A novel method for detecting nine hotspot mutations of deafness genes in one tube.

Deafness is a common sensory disorder. In China, approximately 70% of hereditary deafness originates from four common deafness-causing genes: GJB2, SLC26A4, GJB3, and MT-RNR1. A single-tube rapid detection method based on 2D-PCR technology was established for nine mutation sites in the aforementioned genes, and Sanger sequencing was used to verify its reliability and accuracy. The frequency of hotspot mutations in deafness genes was analysed in 116 deaf students. 2D-PCR identified 27 genotypes of nine loci according to the melting curve of the FAM, HEX, and Alexa568 fluorescence channels. Of the 116 deaf patients, 12.9% (15/116) carried SLC26A4 mutations, including c.919-2A > G and c.2168A > G (allele frequencies, 7.3% and 2.2%, respectively). The positivity rate (29.3%; 34/116) was highest for GJB2 (allele frequency, 15.9% for c.235delC, 6.0% for c.299_300delAT, and 2.6% for c.176-191del16). Sanger sequencing confirmed the consistency of results between the detection methods based on 2D-PCR and DNA sequencing. Common pathogenic mutations in patients with non-syndromic deafness in Changzhou were concentrated in GJB2 (c.235delC, c.299_300delAT, and c.176-191del16) and SLC26A4 (c.919-2A > G and c.2168 A > G). 2D-PCR is an effective method for accurately and rapidly identifying deafness-related genotypes using a single-tube reaction, and is superior to DNA sequencing, which has a high cost and long cycle.