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Validating the splicing effect of rare variants in the SLC26A4 gene using minigene assay

BMC Medical Genomics volume 17, Article number: 233 (2024) Cite this article

Abstract

Background

The SLC26A4 gene is the second most common cause of hereditary hearing loss in human. The aim of this study was to utilize the minigene assay in order to identify pathogenic variants of SLC26A4 associated with enlarged vestibular aqueduct (EVA) and hearing loss (HL) in two patients.

Methods

The patients were subjected to multiplex PCR amplification and next-generation sequencing of common deafness genes (including GJB2, SLC26A4, and MT-RNR1), then bioinformatics analysis was performed on the sequencing data to identify candidate pathogenic variants. Minigene experiments were conducted to determine the potential impact of the variants on splicing.

Results

Genetic testing revealed that the first patient carried compound heterozygous variants c.[1149 + 1G > A]; [919–2 A > G] in the SLC26A4 gene, while the second patient carried compound heterozygous variants c.[2089 + 3 A > T]; [919–2 A > G] in the same gene. Minigene experiments demonstrated that both c.1149 + 1G > A and c.2089 + 3 A > T affected mRNA splicing. According to the ACMG guidelines and the recommendations of the ClinGen Hearing Loss Expert Panel for ACMG variant interpretation, these variants were classified as “likely pathogenic”.

Conclusions

This study identified the molecular etiology of hearing loss in two patients with EVA and elucidated the impact of rare variants on splicing, thus contributing to the mutational spectrum of pathogenic variants in the SLC26A4 gene.

Peer Review reports

Introduction

Hearing loss (HL) is one of the most common sensory disorders, affecting over 5% of the world’s population [1], and it has a significant impact on the life quality of affected individuals. Approximately half of HL cases are attributed to genetic factors [2, 3]. In hereditary deafness, about 75–80% is inherited through autosomal recessive inheritance [4], and common pathogenic genes include GJB2 and SLC26A4. Pathogenic variants in the SLC26A4 gene are the leading cause of hereditary hearing loss in humans, second only to the GJB2 gene [5].

The SLC26A4 gene, which is located on chromosome 7, covers about 57 kb of genomic DNA and comprises 21 exons (including the first non-coding exon) [6]. The SLC26A4 gene product, pendrin, is a multipass transmembrane protein [7], which permits the exchange of anions between the cytosol and extracellular space [8,9,10]. Pendrin is primarily expressed in the inner ear, thyroid, and kidney [6, 11]. Variants in the SLC26A4 gene cause hearing loss, which can be non-syndromic autosomal recessive deafness (DFNB4, OMIM #600791) associated with enlarged vestibular aqueduct (EVA) or Pendred syndrome (Pendred, OMIM #605646) [12,13,14]. DFNB4 is characterized by sensorineural hearing loss, combined with EVA or less common cochlear malformation defect [15, 16]. Pendred syndrome is characterized by bilateral sensorineural hearing loss with EVA and an iodine defect that can lead to thyroid goiter [16, 17]. Now, it is known that EVA is associated with variants in the SLC26A4 gene [18] and is a penetrant feature of SLC26A4-related HL [6]. However, there are also some studies suggesting that two other genes, KCNJ10 or FOXI1, are associated with EVA, but this is still debated [15, 19]. Additionally, variants in both the SLC26A4 and EPHA2 genes can also cause Pendred syndrome [20].

The diagnosis of hereditary hearing loss requires audiological, physical examination, ancillary tests (e.g., computed tomography examination of the temporal bone), and molecular genetic analysis, and the latter can be used for various types of syndromic and non-syndromic deafness [21]. Screening for SLC26A4 variants has become an important part of molecular genetic testing for HL, especially for patients with EVA [6]. Next-generation sequencing (NGS) techniques, which are capable of detecting both known and novel causative variants in many genes associated with HL, including SLC26A4, are increasingly used for routine diagnosis of HL [6, 22, 23]. The SLC26A4 gene has been extensively studied, and over 100 different pathogenic variants spanning the entire coding region have been reported [11]. However, the molecular mechanism of SLC26A4 variants in hearing loss has not yet been fully elucidated [24].

