Next-Generation Molecular Investigations in Lysosomal Diseases: Clinical Integration of a Comprehensive Targeted Panel.

Bénédicte Sudrié-Arnaud,Axel Lebas,Lenaig Abily-Donval,Sarah Snanoudj,Hélène Dranguet,Marc Duval-Arnould,Isabelle Marie,Abdellah Tebani,Ivana Dabaj,Soumeya Bekri,Stéphane Marret,Bénédicte Héron,Myriam Vezain

doi:10.3390/diagnostics11020294

Abstract

Diagnosis of lysosomal disorders (LDs) may be hampered by their clinical heterogeneity, phenotypic overlap, and variable age at onset. Conventional biological diagnostic procedures are based on a series of sequential investigations and require multiple sampling. Early diagnosis may allow for timely treatment and prevent clinical complications. In order to improve LDs diagnosis, we developed a capture-based next generation sequencing (NGS) panel allowing the detection of single nucleotide variants (SNVs), small insertions and deletions, and copy number variants (CNVs) in 51 genes related to LDs. The design of the LD panel covered at least coding regions, promoter region, and flanking intronic sequences for 51 genes. The validation of this panel consisted in testing 21 well-characterized samples and evaluating analytical and diagnostic performance metrics. Bioinformatics pipelines have been validated for SNVs, indels and CNVs. The clinical output of this panel was tested in five novel cases. This capture-based NGS panel provides an average coverage depth of 474× which allows the detection of SNVs and CNVs in one comprehensive assay. All the targeted regions were covered above the minimum required depth of 30×. To illustrate the clinical utility, five novel cases have been sequenced using this panel and the identified variants have been confirmed using Sanger sequencing or quantitative multiplex PCR of short fluorescent fragments (QMPSF). The application of NGS as first-line approach to analyze suspected LD cases may speed up the identification of alterations in LD-associated genes. NGS approaches combined with bioinformatics analyses, are a useful and cost-effective tool for identifying the causative variations in LDs.

Highlights

The lysosome is an intracellular organelle characterized by its acidic pH, and its main function consists in degradation of intra or extracellular macromolecules into monomers.This metabolic process is carried out by more than fifty lysosomal enzymes
Twenty-one well-characterized lysosomal disorders (LDs) patients have been included for validation purposes (Supplementary Table S1)
Twenty-seven disease-causing variations and 50 benign variations have been previously identified by Sanger sequencing and were used for validation of the single nucleotide variants (SNVs) and small insertions/deletions sequencing process and the bioinformatics pipeline (Supplementary Tables S1 and S2)

Summary

Introduction

The lysosome is an intracellular organelle characterized by its acidic pH, and its main function consists in degradation of intra or extracellular macromolecules into monomers. This metabolic process is carried out by more than fifty lysosomal enzymes. Over a hundred structural proteins and carriers essential for lysosomal function have been identified [1]. “Lysosomal storage disorders” (LSD) was the conventional term used to describe the group of inborn errors of metabolism (IEMs) related to the absence or failure of substrate degradation or transport, and their subsequent accumulation in the lysosome [2]. In recent years, the lysosome is being viewed as a dynamic structure with multiple roles in nutrient sensing, autophagy, apoptosis, and cellular response to environmental cues. It is a signaling hub that interacts with other organelles [3]

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