[Application of the real-time fluorescence PCR melting curve method in gene screening of non-syndromic hearing loss]. Zhonghua Er Bi Yan Hou Tou Jing Wai Ke Za Zhi 2019 Apr 07;54(4):286-291
Date
04/18/2019Pubmed ID
30991779DOI
10.3760/cma.j.issn.1673-0860.2019.04.009Abstract
Objective: To detect 20 common deafness gene mutations in non-syndromic hearing loss patients in China using the melting curve method, and analyze and summarize the mutation data to explore the clinical value of this method. Methods: The real-time fluorescence PCR melting curve method was used to detect 20 common mutations of four deafness genes(GJB2,GJB3,SLC26A4 and mtDNA) in 492 patients with non-syndromic hearing loss recruited between March 2014 and September 2016 from the Otolaryngology Department of Xiangya Hospital, Central South University(283 males and 209 females, the age ranged from 1 to 48 years old). The Sanger sequencing method was used to compare the sensitivity, specificity, positive predictive value, negative predictive value, and total coincidence rate of the deafness mutation detected by the real-time fluorescence PCR melting curve method. Results: A total of 492 samples were detected. 193 wild-type samples, 93 homozygous mutant samples, 145 heterozygous mutant samples, 59 composite heterozygous mutant samples and 2 samples with unknown mutations were detected using the real-time fluorescence PCR melting curve method within the range of 20 gene mutations, whichwere identical to the Sanger sequencing results.The two samples were detected as unknown mutations by the real-time fluorescent PCR melting curve method were confirmed by Sanger sequencing, including a composite heterozygous mutant sample and a homogenous mutation sample. GJB2 c.235delC and SLC26A4 c.919-2 A>G were the most common hotspot mutations in this study, followed by mtDNA m.1555 A>G. Compared with the Sanger sequencing method, the sensitivity, specificity, positive predictive value, negative predictive value, and total coincidence rate of the real-time fluorescence PCR melting curve method were 100%, the Youden's index was 1.0, and the Kappa value was 1. Conclusions: The real-time fluorescence PCR melting curve method is suitable for the detection of deafness gene mutations. It has the advantages in terms of simple, rapid, high sensitivity and strong specificity and can accurately detect the 20 gene mutations of 4 common deafness genes in Chinese population, which is expected to be used for the clinical detection of deafness genes in the future.
Author List
Liu YL, Jiang XH, Sun J, Mei LY, He CF, Deng YY, Wen J, Feng YAuthor
Ling Mei MD Associate Professor in the Medicine department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
AdolescentAdult
Child
Child, Preschool
China
DNA Mutational Analysis
Deafness
Female
Hearing Loss
Humans
Infant
Male
Middle Aged
Mutation
Real-Time Polymerase Chain Reaction
Sensitivity and Specificity
Young Adult