In this study, clinical phenotype analysis was performed on two patients with EVA, and genetic screening was performed on patients with Multiplex PCR and NGS. The results showed that compound heterozygous SLC26A4 variants (c.[1149 + 1G > A]; [919–2 A > G], c.[2089 + 3 A > T]; [919–2 A > G]) were present in both patients. The c.919–2 A > G was a commonly known pathogenic variant. The splicing variant c.1149 + 1G > A was reported in the ClinVar database, and the other splicing variant c.2089 + 3 A > T was not reported in the related database and literature. In order to determine the pathogenicity of these two splicing variants, we conducted minigene experiments to reveal their effect on splicing.

Materials and methods

Study participants and clinical evaluation

The participants were two patients with hearing loss who visited Peking University People’s Hospital. All experiments were approved by the Ethics Committee of Peking University People’s Hospital (approval No. 2021PHA153-003). Written informed consent was obtained from the patients or their parents. The clinical evaluation conducted in this study included otoscopy, pure-tone audiometry (PTA), tympanometry, distortion product otoacoustic emissions (DPOAE), auditory brainstem response (ABR). Temporal bone CT was also included.

Next-generation sequencing and bioinformatics analysis

A total of 2 mL of peripheral blood were extracted from each of the two patients and their parents. The genetic testing was performed using a test kit developed by the Precision Medicine Center at the Academy of Medical Sciences, Zhengzhou University, primarily focusing on the identification of single nucleotide variants (SNVs) and small indels of common HL genes in Chinese population, including GJB2, SLC26A4, and MT-RNR1. DNA extraction, next-generation sequencing, bioinformatics analysis, and variant interpretation were conducted as in a previous study [23]. In brief, after purity and quality checking of extracted genomic DNA, multiplex PCR enrichment was performed to amplify the exonic regions and the flanking region of the three designed genes, and it was divided into two application rounds. Bioinformatics analysis was performed in the bcbio-nextgen framework (https://github.com/bcbio/bcbio-nextgen), which integrates Burrows-Wheeler Aligner (BWA; version 0.7.17-r1188), GATK Haplotype Caller software (version 4.1.2) for variant calling, and variant annotation tool Vcfanno software (version 0.3.1) with external databases. Clinicians and genetic consultants then interpreted the selected variants in accordance with the standard guidelines for genetic variation interpretation provided by ACMG [25], as well as the specific recommendations from ClinGen’s hearing loss expert group [26]. The variant nomenclature was based on the canonical transcript NM_000441.1 of the SCL26A4 gene.

Minigene analysis

To construct the vectors for minigene experiments of variants c.1149 + 1G > A and c.2089 + 3 A > T, we designed primers using the NCBI Primer-Blast website for the exons 9 and 18 of the SLC26A4 gene and their adjacent intronic regions. The primers (SLC26A4-1049-F1/R1 and SLC26A4-2089-F1/R1) were obtained from Sangon Biotech (China) and were listed in Table 1. Specific primers were used for PCR amplification of the target regions (DNA polymerase, Takara, R050) with gDNA from the proband and normal control group as templates. The purified PCR products and the plasmid pcMINI were digested with restriction endonucleases NotI-HF (NEB, R3189) and KpnI-HF(NEB, R3142) and then ligated by T4 DNA ligase (ThermoFisher, EL0011). The ligation products were transformed into E. coli Stabl3 competent cells (ANGYUBIO, G6009), and positive clones were selected and sequenced for identification (Sangon Biotech). The correctly identified positive recombinant plasmids were used for cell transfection.

Table 1 Primer sequences used in the minigene assay
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Both HEK-293 and HeLa cells were cultured in DMEM (Gibco, 11995065) with 10% FBS (Procell, 164210-50). The four groups of recombinant plasmids and pcMINI control plasmid were separately transfected into HEK-293 and HeLa cells by polyethyleneimine (Polysciences, 23966). After 48 h of transfection, the total mRNA of cells was extracted (SparkJade, AC0202-B), and then RT-PCR using HiScript II One-Step RT-PCR Kit (Vazyme, P162-01) and specific primers, pMini-F/R (Table 1). The PCR products were identified by 1% agarose (BIOWEST, 111935) gel electrophoresis and validated by Sanger sequencing (Sangon Biotech).

