ARR3基因突变致女性特发性X连锁早发高度近视的分子机制研究

Degenerative changes in the retina associated high myopia has become one of the most common causes for irreversible blindness. So far, there is no efficient measure to prevent or cure such changes because of minimal knowledge about the molecular mechanism of high myopia. Previously, we have performed a series of study on the molecular genetics of early onset high myopia. Recently, we found that heterozygous mutations in ARR3, a retina-enriched and cone-specific gene, are a common cause of early onset high myopia, which demonstrate an unusual X-linked female-limited pattern of inheritance. Here in this study, we plan to investigate the molecular mechanism of myopia development associated with ARR3 mutations. First, Arr3 knock out rat models as well as human iPSC-derived three-dimensional retinal cups from members of families with ARR3 mutations will be established. Then, the morphological development, cone-cone and cone-rod spatial array, cone-bipolar synaptic connection will be analyzed by immunohistochemistry, while the proteomics and transcriptomes will be comparatively analyzed. All those analyses will be measured at different time points. Morphological changes and comparative informatics analysis of genome-wide expression will lead to the identification of critical time course of myopia development as well as key molecules associated with ARR3 mutation-induced myopia. These information will be used and verified in ARR3 knock out monkey models in future with the hope of disclosing the molecular pathways of myopia, which might be of great help in searching for targets that may be regulated by drugs. In the meantime, this study will also provide valuable information to disclose the molecular basis of X-linked female-limited pattern of inheritance.

高度近视已成为常见难治性致盲眼病之一，因发病机制不明而缺乏有效的防治措施。我们曾对早发高度近视开展了系列分子遗传学研究，最近发现视锥细胞特异表达基因ARR3突变是单纯性早发高度近视的常见原因之一，并呈特殊的X连锁女性限性遗传。本项目拟以ARR3基因为切入点，通过构建大鼠基因敲除模型和突变家系成员iPSC诱导视杯模型，应用免疫组化、蛋白质组与转录组分析等技术方法，比较分析模型组与对照组眼球与视杯的形态发育、锥杆细胞空间排列及与次级神经元的突触连接、蛋白相互作用、相关基因表达谱变化等，揭示ARR3突变导致近视的重要时间节点、累及组织细胞类型以及差异表达的关键因子，为下一步构建ARR3基因敲除猴模型研究。并最终明确ARR3相关近视信号通路奠定基础，结果对阐明近视发生发展机制及寻找可能的近视药物调控靶点有重要意义，同时对揭示X连锁女性限性遗传这一特殊遗传方式的机制有科学价值。

高度近视是近视的严重类型，其相关并发症居致盲原因的前三位，是防盲的难点和重点之一。明确病因和发病机制是高度近视防控的前提。学龄前发生的早发高度近视主要由遗传导致，基于明确的遗传基础，探索其导致高度近视的分子机制，将对高度近视的防治提供重要的理论依据。项目组前期研究发现ARR3基因突变导致特殊的X连锁女性限性高度近视，为进一步探索相关分子机制，本项目构建了Arr3基因缺陷鼠模型，通过对不同基因型Arr3基因缺陷鼠模型的眼部表型参数检测、视网膜结构形态、以及转录组测序和单细胞测序等分析，发现Arr3杂合缺陷鼠在1月龄时视网膜电流图反应显著低于同月龄野生型小鼠，而视网膜厚度显著高于野生型对照组，提示Arr3杂合突变影响鼠眼球以及感光细胞功能的正常发育过程。通过对鼠视网膜铺片的免疫组织化学染色，显示1月龄杂合缺陷鼠的M-视椎细胞的计数较野生型显著下降。由于杂合缺陷鼠相较野生型和半合突变鼠的转录组测序未发现明确的差异表达基因，因此进一步开展单细胞测序分析，结果发现并验证杂合缺陷鼠Opn1mw及Pcp2的表达低于野生型及半合鼠，且Opn1mw表达降低与M-视锥细胞的数目减少结果相互对应。基于以上结果，初步阐释ARR3基因杂合突变导致女性限性的早发高度近视的可能机制为：Arr3基因杂合突变特异性地对Opn1mw-视锥细胞的数目和功能继而对应的二级神经元亚类的数目发生影响，导致光电信号转导过程产生信号混杂和紊乱，最终发生眼轴增长的病变。提示了早发高度近视发病机制的新方向。. 此外，项目组对早发高度近视的相关基因开展了系列研究，发现ARR3基因突变是早发高度近视最常见的原因，发现BMP4、FZD5、LAMA1及CPSF1，的变异与早发高度近视密切相关。这些结果进一步明确了早发高度近视的基因突变频谱，对高度近视诊疗防的转化应用提供了重要的依据。相关结果已总结发表国际期刊论文5篇、综述1篇，参编近视专著的章节，待发表论文2篇。