Comprehensive Mutation Analysis for Congenital Muscular Dystrophy: A Clinical PCR-Based Enrichment and Next-Generation Sequencing Panel

Autor: Martin Robert Littlejohn, Anne Rutkowski, Madhuri Hegde, Susan Sparks, Lisa Mari Keong, C. Alexander Valencia, Shruti Bhide, Arunkanth Ankala, Devin Rhodenizer, Carsten G. Bönnemann
Rok vydání: 2013
Předmět:
Genotype
DNA Mutational Analysis
Molecular Diagnostic Method
lcsh:Medicine
Muscle disorder
Biology
Polymerase Chain Reaction
Sensitivity and Specificity
Muscular Dystrophies
DNA sequencing
03 medical and health sciences
symbols.namesake
0302 clinical medicine
Genomic Medicine
Diagnostic Medicine
medicine
Humans
Genetic Predisposition to Disease
Genetic Testing
Genome Sequencing
lcsh:Science
030304 developmental biology
Genetic testing
Clinical Genetics
Genetics
Sanger sequencing
0303 health sciences
Multidisciplinary
medicine.diagnostic_test
lcsh:R
High-Throughput Nucleotide Sequencing
Reproducibility of Results
Computational Biology
Genomics
medicine.disease
Clinical Laboratory Sciences
Neurology
Mutation
Mutation (genetic algorithm)
Congenital muscular dystrophy
symbols
Medicine
lcsh:Q
030217 neurology & neurosurgery
Research Article
Zdroj: PLoS ONE
PLoS ONE, Vol 8, Iss 1, p e53083 (2013)
ISSN: 1932-6203
DOI: 10.1371/journal.pone.0053083
Popis: The congenital muscular dystrophies (CMDs) comprise a heterogeneous group of heritable muscle disorders with often difficult to interpret muscle pathology, making them challenging to diagnose. Serial Sanger sequencing of suspected CMD genes, while the current molecular diagnostic method of choice, can be slow and expensive. A comprehensive panel test for simultaneous screening of mutations in all known CMD-associated genes would be a more effective diagnostic strategy. Thus, the CMDs are a model disorder group for development and validation of next-generation sequencing (NGS) strategies for diagnostic and clinical care applications. Using a highly multiplexed PCR-based target enrichment method (RainDance) in conjunction with NGS, we performed mutation detection in all CMD genes of 26 samples and compared the results with Sanger sequencing. The RainDance NGS panel showed great consistency in coverage depth, on-target efficiency, versatility of mutation detection, and genotype concordance with Sanger sequencing, demonstrating the test's appropriateness for clinical use. Compared to single tests, a higher diagnostic yield was observed by panel implementation. The panel's limitation is the amplification failure of select gene-specific exons which require Sanger sequencing for test completion. Successful validation and application of the CMD NGS panel to improve the diagnostic yield in a clinical laboratory was shown.
Databáze: OpenAIRE
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