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

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.

Clinical and Molecular Characterization of Mucopolysaccharidosis Type 3A and 3B in a Turkish Series

“…Genomic DNA of the 15 patients and their non-affected parents was extracted from peripheral blood using QIAamp DNA Blood Mini Kit ® (Qiagen) or the QuickGene-610L platform (Kurabo Biomedical, FujiFilm). SGSH and NAGLU genes were sequenced using LysoGene panel, which includes 52 lysosomal genes [12]. This next-generation sequencing-based method is implemented on an Illumina ® platform (San Diego, CA, USA).…”

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

“…CNVs in MPS-related genes have been characterized and encompass: (i) deletion of exon 14-3′UTR, and duplication of exon 2-intron 12 in IDUA in patients affected by MPS I [ 42 , 43 ]; (ii) deletion of SGSH exons 1–5 in MPS IIIA patients [ 44 ]; (iii) Alu -mediated deletion of NAGLU exons 3–4 in patients with MPS IIIB/Sanfilippo type B syndrome [ 45 , 46 ]; (iv) heterozygous deletion of exon 15 [ 47 ] and homozygous deletion of exons 9–10 in HGSNAT in MPS IIIC or Sanfilippo type C patients [ 43 ]; (v) deletions of GNS exon 1, 2–3, 6–7, 9–14 in patients affected by MPS IIID/Sanfilippo disease type D [ 48 , 49 ]; (vi) deletion of multiple contiguous exons including 1–3, 2–4, 2–5, 3–14, 5–8, 9–14, 10–14, 11–12, and single exon 5 and 13 of GALNS in patients with MPS IVA/Morquio A [ 43 , 50 , 51 , 52 , 53 ]; (vii) ARSB deletion of exons 2–3, exon 4 and exon 5 MPS VI patients [ 54 , 55 , 56 ].…”

Genetic Testing for Rare Diseases

“…Sudrié-Arnaud et al [19] designed a custom gene panel sequencing including 51 genes responsible for lysosomal disorders and validated it in 21 well-characterized patients. The bioinformatic pipelines used were also validated to detect single nucleotide variants, copy number variants and indels.…”