MOLECULAR GENETICS OF PRIMARY COGENITAL GLAUCOMA

原发性先天性青光眼（PCG）是一种遗传性眼病， 未知的眼前节和小梁网发育缺陷 （TM）。PI以前的工作已经清楚地表明，遗传易感性是一个 PCG发展的主要因素。因此，他的目的是进一步确定 并描述这种情况下潜在的分子缺陷。通过使用 多重受影响的家庭，他已经绘制了第一个（GLC 3A在2 p21）， 1 p36上的第二个GLC 3B），以及最近的第三个位点（14q24.3上的GLC 3C 对于这种情况。他还报道了第一系列的突变， 2 p21上GLC 3A位点的细胞色素P4501 B1（CYP 1B 1）。到目前为止他 确定了37个CYP 1B 1突变，并显示87%的家族性和27%的 %的孤立PCG病例仅由该基因编码。基于 3D同源性建模，所有观察到的突变主要影响高度同源性。 这种蛋白质的保守核心结构。用于体外功能分析 CYP 1B 1，他制造了一系列包含正常或 突变体，并在E.杆菌纯化的 正常蛋白已被用于在鸡中产生多克隆抗体。进一步 目前正在使用NIH开发的Cyplb 1基因缺失小鼠， 对这种蛋白质进行了全面的体内功能研究。其他2种蛋白质 由GLC 3B和GLC 3C基因座上的基因编码的基因仍有待鉴定。 因此，这项建议有两个主要的具体目标。第一 目的是：1）-在一些候选基因中寻找突变， 在1 p36的GLC 3B和14q24.3的GLC 3C两个临界区间内; 以及，如果需要的话，2）-启动定位克隆策略， 将导致识别，隔离和描述这两个 推定的GLC 3B和GLC 3C缺陷蛋白。第二个目标是进行 采用两种不同的体外和体外方法对CYP 1B 1进行了综合功能研究， 在体内模型系统中，最终目的是：1）-鉴定一种肿瘤性CYP 1B 1 2）研究其生化特性和表达模式;以及 3）-确定CYP 1B 1在正常和PCG眼发育中的总体作用。一 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GLC 3B和GLC 3C位点。他将使用PCR、凝胶电泳、SSCP、DGGE 和直接测序，以寻找突变;基因桥4辐射杂交 Panel和YAC/BAC/Cosmid克隆，以进一步缩小两个候选克隆的范围。 GLC 3B和GLC 3C的间隔; cDNA克隆。RACE或直接cDNA选择， 从大DNA片段中分离潜在基因;原位杂交， 研究CYP 1B 1在眼组织中表达，最后用GC/MS和HPLC进行体外研究 测定CYP 1B 1产生的代谢物。不断增加的可用性 人类基因组测序中心正在产生的基因组数据将 大大加快了我们识别两个新GLC 3B和GLC 3C的机会 基因. PI功能研究可能阐明确切的病理 导致PCG的机制，并可能提供第一个关键步骤， 了解人眼的复杂性，其胚胎学和功能， 可能最终导致特定的合理的医疗或手术的发展， 治疗这种儿科表型。

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.