Molecular Genetics of Primary Congenital Glaucoma

描述：原发性先天性青光眼(PCG)是一种遗传性隐性疾病，伴有小梁网(TM)和眼前段的发育缺陷。此前，我们定位了GLC3A-C的3个基因座，并首次报道了导致细胞色素P4501B1(CyP1B1)的PCG突变。该基因现已在世界范围内的PCG人群中进行了筛查，据报道有50多个突变。到目前为止，GLC3B-C基因座的基因还没有确定。CYP1B1是一种单加氧酶，属于细胞色素P450(Cyp)家族1-3。CYP1B1基因缺失的小鼠眼压升高，TM存在缺陷。在过去的获奖期内，我们：1)-表明在人和小鼠的子宫发育过程中都存在CYP1-3的8个同源成员；2)-建立了与PCG不完全外显相关的野生型CYP1B1以及G61E和R469W突变的大肠杆菌表达系统；3)-表明G61E和R469W突变编码的全酶活性降低，稳定性改变；以及4)-表明CYP1B1的低酶活性及其诱导性可能是PCG不完全外显的基础。我们目前研究的第一个目标是确定另外两个PCG缺陷基因，并在大量PCG患者中进行筛查。接下来，我们测试了我们关于不完全外显机制的假设，方法是：a)表达E229K、A330F、R368H和D374N的4个CYP1B1突变并测定其酶稳定性和活性；b)在小鼠中产生G61E和R469W突变并确定这些同源基因是否具有与人类突变相似的缺陷；c)在小鼠发育中的小鼠眼睛中检测CYP1B1在子宫和出生后发育过程中的蛋白质谱；G61E和R469W突变小鼠品系的退化，以及与我们的CYP1B1阴性菌落相比眼睛中的发育缺陷的评估。将创造条件，通过饲喂吲哚-3-甲醇诱导CyP1B1的产生，来测试不完全外显的基础；以及e)利用TM酵母双杂交文库鉴定与CyP1B1相互作用的蛋白质，目的是评估CYP1B1蛋白-蛋白质相互作用在PCG病因学中的作用。本研究结束时获得的结果将进一步阐明导致这种表型的机制，即CYP1B1突变或其他2个PCG基因座的缺陷基因。这也可能为开发一种更有效的治疗这种儿童眼病的方法提供进一步的洞察力。

DESCRIPTION: Primary Congenital Glaucoma (PCG) is an inherited recessive condition with developmental defects in the trabecular meshwork (TM) and anterior segments of the eye. Previously, we mapped 3 loci of GLC3A-C and reported a first series of PCG-causing mutations in Cytochrome P4501B1 (CYP1B1). This gene is now screened in a world-wide PCG population and over 50 mutations are reported. Genes at the GLC3B-C loci have not been identified as yet. CYP1B1 is a monooxygenase and a member of Cytochrome P450 (CYP) Families 1-3. A Cyp1b1-null mouse has elevated intraocular pressure and defects in the TM. During the past award period we: 1)-showed that 8 orthologous members of CYP1-3 are present during uterine development in both human and mouse; 2)-created an E. coli expression system for wild-type CYP1B1 and the G61E and R469W mutations that are associated with PCG incomplete penetrance; 3)-Showed that G61E and R469W mutations code for holoenzymes with diminished activity and altered stability and; 4)-Suggested that low enzymatic activity of CYP1B1 plus its inducibility might be the basis for PCG incomplete penetrance. Our first objective for the present study is to identify the other 2 PCG defective genes and to screen them in a large number of PCG patients. Next, we test our hypothesis on mechanism of incomplete penetrance by: a)- expressing 4 CYP1B1 mutations of E229K, A330F, R368H, and D374N and by determining their enzymatic stability and activities; b)-creating the G61E and R469W mutations in mouse Cyp1b1 and by determining whether these orthologs have defects similar to the human mutations; c)-protein profiling of Cyp1b1 in the developing mouse eyes during in utero and postnatal development; degeneration of G61E- and R469W- mutant mouse lines and assessment of developmental defects in the eye as compared to our Cyp1b1-null colony. Conditions will be created to test the basis of incomplete penetrance, through induction of Cyp1b1 by feeding indole-3-carbinol; and e)-ldentification of CYP1B1-interacting proteins by using a specific TM yeast two-hybrid cDNA library, aiming to evaluate the role of CYP1B1 protein-protein interaction in the etiology of PCG. Results obtained by the end of this study should shed additional light on the mechanisms through which mutations in the CYP1B1 or defective genes at other 2 PCG loci lead to this phenotype. This may also provide further insight for development of a more effective therapy for this pediatric ocular condition.