Performance evaluation of the TheraTyper-GJB2 assay for detection of GJB2 gene mutations.
J Mol Diagn
; 16(5): 573-583, 2014 Sep.
Article
em En
| MEDLINE
| ID: mdl-24998936
ABSTRACT
Mutations in the GJB2 gene are the most common cause of congenital hearing loss in many populations. This study describes the development of a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based minisequencing assay, TheraTyper-GJB2, for the detection of c.35delG, c.167delT, and c.235delC mutations in the GJB2 gene. This assay was evaluated for analytic performance, including detection limit, interference, cross-reactivity, and precision, using GJB2 reference standards prepared by site-directed mutagenesis of a molecular clone. The detection limit was as low as 0.040 ng of human genomic DNA per PCR. No cross-reactivity with bacteria and viruses and no negative effects of increased levels of various potential interfering substances was observed. A precision test involving repetitive analysis of 2400 replicates showed 99.9% agreement (2397 of 2,400) with 99.8% (95% CI, 99.7%-99.8%) sensitivity and 100.0% (95% CI, 99.3%-100.0%) specificity. TheraTyper-GJB2 and direct sequencing assays showed 100% concordance for detecting mutations in 1,113 clinical specimens. Overall, TheraTyper-GJB2 showed comparable performance for detecting GJB2 mutations in reference and clinical samples with that of direct sequencing, and easier interpretation of results for analysis of a large quantity of samples. Therefore, the TheraTyper-GJB2 assay will be practically useful for the diagnosis of GJB2 mutations associated with congenital hearing loss with faster, cheaper, more reliable, and high-throughput capability.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Análise de Sequência de DNA
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Conexinas
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Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
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Mutação
Tipo de estudo:
Diagnostic_studies
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Guideline
Limite:
Humans
Idioma:
En
Revista:
J Mol Diagn
Ano de publicação:
2014
Tipo de documento:
Article