title: Comprehensive mutation analysis for congenital muscular dystrophy: a clinical PCR-based enrichment and next-generation sequencing panel.
abstract: 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.
authors: Department of Human Genetics, Emory University School of Medicine, Atlanta, Georgia, USA.
authors: C Alexander
authors: CA
authors: Valencia
authors: Arunkanth
authors: A
authors: Ankala
authors: Devin
authors: D
authors: Rhodenizer
authors: Shruti
authors: S
authors: Bhide
authors: Martin Robert
authors: MR
authors: Littlejohn
authors: Lisa Mari
authors: LM
authors: Keong
authors: Anne
authors: A
authors: Rutkowski
authors: Susan
authors: S
authors: Sparks
authors: Carsten
authors: C
authors: Bonnemann
authors: Madhuri
authors: M
authors: Hegde
mesh_terms: D004252:DNA Mutational Analysis / Q000379:methods*; D020022:Genetic Predisposition to Disease / Q000235:genetics; D005820:Genetic Testing / Q000379:methods; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D009136:Muscular Dystrophies / Q000151:congenital / Q000175:diagnosis / Q000235:genetics*; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity