Abstract 452: Classification And Effect Of Correctors On ABCG8 Sitosterolemia Associated Cytosolic Mutants

Background: Sitosterolemia is a rare form of Familial Hypercholesterolemia (FH) which is unique from other forms of FH due to the accumulation of phytosterols in the plasma and tissues. Sitosterolemia is autosomal recessive and caused by mutations in either the ABCG5 or ABCG8 gene. ABCG5 and ABCG8 form a heterodimer (ABCG5/ABCG8) that functions at the apical surface of hepatocytes and enterocytes to promote cholesterol and phytosterol excretion. Small molecule modulators classified as either correctors or potentiators have therapeutic benefit when used to treat mutants of ABCB4 (PFIC3) or ABCC7 (Cystic Fibrous) that have impaired folding, stability, or activity. There are over 40 missense mutations in ABCG5/ABCG8 which have been clinically confirmed in patients with Sitosterolemia. Methods: We developed a classification system for ABCG5 and ABCG8 mutations based on the underlying molecular defect for ABCG5/ABCG8 dysfunction (maturation, trafficking, activity, etc.). We used site directed mutagenesis to introduce ABCG8 missense mutations located in the N-terminal, cytosolic domain of human ABCG8. Normal and mutant ABCG5/ABCG8 constructs were transiently transfected into a human hepatocyte cell line, Huh-7, and analyzed for heterodimerization, trafficking beyond the ER, and localization at the cell surface. Results: Of the ABCG8 cytosolic mutants studied, R184H, L196Q, L228P, and R263Q failed to traffic beyond the ER to form a stable heterodimer (class II) while P231T, T400K, N409D, N409I, and P415H were trafficking competent. Conclusions: Forty four percent (4 of 9) of cytosolic, ABCG8 mutations that cause Sitosterolemia are due to failures in complex formation and trafficking beyond the ER. Fifty six (5 of 9) percent were trafficking competent, indicating a stable ABCG5/G8 complex, but loss of activity due to impaired ATP hydrolysis or substrate binding. ABCG8 mutants may be potentially restored by proteostatic regulators and/or potentiators & correctors that have shown to be effective in disease-causing mutations in other ABC transporters.