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

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.

Keywords: Exo+ polymerase; Large vestibular aqueduct syndrome; Phosphorothioate modification; Solute carrier family 26 member 4.

Fig. 1

DNA product from Overlapping PCR. M: 100 bp DNA marker; 1: PCR product harboring SLC26A4 gene c.919-2A > G flanking sequence, and the DNA fragment is 360 bp; 2: PCR product harboring SLC26A4 gene c.2168A > G flanking sequence, and the DNA fragment is 444 bp; 3: overlapping PCR products using the product 1 and 2 as temples, and the DNA fragment is 766 bp

Fig. 2

Representative of DNA sequencing of the constructed vector. a DNA sequencing result residing c.919-2A > G site in SLC26A4 gene for A base; b DNA sequencing result residing c.919-2A > G site in SLC26A4 gene for G base; c DNA sequencing result residing c.2168A > G site in SLC26A4 gene for A base; d DNA sequencing result residing c.2168A > G site in SLC26A4 gene for G base

Fig. 3

Discrimination of SLC26A4 gene c.919-2A > G and c.2168A > G variants by Pfu DNA polymerase-mediated phosphorothioate-modified primer extension. M is 50 bp DNA marker; Lanes 1 and 8 is specific primer extension products from the primer of c.919-2A > G site for A allele, Lanes 2 and 7 are specific primer extension products from the primer of c.919-2A > G site for G allele, Lanes 3 and 10 are specific primer extension products from the primer of c.2168A > G site for A allele, Lanes 4 and 9 are specific primer extension products from the primer of c.2168A > G site for G allele, Lanes 5 and 12 are specific primer extension products from primer mixture of c.919-2A > G site for A allele and c.2168A > G site for A allele, Lanes 6 and 11 are specific primer extension products from primer mixture of c.919-2A > G site for G allele and c.2168A > G site for G allele; Lanes 1 to 6 are the PCR products using wild vector as wild template; Lanes 7 to 12 are the PCR products using mutant vector as mutant template. The specific DNA product targeting c.919-2A > G and c.2168A > G loci is 280 bp and 65 bp respectively. Lanes 5, 6, 11 and 12 generate an extra 518 bp nonspecific fragment from a pair of amplification primers consisting of the forward detection primer of c.919-2A > G and the reverse detection primer of c.2168A > G

Fig. 4

The clinical application of Pfu DNA polymerase-mediated 3′ terminal phosphorothioate-modified primer extension on screening the SLC26A4 high-frequency variant. a M is 50 bp DNA marker; Lanes 1, 2, 5 and 6 are specific primer extension products from primer mixtures of c.919-2A > G site for G allele and c.2168A > G site for G allele, which perfectly match mutant template; Lanes 3 and 4 are specific primer extension products from primer mixtures of c.919-2A > G site for A allele and c.2168A > G site for A allele, which perfectly matches wild template. Lanes 1 and 3 represent LVAS case1, and Lanes 2 and 4 represent LVAS case2. Lanes 5 is mutant vector as the positive control; Lanes 6 is wild vector as the negative control. b, c and d Sequence analysis of SLC26A4 gene. b Represents healthy volunteer, c and d respectively represent LVAS case1 and 2