A reverse dot blot assay for the screening of twenty mutations in four genes associated with NSHL in a Chinese population

Background
Congenital deafness is one of the most distressing disorders affecting humanity and exhibits a high incidence worldwide. Most cases of congenital deafness in the Chinese population are caused by defects in a limited number of genes. A convenient and reliable method for detecting common deafness-related gene mutations in the Chinese population is required.
Methods
We developed a PCR-reverse dot blot (RDB) assay for screening 20 hotspot mutations of GJB2, GJB3, SLC26A4, and MT-RNR1, which are common non-syndromic hearing loss (NSHL)–associated genes in the Chinese population. The PCR-RDB assay consists of multiplex PCR amplifications of 10 fragments in the target sequence of the four above-mentioned genes in wild-type and mutant genomic DNA samples followed by hybridization to a test strip containing allele-specific oligonucleotide probes. We applied our method to a set of 225 neonates with deafness gene mutations and 30 normal neonates.
Results
The test was validated through direct sequencing in a blinded study with 100% concordance.
Conclusions
The results demonstrated that our reverse dot blot assay is a reliable and effective genetic screening method for identifying carriers and individuals with NSHL among the Chinese population.

背景
先天性耳聋是困扰人类的最严重疾病之一，在全球范围内发病率较高。中国人群中的绝大多数先天性耳聋病例由少数基因的缺陷所致，因此亟需一种便捷可靠的方法，用于筛查中国人群中常见的耳聋相关基因突变。
方法
本研究开发了一种聚合酶链式反应-反向斑点杂交（PCR-reverse dot blot，RDB）检测方法，用于筛查中国人群中常见非综合征性听力损失（non-syndromic hearing loss，NSHL）相关基因GJB2、GJB3、SLC26A4及MT-RNR1的20个热点突变。该PCR-RDB检测方法通过对上述4个基因的靶序列中的10个片段进行多重PCR扩增，覆盖野生型及突变型基因组DNA样本，随后与携带等位基因特异性寡核苷酸探针的检测试纸条进行杂交。本研究将该方法应用于225例携带耳聋基因突变的新生儿及30例正常新生儿队列。
结果
通过盲法研究结合直接测序进行验证，本检测方法的一致性达100%。
结论
研究结果表明，本研究所开发的反向斑点杂交检测方法是一种可靠有效的遗传筛查手段，可用于在中国人群中识别非综合征性听力损失携带者及患者。