MOLECULAR GENETICS OF PRIMARY COGENITAL GLAUCOMA

原发性青光眼(PCG)是一种遗传性眼病，由 前段和小分子网状结构的未知发育缺陷 (商标)。PI之前的工作清楚地表明，遗传易感性是一种 影响盈科发展的主要因素。因此，他的目标是进一步确定 并描述了这种情况下潜在的分子缺陷。通过使用 乘以受影响的家庭，他已经绘制了第一张地图(2p21上的GLC3A)， 位于1p36上的第二个GLC3B)和最近位于14q24.3上的第三个基因座(GLC3C 在这种情况下。他还报告了第一系列的基因突变 细胞色素P4501B1(细胞色素P4501B1)位于2p21的GLC3A位点。到目前为止，他已经 确定了37个CYP1B1突变，显示87%的家族性和27 分离的PCG病例中有百分比仅由该基因编码。基于 三维同源建模，所有观察到的突变主要影响高度的 该蛋白的核心结构保守。用于体外功能分析 CyP1B1，他已经制作了一系列包含正常或 该蛋白的突变形式，并在大肠杆菌中表达。纯净的 用正常蛋白在鸡体内制备多克隆抗体。进一步 目前正在研究将NIH开发的Cyplbl基因缺失小鼠用于 对该蛋白进行了全面的体内功能研究。另外两种蛋白质 GLC3B和GLC3C基因座的基因编码仍有待鉴定。 因此，这项提议有两个主要的具体目标。第一 目标是：1)-在一些候选基因中寻找突变 在1p36的GLC3B和14q24.3的GLC3C的两个临界区间内； 如果需要，2)-启动位置克隆策略，最终 将导致对两者的鉴定、分离和表征 推测为GLC3B和GLC3C缺陷蛋白。第二个目标是进行 用两种不同的体外和体外实验对细胞色素P1B1的功能进行综合研究 活体模型系统，最终目标：1)-鉴定青光眼细胞色素P1B1 底物；2)-研究其生化特性和表达模式；以及 3)-确定CYP1B1在正常和PCG眼发育中的总体作用。一个 一旦我们确定了另外两个编码基因，也将采用类似的方法 在GLC3B和GLC3C基因座。他将使用聚合酶链式反应，凝胶电泳，SSCP，DGGE 和直接测序寻找突变；GeneBridge 4辐射杂交种 Panel和YAC/BAC/Cosmid克隆进一步缩小两名候选人的范围 GLC3B和GLC3C的间隔期；竞争或直接选择cdna以 从大的DNA片段中分离潜在的基因；原位杂交 研究细胞色素P1B1在眼组织中的表达；最后，用气相色谱/质谱仪和高效液相色谱体外检测 细胞色素P1B1代谢产物的测定。不断增加的可用性 由人类基因组测序中心产生的基因组数据将 极大地加快了我们确定两个新的GLC 3B和GLC 3C的机会 基因。PI功能研究可能阐明其确切的病理机制 导致PCG的机制，并可能在 了解人眼的复杂性及其胚胎学和功能 可能最终导致特定理性内科或外科的发展 对这种儿科表型的治疗。

Primary Cogenital Glaucoma (PCG) is an inherited eye disorder that is caused by unknown developmental defects of the anterior segment and Tabecular Meshwork (TM). The PI previous work has clearly showed that genetic predisposition is a major factor in the development of PCG. Therefore, he aims to further identify and characterize the underlying molecular defects of this condition. By using multiply affected families, he has already mapped the first (GLC3A on 2p21), the second GLC3B on 1p36) and, very recently the third locus (GLC3C on 14q24.3 for this condition. He has also reported the first series of mutations in the Cytochrome P4501B1 (CYP1B1) for the GLC3A locus on 2p21. So far he has identified 37 CYP1B1 mutations and showed that 87 percent of familial and 27 percent of isolated PCG cases are coded by this gene alone. Based on 3D-homology modeling, all of the observed mutations mainly affect the highly conserved core structure of this protein. For in vitro functional analysis of CYP1B1, he has made a series of constructs containing either the normal or mutant forms of this protein and expressed them in the E. coli. The purified normal protein has been used to raise polyclonal antibody in chicken. Further work is underway to use the Cyplbl-null mice, developed at NIH, for a comprehensive in vivo functional study of this protein. The other 2 proteins coded by genes at the GLC3B and GLC3C loci still remain to be identified. Therefore, there are two main specific aims in this proposal. The first objective is: 1)-to search for mutation in a number of candidate genes from within the two critical intervals of the GLC3B on 1p36 and GLC3C on 14q24.3; and, if needs be, 2)-to initiate a positional cloning strategy that eventually would lead to the identification, isolation and characterization of the two putative GLC3B and GLC3C defective proteins. The second objective is to conduct a comprehensive functional study of CYP1B1 by using two different in vitro and in vivo model systems, ultimately aiming: 1)-to identify a glaucomatous CYP1B1 substrate; 2)-to study its biochemical properties and expression patterns; and 3)-to determine the overall CYP1B1 role in normal and PCG eye development. A similar approach will also be adopted once we identify the 2 other coding genes at the GLC3B and GLC3C loci. He will use PCR, gel electrophoresis, SSCP, DGGE and direct sequencing to search for mutation; GeneBridge 4 Radiation Hybrid Panel and YAC/BAC/Cosmid clones to further narrow down the two candidate intervals of GLC3B and GLC3C; cDNA clones. RACE or direct cDNA selection to isolate potential genes from large DNA fragments; In Situ hybridization to study expression of CYP1B1 in eye; and finally, GC/MS and HPLC for in vitro assay of metabolites produced by CYP1B1. The ever-increasing availability of genomic data being produced by the Human Genome Sequencing Centers will immensely accelerate our chance of identifying the two new GLC3B and GLC3C genes. The PI functional study may elucidate the precise pathological mechanisms leading to PCG and may provide the first critical step in understanding the complexity of the human eye, its embryology and function that may eventually lead to the development of specific rational medical or surgical treatment for this pediatric phenotype.