University of Kentucky
Characterization of novel disease-causing mutations in ABCG5 and ABCG8
Grade Level at Time of Presentation
Junior
Major
Biology
Minor
Creative Writing
KY House District #
59
KY Senate District #
6
Faculty Advisor/ Mentor
Gregory Graf, PhD
Department
Department of Physiology
Abstract
Cardiovascular disease is the leading cause of death throughout the United States and is particularly high in Kentucky, the Ohio River Valley, and Appalachia. Genetics and lifestyle contribute to the risk of cardiovascular disease. Sitosterolemia is a rare form of familial hypercholesterolemia caused by mutations in ABCG5 and ABCG8 and distinguished by the accumulation of phytosterols in plasma and tissue. ABCG5 and ABCG8 form a sterol pump that opposes sterol absorption in the intestine and promotes cholesterol elimination in the liver. ABCG5 and ABCG8 must form a dimer within the endoplasmic reticulum in order to traffic to the cell surface to pump cholesterol. Whole genome and exome sequencing suggest there are over 2000 likely pathogenic mutants of ABCG5 and ABCG8, but only 60 have been clinically confirmed. In collaboration with the Weill Cornell Medical Center, whole genome sequencing was performed in patients with elevated risk for cardiovascular disease, including hypercholesterolemia. 3 patients were identified with elevated phytosterols and novel variants in either ABCG5 or ABCG8 (ABCG8_G216D, ABCG5_R446Q, and ABCG5_Q392P). However, the impact of those variants on G5/G8 formation and trafficking is unknown. To determine this impact, the variants were generated by long-range PCR site-directed mutagenesis and confirmed by Sanger sequencing. Plasmids encoding native and variant ABCG5 and ABCG8 were co-transfected into human Huh7 hepatocytes and cell lysates analyzed by SDS-PAGE and immunoblot analysis. Maturation of G5G8 was assessed by electrophoretic mobility of ABCG5 ABCG8. Assessment of these mutations demonstrates that these novel, likely pathogenic alleles associated with the clinical presentation of Sitosterolemia do not interfere and may enhance maturation of G5G8. However, two appear to disrupt trafficking of the G5G8 transporter to the cell surface. Further analysis of the structure-function relationships of these mutants reveals the possibility for rescue with small molecule chaperones effective in the treatment of Cystic Fibrosis.
Characterization of novel disease-causing mutations in ABCG5 and ABCG8
Cardiovascular disease is the leading cause of death throughout the United States and is particularly high in Kentucky, the Ohio River Valley, and Appalachia. Genetics and lifestyle contribute to the risk of cardiovascular disease. Sitosterolemia is a rare form of familial hypercholesterolemia caused by mutations in ABCG5 and ABCG8 and distinguished by the accumulation of phytosterols in plasma and tissue. ABCG5 and ABCG8 form a sterol pump that opposes sterol absorption in the intestine and promotes cholesterol elimination in the liver. ABCG5 and ABCG8 must form a dimer within the endoplasmic reticulum in order to traffic to the cell surface to pump cholesterol. Whole genome and exome sequencing suggest there are over 2000 likely pathogenic mutants of ABCG5 and ABCG8, but only 60 have been clinically confirmed. In collaboration with the Weill Cornell Medical Center, whole genome sequencing was performed in patients with elevated risk for cardiovascular disease, including hypercholesterolemia. 3 patients were identified with elevated phytosterols and novel variants in either ABCG5 or ABCG8 (ABCG8_G216D, ABCG5_R446Q, and ABCG5_Q392P). However, the impact of those variants on G5/G8 formation and trafficking is unknown. To determine this impact, the variants were generated by long-range PCR site-directed mutagenesis and confirmed by Sanger sequencing. Plasmids encoding native and variant ABCG5 and ABCG8 were co-transfected into human Huh7 hepatocytes and cell lysates analyzed by SDS-PAGE and immunoblot analysis. Maturation of G5G8 was assessed by electrophoretic mobility of ABCG5 ABCG8. Assessment of these mutations demonstrates that these novel, likely pathogenic alleles associated with the clinical presentation of Sitosterolemia do not interfere and may enhance maturation of G5G8. However, two appear to disrupt trafficking of the G5G8 transporter to the cell surface. Further analysis of the structure-function relationships of these mutants reveals the possibility for rescue with small molecule chaperones effective in the treatment of Cystic Fibrosis.