聋病基因HARS2的听觉功能及致聋机制研究

Mutations of gene HARS2 can cause not only nonsyndromic but also syndromic hearing loss. HARS2, which encodes the mitochondrial histidyl tRNA synthetase, highly expresses in mitochondria, and functions in vivo as a homodimer. To date, functional study of HARS2 is very rare, the pathogenic mechanism of HARS2 are not clear, no animal models has been used in functional study of HARS2. Previously we identified 4 novel missense mutations of HARS2 with compound heterozygous mutations co-segregated with deafness in two autosomal recessive deafness families respectively. In this study, on the basis of sufficient previously outcomings, we propose to identify the pathogenic mechanism of novel missense mutations of HARS2 by analyses of protein expression, dimerization, and enzyme activity of HARS2 mutants in vitro. Further, we propose to generate the knock-out zebrafish model for orthologous gene hars and knock-in models for missense mutations of hars. By comparative study of auditory physiology, function, micromorphology of the inner ear and the lateral line system in the knock-out and the knock-in zebrafish models in vivo, we aim to identify the key role of HARS2 in auditory pathway and the pathogenic mechanism of its different types of mutations. The proposed study may provide important insights into the mechanism of hearing and deafness from multiple levels, and may improve the diagnosis, intervention and prevention of the associated hearing impairment.

HARS2基因突变既可引起非综合征型耳聋，又可引起综合征型耳聋。HARS2编码组氨酰tRNA合成酶，高表达于线粒体中，并以二聚体方式发挥功能。迄今，HARS2的功能研究甚少，致聋机制不明，未见利用动物模型进行功能研究的报道。前期，本课题组在两个常染色体隐性遗传耳聋家系中发现四个HARS2新发错义突变，在各自的家系中分别以复合杂合突变型与耳聋表型共分离。本课题拟在充分的前期基础上，体外研究突变体蛋白的表达量、表达位置、二聚体化及酶活性的变化，以验证HARS2新发错义突变的致病性和致聋机理；同时，构建同源基因hars基因敲除和错义突变敲入斑马鱼模型，在体研究不同突变体模型的听觉生理学、内耳器官及侧线系统的显微形态学及功能学变化，以明确HARS2在听觉功能途径中所发挥的关键作用及其不同类型突变的致聋机制。本研究有望在多个层面更深入的理解听觉的发生和发病机制，促进相关聋病的诊断、干预和预防。

HARS2基因突变既可引起非综合征性耳聋，又可引起综合征性耳聋。HARS2编码氨酰tRNA合成酶，高表达于线粒体中，并以二聚体方式发挥功能。迄今，HARS2的功能研究甚少，致聋机制不明，未见利用动物模型进行功能研究的报道。本研究按计划同时开展体外和体内功能研究，其中体内研究额外增加小鼠模式动物研究验证斑马鱼研究结果。体外实验暂未发现有价值研究结果。斑马鱼dtd1基因研究发现如下：1）野生型dtd1表达于斑马鱼神经系统、侧线神经丘及内耳毛细胞；2）MO敲降dtd1（dtd1-mo）后躯干毛细胞簇、内耳毛细胞、侧线神经丘L1和L3毛细胞数量减少；野生型mRNA可以部分挽救，而点突变mRNA挽救失败。3）dtd1-mo斑马鱼听力下降；4）dtd1-mo斑马鱼内耳毛细胞静纤毛数量减少，且分布不均匀；5）dtd1-mo斑马鱼平衡功能异常，野生型mRNA可以部分挽救。小鼠hars2基因模式动物研究发现如下：1）野生型hars2表达于内耳毛细胞和螺旋神经节；2）hars2基因内耳条件性敲除（hars2-CKO）小鼠内耳发育正常；3）hars2-CKO小鼠随年龄增长毛细胞从底圈向顶圈逐渐丢失，与听力由高频向全频下降渐降符合；4）线粒体肿胀所致功能障碍可能为hars2-CKO小鼠听力下降的原因。本研究利用斑马鱼和小鼠两种模式动物从体内层面充分探索HARS2基因的内耳功能及致病机制，深入的理解其在听觉中的发生和发病机制，促进相关聋病的诊断、干预和预防，为基因治疗提供理论依据。