Hermansky-Pudlak Syndromes (HPS) is a group of recessively inherited, and presently noncurable, diseases which cause severe bleeding due to functional abnormalities of platelets. visual and pigmentation deficiencies due to abnormalities in melanosomes and premature death due to fibrotic lung disease. A primary long-term objective is to develop diagnostic procedures and therapies for inherited forms of HPS, which have not yet been molecularly identified. Toward this end, the inherent advantages of inbred mice will be used to clone two mouse HPS genes. A second important long-term objective is to identify new genes involved in the synthesis and trafficking of specialized mammalian subcellular organelles including platelet dense granules and melanosomes. The mouse HPS mutants, cocoa and subtle gray, are of special interest in that they have specific effects on subcellular organelles, affecting platelet dense granules and melanosornes, but not lysosomes. Their molecular identification is expected therefore to lead to identification of genes, which specifically regulate the biogenesis of the former two organelles.
The Specific Aims of the proposal are to: 1) molecularly identify and partially characterize the mouse cocoa (coa) HPS gene; 2) molecularly identify and partially characterize the mouse subtle gray (sut) I-IFS gene; 3) isolate the human homologues of the cloned cocoa and subtle gray HPS genes and test for alterations of these genes in human kindreds; 4) identify and characterize new mouse models for HPS, platelet storage pool deficiency and Griscelli Syndrome.
These aims will be accomplished by positional/candidate cloning approaches including large interspecific mouse backcrosses to obtain high resolution genetic maps and the construction of high resolution physical maps with overlapping contigs of BACs (bacterial artificial chromosomes). Transcripts within critical BACs will be identified by a combination of exon trapping and complete BAC sequencing. Transcripts containing mutations will be identified by qualitative and quantitative approaches including complete cDNA sequencing and ribonuclease protection assays. To identify patients with mutations in the corresponding human genes, the cDNA sequence of the human homologue of each gene and its expression level will be determined in normal individuals and in HPS patients with no mutations in known HPS genes. Each gene will be partially characterized by assays of transcript and protein tissue distribution together with determination of subcellular localization by immunofluorescence techniques. New mouse models of HPS including ashen, buff and a double mutant of pale ear and pearl will be characterized for organelle abnormalities of platelets and other tissues by a battery of hematological and cell biological techniques.
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