Fig. 3

RNA sequencing, performed in addition to WES, determined pathogenic variants in PT41. a The proband has multiple pruritic nodules and vesicles on the lower legs as well as dystrophy of the toenails. The clinical presentation is consistent with EB pruriginosa. b All four disease-associated variants in COL7A1 have CADD scores < 10. c RNA sequencing shows that c.5820 + 4A > G leads to the retention of intron 70 without the deletion of exon 70. d RT-PCR plus Sanger sequencing shows that c.5820 + 4A > G results in the deletion of exon 70 but not the retention of intron 70. e The proband inherited c.5820 + 4A > G from his mother and c.3562G > A from his father. f Staining with LH7.2, which binds to amino acid 733, is comparable between the proband and the control. In contrast, staining with a homemade polyclonal antibody that reacts with the human vWFA2 domain of C7 is reduced in the proband (× 400 magnification). This indicates that while C7 expression is not reduced, c.3562G > A (p.Val1188Met) might have caused major conformational changes in the vWFA2 region that impaired the function of C7