A novel genotyping technique for discriminating LVAS-associated hotspot mutations in SLC26A4 gene

An increasing number of biological and epidemiological evidence suggests that IVS7-2A>G and 2168A>G mutations of solute carrier family 26, member 4 ( SLC26A4 ) gene plays 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 hotspot mutations in SLC26A4 gene. The genotyping technique consists of 3’ terminal exonuclease-resistant phosphorothioate-modied allele specic primer extension mediated by exo + polymerase. In PCR amplication by Pfu polymerase, allelic specic primers perfectly matching wild type allele were extended while no specic products were yielded from primers targeting mutant type allele. Similarly, allelic specic primers perfectly matching mutant type allele were extended and no specic products were observed from primers targeting wild type allele . The clinical application of 3’ terminal phosphorothioate-modied allele specic primers extension mediated by Pfu polymerase identied both homozygous for SLC26A4 gene IVS7-2A>G mutation in two LVAS patients diagnosed as by temporal bone CT scan. The genetic results from this method is consistent with that of DNA sequencing. The data suggest that exo + polymerase-mediated 3’ terminal phosphorothioate-modied primer extension is reliable in the identication of SLC26A4 gene hotspot mutation 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.


Introduction
Large vestibular aqueduct syndrome (LVAS) is an autosomal recessive genetic hearing loss disorder with high incidence which mainly accompanied with progressive and uctuating 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 mutations (Nishio et al. 2016;Chao et al. 2019;Kim et al. 2019). Before clinical application of gene diagnosis, the imageological examination was the only way of LVAS diagnosis and had stated clearly that LVAS is the most common inner ear malformation (Connor et al. 2019;Yang and Liu 2019). But many hospitals lack high-resolution CT equipment or specialist familiar with LVAS, large numbers of LVAS patients are misdiagnosed. In addition, CT scan has been solidly con rmed to increase cancer development by its mutagenesis effect.
A number of studies have shown that SLC26A4 gene mutation has obvious racial speci city, LVAS patients from different races with different mutant hot spots and mutation frequencies (Berrettini et al. 2005;Hu et al. 2007;Lee et al. 2008). For years, the interest in SLC26A4 gene mutation closely associated with Chinese LVAS individuals has been focused on IVS7-2A > G and 2168A > G mutations (Hu et al. 2007;Zhu et al. 2012). Therefore, screening the hot mutation can early diagnose LVAS patient and nd mutation carrier and then taking measures to prevent further hearing loss by keeping LVAS patient and mutation carrier from cold and head injury.
We have previously reported that exo + polymerase-mediated 3' terminal exonuclease-resistant phosphorothioate-modi ed allele speci c primer extension formed a molecular switch sensitive to single nucleotide discrimination (Zhang and Li 2003;Zhang et al. 2005). For 3' allele-speci c primers with phosphorothioate-modi cation, perfect-matched primer turns on and mismatched primers turns off DNA polymerization. Exo + polymerases generated products only from perfect-matched primers and no products from mismatched primers, which provided an unambiguous readout for yes or no in speci c loci mutation detection assay. In this study, we will construct a double PCR technology platforms mediated by mutation sensitive molecular switch for rapid screening the SLC26A4 gene IVS7-2A > G and 2168A > G hotspot mutations and explore strategies for clinical genetic diagnosis.

Reagents
Pfu DNA polymerase with strong 3'→5' exonuclease activity was purchased from TaKaRa Bio Inc.
(Dalian, China). KOD-Plus-Mutagenesis Kit used in this study was obtained from TOYOBO Inc. (Shanghai, China). pMD19-T vector was the product of TaKaRa Bio Inc. (Dalian, China). Both unmodi ed primers and phosphorothioate-modi ed primers were synthesized commercially by TaKaRa Bio Inc. (Dalian, China).

Primer design
There are three types of primers: wild template preparation primers, mutagenesis primers and detection primers. The unmodi ed primers for wild type SLC26A4 gene preparation were designed basing on the information of Genebank database (NC_000007.13). The mutagenesis primers for SLC26A4 gene IVS7-2A>G and 2168A>G mutation through inverse PCR were designed to create point mutations in the corresponding sites according to the Muta Primer 2.0 software. Allelic speci c detection primers targeting wild and mutant type template were both designed with 3' terminal phosphorothioate-modi cation. The 3' terminal upstream to the -2 base of the reverse primers of IVS7-2A>G and the forward primers of 2168A>G were the respective mutation loci. All primers were synthesized by TaKaRa Bio Inc according to the following sequences as illustrated in Table 1.

