Yang Shu, Xie Bing-Lin, Dong Xiao-Ping, Wang Ling-Xiang, Zhu Gang-Hua, Wang Tian, Wu Wei-Jing, Lai Ruo-Sha, Tao Rong, Guan Min-Xin, Chen Fang-Yi, Tan Dong-Hui, Deng Zhong, Xie Hua-Ping, Zeng Yong, Xiao Zi-An, Xie Ding-Hua
Department of Otorhinolaryngology-Head & Neck Surgery, The Second Xiangya Hospital of Central South University, Changsha, Hunan, China.
Laboratory of Animal Nutrition and Human Health, Hunan International Joint Laboratory of Animal Intestinal Ecology and Health, College of Life Science, Hunan Normal University, Changsha, Hunan, China.
Front Mol Neurosci. 2023 Jul 27;16:1079529. doi: 10.3389/fnmol.2023.1079529. eCollection 2023.
The pathogenic gene plays a pivotal role in tip links, which is indispensable for mechanoelectrical transduction in the hair cells. However, the underlying molecular mechanism and signal regulatory networks that influence deafness is still largely unknown.
In this study, a congenital deafness family, whole exome sequencing revealed a new mutation in the pathogenic gene , subsequently; the mutation has been validated using Sanger sequencing method. Then CRISPR/Cas9 technology was employed to knockout zebrafish gene. Startle response experiment was used to compare with wide-type, the response to sound stimulation between wide-type and cdh23. To further illustrate the molecular mechanisms underlying congenital deafness, comparative transcriptomic profiling and multiple bioinformatics analyses were performed.
The YO-PRO-1 assay result showed that in deficient embryos, the YO-PRO-1 signal in inner ear and lateral line neuromast hair cells were completely lost. Startle response experiment showed that compared with wide-type, the response to sound stimulation decreased significantly in mutant larvae. Comparative transcriptomic showed that the candidate genes such as and could affect hearing by regulating ATP production and purine metabolism in a synergetic way with . RT-qPCR results further confirmed the transcriptomics results. Further compensatory experiment showed that ATP treated cdh23 embryos can partially recover the mutant phenotype.
In conclusion, our study may shed light on deciphering the principal mechanism and provide a potential therapeutic method for congenital hearing loss under the condition of mutation.
致病基因在纤毛连接中起关键作用，这对于毛细胞的机械电转导是不可或缺的。然而，影响耳聋的潜在分子机制和信号调控网络在很大程度上仍然未知。
在本研究中，对一个先天性耳聋家族进行全外显子测序，发现致病基因存在一个新突变，随后使用桑格测序法对该突变进行了验证。然后采用CRISPR/Cas9技术敲除斑马鱼的 基因。使用惊吓反应实验比较野生型和cdh23对声音刺激的反应。为了进一步阐明先天性耳聋的分子机制，进行了比较转录组分析和多种生物信息学分析。
YO-PRO-1检测结果表明，在 缺陷的胚胎中，内耳和侧线神经丘毛细胞中的YO-PRO-1信号完全丧失。惊吓反应实验表明，与野生型相比， 突变幼虫对声音刺激的反应显著降低。比较转录组分析表明，诸如 和 等候选基因可通过与 协同调节ATP生成和嘌呤代谢来影响听力。RT-qPCR结果进一步证实了转录组学结果。进一步的补偿实验表明，用ATP处理cdh23胚胎可部分恢复突变表型。
总之，我们的研究可能有助于阐明主要机制，并为 突变情况下的先天性听力损失提供一种潜在的治疗方法。
