Assessing Lysosomal Disorders in the NGS Era: Identification of Novel Rare Variants

Abstract

Lysosomal storage diseases (LSDs) are a heterogeneous group of genetic disorders with variable degrees of severity and a broad phenotypic spectrum, which may overlap with a number of other conditions. While individually rare, as a group LSDs affect a significant number of patients, placing an important burden on affected individuals and their families but also on national health care systems worldwide. Here, we present our results on the use of an in-house customized next-generation sequencing (NGS) panel of genes related to lysosome function as a first-line molecular test for the diagnosis of LSDs. Ultimately, our goal is to provide a fast and effective tool to screen for virtually all LSDs in a single run, thus contributing to decrease the diagnostic odyssey, accelerating the time to diagnosis. Our study enrolled a group of 23 patients with variable degrees of clinical and/or biochemical suspicion of LSD. Briefly, NGS analysis data workflow, followed by segregation analysis allowed the characterization of approximately 41% of the analyzed patients and the identification of 10 different pathogenic variants, underlying nine LSDs. Importantly, four of those variants were novel, and, when applicable, their effect over protein structure was evaluated through in silico analysis. One of the novel pathogenic variants was identified in the GM2A gene, which is associated with an ultra-rare (or misdiagnosed) LSD, the AB variant of GM2 Gangliosidosis. Overall, this case series highlights not only the major advantages of NGS-based diagnostic approaches but also, to some extent, its limitations ultimately promoting a reflection on the role of targeted panels as a primary tool for the prompt characterization of LSD patients.

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

“…This approach allowed for both lowering the costs and enhancing the diagnostic effectiveness. Recent studies reported NGS-based analyses in LD genetic diagnosis [ 8 , 9 , 10 , 11 ]. CNV detection was reported in only one study that included 28 LD genes [ 11 ].…”

“…The analysis of the captured sequence takes into account the clinical context. In this perspective, we defined five overlapping sub-panels for sequence analysis (Figure 1): Organomegaly (27 genes), neurological impairment (38 genes), bone abnormalities (23 genes), neuronal ceroid lipofuscinoses (10 genes), and cherry red spots (8).…”

“…Next generation sequencing (NGS) technologies represent an essential tool for rapid and effective diagnosis of these diseases and may be used in some complex situations prior to multiple and often sequential functional studies. Recent studies highlighted the clinical utility of NGS approach for LD genetic diagnosis [ 8 , 9 , 10 , 11 ]. Here we report on the design, validation and testing of an NGS panel for genes involved in LDs named LysoGene.…”

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

“…Fortunately, the advent of next-generation sequencing (NGS) is revolutionizing the field of both diagnosis and screening, playing a relevant role as a genetic support tool in the diagnosis of LSDs always in combination with biochemical and clinical data [ 11 ]. With NGS, the simultaneous analysis of large numbers of genetic loci and samples can drive down costs and turnaround time; laboratory processes can largely be automated, and a single assay can be used to screen a set of disorders (regardless of whether a biochemical marker is available) [ 12 , 13 ].…”

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

“…In the past decade, the emergence of next generation sequencing (NGS) technologies has proven to be an effective alternative to conventional techniques, in both research and clinical settings, allowing for the simultaneous interrogation of several genes in one single reaction and reducing, considerably, the time and costs for Sanger sequencing of a single gene [9,10]. The introduction of ad hoc designed genetic tests (targeted NGS panels) into diagnostic workflows offers the opportunity for easier identification of LSDs, timely diagnosis, and optimized clinical management, reducing the psychological burden and providing appropriate genetic counseling to parents [4].…”