Targeted Next-Generation Sequencing Validated for Lysosomal Storage Disorders Diagnosis

Lysosomal storage disorders (LSDs) are a group of more than 50 inherited rare disorders characterized by the accumulation of specific undegraded metabolites in the lysosomes. This overstorage is commonly caused by a deficient or absent activity of one of the many lysosomal hydrolases or, in a few cases, by the deficit of other non-enzymatic lysosomal proteins.

Generally, the diagnostic approach includes an accurate clinical evaluation, which leads to the formulation of a suspicion for one or more LSDs. This is followed by biochemical tests, aimed to detect the storage products in body fluids, whose results may orient the following enzymatic analyses. Finally, if an enzyme deficit is detected, genetic analysis is performed on the suspected gene.


Scientists specializing in LSDs from the University of Padova (Padova, Italy) 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.

The investigators used the Ion AmpliSeq platform (Thermo Fisher Scientific, Waltham, MA; USA) for the design of a panel including the selected genes. DNA library preparation was performed according to the Thermo Fisher Scientific Ion AmpliSeq Library Preparation protocol in combination with the Ion AmpliSeq Library kit version 2.0. After DNA quantification using the Qubit dsDNA HS Assay Kit, the libraries were constructed starting from 10 ng of each DNA sample. The first step of target amplification was performed by using our AmpliSeq LSD-Panel Primer pools. The amplicons were then indexed using the Ion Xpress Barcode Adapters kit and purified using AMPure XP magnetic beads (Beckman Coulter, Inc., Brea, CA, USA).

The team identified disease-causing variants 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 and one of them was diagnosed. Conserved intronic fragment analysis, performed in cases unresolved by the first-level analysis, evidenced no candidate intronic variants.

The authors concluded that targeted sequencing is an appealing approach to implement routine diagnostic strategy, given its low sequencing costs and short sequencing time. However, a good coverage must be ensured and, when this is not reached, validation by Sanger sequencing needs to be performed on the proband and on the parents as final step, also to exclude the presence of deletions in cases of homozygous variant finding. The study was published in the April 2020 issue of the Journal of Molecular Diagnostics.