A Comprehensive, Targeted NGS Approach to Assessing Molecular Diagnosis of Lysosomal Storage Diseases.

With over 60 different disorders and a combined incidence occurring in 1:5000-7000 live births, lysosomal storage diseases (LSDs) represent a major public health problem and constitute an enormous burden for affected individuals and their families. Several reasons make the diagnosis of LSDs an arduous task for clinicians, including the phenotype and penetrance variability, the shared signs and symptoms, and the uncertainties related to biochemical enzymatic assay results. Developing a powerful diagnostic tool based on next generation sequencing (NGS) technology may help reduce the delayed diagnostic process for these families, leading to better outcomes for current therapies and providing the basis for more appropriate genetic counseling. Herein, we employed a targeted NGS-based panel to scan the coding regions of 65 LSD-causative genes. A reference group sample (n = 26) with previously known genetic mutations was used to test and validate the entire workflow. Our approach demonstrated elevated analytical accuracy, sensitivity, and specificity. We believe the adoption of comprehensive targeted sequencing strategies into a routine diagnostic route may accelerate both the identification and management of LSDs with overlapping clinical profiles, producing a significant reduction in delayed diagnostic response with beneficial results in the treatment outcome.

Design and Validation of a Custom NGS Panel Targeting a Set of Lysosomal Storage Diseases Candidate for NBS Applications.

Lysosomal storage diseases (LSDs) are a heterogeneous group of approximately 70 monogenic metabolic disorders whose diagnosis represents an arduous challenge for clinicians due to their variability in phenotype penetrance, clinical manifestations, and high allelic heterogeneity. In recent years, the approval of disease-specific therapies and the rapid emergence of novel rapid diagnostic methods has opened, for a set of selected LSDs, the possibility for inclusion in extensive national newborn screening (NBS) programs. Herein, we evaluated the clinical utility and diagnostic validity of a targeted next-generation sequencing (tNGS) panel (called NBS_LSDs), designed ad hoc to scan the coding regions of six genes (GBA, GAA, SMPD1, IDUA1, GLA, GALC) relevant for a group of LSDs candidate for inclusion in national NBS programs (MPSI, Pompe, Fabry, Krabbe, Niemann Pick A-B and Gaucher diseases). A standard group of 15 samples with previously known genetic mutations was used to test and validate the entire flowchart. Analytical accuracy, sensitivity, and specificity, as well as turnaround time and costs, were assessed. Results showed that the Ion AmpliSeq and Ion Chef System-based high-throughput NBS_LSDs tNGS panel is a fast, accurate, and cost-effective process. The introduction of this technology into routine NBS procedures as a second-tier test along with primary biochemical assays will allow facilitating the identification and management of selected LSDs and reducing diagnostic delay.