Molecular Genetics of Primary Congenital Glaucoma

原发性先天性青光眼（PCG）是一种遗传性隐性疾病，在小梁网（TM）和眼睛前段存在发育缺陷。之前，我们绘制了GLC3A-C的3个位点，并报道了细胞色素P4501B1 （CYP1B1）中引起pcg的第一系列突变。该基因现在在世界范围的PCG人群中进行筛选，并报道了50多个突变。GLC3B-C基因座的基因尚未被确定。CYP1B1是一种单加氧酶，是细胞色素P450 （CYP）家族1-3的成员。cyp1b1缺失的小鼠眼压升高，TM有缺陷。在过去的奖励期间，我们：1)-表明CYP1-3的8个同源成员在人和小鼠的子宫发育中都存在；2)-建立了野生型CYP1B1和与PCG不完全外显性相关的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；4)-提示CYP1B1酶活性低加上其诱导性可能是PCG不完全外显的基础。我们本研究的第一个目标是鉴定另外两个PCG缺陷基因，并在大量PCG患者中进行筛选。接下来，我们通过：a)表达4个CYP1B1突变E229K、A330F、R368H和D374N，并通过测定它们的酶稳定性和活性来验证我们关于不完全外显率机制的假设；b)-在小鼠Cyp1b1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)子宫内和出生后发育小鼠眼睛中Cyp1b1蛋白谱；G61E-和R469W-突变小鼠系的变性和眼部发育缺陷的评估，与我们的cyp1b1缺失群体相比。通过注入吲哚-3-甲醇诱导Cyp1b1，创造条件来测试不完全外显率的基础；e)-利用特异性TM酵母双杂交cDNA文库鉴定CYP1B1相互作用蛋白，旨在评估CYP1B1蛋白-蛋白相互作用在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CYP1B1突变或其他2个PCG位点上的缺陷基因导致这种表型的机制。这也可能为开发更有效的儿童眼病治疗方法提供进一步的见解。

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.