Results

Clinical phenotype analysis of the patients 1 and 2

Both patients are from non-consanguineous family, and are the first child of the family (Fgiure 1 A, B). Patient 1 presented with bilateral profound sensorineural hearing loss (Fig. 1C), which started at the age of 2 with no fluctuations. Patient 2 had bilateral severe hearing loss (Fig. 1D), poor language development, and had undergone cochlear implantation. Temporal bone CT scans confirmed bilateral EVA in both patients (Fig. 1E, F). Thyroid disease was ruled out in both patients. Threrefore, these two patients were clinically diagnosed as hearing loss with EVA.

Fig. 1
figure 1

Family pedigree, auditory phenotypes, and magnetic resonance imaging (MRI) of EVA patients 1 and 2. A-B. pedigree of the two patients with EVA. Squares and circles represent males and females, respectively, and P represents proband; C-D. pure-tone audiometry results of two patients. E-F. CT imaging of two patients showing bilateral enlarged vestibular aqueduct

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Genetic analysis revealed common and rare SLC26A4 variants

We conducted multiplex PCR and NGS of common hearing loss genes aimed to screening pathogenic variants associated with EVA and hearing loss in the two patients, revealing that both patients have variants in the SLC26A4 gene.

Patient 1 carries compound heterozygous variants in SLC26A4 (NM_000441.1) c.1149 + 1G > A and c.919–2 A > G, inherited from the father and mother, respectively. The c.919–2 A > G variant is a known pathogenic variant, and the c.1149 + 1G > A variant was not found in the gnomAD and ExAC databases in the general population (ACMG PM2_Supporting evidence). Multiple software predictions suggest that this variant has a damaging effect on the gene or gene product (dbscSNV score: 0.9999, MaxEntScan score: 8.182, ACMG PP3 evidence). It has been confirmed that the variant c.1149 + 1G > A is arranged in a trans with the variant c.919–2 A > G, and the latter is a known pathogenic variant (PM3 evidence). Therefore, according to the ACMG guidelines, this variant is classified as a Variant of Uncertain Significance (VUS) (Table 2).

Table 2 Pathogenic variants in the SLC26A4 gene found in the two patients
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Patient 2 carries compound heterozygous variants in SLC26A4 (NM_000441.1) c.2089 + 3 A > T and c.919–2 A > G, inherited from the father and mother, respectively. The c.2089 + 3 A > T variant was not found in the gnomAD and ExAC databases in the general population (ACMG PM2_Supporting evidence). Multiple software predictions indicate that this variant has a deleterious impact on the gene or gene product (dbscSNV score: 0.9994, MaxEntScan score: 3.6843, ACMG PP3 evidence). Confirmation has been made that variant c.1149 + 1G > A is in a trans with variant c.919–2 A > G, and the latter variant is known to be pathogenic (PM3 evidence). Therefore, according to the ACMG guidelines, this variant is classified as VUS (Table 2).

The variant SLC26A4 c.1149 + 1G > A has been reported in ClinVar (ClinVar RCV003472912.1) but has no functional studies. The SLC26A4 c.2089 + 3 A > T is a novel variant and has not been reported. In order to further investigate the impact of these two variants on mRNA splicing, we will conduct functional studies using minigene experiments.

Minigene experiments confirmed the splicing effect of rare variants

The variants SLC26A4 c.1149 + 1G > A and c.2089 + 3 A > T are located in the downstream introns of exon 9 and exon 18 of the SLC26A4 gene, respectively. Therefore, we constructed wild-type and variant fragments of exon 9 and exon 18, along with their partial upstream and downstream intronic sequences (including the variant sites), in the pcMINI vector. The newly constructed vector sequence pattern is ExonA-IntronA- inserted fragment-IntronB-ExonB, with pcMINI vector as the negative control (the corresponding sequence is ExonA-IntronA-IntronB-ExonB). These constructs were then transfected into HEK-293 and HeLa cells. Finally, total RNA was extracted from the samples for RT-PCR, and the products were analyzed by agarose gel electrophoresis and Sanger sequencing.

