Amplicon-based next-generation sequencing: An effective approach for the molecular diagnosis of epidermolysis bullosa

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 biopsy 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 diagnosis and treatment of EB, the assessment of the mutational landscape is now a fundamental step for developing a comprehensive diagnostic path. We aimed to develop a customized, cost-effective amplicon panel for the complete and accurate sequencing of all the pathogenic genes already identified in EB, and to minimize the processing time required for the execution of the test and to refine the analysis pipeline to achieve cost-effective results from the perspective of a routine laboratory set-up. 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. Materials and methods We developed an EB disease-comprehensive AmpliSeq panel to accomplish the NGS on an Ion Torrent Personal Genome Machine platform. The panel was performed on 10 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 10 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 cost- and time-effective 72-h procedure. What's already known about this topic? Skin microscopy and Sanger sequencing (SS) are valuable diagnostic tools for the diagnosis of epidermolysis bullosa (EB), although in some cases provide suboptimal specificity and sensitivity for an accurate diagnosis and are labour intensive and expensive. Whole-exome sequencing (WES) is an extremely effective technique able to refine and improve the diagnosis of EB, overcoming the limits of skin microscopy and SS. WES is a time-consuming procedure, is not yet cost-effective and needs particularly intensive data management. What does this study add? Amplicon-based targeted next-generation sequencing (NGS) is an extremely accurate diagnostic tool. Amplicon-based targeted NGS meets the needs of medium-sized diagnostics laboratories, offering a very fast and cost-effective sample processing of EB. Amplicon-based NGS can be fully disease-customized and simplifies data interrogation managing