Targeted Re-Sequencing Emulsion PCR Panel for Myopathies: Results in 94 Cases

Article type: Research Article

Authors: Punetha, Jaya^{a; b} | Kesari, Akanchha^{a; 1} | Uapinyoying, Prech^{a; b} | Giri, Mamta^a | Clarke, Nigel F.^c | Waddell, Leigh B.^c | North, Kathryn N.^{c; d} | Ghaoui, Roula^c | O’Grady, Gina L.^c | Oates, Emily C.^c | Sandaradura, Sarah A.^c | Bönnemann, Carsten G.^e | Donkervoort, Sandra^e | Plotz, Paul H.^f | Smith, Edward C.^g | Tesi-Rocha, Carolina^{a; 2} | Bertorini, Tulio E.^h | Tarnopolsky, Mark A.ⁱ | Reitter, Bernd^j | Hausmanowa-Petrusewicz, Irena^k | Hoffman, Eric P.^{a; b; *}

Affiliations: [a] Research Center for Genetic Medicine, Children’s National Medical Center, Washington DC, USA | [b] Department of Integrative Systems Biology, The George Washington University School of Medicine and Health Sciences, Washington, DC, USA | [c] INMR, The Children’s Hospital at Westmead & Discipline of Paediatrics and Child Health, University of Sydney, Sydney, Australia | [d] Murdoch Childrens Research Institute, Melbourne, Australia; Department of Paediatrics, Faculty of Medicine, University of Melbourne, Melbourne, Australia | [e] National Institute of Neurological Disorders and Stroke/NIH, Porter Neuroscience Research Center, Bethesda, MD, USA | [f] National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD, USA | [g] Department of Pediatrics, Division of Pediatric Neurology, Duke University Medical Center, Durham, NC, USA | [h] Department of Neurology, The University of Tennessee Health Science Center, Memphis, TN, USA | [i] Departments of Pediatrics and Medicine, McMaster University, Neuromuscular Disease Clinic, Health Sciences Centre, ON, Canada | [j] Children’s Hospital, Johannes Gutenberg University, Mainz, Germany | [k] Neuromuscular Unit, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland

Correspondence: [*] Correspondence to: Eric P Hoffman, PhD, Children’s National Medical Center, 111 Michigan Ave NW, Washington DC 20010, USA. Tel.: +1 202 476 6011; Fax: +1 202 476 6014; E-mail: [email protected].

Note: [1] Present address: Miami Children’s Hospital, Miami Genetic Laboratory, Miami, FL, USA.

Note: [2] Present address: Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Palo Alto, CA, USA.

Abstract: Background: Molecular diagnostics in the genetic myopathies often requires testing of the largest and most complex transcript units in the human genome (DMD, TTN, NEB). Iteratively targeting single genes for sequencing has traditionally entailed high costs and long turnaround times. Exome sequencing has begun to supplant single targeted genes, but there are concerns regarding coverage and needed depth of the very large and complex genes that frequently cause myopathies. Objective: To evaluate efficiency of next-generation sequencing technologies to provide molecular diagnostics for patients with previously undiagnosed myopathies. Methods: We tested a targeted re-sequencing approach, using a 45 gene emulsion PCR myopathy panel, with subsequent sequencing on the Illumina platform in 94 undiagnosed patients. We compared the targeted re-sequencing approach to exome sequencing for 10 of these patients studied. Results: We detected likely pathogenic mutations in 33 out of 94 patients with a molecular diagnostic rate of approximately 35%. The remaining patients showed variants of unknown significance (35/94 patients) or no mutations detected in the 45 genes tested (26/94 patients). Mutation detection rates for targeted re-sequencing vs. whole exome were similar in both methods; however exome sequencing showed better distribution of reads and fewer exon dropouts. Conclusions: Given that costs of highly parallel re-sequencing and whole exome sequencing are similar, and that exome sequencing now takes considerably less laboratory processing time than targeted re-sequencing, we recommend exome sequencing as the standard approach for molecular diagnostics of myopathies.

Keywords: Muscular dystrophy, myopathies, molecular diagnostic testing, high-throughput DNA sequencing, massively-parallel sequencing, Exome, molecular diagnostics, genetic screening

DOI: 10.3233/JND-160151

Journal: Journal of Neuromuscular Diseases, vol. 3, no. 2, pp. 209-225, 2016