Targeted Re-Sequencing Emulsion PCR Panel for Myopathies

Results in 94 Cases

Jaya Punetha, Akanchha Kesari, Prech Uapinyoying, Mamta Giri, Nigel F. Clarke, Leigh B. Waddell, Kathryn N. North, Roula Ghaoui, Gina L. O'Grady, Emily C. Oates, Sarah A. Sandaradura, Carsten G. Bönnemann, Sandra Donkervoort, Paul H. Plotz, Edward C. Smith, Carolina Tesi-Rocha, Tulio Bertorini, Mark A. Tarnopolsky, Bernd Reitter, Irena Hausmanowa-Petrusewicz & 1 others Eric P. Hoffman

Research output: Contribution to journalArticle

5 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)209-225
Number of pages17
JournalJournal of Neuromuscular Diseases
Volume3
Issue number2
DOIs
StatePublished - Jan 1 2016

Fingerprint

Exome
Muscular Diseases
Emulsions
Molecular Pathology
Polymerase Chain Reaction
Genes
Costs and Cost Analysis
Mutation
Gene Targeting
Human Genome
Mutation Rate
Exons
Technology

All Science Journal Classification (ASJC) codes

  • Neurology
  • Clinical Neurology

Cite this

Punetha, J., Kesari, A., Uapinyoying, P., Giri, M., Clarke, N. F., Waddell, L. B., ... Hoffman, E. P. (2016). Targeted Re-Sequencing Emulsion PCR Panel for Myopathies: Results in 94 Cases. Journal of Neuromuscular Diseases, 3(2), 209-225. https://doi.org/10.3233/JND-160151

Targeted Re-Sequencing Emulsion PCR Panel for Myopathies : Results in 94 Cases. / Punetha, Jaya; Kesari, Akanchha; Uapinyoying, Prech; Giri, Mamta; Clarke, Nigel F.; Waddell, Leigh B.; North, Kathryn N.; Ghaoui, Roula; O'Grady, Gina L.; Oates, Emily C.; Sandaradura, Sarah A.; Bönnemann, Carsten G.; Donkervoort, Sandra; Plotz, Paul H.; Smith, Edward C.; Tesi-Rocha, Carolina; Bertorini, Tulio; Tarnopolsky, Mark A.; Reitter, Bernd; Hausmanowa-Petrusewicz, Irena; Hoffman, Eric P.

In: Journal of Neuromuscular Diseases, Vol. 3, No. 2, 01.01.2016, p. 209-225.

Research output: Contribution to journalArticle

Punetha, J, Kesari, A, Uapinyoying, P, Giri, M, Clarke, NF, Waddell, LB, North, KN, Ghaoui, R, O'Grady, GL, Oates, EC, Sandaradura, SA, Bönnemann, CG, Donkervoort, S, Plotz, PH, Smith, EC, Tesi-Rocha, C, Bertorini, T, Tarnopolsky, MA, Reitter, B, Hausmanowa-Petrusewicz, I & Hoffman, EP 2016, 'Targeted Re-Sequencing Emulsion PCR Panel for Myopathies: Results in 94 Cases', Journal of Neuromuscular Diseases, vol. 3, no. 2, pp. 209-225. https://doi.org/10.3233/JND-160151
Punetha J, Kesari A, Uapinyoying P, Giri M, Clarke NF, Waddell LB et al. Targeted Re-Sequencing Emulsion PCR Panel for Myopathies: Results in 94 Cases. Journal of Neuromuscular Diseases. 2016 Jan 1;3(2):209-225. https://doi.org/10.3233/JND-160151
Punetha, Jaya ; Kesari, Akanchha ; Uapinyoying, Prech ; Giri, Mamta ; Clarke, Nigel F. ; Waddell, Leigh B. ; North, Kathryn N. ; Ghaoui, Roula ; O'Grady, Gina L. ; Oates, Emily C. ; Sandaradura, Sarah A. ; Bönnemann, Carsten G. ; Donkervoort, Sandra ; Plotz, Paul H. ; Smith, Edward C. ; Tesi-Rocha, Carolina ; Bertorini, Tulio ; Tarnopolsky, Mark A. ; Reitter, Bernd ; Hausmanowa-Petrusewicz, Irena ; Hoffman, Eric P. / Targeted Re-Sequencing Emulsion PCR Panel for Myopathies : Results in 94 Cases. In: Journal of Neuromuscular Diseases. 2016 ; Vol. 3, No. 2. pp. 209-225.
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AU - Punetha, Jaya

AU - Kesari, Akanchha

AU - Uapinyoying, Prech

AU - Giri, Mamta

AU - Clarke, Nigel F.

AU - Waddell, Leigh B.

AU - North, Kathryn N.

AU - Ghaoui, Roula

AU - O'Grady, Gina L.

AU - Oates, Emily C.

AU - Sandaradura, Sarah A.

AU - Bönnemann, Carsten G.

AU - Donkervoort, Sandra

AU - Plotz, Paul H.

AU - Smith, Edward C.

AU - Tesi-Rocha, Carolina

AU - Bertorini, Tulio

AU - Tarnopolsky, Mark A.

AU - Reitter, Bernd

AU - Hausmanowa-Petrusewicz, Irena

AU - Hoffman, Eric P.

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N2 - 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.

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