Figure 1

Compound heterozygous mutations in patient (EB) fibroblasts lead to reduced C7 expression. (a) c.5047 C > T on allele 1 is a nonsense mutation which changes Arginine to a stop codon, leading to a truncated mRNA transcript which is degraded by nonsense-mediated decay. This typically leads to no C7 produced from the affected allele and the blistering phenotype. (b) Schematic of the 2 alleles of the COL7A1 gene in patient EB, illustrating compound heterozygosity. The c.5047 C > T mutation on allele 1 is in Exon 54 and is the mutation of interest. The c.7344 + 1 G > A mutation on allele 2 is in Intron 95 of the gene. (c) Sanger sequencing of DNA isolated from EB fibroblasts confirms the nonsense mutation in Exon 54 in comparison to wild-type sequences from Wild Type fibroblast (WT) controls. (d) Immunostaining for C7 reveals markedly reduced expression in EB fibroblasts compared to normal. Representative images taken at 40× magnification shown. Scale bar is 20 µm. (e) Quantification of relative immunofluorescence (IF) intensity of C7 demonstrates a significant reduction in C7 in EB cells compared to WT. t-test performed, p value < 0.0001. Western Blot analysis found EB fibroblasts secrete approximately 50% of normal levels of full length C7 into cell medium (f) and produce 50% of normally expressed full-length C7 in the cell lysate (g). Total protein loading was controlled using Ponceau S for cell lysate and cell medium. 3 technical replicates and t test performed showing significance (p value < 0.05). Full blots are presented in Supplementary Fig. S1b–e.