title: Performance evaluation of the TheraTyper-GJB2 assay for detection of GJB2 gene mutations.
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.
authors.affiliation: Research and Development Center, GeneMatrix, Inc., Seongnam, South Korea.
authors.forename: Ji-Yong
authors.initials: JY
authors.lastname: Chun
authors.affiliation: Research and Development Center, GeneMatrix, Inc., Seongnam, South Korea.
authors.forename: Soo-Kyung
authors.initials: SK
authors.lastname: Shin
authors.affiliation: Research and Development Center, GeneMatrix, Inc., Seongnam, South Korea.
authors.forename: Kyung Tae
authors.initials: KT
authors.lastname: Min
authors.affiliation: Research and Development Center, GeneMatrix, Inc., Seongnam, South Korea.
authors.forename: Woojae
authors.initials: W
authors.lastname: Cho
authors.affiliation: Research and Development Center, GeneMatrix, Inc., Seongnam, South Korea.
authors.forename: Jaeil
authors.initials: J
authors.lastname: Kim
authors.affiliation: Research and Development Center, GeneMatrix, Inc., Seongnam, South Korea.
authors.forename: Soo-Ok
authors.initials: SO
authors.lastname: Kim
authors.affiliation: Research and Development Center, GeneMatrix, Inc., Seongnam, South Korea. Electronic address: sunphong@genematrix.net.
authors.forename: Sun Pyo
authors.initials: SP
authors.lastname: Hong
mesh_terms: D000072259:Connexin 26; D017630:Connexins / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods; D059788:Dried Blood Spot Testing; D034381:Hearing Loss / Q000151:congenital / Q000175:diagnosis / Q000235:genetics; D006801:Humans; D009154:Mutation; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D017630:Connexins; C000607042:GJB2 protein, human; D000072259:Connexin 26