NanoLuc reporters identify COL4A5 nonsense mutations susceptible to drug-induced stop codon readthrough

iScience

Michel Roberge¹  Hirofumi Kai²  Kohei Omachi³  Jeffrey H. Miner³ 

[1] Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada;Department of Molecular Medicine, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan;Division of Nephrology, Washington University School of Medicine, 4523 Clayton Avenue, St. Louis, MO 63110, USA;

关键词: Biochemistry;    Molecular biology;    Molecular medicine;   

DOI  :  

来源: DOAJ

【 摘 要 】

Summary: Alport syndrome, a disease of kidney, ear, and eye, is caused by pathogenic variants in the COL4A3, COL4A4, or COL4A5 genes encoding collagen α3α4α5(IV) of basement membranes. Collagen IV chains that are truncated due to nonsense variants/premature termination codons (PTCs) cannot assemble into heterotrimers or incorporate into basement membranes. To investigate the feasibility of PTC readthrough therapy for Alport syndrome, we utilized two NanoLuc reporters in transfected cells: full-length for monitoring translation, and a split version for assessing readthrough product function. Full-length assays of 49 COL4A5 nonsense variants identified eleven as susceptible to PTC readthrough using various readthrough drugs. In split-NanoLuc assays, the predicted missense α5(IV) readthrough products of five nonsense mutations could heterotrimerize with α3(IV) and α4(IV). Readthrough was also observed in kidney cells from an engineered Col4a5 PTC mouse model. These results suggest that readthrough therapy is a feasible approach for a fraction of patients with Alport syndrome.

【 授权许可】

Unknown