Hearing loss and pathological changes of cochlea in a mouse model carrying compound heterozygous variants in the Gjb2 non-coding region

Abstract

Background: GJB2 gene variants are the most important cause of sensorineural hearing loss. A large number of clinical studies have focused on coding region variants, and a significant proportion of patients with single coding region variants have unexplained clinical phenotypes. Current animal models consist mainly of conditional knockout mice and a small number of the mouse models with point variants.

Objective: To investigate the underlying deafness-inducing mechanisms in the mouse models with a point variant and compound heterozygous variants in noncoding region of the Gjb2 gene.

Method: The CRISPR-Cas9 technology was utilized to develop the mouse models carrying Gjb2 c.IVS1+1G>A variant. The Gjb2^IVS1+1G>A/WT mice were crossed with Cx26 conditional knockout mice (Gjb2^loxP/loxP; Rosa26^CreER) to obtain the Gjb2^{IVS1+1G>A/−} mice. Genotyping and Sanger sequencing were used to identify the mouse models. The change in hearing thresholds was detected by auditory brainstem response (ABR). Hair cells and spiral ganglion neurons (SGNs) were quantitatively estimated by using whole-mount cochlear preparations. Immunofluorescence staining was performed to observe the morphology of Cx26 gap junction plaques (GJPs) among cochlear supporting cells and monitor the accumulation of reactive oxygen species (ROS). A glucose analog was injected to assess the glucoseuptake capacity of outer hair cells.

Result: During the observation period, Gjb2^{IVS1+1G>A/−} mice showed late-onset hearing loss. At postnatal day 20 (P20), the Gjb2^{IVS1+1G>A/−} mice did not show significant loss of hair cells and SGNs. The Cx26 GJPs showed fragmentation. The ability of the outer hair cells to uptake glucose decreased, and the accumulation of ROS in the cochlea increased.

Conclusion: We speculated that fragmented GJPs leading to impaired materials supply and oxidative stress accumulation may contribute to hearing loss. Our study confirmed the pathogenicity of c.IVS1+1G>A variant and laid the foundation for explaining the clinical phenotype of patients.

Keywords

GJB2 / hearing loss / IVS1þ1G>A / metabolic product transport impairment

References

[1] Butcher E, Dezateux C, Cortina-Borja M, Knowles RL. Prevalence of permanent childhood hearing loss detected at the universal newborn hearing screen: systematic review and meta-analysis. PLoS One. 2019; 14 (7): e0219600.

[2] Chadha S, Kamenov K, Cieza A. The world report on hearing, 2021. Bull World Health Organ. 2021; 99 (4): 242- 242A.

[3] Lieu JEC, Kenna M, Anne S, Davidson L. Hearing loss in children: a review. JAMA. 2020; 324 (21): 2195- 2205.

[4] Koffler T, Ushakov K, Avraham KB. Genetics of hearing loss: syndromic. Otolaryngol Clin. 2015; 48 (6): 1041- 1061.

[5] Sheffield AM, Smith RJH. The epidemiology of deafness. Cold Spring Harbor Perspectives In Medicine. 2019; 9 (9): a033258.

[6] Smith RJH, Bale JF, White KR. Sensorineural hearing loss in children. Lancet. 2005; 365 (9462): 879- 890.

[7] Shearer AE, Hildebrand MS, Schaefer AM, Smith RJH. Genetic hearing loss overview. In: Adam MP, et al., eds. GeneReviews(®). University of Washington, Seattle Copyright © 1993-2024, University of Washington, Seattle. GeneReviews is a registered trademark of the University of Washington, Seattle. All rights reserved; 1993.

[8] Chan DK, Chang KW. GJB2-associated hearing loss: systematic review of worldwide prevalence, genotype, and auditory phenotype. Laryngoscope. 2014; 124 (2): E34- E53.

[9] Wu X, Tang W, Muly EC, Zhang L, Lin X. Expression pattern of Connexin 26 and Connexin 30 in mature cochlea of the monkey. Biochem Biophys Res Commun. 2019; 518 (2): 357- 361.

[10] Liu W, Boström M, Kinnefors A, Rask-Andersen H. Unique expression of connexins in the human cochlea. Hear Res. 2009; 250 (1-2): 55- 62.

[11] Posukh OL, Maslova EA, Danilchenko VY, Zytsar MV, Orishchenko KE. Functional consequences of pathogenic variants of the GJB2 gene (Cx26) localized in different Cx26 domains. Biomolecules. 2023; 13 (10): 1521.

