Validating the Splicing Effect of Rare Variants in the SLC26A4 Gene Using Minigene Assay

Overview

Journal BMC Med Genomics

Publisher Biomed Central

Specialty Genetics

Date 2024 Sep 28

PMID 39334476

Authors

Yixin Zhao

Yan Long

Tao Shi

Xin Ma

Chengyu Lian

Hanjun Wang

Hongen Xu

Lisheng Yu

Xiaotao Zhao

Affiliations

Soon will be listed here.

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.

References

Albert S, Blons H, Jonard L, Feldmann D, Chauvin P, Loundon N . 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. DOI: 10.1038/sj.ejhg.5201611. View

Danilchenko V, Zytsar M, Maslova E, Posukh O . Selection of Diagnostically Significant Regions of the Gene Involved in Hearing Loss. Int J Mol Sci. 2022; 23(21). PMC: 9655724. DOI: 10.3390/ijms232113453. View

Tian Y, Xu H, Liu D, Zhang J, Yang Z, Zhang S . Increased diagnosis of enlarged vestibular aqueduct by multiplex PCR enrichment and next-generation sequencing of the SLC26A4 gene. Mol Genet Genomic Med. 2021; 9(8):e1734. PMC: 8404235. DOI: 10.1002/mgg3.1734. View

Pera A, Dossena S, Rodighiero S, Gandia M, Botta G, Meyer G . 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. PMC: 2584577. DOI: 10.1073/pnas.0805831105. View

Yuan Y, You Y, Huang D, Cui J, Wang Y, Wang Q . Comprehensive molecular etiology analysis of nonsyndromic hearing impairment from typical areas in China. J Transl Med. 2009; 7:79. PMC: 2754984. DOI: 10.1186/1479-5876-7-79. View

Kim M, Kim S, Ryu N, Ma J, Kim Y, Jung J . Gene therapy for hereditary hearing loss by SLC26A4 mutations in mice reveals distinct functional roles of pendrin in normal hearing. Theranostics. 2019; 9(24):7184-7199. PMC: 6831294. DOI: 10.7150/thno.38032. View

Hilgert N, Smith R, Van Camp G . Forty-six genes causing nonsyndromic hearing impairment: which ones should be analyzed in DNA diagnostics?. Mutat Res. 2008; 681(2-3):189-196. PMC: 2847850. DOI: 10.1016/j.mrrev.2008.08.002. View

Krawczak M, Thomas N, Hundrieser B, Mort M, Wittig M, Hampe J . Single base-pair substitutions in exon-intron junctions of human genes: nature, distribution, and consequences for mRNA splicing. Hum Mutat. 2006; 28(2):150-8. DOI: 10.1002/humu.20400. View

Dai X, Li J, Hu X, Ye J, Cai W . SLC26A4 Mutation Promotes Cell Apoptosis by Inducing Pendrin Transfer, Reducing Cl Transport, and Inhibiting PI3K/Akt/mTOR Pathway. Biomed Res Int. 2022; 2022:6496799. PMC: 9444440. DOI: 10.1155/2022/6496799. View

10.

Alper S, Sharma A . The SLC26 gene family of anion transporters and channels. Mol Aspects Med. 2013; 34(2-3):494-515. PMC: 3602804. DOI: 10.1016/j.mam.2012.07.009. View

11.

Klarov L, Pshennikova V, Romanov G, Cherdonova A, Solovyev A, Teryutin F . 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). PMC: 9740095. DOI: 10.3390/ijms232315372. View

12.

Honda K, Griffith A . Genetic architecture and phenotypic landscape of SLC26A4-related hearing loss. Hum Genet. 2021; 141(3-4):455-464. DOI: 10.1007/s00439-021-02311-1. View

13.

Al-Ani R . Various aspects of hearing loss in newborns: A narrative review. World J Clin Pediatr. 2023; 12(3):86-96. PMC: 10278076. DOI: 10.5409/wjcp.v12.i3.86. View

14.

Ito T, Choi B, King K, Zalewski C, Muskett J, Chattaraj P . SLC26A4 genotypes and phenotypes associated with enlargement of the vestibular aqueduct. Cell Physiol Biochem. 2011; 28(3):545-52. PMC: 3709178. DOI: 10.1159/000335119. View

15.

Mey K, Muhamad A, Tranebjaerg L, Rendtorff N, Rasmussen S, Bille M . Association of SLC26A4 mutations, morphology, and hearing in pendred syndrome and NSEVA. Laryngoscope. 2019; 129(11):2574-2579. DOI: 10.1002/lary.27319. View

16.

Sakuma N, Moteki H, Takahashi M, Nishio S, Arai Y, Yamashita Y . 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. PMC: 4819760. DOI: 10.1038/jhg.2015.143. View

17.

Landa P, Differ A, Rajput K, Jenkins L, Bitner-Glindzicz M . 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. PMC: 3765178. DOI: 10.1186/1471-2350-14-85. View

18.

Wu T, Cui L, Mou Y, Guo W, Liu D, Qiu J . A newly identified mutation (c.2029 C > T) in SLC26A4 gene is associated with enlarged vestibular aqueducts in a Chinese family. BMC Med Genomics. 2022; 15(1):49. PMC: 8898487. DOI: 10.1186/s12920-022-01200-4. View

19.

Miyagawa M, Nishio S, 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. PMC: 4521295. DOI: 10.1038/jhg.2014.12. View

20.

Liu Y, Wang L, Feng Y, He C, Liu D, Cai X . A New Genetic Diagnostic for Enlarged Vestibular Aqueduct Based on Next-Generation Sequencing. PLoS One. 2016; 11(12):e0168508. PMC: 5173027. DOI: 10.1371/journal.pone.0168508. View