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

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