Hermansky-Pudlak syndrome (HPS; MIM 203300) is a genetically heterogeneous disorder characterized by oculocutaneous albinism, prolonged bleeding and pulmonary fibrosis due to abnormal vesicle trafficking to lysosomes and related organelles, such as melanosomes and platelet dense granules [1][2][3] . In mice, at least 16 loci are associated with HPS 4-6 , including sandy (sdy ; ref. 7 ). Here we show that the sdy mutant mouse expresses no dysbindin protein owing to a deletion in the gene Dtnbp1 (encoding dysbindin) and that mutation of the human ortholog DTNBP1 causes a novel form of HPS called HPS-7. Dysbindin is a ubiquitously expressed protein that binds to α-and β-dystrobrevins, components of the dystrophin-associated protein complex (DPC) in both muscle and nonmuscle cells 8 . We also show that dysbindin is a component of the biogenesis of lysosome-related organelles complex 1 ), which regulates Correspondence should be addressed to R.T.S (richard.swank@roswellpark.org). 11 These authors contributed equally to this work.Note: Supplementary information is available on the Nature Genetics website. Competing Interests Statement:The authors declare that they have no competing financial interests. We previously showed 7 that the sdy mutant mouse is a valid model for human HPS and localized the gene sdy to mouse chromosome 13. Here we genotyped 20 microsatellite markers in 1,250 progeny of sdy backcrosses to localize sdy to the 2.2-cM interval between D13Mit244 and D13Mit267 (Fig. 1). We identified the sdy interval within a 28-Mb scaffold (Celera Discovery System) containing two known genes, Jmj and Dtnbp1 (Fig. 1b). We used PCR products of D13Mit179 and the Dtnbp1 cDNA as probes to generate a BAC contig covering the sdy interval (Fig. 1b). NIH Public AccessNorthern-blot analysis and sequencing of RT-PCR products of Jmj identified no abnormalities in sdy mutants, but truncated genomic PCR products (Fig. 2a) and mRNA ( Fig. 2b) of Dtnbp1 were apparent in sdy tissues. Sequencing of RT-PCR products showed that exons 6 and 7 (156 bp) of Dtnbp1 were deleted in mutant mice, resulting in the loss of 52 amino acids from position 119-172 of the dysbindin protein ( Supplementary Fig. 1 online). Genomic sequencing showed that this results from a large deletion (38,129 bp) from nucleotide 3,701 of intron 5 to nucleotide 12,377 of intron 7. This deletion was not found in twelve other inbred mouse strains (Fig. 2a), including coisogenic DBA/2J, indicating that it was not a strain-specific polymorphism. This in-frame deletion creates a 1.5-kb mutant dysbindin transcript ( Fig. 2b) and abolishes expression of the 51-kDa dysbindin 8 protein in sdy/sdy mice ( Fig. 2c). Expression of dysbindin is restored in sdy/sdy transgenic mice containing BAC54F9 (Fig. 2c). Platelet serotonin levels of six of these transgenics were normal (>1.12 μg per 10 9 platelets), whereas all five sdy/sdy litter-mates without BAC54F9 had platelet serotonin levels of <0.06 μg per 10 9 platelets. sdy/sdy progeny containing the BAC transgene had dar...

Few molecular events important to platelet biogenesis have been identified. Mice homozygous for the spontaneous, recessive mutation gunmetal (gm) have prolonged bleeding, thrombocytopenia, and reduced platelet ␣-and ␦-granule contents. Here we show by positional cloning that gm results from a G3 A substitution mutation in a splice acceptor site within the ␣-subunit of Rab geranylgeranyl transferase (Rabggta), an enzyme that attaches geranylgeranyl groups to Rab proteins. Most Rabggta mRNAs from gm tissues skipped exon 1 and lacked a start codon. Rabggta protein and Rab geranylgeranyl transferase (GGTase) activity were reduced 4-fold in gm platelets. Geranylgeranylation and membrane association of Rab27, a Rab GGTase substrate, were significantly decreased in gm platelets. These findings indicate that geranylgeranylation of Rab GTPases is critical for hemostasis. Rab GGTase inhibition may represent a new treatment for thrombocytosis and clotting disorders. P latelets play a pivotal role in acute myocardial infarction, unstable angina, deep venous thrombosis, and stroke. About 4 million individuals are hospitalized each year in the United States with these disorders. Treatment with agents that prevent platelet activation reduces the risk of myocardial infarction and stroke by about 30% and death by about 15% (1). Novel platelet antagonists are needed, however, because platelet activation is not always inhibited by current drugs.Mice homozygous for gunmetal (gm), a spontaneous, recessive mutation, have prolonged bleeding caused by defects in platelets and megakaryocytes (2, 3). These mice also have macrothrombocytopenia and reduced platelet ␣-and ␦-granule contents (storage pool deficiency, SPD). Megakaryocytes, the progenitors of platelets, are more plentiful in gm mice, but have abnormal intracellular membranes, increased emperipolesis, and decreased platelet synthesis. In addition, gm homozygotes have partial cutaneous albinism (Fig. 1). These features closely resemble the rare human disorders gray platelet syndrome (GPS) and platelet ␣,␦-SPD. Positional cloning of gm was undertaken both to shed light on the mechanism of disease in platelet SPD disorders and to identify a possible target for antiplatelet drug development. Materials and MethodsMice and Genetic Mapping. Mice obtained from The Jackson Laboratory were bred at Roswell Park Cancer Institute. Backcross mice were phenotyped by coat color and genotyped for simple sequence length polymorphisms (SSLPs) and restriction fragment length polymorphism (RFLP) using standard techniques. Linkage relationships were determined by segregation analysis, and best locus order was decided by minimizing crossovers and eliminating double crossovers (4). Inverse Repetitive Element-Direct cDNA Selection. Expressed sequences were isolated from YACs by inverse repetitive elementdirect cDNA selection (6). Briefly, outward-oriented, biotinlabeled, B1 repetitive element-specific primers were used for long-range PCR of YAC clones (5). Denatured PCR products were incubated with rib...

Background: Chediak-Higashi syndrome (CHS) is an inherited immunodeficiency disease characterized by giant lysosomes and impaired leukocyte degranulation. CHS results from mutations in the lysosomal trafficking regulator (LYST) gene, which encodes a 425-kD cytoplasmic protein of unknown function. The goal of this study was to identify proteins that interact with LYST as a first step in understanding how LYST modulates lysosomal exocytosis. Materials and Methods: Fourteen cDNA fragments, covering the entire coding domain of LYST, were used as baits to screen five human cDNA libraries by a yeast twohybrid method, modified to allow screening in the activation and the binding domain, three selectable markers, and more stringent confirmation procedures. Five of the interactions were confirmed by an in vitro binding assay.