Amplicon-based Next Generation Sequencing: an effective approach to molecular diagnosis of Epidermolysis Bullosa

Epidermolysis Bullosa (EB) is caused by mutations in genes encoding for proteins of the epidermal–dermal junction assembly. Due to the extreme clinical/genetic heterogeneity of the disease, current methods in EB diagno- stics comprise immunohistochemistry on bioptic samples and transmission electron microscopy followed by single candidate gene Sanger Sequencing (SS) that therefore represents the final phase of a labour intensive and ex- pensive clinical pathway. Methods: Participants in a cross sectional study included individuals with Muenke syndrome (P250R mutation in FGFR3) and their mutation negative siblings. Participants completed validated assessments of executive functio- ning (Behavior Rating Inventory of Executive Function; BRIEF) and adaptive behavior skills (Adaptive Behavior Assessment System; ABAS-II). According to the recently published recommendations for diagnosis and treatment in EB, the assessment of mutational landscape is instead a fun- damental step to a comprehensive diagnosis path; Next Generation Sequen- cing (NGS), throughout parallel ultra-deep sequencing of many genes, would represent a proper method for reducing timing and costs in EB diagnostics. We developed an EB disease-comprehensive amplicon panel (AmpliSeq pa- nel), to accomplish NGS onto Ion Torrent PGM platform. The panel was dealt on ten patients with known genetic diagnosis, and then employed in eight family trios with unknown molecular footprinting. Results: Forty-four FGFR3 mutation positive individuals, median age 9, range 6 months to 52 years were evaluated with the BRIEF and ABAS-II. Additio- nally, 10 unaffected siblings were used as controls. For the General Executive Composite scale of the BRIEF, 32.1% of the cohort had scores greater than +1.5 SD, signifying “Potential Clinical Significance.” For the General Adaptive Composite of the ABAS-II, 28.2% of affected individuals scored in the “Ex- tremely Low” category” (3rd -8th percentile of normative population) and 53.9% were below the “Average” category (less than the 25th percentile). Multiple regression analysis showed that the presence of craniosynostosis was not a predictor (P = 0.7) of BRIEF and ABAS-II scores. The AmpliSeq panel, obtaining a proof of concept of the sensitivity, specificity, and accuracy of this kind of procedure, showed successful in finding the causative mutations in all the ten patients with known mutations, fully confirming SS data. Besides, showing consistent with the clinical diagnosis, it was effective in trios, identifying all the variants, even the ones SS missed or in case of de novo mutations. NGS