The lysosomal storage disorders (LSD) are a diverse group of inherited conditions caused by the defective function of specific lysosomal proteins. It is estimated that there are more than 50 different types. The disease course of these conditions is generally progressive, but the age of onset, nature and severity of symptoms can be variable between the disorders. Common symptoms of lysosomal disorders include hepatosplenomegaly, coarsening of facial features, short stature, corneal clouding, dysostosis multiplex and neurological regression. The combined prevalence of LSDs is estimated to be 1 in 7,000 births.

Sequencing is performed on genomic DNA using an Agilent targeted sequence capture method to enrich for the exome. Direct sequencing of the amplified captured regions was performed using 2X150bp reads on Illumina next generation sequencing (NGS) systems. A base is considered to have sufficient coverage at 20X and an exon is considered fully covered if all coding bases plus three nucleotides of flanking sequence on either side are covered at 20X or more. A list of these regions, if any, is available upon request. Alignment to the human reference genome (GRCh37) is performed and annotated variants are identified in the targeted region. Variants reviewed have a minimum coverage of 8X and an alternate allele frequency of 20% or higher. Indel and single nucleotide variants (SNVs) may be confirmed by Sanger sequence analysis before reporting at director discretion. This assay cannot detect variants in regions of the exome that are not covered, such as deep intronic, promoter and enhancer regions, areas containing large numbers of tandem repeats, and variants in mitochondrial DNA. Copy number variation (CNV) analysis detects deletions and duplications; in some instances, due to the size of the exons, sequence complexity, or other factors, not all CNVs may be analyzed or may be difficult to detect. When reported, copy number variant size is approximate. Actual breakpoint locations may lie outside of the targeted regions. CNV analysis will not detect tandem repeats, balanced alterations (reciprocal translocations, Robertsonian translocations, inversions, and balanced insertions), methylation abnormalities, triploidy, and genomic imbalances in segmentally duplicated regions. This assay is not designed to detect mosaicism; possible cases of mosaicism may be investigated at the discretion of the laboratory director. Primary data analysis is performed using Illumina bcl2fastq converter v2.19. Secondary analysis is performed using Illumina DRAGEN Bio-IT Platform v.3.10.8. Tertiary data analysis is performed using SnpEff v5.0 and Revvity Omics’ internal ODIN v.1.01 software. CNV and absence of heterozygosity are assessed using BioDiscovery’s NxClinical v6.1 software.

Long-range PCR is performed to capture the true GBA genomic sequence from this individual’s genomic DNA to avoid pseudogene contaminants. PCR product was utilized to prepare the library and further sequenced using NGS technology on an Illumina system with 150 base pair paired-end reads. Indels and SNVs are confirmed by Sanger sequence analysis before reporting at director discretion. This analysis cannot detect variants in regions not analyzed such as promoters, deep intronic regions, or long repetitive regions. CNV analysis was assessed using MLPA or by manual review of NGS reads using IGV viewer. This analysis cannot determine the location or orientation of a duplication. Copy neutral gene conversion or fusion, complex rearrangement, or large deletion including the entire GBA gene with breakpoint(s) outside the targeted region may not be detected by this assay. This assay is not designed to detect mosaicism; possible cases of mosaicism may be investigated at the discretion of the laboratory director. Primary data analysis is performed using Illumina bcl2fastq converter v2.19. Secondary analysis is performed using Illumina DRAGEN Bio-IT Platform v.3.10.8. Tertiary data analysis is performed using SnpEff v5.0 and Revvity Omics’ internal ODIN v.1.01 software.