Zhou Chen, Zou Xiangman, Peng Cuiying, Gao Guoqiang, Guo Zifen
Institute of Pharmacy and Pharmacology, University of South China, Hengyang, Hunan, China.
Hunan Province Cooperative Innovation Center for Molecular Target New Drug Study, Hengyang, Hunan, China.
AMB Express. 2020 Sep 15;10(1):166. doi: 10.1186/s13568-020-01102-7.
An increasing number of biological and epidemiological evidence suggests that c.919-2A > G and c.2168A > G variants of solute carrier family 26, member 4 (SLC26A4) gene play a critical role in the development of large vestibular aqueduct syndrome (LVAS). In this study, we developed a rapid genotyping method for discriminating LVAS-associated high-frequency variants in SLC26A4 gene. The genotyping technique consists of 3' terminal exonuclease-resistant phosphorothioate-modified allele specific primer extension mediated by exo polymerase. In PCR amplification by Pfu polymerase, allelic specific primers perfectly matching wild type allele were extended while no specific products were yielded from primers targeting variant allele. Similarly, allelic specific primers perfectly matching variant allele were extended and no specific products were observed from primers targeting wild type allele. The clinical application of 3' terminal phosphorothioate-modified allele specific primer extension mediated by Pfu polymerase identified both homozygous for SLC26A4 gene c.919-2A > G variant in two patients clinically diagnosed as LVAS by temporal bone CT scan. The genetic results from this method are consistent with that of DNA sequencing. The data suggest that exo polymerase-mediated 3' terminal phosphorothioate-modified primer extension is reliable in the identification of SLC26A4 gene high-frequency variant prior to high-resolution CT scan. The method is extremely suitable for quickly molecular etiologic screening and early diagnosis and aggressive prevention therapy of LVAS.
越来越多的生物学和流行病学证据表明，溶质载体家族26成员4（SLC26A4）基因的c.919-2A＞G和c.2168A＞G变异在大前庭导水管综合征（LVAS）的发生发展中起关键作用。在本研究中，我们开发了一种快速基因分型方法，用于鉴别SLC26A4基因中与LVAS相关的高频变异。该基因分型技术由3'末端抗外切核酸酶的硫代磷酸酯修饰的等位基因特异性引物延伸组成，由外切聚合酶介导。在Pfu聚合酶进行的PCR扩增中，与野生型等位基因完美匹配的等位基因特异性引物得以延伸，而针对变异等位基因的引物未产生特异性产物。同样，与变异等位基因完美匹配的等位基因特异性引物得以延伸，而针对野生型等位基因的引物未观察到特异性产物。由Pfu聚合酶介导的3'末端硫代磷酸酯修饰的等位基因特异性引物延伸的临床应用，在两名经颞骨CT扫描临床诊断为LVAS的患者中鉴定出均为SLC26A4基因c.919-2A＞G变异的纯合子。该方法的基因检测结果与DNA测序结果一致。数据表明，外切聚合酶介导的3'末端硫代磷酸酯修饰的引物延伸在高分辨率CT扫描前鉴定SLC26A4基因高频变异方面是可靠的。该方法极其适合于LVAS的快速分子病因学筛查、早期诊断及积极的预防治疗。
