ISOLATION & CHARACTERIZATION OF HRG4, A NEW RETINAL GENE

描述(摘自申请者的摘要)：新的视网膜基因已经 在调查员的实验室中进行识别和研究，以确定 遗传性失明视网膜疾病的病因，研究这些基因如何 引发疾病，并利用这些信息找到最好的治疗或治愈方法 对他们来说。到目前为止，已经证明：(1)晚发型患者 视锥-视杆细胞营养不良症感光基因HRG4有无意义密码子突变 在研究人员的实验室中确认；(2)转基因小鼠模型 携带与患者相同的突变显示ERG异常，一致 光感受器突触的神经传递问题和迟发性 视网膜变性；(3)视杆细胞和视锥细胞表达hrg4。 突触，使其成为第一个与视网膜有关的突触蛋白 (4)HRG4与C. 有协调、进食和化学感官问题的优雅动物。为 在HRG4研究的基础上，提出了4个目标。特定目标1将测试 HRG4在突触小泡周期中发挥重要作用的假说 这构成了突触神经传递的基础。突触的现状 囊泡周期蛋白及其相互作用将在 转基因模型小鼠与正常小鼠。目标2是确定 HRG4与之相互作用的伙伴或靶蛋白，以阐明 HRG4的功能。直接结合试验、免疫共沉淀试验 使用重组的HRG4，并采用酵母双杂交策略。目标 3构建HRG4基因靶向(敲除)小鼠模型并研究 该基因零表达对骨肉瘤表型及机制的影响 表型的起源。这一目标的目的是(I)调查 HRG4的功能和致病机制及(II)建立小鼠肾移植动物模型 通过转基因可以进行哪些HRG4的结构和功能研究 在干净的背景下表达，并且最终可以用来 探讨视网膜退行性变的机制及治疗方法。目标4是 通过(A)筛查视网膜病变患者的HRG4基因突变 寻找视网膜病变已被映射到17q11的患者并进行筛查 (B)搜索患有神经纤维瘤病的患者，映射到 17q11.2和视网膜病变，并进行筛查以确定其存在 一种连续染色体综合征和HRG4参与的 视网膜病变；(C)利用变性进行快速随机突变筛查 未定位病例的梯度凝胶电泳法或异源双链分析 视网膜病变；(D)研究HRG4突变可能的相互作用 和其他视网膜变性基因；和(E)进行 共分离分析和筛选正常DNA中发现的任何突变 以确定致病关系，确立其意义。 HRG4用于视网膜疾病。

DESCRIPTION (Adapted from applicant's abstract): New retinal genes have been identified and studied in the investigator's laboratory in order to identify etiologies for hereditary blinding retinal diseases, to study how these genes cause disease, and to use this information to find the best treatment or cure for them. To date, it has been demonstrated that: (1) a patient with late-onset cone-rod dystrophy has a nonsense codon mutation in HRG4, a photoreceptor gene identified in the investigator's laboratory; (2) a transgenic mouse model carrying the same mutation as the patient showed an ERG abnormality, consistent with a problem in neurotransmission at the photoreceptor synapse and late-onset retinal degeneration; (3) HRG4 is present in the rod and cone photoreceptor synapses, making it the first synaptic protein implicated in retinal degeneration; and (4) HRG4 is homologous to UNC119, a mutated protein in C. elegans with problems of coordination, feeding, and chemosensation. For continuation of the HRG4 study, 4 aims are proposed. Specific aim 1 will test the hypothesis that HRG4 plays a significant role in the synaptic vesicle cycle which forms the basis of synaptic neurotransmission. The status of synaptic vesicle cycle proteins and their interactions will be compared in the transgenic model versus normal mice. Aim 2 is to determine the identity of the partner or target protein with which HRG4 interacts in order to elucidate the function of HRG4. Direct binding assays, co-immunoprecipitation experiments using the recombinant HRG4, and the yeast two-hybrid strategy will be used. Aim 3 is to construct an HRG4 gene-targeted (knock-out) mouse model and to study the effect of null expression of this gene on the phenotype and mechanism of genesis of the phenotype. The goals of this aim are to (i) investigate the function and pathogenic mechanism of HRG4 and (ii) to obtain an animal model in which structure-function studies of HRG4 can be carried out by transgene expression against a clean background, and which ultimately can be used to investigate the mechanism and the treatment of retinal degeneration. Aim 4 is to screen the HRG4 gene for mutations in patients with retinopathies by (a) searching for patients whose retinopathy has been mapped to 17q11 and screening them, if found; (b) searching for patients with neurofibromatosis mapping to 17q11.2 and retinopathy and screening them in order to determine the presence of a contiguous chromosome syndrome and the involvement of HRG4 in the retinopathy; (c) conducting rapid random mutational screening using denaturing gradient gel electrophoresis or heteroduplex analysis on unmapped cases of retinopathy; (d) investigating the possible interaction of mutations in HRG4 and other retinal degeneration genes by screening both; and (e) performing co-segregation analysis and screening of normal DNA for any mutations found in order to confirm the pathogenic relationship and establish the significance of HRG4 for retinal diseases.