Subtilisin/Kexin Type 9) as a Therapeutic Alternative to Genome Editing. Arterioscler Thromb Vasc Biol 37:1741-1747
Pashos, Evanthia E; Park, YoSon; Wang, Xiao et al. (2017) Large, Diverse Population Cohorts of hiPSCs and Derived Hepatocyte-like Cells Reveal Functional Genetic Variation at Blood Lipid-Associated Loci. Cell Stem Cell 20:558-570.e10
Wang, Xiao; Raghavan, Avanthi; Chen, Tao et al. (2016) CRISPR-Cas9 Targeting of PCSK9 in Human Hepatocytes In Vivo-Brief Report. Arterioscler Thromb Vasc Biol 36:783-6