Molecular Genetics of Primary Congenital Glaucoma

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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.