Cas9-targeted long-read sequencing of the entire COL7A1 genomic region uncovers revertant mosaicism in recessive dystrophic epidermolysis bullosa

Summary: Abstract Body: Revertant mosaicism (RM) is a phenomenon in which disease-causing germline variants are spontaneously corrected in somatic cells. Epidermolysis bullosa (EB) is a group of congenital skin fragility disorders caused by variants in genes encoding epidermal basement membrane zone proteins. Some EB patients develop clinically intact skin spots resulting from RM (revertant skin), which do not show skin fragility, highlighting epidermal autografts derived from these spots as a promising therapeutic option. However, conventional techniques have difficulty confirming RM in these clinically revertant skin spots. Nanopore Cas9-targeted sequencing (nCATS) enables the enrichment and sequencing of specific genomic DNA (gDNA) regions by combining Cas9 cleavage, adapter ligation, and MinION sequencing. Recently, our laboratory confirmed RM in clinically revertant skin from a recessive dystrophic EB (RDEB) patient using this technique. We established an nCATS strategy to enrich the entire 31-kb COL7A1 genomic region for the phasing of its variants. This region was successfully enriched in gDNA obtained from peripheral blood of healthy individuals and RDEB patients. We then applied this method to gDNA extracted from autologous cultured epidermal sheets derived from clinically revertant skin in three RDEB patients. The 31-kb nCATS approach succeeded in phasing the COL7A1 variants and identified RM-induced wild-type allele reads in at least one patient. In contrast, immunofluorescence studies failed to verify RM because COL7 staining was seen in both the clinically revertant skin and the lesional skin. This failure is likely due to leaky protein expression associated with missense or splice-site COL7A1 variants. Our study illuminates nCATS as a feasible strategy for detecting RM and supports the development of epidermal autografts for EB therapy. Takashi Seo¹, Shota Takashima¹, Daiki Kumakura^{2, 3}, Shinji Nakaoka², Takuma Nohara¹, Mika Watanabe¹, Hideyuki Ujiie¹, Ken Natsuga¹ 1. Dermatology, Hokkaido Daigaku, Sapporo, Hokkaido Prefecture, Japan. 2. Laboratory of Mathematical Biology, the Graduate School of Life Science, Hokkaido Daigaku, Sapporo, Hokkaido Prefecture, Japan. 3. iTEMS, Rikagaku Kenkyujo, Wako, Saitama Prefecture, Japan. Genetic Disease, Gene Regulation, Gene Therapy & Epigenetics