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. 2014 Apr 25:9:59.
doi: 10.1186/1750-1172-9-59.

Assessment of a targeted resequencing assay as a support tool in the diagnosis of lysosomal storage disorders

Ana Fernández-Marmiesse  1 , Marcos Morey, Merce Pineda, Jesús Eiris, Maria Luz Couce, Manuel Castro-Gago, Jose Maria Fraga, Lucia Lacerda, Sofia Gouveia, Maria Socorro Pérez-Poyato, Judith Armstrong, Daisy Castiñeiras, Jose A Cocho
Affiliations

Assessment of a targeted resequencing assay as a support tool in the diagnosis of lysosomal storage disorders

Ana Fernández-Marmiesse et al. Orphanet J Rare Dis. .

Abstract

Background: With 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.

Methods: We 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.

Results: We correctly diagnosed 18 positive blinded controls, provided genetic diagnosis to 25 potential LSD patients, and ended with 18 diagnostic odysseys.

Conclusion: We 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.

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Figures

Figure 1
Figure 1
Global flowchart of the filtering pipeline used for selection of most likely pathogenic mutations, starting from SOLiD4 raw data. (NRA: non-reference allele).
Figure 2
Figure 2
Heterozygous macrodeletions detected by LSD-NGS. A) GLB1 exon 5 deletion; B) CLN3 exon 7 + 8 deletion. The area of the coverage peak for each deleted exon is half that seen in the corresponding control sample.
Figure 3
Figure 3
Diagnosis achieved using NGS-LSD. (1x: one mutation found; 2x: two mutations found in the same gene; VCS: variant calling software; ??: not confirmed.)
See this image and copyright information in PMC

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