HKDC1 在视网膜色素变性病变中的功能研究

Retinitis Pigmentosa (RP) is an inheritable retinal disease that can lead to blindness. RP affects more than 3 million people in China, and there is no effective treatment of RP. One key aspect of RP therapy research is to identify causative genes and the molecular mechanisms underlying the disease. Our previous work identified HKDC1 as an arRP candidate gene through whole exome sequencing analysis of a cohort of RP families. However, the molecular functions of HKDC1 in the retina remain elusive. This proposal aims to investigate the roles of HKDC1 in RP pathogenesis using Hkdc1 knockout mice generated in our previous study. Transcriptomics and proteomics analysis will be applied to Hkdc1 KO retina to investigate gene expression profile changes, metabolic changes in the mutant retina. ERG test will be applied to examine the physiological function of the mutant retina. Expression and localization of phototransduction proteins will also be examined by immunostaining. The function of HKDC1 in glycolysis in the retina will be defined and its roles in pathogenesis of RP will be identified. Another aim is to explore the interaction of HKDC1 with its homologous gene HK1, a gene involved in autosomal dominant RP. The roles of hexokinase in RP will be identified. The interaction of HKDC1 with RanBP2 will also be investigated. Our study might provide new direction of RP therapy development by investigating the roles of HKDC1 in the retina and the molecular mechanisms underlying the disease pathogenesis.

视网膜色素变性疾病(Retinitis Pigmentosa,RP)是严重的致盲性眼病, 在我国影响300多万患者，目前尚无有效治疗方法。RP的干预治疗研究关键在于鉴定出疾病基因并阐明其致病分子机制。前期通过RP家系研究初步鉴定了一个常染色体隐性RP基因HKDC1, 但其生物功能尚不清楚。本研究拟通过多种技术方法分析自主构建的Hkdc1基因敲除小鼠模型（KO），转录组、蛋白质学研究KO视网膜基因表达、代谢变化，ERG检测KO视网膜生理功能，免疫染色分析光转导蛋白表达定位变化。明确HKDC1在视网膜糖代谢中功能，阐明其在视网膜色素变性病变中的作用，进一步探索与另一个RP基因HK1的协同作用，明确视网膜糖代谢异常与RP病变的关系，研究HKDC1与RanBP2相互作用，了解其致病机制。通过HKDC1在视网膜中的功能及其在RP发病过程中的致病机制研究，为视网膜色素变性疾病的干预寻找新路。

视网膜色素变性疾病(Retinitis Pigmentosa,RP)是严重的致盲性眼病, 在我国影响300 多万患者，目前尚无有效治疗方法。RP的干预治疗研究关键在于鉴定出疾病基因并阐明其致病分子机制。本课题利用前期收集的视网膜色素变性加息样本，通过全外显子测序技术，鉴定了RP 候选基因HKDC1。T58M突变蛋白表达、定位与野生型无差别；己糖激酶活性检测表明突变HKDC1蛋白丧失了40%左右的活性，提示该突变是功能丧失突变。运用CRISPR/Cas9技术构建了Hkdc1敲除小鼠(Hkdc1 KO)，9个月龄KO视网膜ERG 暗反应灵敏度降低，a波，b波均降低。免疫荧光染色分析发现视紫红质蛋白定位异常。明确了Hkdc1 缺失小鼠视网膜病理变化特征：Hkdc1敲除模型视网膜在17个月时感光细胞外节变短，外核层细胞数目减少，厚度变薄。RT-PCR、免疫印迹和免疫荧光染色分析发现Hkdc1 突变视网膜中Hk1的表达升高。视杆细胞特异敲除Hk1导致视杆细胞进行性病变，外节变短，ONL厚度变薄。视锥细胞特异敲除Hk1导致视锥细胞进行性病变，数目减少。深度挖掘外显子测序数据，和印度阿拉汶眼科医院P. Sandaresan教授合作，我们还在一个近亲结婚大家系鉴定了另外一个视网膜色素变性基因AHR。此突变为剪接突变，导致突变蛋白变短，丧失了生物活性。我们构建了Ahr视网膜组细胞特异敲除模型，发现敲除鼠出现Ahr 敲除模型视网膜ERG 暗反应波幅降低，感光细胞死亡。研究成果获得国家发明专利授权6项；发表SCI研究论文17篇，包括Human Molecular Genetics, Cell Death and Differentiation, Journal of Neurochemistry 等专业杂志。项目负责人获得四川省科技进步一等奖（2018年，排名第四）；培养了四名研究生。