Early detection of lysosomal diseases by screening of cases of idiopathic splenomegaly and/or thrombocytopenia with a next‐generation sequencing gene panel

Muñoz, Gloria; García-Seisdedos, David; Ciubotariu, Crina; Piris-Villaespesa, Miguel; Gandía, Marta; Martin‐Moro, Fernando; Gutiérrez-Solana, Luis González; Morado, Marta; López‐Jiménez, Javier; Sánchez-Herranz, Antonio; Villarrubia, Jesús; Castillo, Francisco J. del

doi:10.1002/jmd2.12078

Cited by 5 publications

(6 citation statements)

References 37 publications

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“…In particular, our results indicate that the use of methods suitable for the detection of CNV due to deleted or recombinant alleles became mandatory when mutations in homozygosity were identified by CES. Moreover, this approach should be considered when using NGS for the study of conditions associated with mutations in different genes, including GBA , such as Parkinson’s disease [ 33 ], dementia with Lewy bodies [ 34 ], lysosomal diseases [ 35 ], and inherited platelets disorders [ 36 ]. Finally, other genes that are characterized by the presence of high homologous sequences or pseudogenes might benefit from the adoption of this integrated molecular strategy.…”

Section: Discussionmentioning

confidence: 99%

Accurate Molecular Diagnosis of Gaucher Disease Using Clinical Exome Sequencing as a First-Tier Test

Zampieri

Cattarossi

Pavan

et al. 2021

IJMS

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Gaucher disease (GD) is an autosomal recessive lysosomal disorder due to beta-glucosidase gene (GBA) mutations. The molecular diagnosis of GD is complicated by the presence of recombinant alleles originating from a highly homologous pseudogene. Clinical exome sequencing (CES) is a rapid genetic approach for identifying disease-causing mutations. However, copy number variation and recombination events are poorly detected, and further investigations are required to avoid mis-genotyping. The aim of this work was to set-up an integrated strategy for GD patients genotyping using CES as a first-line test. Eight patients diagnosed with GD were analyzed by CES. Five patients were fully genotyped, while three were revealed to be homozygous for mutations that were not confirmed in the parents. Therefore, MLPA (multiplex ligation-dependent probe amplification) and specific long-range PCR were performed, and two recombinant alleles, one of them novel, and one large deletion were identified. Furthermore, an MLPA assay performed in one family resulted in the identification of an additional novel mutation (p.M124V) in a relative, in trans with the known p.N409S mutation. In conclusion, even though CES has become extensively used in clinical practice, our study emphasizes the importance of a comprehensive molecular strategy to provide proper GBA genotyping and genetic counseling.

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Section: Discussionmentioning

confidence: 99%

Accurate Molecular Diagnosis of Gaucher Disease Using Clinical Exome Sequencing as a First-Tier Test

Zampieri

Cattarossi

Pavan

et al. 2021

IJMS

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“…This approach allowed for both lowering the costs and enhancing the diagnostic effectiveness. Recent studies reported NGS-based analyses in LD genetic diagnosis [ 8 , 9 , 10 , 11 ]. CNV detection was reported in only one study that included 28 LD genes [ 11 ].…”

Section: Discussionmentioning

confidence: 99%

“…Recent studies reported NGS-based analyses in LD genetic diagnosis [ 8 , 9 , 10 , 11 ]. CNV detection was reported in only one study that included 28 LD genes [ 11 ]. Of note, the present work enabled the analysis of CNVs, not reachable by Sanger sequencing, for all the included 51 LD-related genes.…”

Section: Discussionmentioning

confidence: 99%

“…Next generation sequencing (NGS) technologies represent an essential tool for rapid and effective diagnosis of these diseases and may be used in some complex situations prior to multiple and often sequential functional studies. Recent studies highlighted the clinical utility of NGS approach for LD genetic diagnosis [ 8 , 9 , 10 , 11 ]. Here we report on the design, validation and testing of an NGS panel for genes involved in LDs named LysoGene.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2021

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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.

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“…Such NGS strategies allow the identification of single base pair variants and recombinant alleles (excluding the Recdelta55) with high specificity and sensitivity [167,169]. Conversely, analysis of the GBA1 gene as part of gene panels using well designed NGS strategies that consider the presence of the pseudogene, allows only the identification of point mutations, while fail to identify both large deletions and recombinant alleles due misalignment of reads with the homologous pseudogene [170][171][172][173][174].…”

Section: How Should Molecular Testing Be Performed?mentioning

confidence: 99%

Patient centered guidelines for the laboratory diagnosis of Gaucher disease type 1

Dardis

Michelakakis

Rozenfeld

et al. 2022

Orphanet J Rare Dis

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Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder due to the deficient activity of the acid beta-glucosidase (GCase) enzyme, resulting in the progressive lysosomal accumulation of glucosylceramide (GlcCer) and its deacylated derivate, glucosylsphingosine (GlcSph). GCase is encoded by the GBA1 gene, located on chromosome 1q21 16 kb upstream from a highly homologous pseudogene. To date, more than 400 GBA1 pathogenic variants have been reported, many of them derived from recombination events between the gene and the pseudogene. In the last years, the increased access to new technologies has led to an exponential growth in the number of diagnostic laboratories offering GD testing. However, both biochemical and genetic diagnosis of GD are challenging and to date no specific evidence-based guidelines for the laboratory diagnosis of GD have been published. The objective of the guidelines presented here is to provide evidence-based recommendations for the technical implementation and interpretation of biochemical and genetic testing for the diagnosis of GD to ensure a timely and accurate diagnosis for patients with GD worldwide. The guidelines have been developed by members of the Diagnostic Working group of the International Working Group of Gaucher Disease (IWGGD), a non-profit network established to promote clinical and basic research into GD for the ultimate purpose of improving the lives of patients with this disease. One of the goals of the IWGGD is to support equitable access to diagnosis of GD and to standardize procedures to ensure an accurate diagnosis. Therefore, a guideline development group consisting of biochemists and geneticists working in the field of GD diagnosis was established and a list of topics to be discussed was selected. In these guidelines, twenty recommendations are provided based on information gathered through a systematic review of the literature and two different diagnostic algorithms are presented, considering the geographical differences in the access to diagnostic services. Besides, several gaps in the current diagnostic workflow were identified and actions to fulfill them were taken within the IWGGD. We believe that the implementation of recommendations provided in these guidelines will promote an equitable, timely and accurate diagnosis for patients with GD worldwide.

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Early detection of lysosomal diseases by screening of cases of idiopathic splenomegaly and/or thrombocytopenia with a next‐generation sequencing gene panel

Cited by 5 publications

References 37 publications

Accurate Molecular Diagnosis of Gaucher Disease Using Clinical Exome Sequencing as a First-Tier Test

Accurate Molecular Diagnosis of Gaucher Disease Using Clinical Exome Sequencing as a First-Tier Test

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

Patient centered guidelines for the laboratory diagnosis of Gaucher disease type 1

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