ISOLATION & CHARACTERIZATION OF HRG4, A NEW RETINAL GENE

描述（改编自申请人的摘要）：新的视网膜基因已经被发现。 在研究者的实验室中识别和研究，以识别 遗传性致盲性视网膜疾病的病因，研究这些基因如何 并利用这些信息来寻找最佳治疗或治愈方法 为他们迄今为止，已经证明：（1）晚发性 视锥-视杆细胞营养不良在感光基因HRG 4中存在无义密码子突变 在研究者实验室中鉴定;（2）转基因小鼠模型 携带与患者相同的突变，显示ERG异常，符合 感光器突触的神经传递有问题， 视网膜变性;（3）HRG 4存在于视杆细胞和视锥细胞中 突触，使其成为第一个涉及视网膜神经元的突触蛋白。 HRG 4与C. 有协调、进食和化学感觉问题的秀丽线虫。为 HRG 4研究的继续，提出了4个目标。具体目标1将测试 HRG 4在突触囊泡周期中起重要作用的假说 形成了突触神经传递的基础。突触状态 囊泡周期蛋白及其相互作用将在 转基因小鼠与正常小鼠的比较。目标2是确定 HRG 4与之相互作用的伴侣或靶蛋白，以阐明HRG 4的功能。 HRG 4的功能。直接结合试验、免疫共沉淀试验 使用重组HRG 4，和酵母双杂交策略。目的 3是构建HRG 4基因靶向（敲除）小鼠模型，并研究 该基因的无效表达对表型的影响和机制 表型的起源。这一目标的目标是：（一）调查 HRG 4的功能和致病机制，以及（ii）获得动物模型， HRG 4的哪些结构-功能研究可以通过转基因进行 在一个干净的背景下表达，最终可以用来 探讨视网膜变性的发病机制及治疗方法。目标4是 通过以下步骤筛选视网膜病患者中HRG 4基因的突变：（a） 搜索视网膜病变已映射到17 q11的患者并进行筛查 （B）搜索映射到神经纤维瘤病的患者， 17q11.2和视网膜病变，并对其进行筛查，以确定是否存在 相邻染色体综合征和HRG 4参与 视网膜病变;（c）使用变性进行快速随机突变筛查 梯度凝胶电泳或异源双链体分析， 视网膜病变;（d）研究HRG 4中突变的可能相互作用 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，并建立意义 HRG 4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.