As shown in Figs. 2 and 3, the results indicate that in both HEK-293 and HeLa cells, the size of the variants RT-PCR products for c.1149 + 1 and c.2089 + 3, consistent with the pcMINI product (ExonA-ExonB), were smaller than the products of wild-type. Sequencing results also showed that the wild-type RT-PCR product of c.1149 + 1 contained the normal splicing sequence with exon 9 (ExonA-Exon9-ExonB). However, the c.1149 + 1G > A variant caused abnormal splicing, resulting in complete skipping of exon 9 (Fig. 2). Similarly, the wild-type c.2089 + 3 formed the normal splicing sequence with exon 18 (ExonA-Exon18-ExonB), but the c.2089 + 3 A > T variant caused abnormal splicing, leading to complete skipping of exon 18 (Fig. 3).

Fig. 2
figure 2

The effect of the variant c.1149 + 1G > A on splicing by minigene experiment. (A) Design of minigene experiment and transcript products in c.1149 + 1G wild type and c.1149 + 1G > A; (B) The gel electrophoresis of RT-PCR products using pMini-F/R primers showed a band of 537 bp in the c.1149 + 1 wild-type and a band of 389 bp in c.1149 + 1G > A; (C) Sanger sequencing results of 537 bp and 389 bp PCR products showed two different splicing patterns

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Fig. 3
figure 3

The effect of the variant c.2089 + 3 A > T on splicing by minigene experiment. (A) Design of minigene experiment and transcript products in c.2089 + 3 A wild type and c.2089 + 3 A > T; (B) The gel electrophoresis of RT-PCR products using pMini-F/R primers showed a band of 444 bp in the c.2089 + 3 A wild-type and a band of 389 bp in the c.2089 + 3 A > T; (C) Sanger sequencing results of 444 bp and 389 bp PCR products showed two different splicing pattern

Full size image

Based on these results, it can be concluded that the c.1149 + 1G > A and c.2089 + 3 A > T variants in the SLC26A4 gene can cause abnormal mRNA splicing of SLC26A4 (PS3) and can be classified as “likely pathogenic” (PM2_supporting + PP4 + PM3 + PS3, Table 2).

Discussion

In this study, we first performed multiplex PCR enrichment and next-generation sequencing of the exon and flanking regions of common deafness genes on two patients with EVA. We identified compound heterozygous pathogenic variants c.1149 + 1G > A and c.919–2 A > G in patient 1, and c.2089 + 3 A > T and c.919–2 A > G in patient 2. The c.919–2 A > G variant detected in both patients was located in intron 7, which is a common pathogenic variant in Asian populations, and can lead to the skipping of exon 8, resulting in the formation of a premature stop codon at amino acid position 311 and ultimately generating a truncated form of the pendin protein. The c.1149 + 1G > A and c.2089 + 3 A > T variants are intronic variants that have not been functionally validated. In minigene experiments, we found that both intronic variants resulted in abnormal splicing of the gene. The c.1149 + 1G > A variant led to the skipping of exon 9, while the c.2089 + 3 A > T variant led to the skipping of exon 18. This study identified the molecular etiology of hearing loss in two patients with EVA and elucidated the impact of rare variants on splicing, thus contributing to the mutational spectrum of pathogenic variants in the SLC26A4 gene.

Both DFNB4 and Pendred syndrome are inherited as an autosomal recessive trait. Thus, a genetic testing is expected to detect two mutant alleles (M2), either as homozygous or compound heterozygous variants of SLC26A4. In recent years, many pathogenic variants of SLC26A4 have been found with the majority of patients being compound heterozygotes [27]. Previous studies have shown that the relative proportion of M2 genotypes (some EVA patients with only one detectable mutant allele (M1), or with no mutant alleles (M0)) is variable in different populations [16]. In East Asia, including China, Korea, or Japan, 65–95% of EVA patients can detect biallelic variants in the SLC26A4 gene [18, 23, 28]. In Caucasian individuals with EVA, the proportion of M2 genotypes is much less, approximately 25% [29]. This may suggest the existence of more genetic factors for European-Caucasian EVA patients [16], as not all patients diagnosed with EVA have biallelic SLC26A4 variants [30]. On the other hand, this also highlights the importance of the functional study of variants of uncertain significance in making conclusive genetic tests.

