Monday, 3 September 2012

A couple of interesting summer papers...

We are back from summer vacations. Here are a couple of papers published in August that are worth reading.
The first one is a study showing how NGS can be a useful and powerful method to improve diagnosis in syndromes with complex clinical presentation. The second paper illustrates the use of Solid and IonTorrent NGS platforms in diagnostic routine for the screening of BRCA mutations.

Exploring the utility of Whole-Exome Sequencing as a diagnostic tool in a child with Atypical Episodic Muscle Weakness.
Clin Genet. 2012 Aug 17

Hanchard NA, Murdock DR, Magoulas PL, Bainbridge M, Muzny D, Wu Y, Wang M, McGuire AL, Lupski JR, Gibbs RA, Brown CW.
Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, Texas, 77030, U.S.A.; Texas Children's Hospital, 6621 Fannin Street,, Houston, Texas, 77030, U.S.A.

The advent of whole-exome next-generation sequencing (WES) has been pivotal for the molecular characterization of Mendelian disease; however, the clinical applicability of WES has remained relatively unexplored. We describe our exploration of WES as a diagnostic tool in a three-year old female patient with a two-year history of episodic muscle weakness and paroxysmal dystonia who presented following a previous extensive but unrevealing diagnostic work-up. WES was performed on the proband and her two parents. Parental exome data was used to filter potential de novo genomic events in the proband and suspected variants were confirmed using di-deoxy sequencing. WES revealed a de novo non-synonymous mutation in exon 21 of the calcium channel gene CACNA1S that has been previously reported in a single patient as a rare cause of atypical hypokalemic periodic paralysis. This was unexpected, as the proband's original differential diagnosis had included hypokalemic periodic paralysis, but clinical and laboratory features were equivocal, and standard clinical molecular testing for hypokalemic periodic paralysis and related disorders was negative. This report highlights the potential diagnostic utility of WES in clinical practice, with implications for the approach to similar diagnostic dilemmas in the future.

Development of a Next-Generation Sequencing Method for BRCA Mutation Screening: A Comparison between a High-Throughput and a Benchtop Platform.
J Mol Diagn. 2012 Aug 22.

Chan M, Mo Ji S, Yeo ZX, Gan L, Yap E, Yap YS, Ng R, Tan PH, Ho GH, Ang P, Lee AS.
Division of Medical Sciences, National Cancer Centre, Singapore, Republic of Singapore.

In a clinical setting, next-generation sequencing (NGS) approaches for the enrichment and resequencing of DNA targets may have limitations in throughput, cost, or accuracy. We evaluated an NGS workflow for targeted DNA sequencing for mutation detection. Targeted sequence data of the BRCA1 and BRCA2 genes, generated using a PCR-based, multiplexed NGS approach using the SOLiD 4 (n = 24) and Ion Torrent PGM (n = 20) next-generation sequencers, were evaluated against sequence data obtained by Sanger sequencing. The overall sensitivity for SOLiD and PGM were 97.8% (95% CI = 94.7 to 100.0) and 98.9% (95% CI = 96.8 to 100.0) respectively, using automated variant calling. The specificity for the SOLiD platform was high, at 100.0% (95% CI = 99.3 to 100.0). PGM correctly identified all 3 indels, but 68 false-positive indels were also called. Equimolar normalization of amplicons was not necessary for successful NGS. Both platforms are highly amenable to scale-up (>384 or >16 samples per run for SOLiD and PGM, respectively), potentially reducing the reagent cost for BRCA testing to <US$200. Only 325 ng of DNA per patient is required, with similar coverage and accuracy obtained using DNA derived from peripheral blood or buccal wash samples. The strategy described is accurate and easy to incorporate into conventional workflow, and shows potential for mutation screening of clinically important gene targets in genetic disorders.

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