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 further knowledge about 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 are studying 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.
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