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

• Type of Material: Article
• Authors: C. Alexander Valencia, University of Cincinnati; Arunkanth Ankala, Emory University; Devin Rhodenizer, Emory University; Shruti Bhide, Emory University; Martin Robert Littlejohn, Emory University; Lisa Mari Keong, Emory University; Anne Rutkowski, Cure CMD and Kaiser SCPMG; Susan Sparkes, Carolinas Medical Center; Carsten Bonnemann, National Institute of Neurological Disorders and Stroke; Madhuri Hegde, Emory University
• Language: English
• Date: 2013-01-11
• Publisher: Public Library of Science
• Publication Version: Final Published Version
• Copyright Statement: © 2013 Valencia et al.
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0053083
• Title of Journal or Parent Work: PLoS ONE
• ISSN: 1932-6203
• Volume: 8
• Issue: 1
• Start Page: e53083
• End Page: e53083
• Grant/Funding Information: This research was supported in part by PHS Grants (UL1 RR025008, KL2 R0025009, or TL1 RR025010) from the Clinical and Translational Science Award Program, National Institutes of Health (NIH), National Center for Research Resources.; CB is supported by the intramural program of the National Institute of Neurological Disorders and Stroke, NIH.
• Research Categories: Biology, Genetics; Biology, Neuroscience

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.