Norrin/FZD4信号传导通路对家族性渗出性玻璃体视网膜病变作用机制的研究

Familial exudative vitreoretinopathy (FEVR) is a hereditary blinding disorder that features defects in retinal vascular development. The mutations in the genes encoding the Wnt receptor pair, frizzled 4 (FZD4), norrie disease (NDP)and low-density-lipoprotein receptor-related protein 5 (LRP5), have been shown to cause FEVR. The phenotype of these patients suggested a synergistic effect of the two mutations in the independent FEVR-causing genes. The profile of the mutations obtained in the current study further illustrates the complexity of the disease and provides a better understanding of the spectrum, frequencies, and genotype-phenotype correlation. In FEVR, the pathologic growth of new blood vessels ultimately leads to bleeding and/or detachment and scarring of the neural retina. In humans the retinal vasculature is readily visualized at high resolution by fluorescein angiography. Disorders of vascular structure and function play a central role in FEVR. Mutations in the Frizzled-4 (Fz4) receptor, Lrp5 co receptor, or Norrin ligand cause retinal hypovascularization, but the mechanisms by which Norrin/Fz4/Lrp signaling controls vascular development have not been de?ned. We'll use mouse genetic and cell culture models, show that loss of Fz4 signaling in endothelial cells causes defective vascular growth, which leads to chronic but reversible silencing of retinal neurons. Loss of Fz4 in all endothelial cells disrupts the blood brain barrier in the cerebellum, whereas excessive Fz4 signaling disrupts embryonic angio-genesis. Sox17, a transcription factor that is upregulated by Norrin/Fz4/Lrp signaling, plays a central role in inducing the angiogenic program controlled by Norrin/Fz4/Lrp. These experiments will establish a cellular basis for retinal hypovascularization diseases due to insufficient Frizzled signaling, and they suggest a broader role for Frizzled signaling in vascular growth, remodeling, maintenance, and disease. These observations raise a series of interrelated questions that are central to understanding the cellular basis and functional signi?cance of Norrin/Fz4/Lrp signaling. Is the primary site of Fz4 signaling-and hence the primary cellular defect in inherited diseases in which this system is impaired-neuronal, glial, or vascular? At the physiological and behavioral levels, what aspects of visual function are impaired by vascular defects that result from loss of Fz4 signaling? What cellular processes in vascular development require Fz4 signaling in the intact animal and in cell culture? Is Fz4 signaling a general feature of vascular development outside of the retina? Does Fz4 signaling alter gene expression, and, if so, how does this relate to vascular development? In the this study, we will answer these questions by studying the effects of gain and/or loss of Fz4 signaling in vivo and in cell culture.

家族性渗出性玻璃体视网膜病变（FEVR）是儿童致盲性眼病，是造成儿童失明和弱视的重要原因之一，为一种复杂遗传性疾病，我们前期研究结果发现中国FEVR患者发病具有特异性并可能存在FZD4、NDP和LRP5之间多基因交互作用。本研究拟在前期研究建立带有稳定结合Wnt信号传导途径报导质体Super TopFlash检测细胞活性和质粒稳定表达基础上通过体外细胞功能学研究FZD4、LRP5、NDP和TSPAN12基因的各种突变对Norrin/FZD4信号通路的影响及突变蛋白对细胞功能的影响，研究FZD4基因突变在体内对信号通路影响，揭示该信号通路视网膜血管发育的关系并寻找相关可能的的治疗靶点。

家族性渗出性玻璃体视网膜病变（FEVR）是儿童致盲性眼病，是造成儿童失明和弱视的重要原因之一，为一种复杂遗传性疾病，我们研究结果发现中国FEVR患者发病具有特异性并可能存在FZD4、NDP和LRP5之间多基因交互作用。课题组对36个中国人的FEVR家系，进行了全面的临床资料的收集并设计了FZD4、LRP5和NDP基因扩增引物，并摸索了所有引物的PCR扩增条件，并对其中的28个不同临床表型的FEVR家系进行了FZD4、LRP5和NDP基因的筛查和突变位点的寻找，发现了三十多个FZD4、LRP5和NDP基因突变位点，其中有二十二个为世界首次发现，同时我们研究结果显示：1.在中国FEVR患者中致病性突变多为错义突变；2.在同一FEVR患者DNA样本中可以同时存在两个或者多个基因突变；3.携带有多个突变患者的临床表型较单纯携带一个突变患者的临床表型更为严重。中国人FEVR患者中致病性突变谱与其他人种FEVR致病性突变谱具有显著差别。课题组初步建立报导质体Super TopFlash检测细胞活性和质粒的稳定表达；研究NDP基因突变对Norrin/FZD4信号通路的影响以及突变蛋白对于细胞功能的影响。结果表明中国FEVR患者发病具有很强的遗传特异性，FZD4基因不同位点的致病性突变对Norrin/FZD4通路作用影响不同，并存在Norrin/FZD4通路相关基因之间多基因交互作用。