Vector construction and identi cation
Overlapping PCR generated SLC26A4 gene fragment harboring IVS7-2A>G and 2168A>G anking sequence, which was subsequently added "A" base and then inserted into the pMD19-T vector followed by transforming into E.coli JM109 competent cells. After 37℃ incubation for 12 h, some clones were formed on the LB-agar plates containing X-Gal, IPTG and Ampicillin. White clones were randomly picked up for bacilli PCR ampli cation reaction with the M13 primers as an initial identi cation of the inserted DNA overlapped fragment by their sizes, and 1.0 ml bacilli of each positive clone was send to Invitrogen Biotechnology Co. Ltd. (Shanghai, China) for DNA sequencing.
Inverse PCR according to the manufacturer's protocol was performed twice for site-directed mutagenesis of IVS7-2A>G and 2168A>G mutation in SLC26A4 gene. Following the degradation of methylated DNA template by DpnI endonuclease, the mutant circular PCR product was transformed into E.coli JM109 competent cells again. Similarly, the SLC26A4 gene IVS7-2A>G and 2168A>G mutant template was also con rmed by a sequence analysis.
Extension of phosphorothioate-modi ed primers by exo + polymerase Two-directional primer extension was setup. Following denaturation at 95°C for 2 min, the primer extension was carried out for 30 cycles as follows: 30 sec for denaturation at 95°C, 30 sec for annealing at 60°C, and 20 sec for extension at 72°C. After the 30 cycles, an extra extension of 2 min was done before the reaction mixture was cooled down to 4°C. The primer extension reaction was performed in a total volume of 30 µL with 20 pg of template, 0.2 mmol/L dNTP, 10 ku/L of polymerase, 10 pmol/µL of both sense and antisense primers, and 2.5µL of the 10x Pfu polymerase reaction buffer which provides a nal concentration of 10 mmol/L KCl, 20 mmol/L Tris-HCl (pH 8.8 at 25 °C), 10 mmol/L (NH 4 ) 2 SO 4 , 2 mmol/L MgSO 4 , 0.1% Triton X-100. Electrophoresis in a 2.0% agarose EtBr gel at 8 V/cm in 0.5x TBE buffer was used to check whether the two-directional primer extension products were produced or not.
Hot mutations screening on clinical LVAS patient 2.0 ml of peripheral vein blood was obtained from two cases from the second a liated hospital of University of South China, who were diagnosed as LVAS by the high-resolution temporal bone CT imageological examination, and the human genomic DNA from the peripheral blood lymphocytes was extracted using phenol-chloroform extraction method. Pfu DNA polymerase combining with 3' terminal phosphorothioate-modi ed primers targeting IVS7-2A>G and 2168A>G mutation was utilized to screen the SLC26A4 gene on LVAS patients whether harboring either IVS7-2A>G or 2168A>G hot mutation. Furthermore, another PCR ampli cation was also carried out to separately get exon7+8 and exon19 fragment in SLC26A4 gene, which is suitable product including IVS7-2A>G and 2168A>G locus, and 1.0 ml of the puri ed primer extended products were further used for sequence analysis from Invitrogen Biotechnology Co. Ltd. (Shanghai, China).

Results
Construction and identi cation of vector PCR-generated wild DNA fragment separately harboring SLC26A4 gene IVS7-2A>G and 2168A>G anking sequence were successfully overlapped ( Fig. 1). After the overlapped DNA fragment was inserted into the pMD19-T vector and subsequently transformed into E.coli JM109 competent cells, a number of white clones were formed on the X-Gal/IPTG/ampicillin/LB-agar plates and randomly picked up for bacilli PCR ampli cation with the M13 primers to obtain positive clones. DNA sequencing not only con rmed the correctness of wild vector sequence as wild type template (Fig. 2a, 2c), and also documented the mutagenesis of IVS7-2A>G and 2168A>G through inverse PCR: the existence of SLC26A4 gene IVS7-2A→G and 2168A→G mutation in the mutant vector as mutant type template (Fig. 2b, 2d).