The SLC26A4 c.1149 + 1G > A and c.2089 + 3 A > T variants found in this study are intronic ones that have not been functionally investigated. Intronic variants can lead to monogenic disease at the splicing level [31], and exon skipping or intron retention are the preferred consequences [32]. Among the splicing variants (accounting for about 10% of pathogenic variants) recorded in HGMD, approximately 90% occur within the 5 exonic and 15 intronic nucleotides flanking the exon-intron junction [33]. Splicing-related genetic variants are likely to constitute a significant proportion of genetic alterations in hereditary diseases and potential genetic predisposition to cancer [32].

SLC26A4 variants in the Chinese population exhibit a wider variant spectrum, distributed across all exons and their flanking sequences [34, 35]. Compared to other ethnicities, SLC26A4 variants in the Chinese population demonstrate greater diversity [18], with a few most common variants (e.g., c.919–2 A > G and c.2168 A > G) and many rare variants. Our study highlights the importance of functional study of rare variants, improving diagnostic efficiency [36], and providing effective risk assessment and genetic counseling for hearing loss patients and their families [37].

Conclusions

In this study, compound heterozygous variants (c.1149 + 1G > A/c.919–2 A > G) and (c.2089 + 3 A > T/c.919–2 A > G) were identified in two patients with EVA, further strengthening the association between SLC26A4 variants and EVA. Additionally, minigene splicing analysis classified the variants c.1149 + 1G > A and c.2089 + 3 A > T as “likely pathogenic”, enriching the mutational spectrum of the SLC26A4 gene.

Data availability

We have submitted the two splicing variants of SLC26A4 to ClinVar, and their corresponding accession numbers are SCV005049548 and SCV005049549.https://www.ncbi.nlm.nih.gov/clinvar/variation/3238647/?oq=SCV005049549https://www.ncbi.nlm.nih.gov/clinvar/variation/2678936/?oq=SCV005049548.

Abbreviations

ABR:

Auditory brainstem response

DPOAE:

Distortion product otoacoustic emissions

EVA:

Enlarged vestibular aqueduct

HL:

Hearing loss

M1:

One detectable mutant allele

M2:

Two mutant alleles

NGS:

Next-generation sequencing

PTA:

Pure-tone audiometry

SNVs:

Tympanometry, single nucleotide variants

VUS:

Variant of Uncertain Significance

References

  1. Deafness. and hearing loss [https://www.who.int/news-room/fact-sheets/detail/deafness-and-hearing-loss]

  2. Morton CC, Nance WE. Newborn hearing screening–a silent revolution. N Engl J Med. 2006;354(20):2151–64.

    Article  CAS  PubMed  Google Scholar 

  3. Al-Ani RM. Various aspects of hearing loss in newborns: a narrative review. World J Clin Pediatr. 2023;12(3):86–96.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Sakuma N, Moteki H, Takahashi M, Nishio SY, Arai Y, Yamashita Y, Oridate N, Usami S. An effective screening strategy for deafness in combination with a next-generation sequencing platform: a consecutive analysis. J Hum Genet. 2016;61(3):253–61.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Hilgert N, Smith RJ, Van Camp G. Forty-six genes causing nonsyndromic hearing impairment: which ones should be analyzed in DNA diagnostics? Mutat Res. 2009;681(2–3):189–96.

    Article  CAS  PubMed  Google Scholar 

  6. Danilchenko VY, Zytsar MV, Maslova EA, Posukh OL. Selection of diagnostically significant regions of the SLC26A4 gene involved in hearing loss. Int J Mol Sci 2022, 23(21).

  7. Choi BY, Kim HM, Ito T, Lee KY, Li X, Monahan K, Wen Y, Wilson E, Kurima K, Saunders TL, et al. Mouse model of enlarged vestibular aqueducts defines temporal requirement of Slc26a4 expression for hearing acquisition. J Clin Invest. 2011;121(11):4516–25.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Dossena S, Rodighiero S, Vezzoli V, Nofziger C, Salvioni E, Boccazzi M, Grabmayer E, Bottà G, Meyer G, Fugazzola L, et al. Functional characterization of wild-type and mutated pendrin (SLC26A4), the anion transporter involved in Pendred syndrome. J Mol Endocrinol. 2009;43(3):93–103.

