Setup and Validation of a Targeted Next-Generation Sequencing Approach for the Diagnosis of Lysosomal Storage Disorders

Abstract

Lysosomal storage disorders (LSDs) are monogenic diseases, due to accumulation of specific undegraded substrates into lysosomes. LSD diagnosis could take several years because of both poor knowledge of these diseases and shared clinical features. The diagnostic approach includes clinical evaluations, biochemical tests, and genetic analysis of the suspected gene. In this study, we 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. Disease-causing variants were identified 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: one of them was diagnosed. Conserved intronic fragment analysis, performed in cases unresolved by the first-level analysis, evidenced no candidate intronic variants. Targeted sequencing has low sequencing costs and short sequencing time. However, a coverage >60× to 80× must be ensured and/or Sanger validation should be performed. Moreover, it must be supported by a thorough clinical phenotyping. Lysosomal storage disorders (LSDs) are monogenic diseases, due to accumulation of specific undegraded substrates into lysosomes. LSD diagnosis could take several years because of both poor knowledge of these diseases and shared clinical features. The diagnostic approach includes clinical evaluations, biochemical tests, and genetic analysis of the suspected gene. In this study, we 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. Disease-causing variants were identified 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: one of them was diagnosed. Conserved intronic fragment analysis, performed in cases unresolved by the first-level analysis, evidenced no candidate intronic variants. Targeted sequencing has low sequencing costs and short sequencing time. However, a coverage >60× to 80× must be ensured and/or Sanger validation should be performed. Moreover, it must be supported by a thorough clinical phenotyping. Lysosomal storage disorders (LSDs) are a group of >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. Although singularly considered rare, the combined birth prevalence of LSD is estimated from 7.5 to 23.5 per 100,000 live births.1Kingma S.D.K. Bodamer O.A. Wijburg F.A. Epidemiology and diagnosis of lysosomal storage disorders: challenges of screening.Best Pract Res Clin Endocrinol Metab. 2015; 29: 145-157Crossref PubMed Scopus (58) Google Scholar Clinical signs and symptoms may occur from the prenatal period to adulthood, and may develop at different progression rate, according to the pathology, leading to a wide spectrum of disease forms, from mild to extremely severe, that in most cases affect the neurologic compartment.2Greiner-Tollersrud O.K. Berg T. Lysosomal storage disorders.in: Lysosomes: Medical Intelligence Unit. Springer, Boston, MA2005: 60-73Crossref Google Scholar 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.3Filocamo M. Morrone A. Lysosomal storage disorders: molecular basis and laboratory testing.Hum Genomics. 2011; 5: 156-169Crossref PubMed Scopus (69) Google Scholar Finally, if an enzyme deficit is detected, genetic analysis is performed on the suspected gene. However, this diagnostic route presents several limitations. In fact, some LSDs often share clinical signs and symptoms with other LSDs or different disorders; thus, their identification requires deep clinical expertise. Moreover, the above-mentioned biochemical methods are laborious, and they are often subject to high variability. Specifically, the execution of multiple enzyme assays may be expensive, and fluorogenic substrates to perform them may present scarce availability. Finally, disorders present with of storage this may the diagnosis in some cases, could be and take several years or could be A. M. M. M. of a targeted as a tool in the diagnosis of lysosomal storage PubMed Scopus Google A. A. M. 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