Abstract
BackgroundWith over 50 different disorders and a combined incidence of up to 1/3000 births, lysosomal storage diseases (LSDs) constitute a major public health problem and place an enormous burden on affected individuals and their families. Many factors make LSD diagnosis difficult, including phenotype and penetrance variability, shared signs and symptoms, and problems inherent to biochemical diagnosis. Developing a powerful diagnostic tool could mitigate the protracted diagnostic process for these families, lead to better outcomes for current and proposed therapies, and provide the basis for more appropriate genetic counseling.MethodsWe have designed a targeted resequencing assay for the simultaneous testing of 57 lysosomal genes, using in-solution capture as the enrichment method and two different sequencing platforms. A total of 84 patients with high to moderate-or low suspicion index for LSD were enrolled in different centers in Spain and Portugal, including 18 positive controls.ResultsWe correctly diagnosed 18 positive blinded controls, provided genetic diagnosis to 25 potential LSD patients, and ended with 18 diagnostic odysseys.ConclusionWe report the assessment of a next–generation-sequencing-based approach as an accessory tool in the diagnosis of LSDs, a group of disorders which have overlapping clinical profiles and genetic heterogeneity. We have also identified and quantified the strengths and limitations of next generation sequencing (NGS) technology applied to diagnosis.
Highlights
Lysosomal storage disorders (LSDs) are rare diseases with a combined incidence of ~1 in 1500 to 7000 live births [1,2]
Tests for elevated levels of secreted substrate material are routinely used to examine the pattern of glycosaminoglycans and oligosaccharides in patients suspected of having mucopolysaccharidoses (MPS) or disorders that present with oligosacchariduria
Enzyme activity detected in blood spots, either individually or simultaneously, is useful in the diagnosis of a small number of lysosomal storage diseases (LSDs), but needs verification with a second type of assay; while measurement of enzyme activity in leukocytes and plasma serves this purpose for most LSDs, a proportion of cases may not be detected using this method
Summary
Ethics statement The study protocol adhered to the tenets of the Declaration of Helsinki and was approved by the local Ethics Committee (Comité Ético de Investigación Clínica de Galicia - CEIC). HiSeq2000 platform The library preparation for capturing of selected DNA regions was performed according to the SureSelect XT Target Enrichment System protocol for Illumina pairedend sequencing (Agilent). The DNA with adaptor-modified ends was PCR amplified (6 cycles, Herculase II fusion DNA polymerase) with SureSelect primers, quality controlled using the DNA 7500 assay specific for a library size of 250–350 bp, and hybridized for 24 hr at 65°C. The final library size and concentration was determined using an Agilent 2100 Bioanalyzer 7500 chip and sequenced on an Illumina HiSeq 2000 platform with a paired-end run of 2 × 76 bp, following the manufacturer’s protocol. Variant calling was performed using two software programs in parallel: the diBayes alignment algorithm embeded in the Bioscope suite [18] and the Genome Analysis Toolkit (GATK) v1.5, a software package developed at the Broad Institute (Cambridge, MA) to analyze next-generation resequencing data [19]. To evaluate the possible effect of synonymous variant in gene splicing we used the Human Splicer Finding web tool [30]
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