Joseph A.C. Wadsworth Research Professor of Ophthalmology
Professor of Pathology
Corneal Diseases, Inherited Diseases of the Eye, and Ophthalmic Pathology
Duke Eye Center
Several corneal dystrophies [granular corneal dystrophy (GCD), lattice corneal dystrophy (LCD) types I and IIA, combined GCD-LCD type I, Thiel-Behnke dystrophy and Reis Bücklers dystrophy I] have been found to result from mutations in TGFBI (BIGH3).
These conditions are characterized by the deposition within the cornea of proteins that react with antibodies to TGFBIP (the protein product of the TGFBI gene). For each dystrophy, the accumulations have a characteristic ultrastructure. This proposal is to purify and characterize TGFBIP from plasma of persons with and without different TGFBI corneal dystrophies and from cell cultures of COS-7, CHO-K1 and baculovirus infected insect cells transfected with vectors containing the mutations that produce TGFBI corneal dystrophies. Wild-type and mutant TGFBIP proteins will be compared to test the hypothesis that the structure of the proteins that accumulate within the cornea in the TGFBI corneal dystrophies are related to the mutated protein and that a relationship exists between different phenotypes and the TGFBI genotypes and between the amount of the mutant protein that is expressed. The nature of the amyloid that accumulates in certain TGFBI corneal dystrophies and the reason for its production will be investigated. We will determine if TGFBI-related amyloid is a specific fragment of mutant TGFBIP. Because TGFBIP interacts with components of the extracellular matrix, we will attempt to identify protein-protein interactions that may account for the accumulations that characterize the TGFBI corneal dystrophies. The biosynthesis of TGFBIP will be characterized in rabbit corneal epithelial cells.
The proposed research is part of an ongoing study of macular corneal dystrophy (MCD) and the CHST6 gene. We will maintain and expand a computerized registry and genealogical database of individuals with MCD for relevant studies. Mutation analyses of the CHST6 gene are being determined in families with and without MCD from different populations using DNA extracted from the peripheral blood or from pathologic archival corneal tissue. This is being done with the intent of differentiating disease-producing mutations in the CHST6 gene from inconsequential single nucleotide polymorphisms (SNPs). Genotype-phenotype correlations are also being performed from this information. The molecular basis for the observed immunophenotypes in
MCD is being sought in relationship to specific mutations in CHST6. To determine the distribution of the manifestations of MCD, we will continue to examine various tissues in affected persons when available. We are mining nucleotide and protein computerized databases to add to our knowledge of CHST6 and the encoded carbohydrate sulfotransferase. The recombinant protein synthesized by mammalian and insect cell lines transfected with vectors containing wild-type CHST6 is being purified and characterized biochemically and enzymatically. The role of CHST6 in the sulfation of specific carbohydrate moieties is being determined. We study the subcellular localization of the expressed CHST6 gene product and are developing model systems to study the pathobiology of MCD in cell culture systems. An attempt is being made to develop an intracellular storage disorder comparable to MCD in cultured keratocytes (corneal fibroblasts) using antisense oligonucleotides targeted against specific sequences in CHST6 mRNA.
R01 (EY08249) /04/01/02-03/31/06/
National Eye Institute
"Macular Corneal Dystrophy - Molecular Pathobiology"
This project focuses on determining the spectrum of mutations in the CHST6 gene that cause macular corneal dystrophy. It also determines how the molecular abnormalities cause all of the manifestations of this dystrophy.
R01 (EY12712) /08/01/99-07/31/04/
National Eye Institute
"Study of TGFBI Wild-type and Mutant Protein" investigates the molecular structure of the protein product of the TGFBI (TGFBI) gene. This protein accumulates in the cornea in persons with several corneal dystrophies and may cause severe visual impairment. The goal is to purify and characterize wild-type and mutant transforming growth factor beta-induced protein and gain a better understanding of the fundamental steps that lead to the debilitating conditions in which the mutant protein accumulates in the cornea.