Setup and Validation of a Targeted Next-Generation Sequencing Approach for the Diagnosis of Lysosomal Storage Disorders

Lysosomal storage disorders (LSDs) are monogenic diseases, due to accumulation of specific undegraded substrates into lysosomes. LSD diagnosis could take several years because of both poor knowledge of these diseases and shared clinical features. The diagnostic approach includes clinical evaluations, biochemical tests, and genetic analysis of the suspected gene. In this study, we evaluated an LSD targeted sequencing panel as a tool capable to potentially reverse this classic diagnostic route. The panel includes 50 LSD genes and 230 intronic sequences conserved among 33 placental mammals. For the validation phase, 56 positive controls, 13 biochemically diagnosed patients, and nine undiagnosed patients were analyzed. Disease-causing variants were identified in 66% of the positive control alleles and in 62% of the biochemically diagnosed patients. Three undiagnosed patients were diagnosed. Eight patients undiagnosed by the panel were analyzed by whole exome sequencing: for two of them, the disease-causing variants were identified. Five patients, undiagnosed by both panel and exome analyses, were investigated through array comparative genomic hybridization: one of them was diagnosed. Conserved intronic fragment analysis, performed in cases unresolved by the first-level analysis, evidenced no candidate intronic variants. Targeted sequencing has low sequencing costs and short sequencing time. However, a coverage >60× to 80× must be ensured and/or Sanger validation should be performed. Moreover, it must be supported by a thorough clinical phenotyping.