A BLOC-1 mutation screen reveals a novel BLOC1S3 mutation in Hermansky-Pudlak Syndrome type 8

Hermansky-Pudlak Syndrome (HPS) is a genetically heterogeneous disorder of lysosome-related organelle biogenesis and is characterized by oculocutaneous albinism and a bleeding diathesis. Over the past decade, we screened 250 patients with HPS-like symptoms for mutations in the genes responsible for HPS subtypes 1-6. We identified 38 individuals with no functional mutations, and therefore, we analyzed all eight genes encoding the biogenesis of lysosome-related organelles complex-1 (BLOC-1) proteins in these individuals. Here, we describe the identification of a novel nonsense mutation in BLOC1S3 (HPS-8) in a 6-yr-old Iranian boy. This mutation caused nonsense-mediated decay of BLOC1S3 mRNA and destabilized the BLOC-1 complex. Our patient's melanocytes showed aberrant localization of TYRP1, with increased plasma membrane trafficking. These findings confirm a common cellular defect for HPS patients with defects in BLOC-1 subunits. We identified only two patients with BLOC-1 defects in our cohort, suggesting that other HPS genes remain to be identified.     

Disorders of vesicles of lysosomal lineage: the Hermansky-Pudlak syndromes

Hermansky-Pudlak syndrome (HPS) has evolved into a group of genetically distinct disorders characterized by oculocutaneous albinism, a storage pool deficiency, and impaired formation or trafficking of intracellular vesicles. HPS-1 results from mutations in the HPS1 gene and affects approximately 400 individuals in northwest Puerto Rico due to a 16-bp duplication in exon 15. Another 13 mutations have been reported in non-Puerto Ricans. HPS1 codes for a 79.3 kDa cytoplasmic protein of unknown function. HPS-1 patients typically develop fatal pulmonary fibrosis in their fourth decade. HPS-2 is caused by mutations in ADTB3A, which codes for the beta3A subunit of the adaptor protein-3 complex, AP3. This coat protein complex has been localized to the TGN as well as to a peripheral endosomal compartment. Evidence indicates that AP3 plays a role in the stepwise process of vesicular trafficking which leads to formation of the melanosomal, platelet dense body and lysosomal compartments. All three known HPS-2 patients had childhood neutropenia and infections. HPS-3 results from mutations in HPS3 and affects central Puerto Ricans homozygous for a 3904-bp deletion removing exon 1. At least 8 non-Puerto Rican patients have other HPS3 mutations, including an IVS5+1G->A splicing mutation in five Ashkenazi Jewish patients. HPS3 codes for a 113.7 kDa protein of unknown function. HPS-3 manifests with mild hypopigmentation and bleeding. All types of HPS are diagnosed by whole mount electron microscopic demonstration of absent platelet dense bodies, and molecular diagnoses are available for the Puerto Rican HPS1 and HPS3 founder mutations. Mouse and Drosophila models provide candidates for new genes causing HPS in humans. These genes will reveal the pathways by which specialized vesicles of lysosomal lineage arise within cells.     

Membranous complexes characteristic of melanocytes derived from patients with Hermansky-Pudlak syndrome type 1 are macroautophagosomal entities of the lysosomal compartment

Hermansky-Pudlak syndrome (HPS) is an autosomal recessive disorder resulting from mutations in a family of genes required for efficient transport of lysosomal-related proteins from the trans-Golgi network to a target organelle. To date, there are several genetically distinct forms of HPS. Many forms of HPS exhibit aberrant trafficking of melanosome-targeted proteins resulting in incomplete melanosome biogenesis responsible for oculocutaneous albinism observed in patients. In HPS-1, melanosome-targeted proteins are localized to characteristic membranous complexes, which have morphologic similarities to macroautophagosomes. In this report, we evaluated the hypothesis that HPS-1-specific membranous complexes comprise a component of the lysosomal compartment of melanocytes. Using indirect immunofluorescence, an increase in co-localization of misrouted tyrosinase with cathepsin-L, a lysosomal cysteine protease, occurred in HPS-1 melanocytes. In addition, ribophorin II, an integral endoplasmic reticulum protein that is also a component of macroautophagosomes, and LC3, a specific marker of macrophagosomes, demonstrated localization to membranous complexes in HPS-1 melanocytes. At the electron microscopic level, the membranous complexes exhibited acid phosphatase activity and localization of exogenously supplied horseradish peroxidase (HRP)-conjugated gold particles, indicating incorporation of lysosomal and endosomal components to membranous complexes, respectively. These results confirm that membranous complexes of HPS-1 melanocytes are macroautophagosomal representatives of the lysosomal compartment.     

