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

Abstract

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.

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

“…Developing a powerful diagnostic tool could mitigate the delayed diagnostic process for affected families, leading to better outcomes for current therapies and providing the basis for more appropriate genetic counseling. Many recent reports have emphasized the high clinical utility of NGS technologies and targeted gene panels in the diagnosis of suspected LSDs and their potential to reduce diagnostic delay [11][12][13][14][15][16][17].…”

Detection of Structural Variants by NGS: Revealing Missing Alleles in Lysosomal Storage Diseases

“…Specific user-friendly web-applications, adapted for custom-designed tNGS panels and dedicated to CNVs detection, have been recently developed to perform the analysis of SNVs, CNVs and indels in a single integrated workflow. In Figure 1 and Figure 2 , we show examples of CNVs detection by using tNGS with two panels we have previously used to scan the coding regions of genes implicated in LSDs [ 5 , 129 ]. Deep sequencing data were analyzed with the user-friendly web-application Ion Reporter Software developed by Ion Torrent, which relies on normalized read coverage (DoC) across amplicons to predict also the copy number state, corrected for GC bias and compared with a baseline coverage constructed by the user from control samples with known ploidy.…”

“…Figure 2 shows two further genomic losses involving GALC (panel a) and GAA (panel b) exons present in DNA samples from patients affected by Krabbe and Pompe diseases, respectively. Sequencing reads were generated by using a custom developed NGS panel targeting six LSDs-related genes, previously described in [ 5 ]. The first CNV (panel a) is a homozygous deletion of ~16 kb in GALC (ploidy state 0) resulting in the loss of exons 11–17, successfully identified by the Ion Reporter Software developed by Ion Torrent.…”

“…From a genetic point of view, LSDs are Mendelian monogenic disorders, inherited mainly in an autosomal recessive or in an X-linked manner (i.e., Fabry, Hunter, and Danon diseases), and are caused by mutations in genes encoding for acid hydrolases, integral membrane proteins, activators and transporter proteins, or other accessory proteins related to lysosomal function [ 2 , 5 ]. Although biochemical enzyme assays are considered the gold standard for diagnosis of symptomatic patients, genotyping is a requirement for inclusion in enzyme replacement programs and is a prerequisite for carrier tests in blood relatives and DNA-based prenatal diagnosis.…”

The Utility of Genomic Testing for Hyperphenylalaninemia

“…However, traditional differential diagnosis methods are time consuming and are nowadays inadequate to capture the extensive genetic heterogeneity of HPA. In this context, high-throughput technologies, such as multiplex ligation-dependent probe amplification, DNA microarray and next-generation sequencing (NGS), allow the simultaneous analysis of multiple genetic variants associated with this heterogeneous disorder and, thus, optimize patient care and management [8][9][10][11][12][13].…”