Rapid and Reliable Detection of Nonsyndromic Hearing Loss Mutations by Multicolor Melting Curve Analysis

Abstract

Hearing loss is a common birth defect worldwide. The GJB2, SLC26A4, MT-RNR1 and MT-TS1 genes have been reported as major pathogenic genes in nonsyndromic hearing loss. Early genetic screening is recommended to minimize the incidence of hearing loss. We hereby described a multicolor melting curve analysis (MMCA)-based assay for simultaneous detection of 12 prevalent nonsyndromic hearing loss-related mutations. The three-reaction assay could process 30 samples within 2.5 h in a single run on a 96-well thermocycler. Allelic types of each mutation could be reproducibly obtained from 10 pg ~100 ng genomic DNA per reaction. For the mitochondrial mutations, 10% ~ 20% heteroplasmic mutations could be detected. A comparison study using 501 clinical samples showed that the MMCA assay had 100% concordance with both SNaPshot minisequencing and Sanger sequencing. We concluded that the MMCA assay is a rapid, convenient and cost-effective method for detecting the common mutations, and can be expectedly a reliable tool in preliminary screening of nonsyndromic hearing loss in the Chinese Han population.

Figure 1. The workflow of MMCA assay for the detection of nonsyndromic hearing loss-associated mutations.

Genomic DNA was extracted from the clinical samples and then added into three PCR reactions (each reaction contains the primers and the detection probes labeled three different labeled fluorophores of FAM, HEX or ROX), and subsequently PCR amplification and melting curve analysis were performed on CFX96^TM real-time system. The graphic information containing T_m values was automatically produced by the software. The genotypes of GJB2, MT-RNR1, MT-TS1, SLC26A4 and GJB3 could be confirmed by the values of T_m and ΔT_m. Black lines and colored lines represent for the melting peaks of the wild-type and their corresponding mutations types, respectively.

Figure 2. Genotyping results of nonsyndromic hearing loss-associated mutations by the MMCA assay.

Melting curves and corresponding genotypes (box) of the 12 mutations and wild-type plasmid DNA templates are given according to the detection channels, shown by the corresponding fluorophores. (a–c) Represent the three reactions, respectively. Gray dotted lines indicate no-template control.

Figure 3. Melting curves for different percentages of mtDNA mutations.

Melting curves were from mimic mitochondrial plasmid DNA samples with different percentages (from 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, to 100%) of A1555G (a), C1494T (b), and G7444A/A7445G mutation (c). Both wild-type and mutant-type templates were plasmid DNAs and the concentration was set at 1 × 10⁴ copies per reaction.