[12] Chen S, Sun Y, Lin X, Kong W. Down regulated connexin26 at different postnatal stage displayed different types of cellular degeneration and formation of organ of Corti. Biochem Biophys Res Commun. 2014; 445 (1): 71- 77.

[13] Liu XZ, Jin Y, Chen S, et al. F-actin dysplasia involved in organ of Corti deformity in Gjb2 knockdown mouse model. Front Mol Neurosci. 2021; 14: 808553.

[14] Chen S, Xie L, Xu K, et al. Developmental abnormalities in supporting cell phalangeal processes and cytoskeleton in the Gjb2 knockdown mouse model. Dis Model Mech. 2018; 11 (2).

[15] Xu K, Chen S, Bai X, et al. Degradation of cochlear Connexin26 accelerate the development of age-related hearing loss. Aging Cell. 2023; 22 (11): e13973.

[16] Wang, X, Chen, S, Qiu, Y, et al., PARP inhibitor rescues hearing and hair cell impairment in Cx26-null mice. View. 2023: 4 (6): 20230066.

[17] Lin X, Li G, Zhang Y, et al. Hearing consequences in Gjb2 knock-in mice: implications for human p.V37I mutation. Aging. 2019; 11 (18): 7416- 7441.

[18] Li Q, Cui C, Liao R, et al. The pathogenesis of common Gjb2 mutations associated with human hereditary deafness in mice. Cell Mol Life Sci. 2023; 80 (6): 148.

[19] Chen Y, Hu L, Wang X, et al. Characterization of a knock-in mouse model of the homozygous p.V37I variant in Gjb2. Sci Rep. Sci Rep. 2016; 6 (1): 33279.

[20] Inoshita A, Iizuka T, Okamura HO, et al. Postnatal development of the organ of Corti in dominant-negative Gjb2 transgenic mice. Neuroscience. 2008; 156 (4): 1039- 1047.

[21] Shahin H, Walsh T, Sobe T, et al. Genetics of congenital deafness in the Palestinian population: multiple connexin 26 alleles with shared origins in the Middle East. Hum Genet. 2002; 110 (3): 284- 289.

[22] Snoeckx RL, Hassan DM, Kamal NM, Van Den Bogaert K, Van Camp G. Mutation analysis of the GJB2 (connexin 26) gene in Egypt. Hum Mutat. 2005; 26 (1): 60- 61.

[23] Green GE, Scott DA, McDonald JM, Woodworth GG, Sheffield VC, Smith RJ. Carrier rates in the midwestern United States for GJB2 mutations causing inherited deafness. JAMA. 1999; 281 (23): 2211- 2216.

[24] Bajaj Y, Sirimanna T, Albert DM, Qadir P, Jenkins L, Bitner-Glindzicz M. Spectrum of GJB2 mutations causing deafness in the British Bangladeshi population. Clin Otolaryngol: Official Journal of ENT-UK; Official Journal of Netherlands Society For Oto-Rhino-Laryngology & Cervico-Facial Surgery. 2008; 33 (4): 313- 318.

[25] Minárik G, Tretinárová D, Szemes T, Kádasi Ľ. Prevalence of DFNB1 mutations in Slovak patients with non-syndromic hearing loss. Int J Pediatr Otorhinolaryngol. 2012; 76 (3): 400- 403.

[26] Yuan Y, Yu F, Wang G, et al. Prevalence of the GJB2 IVS1+1G >A mutation in Chinese hearing loss patients with monoallelic pathogenic mutation in the coding region of GJB2. J Transl Med. 2010; 8 (1): 127.

[27] Chen S, Xu K, Xie L, et al. The spatial distribution pattern of Connexin26 expression in supporting cells and its role in outer hair cell survival. Cell Death Dis. 2018; 9 (12): 1180.

[28] Gabriel HD, Jung D, Bützler C, et al. Transplacental uptake of glucose is decreased in embryonic lethal connexin26-deficient mice. J Cell Biol. 1998; 140 (6): 1453- 1461.

[29] Xu K, Chen S, Xie L, et al. Local macrophage-related immune response is involved in cochlear epithelial damage in distinct Gjb2-related hereditary deafness models. Front Cell Dev Biol. 2020; 8: 597769.

