Performance evaluation of the TheraTyper-GJB2 assay for detection of GJB2 gene mutations

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.