A novel genotyping technique for discriminating LVAS-associated high-frequency variants in SLC26A4 gene

Chen Zhou,Cuiying Peng,Guoqiang Gao,Xiangman Zou,Zifen Guo

doi:10.1186/s13568-020-01102-7

Abstract

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.

Highlights

Large vestibular aqueduct syndrome (LVAS) is an autosomal recessive genetic hearing loss disorder with a high incidence which is mainly accompanied by progressive and fluctuating hearing loss (Tong et al 1997; Claros et al 2017)
It is well known that the solute carrier family 26, member 4 (SLC26A4) gene plays a critical role in the development of LVAS, and about 90% of LVAS is closely attributed to SLC26A4 gene variants (Nishio et al 2016; Chao et al 2019; Kim et al 2019)
We have previously reported that exo+ polymerasemediated 3′ terminal exonuclease-resistant phosphorothioate-modified allele specific primer extension formed a molecular switch sensitive to single nucleotide discrimination (Zhang and Li 2003; Zhang et al 2005)

Summary

Introduction

Large vestibular aqueduct syndrome (LVAS) is an autosomal recessive genetic hearing loss disorder with a high incidence which is mainly accompanied by progressive and fluctuating hearing loss (Tong et al 1997; Claros et al 2017). It is well known that the solute carrier family 26, member 4 (SLC26A4) gene plays a critical role in the development of LVAS, and about 90% of LVAS is closely attributed to SLC26A4 gene variants (Nishio et al 2016; Chao et al 2019; Kim et al 2019). Previous studies revealed that SLC26A4 gene variant has obvious racial specificity, LVAS patients from different races with unique variant spectra and different variant frequencies (Berrettini et al 2005; Hu et al 2007; Zhou et al AMB Expr (2020) 10:166. The interest in SLC26A4 gene variant closely associated with Chinese LVAS individuals has been focused on c.919-2A > G and c. Screening the high-frequency variant can early diagnose LVAS patients and find variant carriers and taking measures to prevent further hearing loss by keeping LVAS patients from cold and head injury

Methods

Results

Conclusion