[30] Chang Q, Tang W, Ahmad S, Zhou B, Lin X. Gap junction mediated intercellular metabolite transfer in the cochlea is compromised in connexin30 null mice. PLoS One. 2008; 3 (12): e4088.

[31] Fetoni AR, Zorzi V, Paciello F, et al. Cx26 partial loss causes accelerated presbycusis by redox imbalance and dysregulation of Nfr2 pathway. Redox Biol. 2018; 19: 301- 317.

[32] Snoeckx RL, Huygen PLM, Feldmann D, et al. GJB2 mutations and degree of hearing loss: a multicenter study. Am J Hum Genet. 2005; 77 (6): 945- 957.

[33] Beermann J, Piccoli MT, Viereck J, Thum T. Non-coding RNAs in development and disease: background, mechanisms, and therapeutic approaches. Physiol Rev. 2016; 96 (4): 1297- 1325.

[34] Ramzan M, Duman D, Hendricks LCP, et al. Genome sequencing identifies coding and non-coding variants for non-syndromic hearing loss. J Hum Genet. 2023; 68 (10): 657- 669.

[35] Trigila AP, Pisciottano F, Franchini LF. Hearing loss genes reveal patterns of adaptive evolution at the coding and non-coding levels in mammals. BMC Biol. 2021; 19 (1): 244.

[36] da Silva-Costa SM, Coeli FB, Lincoln-de-Carvalho CR, et al. Screening for the GJB2 c.-3170 G>A (IVS 1+1 G>A) mutation in Brazilian deaf individuals using multiplex ligation-dependent probe amplification. Genet Test Mol Biomark. 2009; 13 (5): 701- 704.

[37] Bonyadi M, Fotouhi N, Esmaeili M. Prevalence of IVS1+1G>A mutation among Iranian Azeri Turkish patients with autosomal recessive non-syndromic hearing loss (ARNSHL). Int J Pediatr Otorhinolaryngol. 2011; 75 (12): 1612- 1615.

[38] Kashef A, Nikzat N, Bazzazadegan N, et al. Finding mutation within non-coding region of GJB2 reveals its importance in genetic testing of hearing loss in Iranian population. Int J Pediatr Otorhinolaryngol. 2015; 79 (2): 136- 138.

[39] Zeinali S, Davoudi-Dehaghani E, Azadmehr S, et al. GJB2 c.-23+1G>A mutation is second most common mutation among Iranian individuals with autosomal recessive hearing loss. Eur Arch Otorhinolaryngol: Official Journal of the European Federation of OtoRhino-Laryngological Societies (EUFOS): Affiliated With the German Society For Oto-Rhino-Laryngology - Head and Neck Surgery. 2015; 272 (9): 2255- 2259.

[40] Seeman P, Sakmaryová I. High prevalence of the IVS 1 + 1 G to A/GJB2 mutation among Czech hearing impaired patients with monoallelic mutation in the coding region of GJB2. Clin Genet. 2006; 69 (5): 410- 413.

[41] Barashkov NA, Dzhemileva LU, Fedorova SA, et al. Autosomal recessive deafness 1A (DFNB1A) in Yakut population isolate in Eastern Siberia: extensive accumulation of the splice site mutation IVS1+1G>A in GJB2 gene as a result of founder effect. J Hum Genet. 2011; 56 (9): 631- 639.

[42] Padma G, Ramchander PV, Nandur UV, Padma T. GJB2 and GJB6 gene mutations found in Indian probands with congenital hearing impairment. J Genet. 2009; 88 (3): 267- 272.

[43] Yuan Y, You Y, Huang D, et al. Comprehensive molecular etiology analysis of nonsyndromic hearing impairment from typical areas in China. J Transl Med. 2009; 7 (1): 79.

[44] Hişmi BO, Yılmaz ST, İncesulu A, Tekin M. Effects of GJB2 genotypes on the audiological phenotype: variability is present for all genotypes. Int J Pediatr Otorhinolaryngol. 2006; 70 (10): 1687- 1694.

[45] Barashkov NA, Teryutin FM, Pshennikova VG, et al. Age-related hearing impairment (ARHI) associated with GJB2 single mutation IVS1+1G>A in the Yakut population isolate in eastern Siberia. PLoS One. 2014; 9 (6): e100848.

[46] Mammano F. Inner ear connexin channels: roles in development and maintenance of cochlear function. Cold Spring Harbor Perspectives In Medicine. 2019; 9 (7): a033233.

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