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

Alport syndrome, a disease of kidney, ear, and eye, is caused by pathogenic variants in the COL4A3 , COL4A4 , or COL4A5 genes encoding collagen a3a4a5(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 alpha 5(IV) readthrough products of five nonsense mutations could heterotrimerize with alpha 3(IV) and alpha 4(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.

Automated summary

Experimental results suggest that PTC readthrough therapy is a feasible approach for a fraction of patients with Alport syndrome, as readthrough drugs can facilitate the functional restoration of coding products from certain nonsense variants.