    Article  CAS  PubMed  Google Scholar 

  9. Alper SL, Sharma AK. The SLC26 gene family of anion transporters and channels. Mol Aspects Med. 2013;34(2–3):494–515.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Pera A, Dossena S, Rodighiero S, Gandia M, Botta G, Meyer G, Moreno F, Nofziger C, Hernandez-Chico C, Paulmichl M. Functional assessment of allelic variants in the SLC26A4 gene involved in Pendred syndrome and nonsyndromic EVA. Proc Natl Acad Sci U S A. 2008;105(47):18608–13.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Albert S, Blons H, Jonard L, Feldmann D, Chauvin P, Loundon N, Sergent-Allaoui A, Houang M, Joannard A, Schmerber S, et al. SLC26A4 gene is frequently involved in nonsyndromic hearing impairment with enlarged vestibular aqueduct in caucasian populations. Eur J Hum Genet. 2006;14(6):773–9.

    Article  CAS  PubMed  Google Scholar 

  12. Kim M-A, Kim SH, Ryu N, Ma J-H, Kim Y-R, Jung J, Hsu C-J, Choi JY, Lee K-Y, Wangemann P, et al. Gene therapy for hereditary hearing loss by SLC26A4 mutations in mice reveals distinct functional roles of pendrin in normal hearing. Theranostics. 2019;9(24):7184–99.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Park HJ, Shaukat S, Liu XZ, Hahn SH, Naz S, Ghosh M, Kim HN, Moon SK, Abe S, Tukamoto K, et al. Origins and frequencies of SLC26A4 (PDS) mutations in east and south asians: global implications for the epidemiology of deafness. J Med Genet. 2003;40(4):242–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Bassot C, Minervini G, Leonardi E, Tosatto SC. Mapping pathogenic mutations suggests an innovative structural model for the pendrin (SLC26A4) transmembrane domain. Biochimie. 2017;132:109–20.

    Article  CAS  PubMed  Google Scholar 

  15. Klarov LA, Pshennikova VG, Romanov GP, Cherdonova AM, Solovyev AV, Teryutin FM, Luginov NV, Kotlyarov PM, Barashkov NA. Analysis of SLC26A4, FOXI1, and KCNJ10 gene variants in patients with incomplete partition of the cochlea and enlarged vestibular aqueduct (EVA) anomalies. Int J Mol Sci 2022, 23(23).

  16. Honda K, Griffith AJ. Genetic architecture and phenotypic landscape of SLC26A4-related hearing loss. Hum Genet. 2022;141(3–4):455–64.

    Article  CAS  PubMed  Google Scholar 

  17. Ito T, Choi BY, King KA, Zalewski CK, Muskett J, Chattaraj P, Shawker T, Reynolds JC, Butman JA, Brewer CC, et al. SLC26A4 genotypes and Phenotypes Associated with enlargement of the vestibular aqueduct. Cell Physiol Biochem. 2011;28(3):545–52.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Wang QJ, Zhao YL, Rao SQ, Guo YF, Yuan H, Zong L, Guan J, Xu BC, Wang DY, Han MK, et al. A distinct spectrum of SLC26A4 mutations in patients with enlarged vestibular aqueduct in China. Clin Genet. 2007;72(3):245–54.

    Article  PubMed  Google Scholar 

  19. Priya Landa A-MD, Rajput K. Lucy Jenkins and Maria Bitner-Glindzicz Lack of significant association between mutations of KCNJ10 or FOXI1 and SLC26A4 mutations in pendred syndrome/enlarged vestibular aqueducts. BMC Med Genet. 2013;14:85.

    Article  PubMed  PubMed Central  Google Scholar 

  20. Li M, Nishio SY, Naruse C, Riddell M, Sapski S, Katsuno T, Hikita T, Mizapourshafiyi F, Smith FM, Cooper LT, et al. Digenic inheritance of mutations in EPHA2 and SLC26A4 in Pendred syndrome. Nat Commun. 2020;11(1):1343.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Kochhar A, Hildebrand MS, Smith RJ. Clinical aspects of hereditary hearing loss. Genet Med. 2007;9(7):393–408.

