State Key Laboratory of Cellular Stress Biology, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, Engineering Research Centre of Molecular Diagnostics, Ministry of Education, School of Life Sciences, Xiamen University, Xiamen, Fujian 361102, China.
Department of Otorhinolaryngology, Zhongshan Hospital of Xiamen, Xiamen University, Xiamen, Fujian, 361004, China.
Sci Rep. 2017 Feb 22;7:42894. doi: 10.1038/srep42894.
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.
听力损失是一种常见的出生缺陷，在全球范围内都有发生。GJB2、SLC26A4、MT-RNR1 和 MT-TS1 等基因已被报道为非综合征型听力损失的主要致病基因。建议进行早期基因筛查，以最大程度地降低听力损失的发生率。本研究描述了一种基于多重荧光熔解曲线分析（MMCA）的方法，用于同时检测 12 种常见的非综合征型听力损失相关突变。该三反应检测可以在单个 96 孔热循环仪中在 2.5 小时内处理 30 个样本。每个反应可从 10pg100ng 基因组 DNA 中重复获得每个突变的等位基因类型。对于线粒体突变，可检测到 10%20%的异质性突变。使用 501 个临床样本进行的比较研究表明，MMCA 检测与 SNaPshot 微测序和 Sanger 测序具有 100%的一致性。我们得出结论，MMCA 检测是一种快速、方便且具有成本效益的方法，可用于检测常见突变，有望成为中国汉族人群非综合征型听力损失初步筛查的可靠工具。
