Background: Epidermolysis Bullosa (EB) is caused by mutations in genes that encode proteins belonging to the epidermal-dermal
junction assembly. Due to the extreme clinical/genetic heterogeneity of the disease, the current methods available
for diagnosing EB involve immunohistochemistry of bioptic samples and transmission electron microscopy followed
by single candidate gene Sanger Sequencing (SS), which are labour intensive and expensive clinical pathways.
Objectives: According to the recently published recommendations for the EB diagnosis and treatment, the assessment of
the mutational landscape is now a fundamental step for developing a comprehensive diagnostic path. Next-Generation
Sequencing (NGS) via the parallel ultra-deep sequencing of many genes represents a proper method for reducing the
processing time and costs of EB diagnostics.
Methods: We developed an EB disease-comprehensive AmpliSeq panel to accomplish the NGS on the Ion Torrent PGM
platform. The panel was performed on ten patients with known genetic diagnoses and was then employed in eight family
trios with unknown molecular footprints.
Results: The panel was successful in finding the causative mutations in all ten of the patients with known mutations, fully
confirming the SS data and providing proof of concept of the sensitivity, specificity, and accuracy of this procedure. In
addition to being consistent with the clinical diagnosis, it was also effective in the trios, identifying all of the variants, including
ones that the SS missed or de novo mutations.
Conclusions: The NGS and AmpliSeq were shown to be an effective approach for the diagnosis of EB, resulting in a costand
time-effective 72-hour procedure