    Article  PubMed  Google Scholar 

  22. Liu Y, Wang L, Feng Y, He C, Liu D, Cai X, Jiang L, Chen H, Liu C, Wu H, et al. A New Genetic Diagnostic for enlarged vestibular aqueduct based on next-generation sequencing. PLoS ONE. 2016;11(12):e0168508.

    Article  PubMed  PubMed Central  Google Scholar 

  23. Tian Y, Xu H, Liu D, Zhang J, Yang Z, Zhang S, Liu H, Li R, Tian Y, Zeng B et al. Increased diagnosis of enlarged vestibular aqueduct by multiplex PCR enrichment and next-generation sequencing of the SLC26A4 gene. Mol Genet Genom Med 2021:e1734.

  24. Dai X, Li J, Hu X, Ye J, Cai W, Ramkumar V. SLC26A4 mutation promotes cell apoptosis by inducing pendrin transfer, reducing Cl- Transport, and inhibiting PI3K/Akt/mTOR pathway. Biomed Res Int. 2022;2022:1–9.

    CAS  Google Scholar 

  25. Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, Grody WW, Hegde M, Lyon E, Spector E, Voelkerding K, Rehm HL. ACMG Laboratory Quality Assurance Committee. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Sci. 2015;17(5):405–24.

    Google Scholar 

  26. Oza AM, DiStefano MT, Hemphill SE, Cushman BJ, Grant AR, Siegert RK, Shen J, Chapin A, Boczek NJ, Schimmenti LA, Murry JB, Hasadsri L, Nara K, Kenna M, Booth KT, Azaiez H, Griffith A, Avraham KB, Kremer H, Rehm HL, Amr SS. Abou Tayoun AN: ClinGen Hearing Loss Clinical Domain Working Group. Expert specification of the ACMG/AMP variant interpretation guidelines for genetic hearing loss. Hum Mutat. 2018;39(11):1593–613.

    Article  PubMed  PubMed Central  Google Scholar 

  27. Mey K, Muhamad AA, Tranebjaerg L, Rendtorff ND, Rasmussen SH, Bille M, Caye-Thomasen P. Association of SLC26A4 mutations, morphology, and hearing in pendred syndrome and NSEVA. Laryngoscope. 2019;129(11):2574–9.

    Article  CAS  PubMed  Google Scholar 

  28. Miyagawa M, Nishio SY, Usami S. Mutation spectrum and genotype-phenotype correlation of hearing loss patients caused by SLC26A4 mutations in the Japanese: a large cohort study. J Hum Genet. 2014;59(5):262–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Chattaraj P, Munjal T, Honda K, Rendtorff ND, Ratay JS, Muskett JA, Risso DS, Roux I, Gertz EM, Schaffer AA, et al. A common SLC26A4-linked haplotype underlying non-syndromic hearing loss with enlargement of the vestibular aqueduct. J Med Genet. 2017;54(10):665–73.

    Article  CAS  PubMed  Google Scholar 

  30. Danilchenko VY, Zytsar MV, Maslova EA, Bady-Khoo MS, Barashkov NA, Morozov IV, Bondar AA, Posukh OL. Different rates of the SLC26A4-Related hearing loss in two indigenous peoples of Southern Siberia (Russia). Diagnostics (Basel) 2021, 11(12).

  31. Nielsen KB, Sørensen S, Cartegni L, Corydon TJ, Doktor TK, Schroeder LD, Reinert LS, Elpeleg O, Krainer AR, Gregersen N, et al. Seemingly neutral polymorphic variants May Confer immunity to splicing-inactivating mutations: a synonymous SNP in exon 5 of MCAD protects from deleterious mutations in a flanking exonic splicing enhancer. Am J Hum Genet. 2007;80(3):416–32.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Krawczak M, Thomas NST, Hundrieser B, Mort M, Wittig M, Hampe J, Cooper DN. Single base-pair substitutions in exon-intron junctions of human genes: nature, distribution, and consequences for mRNA splicing. Hum Mutat. 2007;28(2):150–8.

