507 Understanding mechanisms that govern premature termination codon read-through in human skin diseases

Up to 30% of mutant alleles contributing to genetic diseases harbor a premature termination codon (PTC). As result there is lack of functional protein synthesized and this often leads to severe clinical consequences, as is the case with recessive dystrophic epidermolysis bullosa (RDEB) where approximately 43% of patients harbor PTC mutations in COL7A1. RDEB is a rare genetic skin disease characterized by trauma induced skin blistering and extra-cutaneous complications. The aim of this study is to understand mechanisms determining endogenous and pharmacologically enhanced read-through of PTC mutations in RDEB patient derived skin cells. Unraveling the mechanisms which determine and allow for read-through induced protein synthesis would be a valuable tool in designing therapies for genetic diseases caused by PTC mutations. We evaluated two compounds which were shown to induce read-through – amlexanox and gentamicin – in RDEB patient skin cells harboring PTCs. We show by Western blotting that increase in full length type VII collagen is only evident in cells which initially exhibit endogenous read-through, detected as trace amount of protein. These data suggest that endogenous read-through, even at low level, is a pre-requisite for amlexanox or gentamicin induced read-through of PTCs. Furthermore, the data show that efficiency of read-through protein synthesis correlates with increased phosphorylation of up-frameshift protein 1 (UPF1). UPF1 is an ATP driven RNA helicase critical for initiation of non-sense mediated mRNA decay (NMD). Read-through efficiency also correlated with an increase in PTC containing transcripts after treatment. This study is the first to elucidate mechanisms that govern read-through of PTC in human skin cells and forms the basis for development of future therapeutic approaches for genetic diseases caused by PTC mutations.