Nucleotide Discrimination by exo + polymerase
The data as illustrated in Table 2 and Fig. 3 clearly showed that IVS7-2A>G and 2168A>G allelic speci c primers perfectly matching wild type allele template were extended while no products were produced from point-mutated LVAS-associated mutant type allele template, and also allelic speci c primers perfectly matching point-mutated LVAS-associated mutant type allele template were extended but no products were observed from wild type allele template. And furthermore, we carried out double PCR for SLC26A4 gene IVS7-2A>G and 2168A>G mutations identi cation, and similarly the matched primers extension was extended while mismatched primers was not. Since 3'→5' exonuclease activity of high-delity DNA polymerase can e ciently detect and proofread the mismatched base of the allele speci c primers, once high-delity DNA polymerase combining with 3' terminal exonuclease-resistant phosphorothioatemodi ed allele speci c primers, the perfect-matched primer turned on and the mismatched primers turned off DNA polymerization. That provides an unambiguous readout for yes or no in single nucleotide discrimination: only the perfect-matched phosphorothioate-modi ed primer extension mediated by pfu DNA polymerase generated speci c products and the mismatched primers extension not.
Clinical application on screening the SLC26A4 hotspots mutation Pfu DNA polymerase-mediated phosphorothioate-modi ed primer extension was used to identify the SLC26A4 gene mutation on the LVAS patients who had been diagnosed as LVAS by temporal bone CT scan. The two LVAS patients both harbored IVS7-2A>G mutation, and were both homozygous for IVS7-2A>G mutation con rmed by DNA sequencing (Fig.4).

Discussion
LVAS is frequently seen in children and teenagers, and congenital or acquired sensorineural hearing loss is its main manifestation. Now high-resolution temporal bone CT and MRI imageological examination are still the gold standard and a prerequisite for correct diagnosis of LVAS. However, most domestic hospitals not only lack the requirement suitable for imageological diagnosis but also have no technologies that seem appropriate. As a result, most LVAS patients cannot receive timely correct diagnosis so that the doctor cannot provide the effective treatment and prevention advice to him. Thankfully, with the clinical application of deafness-associated gene mutation screening, it has demonstrated the particular advantages in the etiologic diagnosis of deafness.
Numerous studies indicate that IVS7-2A > G and 2168A > G mutations in SLC26A4 gene are the hot mutations associated with Chinese LVAS individuals (Hu et al. 2007;Jiang et al. 2010;Li et al. 2011), which is similar to other patients in East Asia (Shin et al. 2012;Tsukamoto et al. 2003)and bene ts SLC26A4 gene hotspot mutation screening in clinical application. Remarkably, none of treatment can effectively cure LVAS patients from etiology to treatment until now, so early diagnosis and aggressive prevention therapy have great practical signi cance on preventing hearing further decline. Detecting LVASassociated hotspot mutation IVS7-2A > G and 2168A > G is extremely useful in the early diagnosis and intervention of LVAS, and the current research has focused on how to quickly screen SLC26A4 gene mutation in order to foundationally prevent the occurrence of LVAS. Prohibiting either IVS7-2A > G or 2168A > G mutation carrier from marrying another one with the same mutation is thus the most effective way to protect the occurrence of LVAS.
In general, exo + polymerase-mediated phosphorothioate-modi ed primer extension is easy to operate, economic and reliable, and these advantageous features make it very suitable for clinical genetic detection and mutation screening. So in this study we applied the mutation sensitive molecular switch consisting of 3' terminal exonuclease-resistant phosphorothioate-modi ed allele speci c primers combining exo + polymerase in single base discrimination of IVS7-2A > G and 2168A > G mutations in SLC26A4 gene .
In our present study, the data from DNA sequence analysis proved that large amounts of wild and mutant type template were successfully prepared by inserting the SLC26A4 gene overlapped PCR products into pMD19-T vector and inverse PCR respectively. The two-directional primer extension showed that exo + polymerases, combining 3' terminal phosphorothioate-modi ed mismatched primers, work as an offswitch in DNA polymerization, the perfect match primer turned on and the mismatched primers turned off DNA polymerization respectively. So only allelic speci c primers perfectly matching template yielded speci c PCR product while allelic speci c primers mismatching template not, which provided an unambiguous readout for yes or no in single nucleotide discrimination. The consequence of the offswitch effect resulted from the exonuclease-resistant property of the phosphorothioate-modi cation that blocked mismatched primers excision during 3'→5' exonuclease proofreading procedure.
But it was important to remind that duplex PCR generated another 518 bp nonspeci c fragment regardless of primers perfectly matching constructed vector template or not, which was the product from the forward primer of IVS7-2A > G and the reverse primer of 2168A > G. Of course, owing to ineffective ampli cation when using genomic DNA as template, the nonspeci c product was not yielded. So when we applied Pfu DNA polymerase-mediated phosphorothioate-modi ed primer extension simultaneously screening the SLC26A4 gene on LVAS patients diagnosed as LVAS by temporal bone CT scan whether harboring IVS7-2A > G or 2168A > G mutation, we could only observe speci c PCR product without nonspeci c fragment. Actually, when using primer mixtures of IVS7-2 A > G site for G allele and 2168A > G site for G allele, only speci c primer of IVS7-2 A > G site for G allele yielded speci c PCR product, which showed that the two LVAS patients both harbored IVS7-2A > G mutation. For further identi cation the allele type of LVAS patients, the double PCR from primer mixtures of IVS7-2 A > G site for A allele and 2168A > G site for A allele were carried out again. On the contrary, not speci c primer of IVS7-2 A > G site for A allele but 2168A > G site for A allele yielded speci c PCR product. Thus it showed that both of LVAS case1 and 2 case were homozygous for IVS7-2A > G mutation. Moreover, DNA sequencing also con rmed the reliability of exo + polymerase-mediated phosphorothioate-modi ed primer extension in the identi cation SLC26A4 hotspot IVS7-2A > G and 2168A > G mutation prior to high resolution CT scan. The method is extremely suitable for quickly molecular etiologic screening. As an important testing means, genetic diagnosis of IVS7-2A > G and 2168A > G mutations and imageological examination are complementary with each other, and can improve early diagnosis and deepen aggressive prevention therapy of LVAS.
Therefore, an easy, cheap and fast molecular diagnostics of IVS7-2A > G and 2168A > G mutations in SLC26A4 gene will greatly bene t patients. Through the wide application in clinical otology, genetic diagnostic techniques can guide pre-lingual deaf patients as soon as possible to make use of residual hearing with hearing aids or implanted electronic cochlea for preventing variable dumb and avoiding any further damage to hearing of deafness patients theirselves; More importantly, it can also be used for the assessment of the risk of post-lingual deaf patients, prenatal diagnosis or prognosis, and patients with the marriage, birth genetic information to prevent the occurrence of LVAS patient fundamentally.

