实时PCR熔解曲线技术用于门诊听力损失患者基因筛查OA

Objective To report genetic screening for 20 mutations across four deafness-associated genes using real-time PCR melting curve analysis in outpatients with hearing loss,and to evaluate its potential clinical utility.Methods Saliva or buccal swab samples were collected from 220 outpatients(143 males,77 females)with hearing loss at Women and Children's Hospital of Xiamen University between December 2021 and December 2022.Real-time PCR melting curve was employed to detect mutations of the GJB2,SLC26A4,GJB3,and MT-RNR1 genes,followed by validation using Sanger sequencing.Results Hearing loss among the 220 patients was classified as mild in 103(46.8%),moderate in 31(14.1%),severe in 13(5.9%)and profound in 31(14.1%).Hearing loss was bilateral asymmetric in 42 cases(19.1%).Genomic Specimen DNA concentrations ranged from 6.3 to 141.5 ng/μL(mean 27.5±17.3 ng/μL).Deafness-associated mutations were identified in 34 patients(15.5%),involving the GJB2(18 cases,8.2%),SLC26A4(10 cases,4.6%),GJB3(3 cases,1.4%),MT-RNR1 homoplasmic mutation(2 cases,0.9%),and a GJB2/SLC26A4 mutation(1 case,0.5%).Concordance with Sanger sequencing was 100%.The GJB2 c.235delC and SLC26A4 c.919-2A＞G variants were identified as hotspot mutations among this cohort.Conclusions Real-time PCR melting curve analysis provides several advantages,including simplicity,rapid turnaround time,and automated interpretation.The non-invasive nature of saliva or buccal swab sampling eliminates patient discomfort associated with blood collection while minimizing the risk of infection.This technique shows considerable promise for large-scale application in integrated newborn hearing and deafness genetic screening programs.