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.

Genetic counseling for patients with nonsyndromic hearing impairment directed by gene analysis

“…The results revealed that 36 of the 117 patients (30.77%) carried two deafness-causing mutations, including the GJB2 gene mutations (16.24%), SLC26A4 gene mutations (10.26%) and mitochondrial DNA 12SrRNA 1555 locus mutation (4.27%). The 235delC mutation in the GJB2 gene mutation has been identified at the highest rate in patients in the present study and is also the most common in the Asian population ( 24 ). The present study identified the mitochondrial DNA 12S rRNA 1,555A>G locus mutation in ~4.27% of the patients, which follows the pattern of maternal inheritance ( 11 , 18 ).…”

Establishment of a Flexible Real-Time Polymerase Chain Reaction-Based Platform for Detecting Prevalent Deafness Mutations Associated with Variable Degree of Sensorineural Hearing Loss in Koreans

“…The average of PPV and NPV were obtained from 12 reference articles where methods using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry or real-time PCR were compared with Sanger sequencing in detecting mutations of GJB2 or other human genes. The average PPV and NPV were 96.7% and 92.5%, respectively, and the differences from the lower margin of the 95% CI (δ, the non-inferiority margin) were -4.7% and -6.5% respectively [24–35]. Based on the reference articles, the sizes of positive and normal samples were calculated using the equation below, considering a 10% dropout rate.…”

The role of gene GJB2 and connexin 26 in hearing impairment

“…As mentioned earlier, 50% of hearing impairment cases are caused by genetic factors and most of them are due to mutations in the GJB2 gene. Because GJB2 codes for the instructions of making the protein Cx26, any changes in its nucleotide sequence might either result in a change of amino acids or deleting some of them contributing to the translation of a connexin 26 with an abnormal structure [18]. This irregular structure of the protein, whether it is incomplete or lost an essential bond that connects it with other monomers, it will surely lead it to be a faulty protein as all of its structure components plays a key role in stabilizing it, and so making able to carry its normal function [18].…”

“…According to recent research, the most common connexin 26 mutation is 35delG, and it is a frame shift mutation that involves the deletion of Guanine base at position 35, causing in a shifting of the sequence, and thus resulting in TGA, a premature stop codon. Consequently, the coding sequence is terminated resulting in a shortened Cx26 protein [18].…”