Molecular Genetics of Primary Congenital Glaucoma

原发性先天性青光眼的分子遗传学

• 批准号: 7290999
• 负责人: Mansoor Sarfarazi
• 金额: $ 51.61万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7290999
• 关键词: 14q24.3; 1p36; 2p21; Adult; Affect; Age; Anterior eyeball segment structure; Appearance; Aqueous Humor; Award; Binding; Breeding; CYP1B1 gene; Candidate Disease Gene; Characteristics; Childhood; Ciliary Body; Code; Collection; Conceptions; Condition; Cytochrome P450; Cytochromes; Defect; Development; Embryo; Environmental Risk Factor; Enzyme Kinetics; Enzymes; Escherichia coli; Etiology; Eye; Eye Development; Eye diseases; Family; Female; Fetus; Generations; Genes; Genetic; Genotype; Glaucoma; Grant; Harvest; Hemeproteins; Histologic; Holoenzymes; Homology Modeling; Human; Immunohistochemistry; Indole-3-Carbinol; Inherited; Kinetics; Knockout Mice; Laboratories; Lead; Life; Light; Link; Maps; Missense Mutation; Mixed Function Oxygenases; Modification; Molecular Genetics; Mus; Mutant Strains Mice; Mutation; Newborn Infant; Numbers; Orthologous Gene; Oxidation-Reduction; Patient currently pregnant; Patients; Pattern; Penetrance; Phenotype; Physiologic Intraocular Pressure; Plasmids; Population; Preparation; Process; Proteins; Quartz; Report (account); Reporting; Research Personnel; Role; Screening procedure; Series; Site; Staging; Structure; Structure of sinus venosus of sclera; System; Techniques; Technology; Testing; Trabecular meshwork structure; Wild Type Mouse; Xenobiotics; Yeasts; base; cDNA Library; day; design; disease phenotype; early childhood; embryonic stem cell; eye formation; feeding; in utero; insight; member; migration; mouse model; mouse nonagouti protein; mutant; physical property; polyclonal antibody; postnatal; programs; protein protein interaction; protein structure; pup; tonometry; yeast two hybrid system

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.

产品说明：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）和眼前节中存在发育缺陷。在此之前，我们定位了GLC 3A-C的3个位点，并报道了细胞色素P4501 B1（CYP 1B 1）中导致PCG的第一系列突变。该基因目前在世界范围的PCG人群中进行筛选，报告了超过50种突变。GLC 3B-C位点的基因尚未鉴定。CYP 1B 1是一种单加氧酶，是细胞色素P450（CYP 1B 1）家族1-3的成员。Cyp 1b 1基因敲除小鼠眼内压升高，TM缺陷。在过去的奖励期间，我们：1）-表明8个cyp 1 -3的orthopathic成员存在于人类和小鼠的子宫发育过程中; 2）-创建了一个E。结果表明：（1）G61 E和R469 W突变导致PCG不完全转化;（2）G61 E和R469 W突变导致PCG不完全转化;（3）G61 E和R469 W突变编码的全酶活性降低，稳定性改变;（4）提示CYP 1B 1的低酶活性及其诱导可能是PCG不完全转化的基础。本研究的第一个目的是鉴定其他2个PCG缺陷基因，并在大量PCG患者中进行筛选。接下来，我们通过以下方式检验我们关于不完全转化机制的假设：a）表达E229 K、A330 F、R368 H和D374 N的4种CYP 1B 1突变，并通过测定它们的酶稳定性和活性; B）在小鼠CYP 1b 1中产生G61 E和R469 W突变，并通过测定这些直向同源物是否具有与人突变相似的缺陷; c）-在子宫内和出生后发育期间发育中的小鼠眼睛中Cyp 1b 1的蛋白质谱; G61 E-和R469 W-突变小鼠系的退化和与我们的Cyp 1b 1-空集落相比的眼睛发育缺陷的评估。将创造条件，通过饲喂吲哚-3-甲醇诱导Cyp 1b 1，检测不完全转化的基础; e）使用特异性TM酵母双杂交cDNA文库鉴定CYP 1B 1相互作用蛋白，旨在评价CYP 1B 1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应进一步阐明CYP 1B 1突变或其他2个PCG基因座缺陷基因导致该表型的机制。这也可能为开发针对这种儿科眼部疾病的更有效的治疗方法提供进一步的见解。