Knowledge of genes responsible for inherited diseases which affect platelet function and synthesisis is minimal. Consequently, there is a lack of precise molecular diagnostic tests as well as efficacious treatments. The Hermansky-Pudlak Syndromes (HPS) are a group of recessively inherited diseases of humans that cause prolonged bleeding, platelet storage pool deficiency and lung fibrosis, leading to considerable morbidity and premature death. Recent studies have demonstrated that the disease is multigenic in humans and mice. One of the mouse genes, gunmetal (gm), is of additional interest in that it reduces rates of platelet synthesis due to a mutation in the Rab geranylgeranyl transferase (RabGGTase) gene. The long term goals of this proposal are: a) to utilize the inherent advantages of inbred mice to identify additional HPS genes at the molecular level, b) to apply this knowledge to identify mutations in human HPS patients not yet molecularly defined and c) ultimately to devise effective diagnostic approaches and therapies for the disease. A secondary goal is to contribute to basic knowledge of genes which regulate the synthesis and trafficking of subcellular organelles such as platelet dense granules, lysosomes and melanosomes.
The Specific Aims of the proposal are to: 1) complete the identification and partial characterization of the mouse sandy (sdy) HPS gene; 2) identify and partially characterize the mouse light ear (le) HPS gene; 3) identify and partially characterize the mouse muted (mu) HPS gene; 4) isolate the human homologues of cloned mouse HPS genes and test for alterations of these genes in human HPS kindreds: 5) clarify the mechanism(s) by which decreased RabGGTase activity causes abnormalities in platelet synthesis and organelle function. Large interspecific mouse backcrosses are used to construct high resolution genetic maps of each mouse HPS gene. Physical maps of the critical genetic regions will be constructed by selection of BACs (bacterial artificial chromosomes) which span the critical genetic intervals. Genes within BACs will be identified by exon trapping, cDNA selection and whole BAC sequencing. Candidate genes will be sequenced to detect mutations. The same genes will be analyzed in human HPS patients, not yet molecularly diagnosed, by cDNA sequencing and related techniques to determine if mutations in these genes are the cause of HPS in these patients. The consequences of a reduction in levels of RabGGTase in gunmetal mice will be analyzed by comparing levels of geranylgeranylation of multiple Rab GTPases and by comparing the subcellular distribution of Rabs in tissues of gunmetal and normal mice.
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