MOLECULAR GENETICS OF PRIMARY COGENITAL GLAUCOMA

University Of Connecticut Sch Of Med/Dnt, Farmington CT

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Abstract

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.