    Article  CAS  PubMed  Google Scholar 

  33. Ward AJ, Cooper TA. The pathobiology of splicing. J Pathol. 2009;220(2):152–63.

    Article  Google Scholar 

  34. Wu T, Cui L, Mou Y, Guo W, Liu D, Qiu J, Xu C, Zhou J, Han F, Sun Y. A newly identified mutation (c.2029 C > T) in SLC26A4 gene is associated with enlarged vestibular aqueducts in a Chinese family. BMC Med Genom 2022, 15(1).

  35. Huang S, Han D, Yuan Y, Wang G, Kang D, Zhang X, Yan X, Meng X, Dong M, Dai P. Extremely discrepant mutation spectrum of SLC26A4 between Chinese patients with isolated Mondini deformity and enlarged vestibular aqueduct. J Translational Med. 2011;9:167.

    Article  CAS  Google Scholar 

  36. Lord J, Gallone G, Short PJ, McRae JF, Ironfield H, Wynn EH, Gerety SS, He L, Kerr B, Johnson DS, et al. Pathogenicity and selective constraint on variation near splice sites. Genome Res. 2019;29(2):159–70.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Yuan Y, You Y, Huang D, Cui J, Wang Y, Wang Q, Yu F, Kang D, Yuan H, Han D, et al. Comprehensive molecular etiology analysis of nonsyndromic hearing impairment from typical areas in China. J Translational Med. 2009;7:79.

    Article  Google Scholar 

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Acknowledgements

We sincerely thank all the family members for their participation in this study.

Funding

This study was supported by research and development fund of Peking University People’s Hospital (2147001094).

Author information

Author notes

  1. Yixin Zhao and Yan Long Co-first authors.

Authors and Affiliations

  1. Department of Otorhinolaryngology-Head and Neck Surgery, Peking University People’s Hospital, No.11 Xizhimen South Street, Beijing, 100044, China

    Yixin Zhao, Tao Shi, Xin Ma & Lisheng Yu

  2. Department of Clinical Laboratory, Peking University People’s Hospital, No.11 Xizhimen South Street, Xicheng District, Beijing, 100044, China

    Yan Long & Xiaotao Zhao

  3. Precision Medicine Center, Academy of Medical Science, Zhengzhou University, Daxuebei Road No. 40, Zhengzhou, 450052, China

    Chengyu Lian, Hanjun Wang & Hongen Xu

Authors
  1. Yixin Zhao
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  2. Yan Long
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  3. Tao Shi
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  4. Xin Ma
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  7. Hongen Xu
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  8. Lisheng Yu
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  9. Xiaotao Zhao
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Contributions

Yixin Zhao: Conceptualization, Methodology, Writing-Original draft preparation, Resources; Yan Long: Project administration, Visualization, Writing-Review & Editing; Tao Shi: Data Curation, Validation; Xin Ma: Data Curation, Validation; Chengyu Lian: Data Curation; Hanjun Wang: Methodology, Formal analysis; Hongen Xu: Formal analysis; Lisheng Yu: Conceptualization, Formal analysis, Supervision; Xiaotao Zhao: Conceptualization, Methodology, Formal analysis.

Corresponding authors

Correspondence to Lisheng Yu or Xiaotao Zhao.

Ethics declarations

Ethics approval and consent to participate

This study was performed in line with the principles of the Declaration of Helsinki, and it was approved by the Medical Ethics Committee of Peking University People’s Hospital (Approval No. 2021PHA153-003). The written informed consent to participate in the study was obtained from all the participants and from parents for minors participants in the study.

Consent for publication

The written informed consent was obtained from all participants and parents of the minor participants for the publication of identifying images or other personal or clinical details to be published in this study.

Competing interests

The authors declare no competing interests.

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Zhao, Y., Long, Y., Shi, T. et al. Validating the splicing effect of rare variants in the SLC26A4 gene using minigene assay. BMC Med Genomics 17, 233 (2024). https://doi.org/10.1186/s12920-024-02007-1

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  • DOI: https://doi.org/10.1186/s12920-024-02007-1

Keywords

BMC Medical Genomics

ISSN: 1755-8794

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