Recessive dystrophic epidermolysis bullosa (RDEB) is an intractable skin genetic disease characterized by the skin fragility leading to the recurrent blistering. The mutations in COL7A1 gene, encoding type VII collagen (C7) that forms anchoring fibrils, cause RDEB. RDEB patients are known to show variable prognosis, likely influenced by the position of pathogenic mutations. However, as the mutations in COL7A1 gene distribute across the gene without any apparent hotspots and the majority of RDEB patients harbor the mutations in a compound heterozygous manner, the genotype-phenotype correlation is not fully elucidated. Here, we propose a fast and robust framework to understand how each pathogenic mutation impact of the function of type VII collagen using i-GONAD, a CRISPR based method for rapid genome editing. i-GONAD removes time- and cost-consuming steps in generating genetically modified mice and enables to establish multiple genetically modified mice in a single laboratory. As a proof-of-concept study, we chose three COL7A1 mutations relatively common in Japanese RDEB patients (E2857X, c.5818delC, and 6573+1G>C). Using i-GONAD, we introduced these three mutations to the corresponding locations in the mouse genome. Mice with the 5818delC type mutation in a homozygous manner died soon after birth with blistering. The mice with E2857X and 6573+1G>C type homozygous mutations showed milder RDEB related phenotype. Mice with the c.5818delC/E2857X mutations, most of the RDEB patients have mutations in a compound heterozygous manner, presented an intermediate phenotype between the c.5818delC and E2857X homozygous mice. These phenotypes in each mouse well recapitulated the phenotypes seen in human patients. Together, we have shown that our method is a powerful tool to analyze how the COL7A1 mutations detected in RDEB patients impact on the skin integrity. We believe the proposed strategy will facilitate studies to develop a better RDEB therapy.

ESDR295 - Genotype-phenotype correlation analysis in recessive dystrophic epidermolysis bullosa using in situ germline mouse genome editing technique

The scientific meeting of the European Society for Dermatological Research (ESDR) is held yearly, usually in September. With an international attendance of more than 1000 skin scientists from around the globe, the four-day event includes short oral presentations from peer-reviewed abstracts, keynote lectures from international experts, sponsored symposia, satellite programs, educational events as well as an important focus on posters, networking and building collaborations amongst scientists. For more information about this year’s meeting visit our website.