Gjb2非编码区复合杂合变异体小鼠模型的听力损失和耳蜗病理改变

Xinyu Shi, Xiaozhou Liu, Zhengdong Zhao, Yanjun Zong, Weijia Kong, Yu Sun
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引用次数: 0

摘要

GJB2基因变异是引起感音神经性听力损失的最重要原因。大量的临床研究都集中在编码区变异上,有相当比例的单一编码区变异患者的临床表型无法解释。目前的动物模型主要包括条件敲除小鼠和少量点变异小鼠模型。目的探讨Gjb2基因非编码区点变异体和复合杂合变异体小鼠模型的致聋机制。方法利用CRISPR-Cas9技术建立携带Gjb2 c.IVS1+1G>;A变异的小鼠模型。将Gjb2IVS1+1G>;A/WT小鼠与Cx26条件敲除小鼠(Gjb2loxP/loxP;Rosa26CreER)得到Gjb2IVS1+1G>;A/−小鼠。采用基因分型和Sanger测序对小鼠模型进行鉴定。听觉脑干反应(ABR)检测听力阈值的变化。利用全贴装耳蜗,对毛细胞和螺旋神经节神经元(sgn)进行定量测定。免疫荧光染色观察耳蜗支持细胞间Cx26间隙连接斑块(GJPs)的形态,并监测活性氧(ROS)的积累情况。注射葡萄糖类似物以评估外毛细胞的葡萄糖摄取能力。结果观察期内,Gjb2IVS1+1G>;A/−小鼠表现为晚发型听力损失。在出生后20天(P20), Gjb2IVS1+1G>;A/−小鼠未显示出明显的毛细胞和sgn的损失。Cx26 gj.p.呈现碎片化。外毛细胞吸收葡萄糖的能力下降,耳蜗内ROS的积累增加。结论gjp碎片化导致的材料供应受损和氧化应激积累可能导致听力损失。我们的研究证实了c.IVS1+1G>;A变异的致病性,为解释患者的临床表型奠定了基础。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

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

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

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 non-coding 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 Gjb2IVS1+1G>A/WT mice were crossed with Cx26 conditional knockout mice (Gjb2loxP/loxP; Rosa26CreER) to obtain the Gjb2IVS1+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 glucose-uptake capacity of outer hair cells.

Result

During the observation period, Gjb2IVS1+1G>A/− mice showed late-onset hearing loss. At postnatal day 20 (P20), the Gjb2IVS1+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.

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