Identification of deep intronic variants in junctional epidermolysis bullosa using whole genome sequencing and splicing assays

Abstract Junctional epidermolysis bullosa (JEB) are a group of recessive dermatoses characterized by mucocutaneous fragility and blister formation. We enrolled 69 recessive JEB cases, 13.0% JEB cases remain genetically undiagnosed following an initial whole exome sequencing. In cases carried COL17A1 mutations, this proportion can reach 31.6%. The aim of our study was to genetically diagnosis the JEB cases by employing whole genome sequencing where only one recessive mutation in targeted gene has been identified. Here, four distinct novel deep intronic mutations (c.4156+117G>A, c.2039-104G>A and c.1267+237dupC in COL17A1 gene and c.-38+2T>C promoter mutation in LAMB3 gene) had been identified in six cases. Of these, the c.4156+117G>A variant was found three out of five cases and may be a COL17A1 hotspot deep intronic variants in Chinese JEB. Minigene assays and in vivo studies revealed that these intronic mutations were indeed shown to exert a splicing defect by causes exon skipping or pseudoexon insertion into the transcript. Moreover, the three COL17A1 deep intronic variants demonstrated effect upon mRNA processing in HaCaT cells, not in HEK293 cells. We show that deep intronic mutations are not a rare occurrence, and firstly described to occur in JEB. Our results suggest the importance of selecting the right cell line for mRNA analysis.