Declarations
Authors' contributions Guo Zifen conceived and designed the research. Guo Zifen and Zhou Chen conducted the experiments.
Gao Guoqiang contributed peripheral vein blood of clinical LAVS cases. Guo Zifen and Peng Cuiying analyzed data. Zhou Chen and Xiangman Zou wrote the manuscript. All authors read and approved the manuscript.

Funding
This work was partially funded by the Natural Science Foundation of Hunan Province (No.2018JJ2350) and the key project of Education Department of Hunan Province(No.19A419).

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate
The study was conducted after agreement from the ethics committee of University of South China and with two patients' and a healthy volunteer' informed consent statement.  Discrimination of SLC26A4 gene IVS7-2 A>G and 2168A>G mutations by Pfu DNA polymerase-mediated phosphorothioate-modi ed primer extension. M is 50bp DNA marker, Lanes 1 and 8 is speci c primer extension products from the primer of IVS7-2 A>G site for A allele; Lanes 2 and 7 are speci c primer extension products from the primer of IVS7-2 A>G site for G allele; Lanes 3 and 10 are speci c primer extension products from the primer of 2168A>G site for A allele; Lanes 4 and 9 are speci c primer extension products from the primer of 2168A>G site for G allele; Lanes 5 and 12 are speci c primer extension products from simultaneous primers of IVS7-2 A>G site for A allele and 2168A>G site for A allele; Lanes 6 and 11 are speci c primer extension products from simultaneous primers of IVS7-2 A>G site for G allele and 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.

Figure 4
The clinical application of Pfu DNA polymerase-mediated 3' terminal phosphorothioate-modi ed primer extension on screening the SLC26A4 hotspots mutation. a: M is 50bp DNA marker; Lanes 1, 2, 5 and 6 are speci c primer extension products from primer mixtures of IVS7-2 A>G site for G allele and 2168A>G site for G allele, which perfectly match mutant template; Lanes 3 and 4 are speci c primer extension products from primer mixtures of IVS7-2 A>G site for A allele and 2168A>G site for A allele, which perfectly match wild template. Lanes 1 and 3 represents LVAS case1, and Lanes 2 and 4 represents 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 represents LVAS case1 and 2.