Association of the Hermansky-Pudlak syndrome type-3 protein with clathrin

Background  

Hermansky-Pudlak syndrome (HPS) is a disorder of lysosome-related organelle biogenesis characterized by oculocutaneous albinism and prolonged bleeding. These clinical findings reflect defects in the formation of melanosomes in melanocytes and dense bodies in platelets. HPS type-3 (HPS-3) results from mutations in the HPS3 gene, which encodes a 1004 amino acid protein of unknown function that contains a predicted clathrin-binding motif (LLDFE) at residues 172–176.     

Hermansky-Pudlak syndrome: vesicle formation from yeast to man

The disorders known as Hermansky-Pudlak syndrome (HPS) are a group of genetic diseases resulting from abnormal formation of intracellular vesicles. In HPS, dysfunction of melanosomes results in oculocutaneous albinism, and absence of platelet dense bodies causes a bleeding diathesis. In addition, some HPS patients suffer granulomatous colitis or fatal pulmonary fibrosis, perhaps due to mistrafficking of a subset of lysosomes. The impaired function of specific organelles indicates that the causative genes encode proteins operative in the formation of certain vesicles. Four such genes, HPS1, ADTB3A, HPS3, and HPS4, are associated with the four known subtypes of HPS, i.e. HPS-1, HPS-2, HPS-3, and HPS-4. ADTB3A codes for the beta 3 A subunit of adaptor complex-3, known to assist in vesicle formation from the trans-Golgi network or late endosome. However, the functions of the HPS1, HPS3, and HPS4 gene products remain unknown. These three genes arose with the evolution of mammals and have no homologs in yeast, reflecting their specialized function. In contrast, all four known HPS-causing genes have homologs in mice, a species with 14 different models of HPS, i.e. hypopigmentation and a platelet storage pool deficiency. Pursuit of the mechanism of mammalian vesicle formation and trafficking, impaired in HPS, relies upon investigation of these mouse models as well as studies of protein complexes involved in yeast vacuole formation.     

Characterization of a partial pseudogene homologous to the Hermansky-Pudlak syndrome gene HPS-1; relevance for mutation detection

The HPS-1 gene is the first gene found to be responsible for the autosomal recessive disorder Hermansky-Pudlak syndrome (HPS). HPS is characterized by oculocutaneous albinism, a platelet storage pool deficiency, and ceroid lipofuscinosis. The HPS-1 gene has been mapped to chromosome 10q23.1-23.3 and encodes a 79-kDa protein of unknown function with no homology to any known protein. A sequence database search has revealed that a portion of clone HS 1119A7 shows high sequence similarity to HPS-1 cDNA. By performing sequence alignments and PCR amplification of cDNA from several human tissues, we have shown that part of this clone consists of an unprocessed partial HPS-1 pseudogene located on chromosome 22q12.2-12.3. The pseudogene contains several intact HPS-1 exons and shows 95% sequence homology to the HPS-1 cDNA. Exon 6 of the pseudogene has 100% sequence homology to exon 6 of HPS-1 itself. In the pseudogene, this exon is surrounded by portions of both its normal flanking introns. These data provide the first characterization of an HPS-1 pseudogene, called HPS1-psi1. During amplification of exon 6 of the HPS-1 gDNA for mutation identification, the pseudogene might also be amplified, leading to a false positive for mutation. In addition, amplification of specific parts of the HPS-1 cDNA (e.g., exons 2-5) for mutation detection might lead to false positives for mutations, if the cDNA is contaminated with gDNA. This calls for caution when employing these screening approaches.     

The pallidin (Pldn) gene and the role of SNARE proteins in melanosome biogenesis

This review focuses on the product of the pallidin (Pldn) gene, one of a number of genes that in mice are associated with pigmentation defects and platelet dense granule deficiency. A similar combination of defects is also observed in patients suffering from Hermansky-Pudlak (HPS) and Chediak-Higashi (CHS) syndromes. Pldn encodes a novel, approximately 20-kDa protein that is expressed ubiquitously in mammalian tissues. The pallidin protein was found to bind to syntaxin 13, a member of the syntaxin family of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). As SNARE proteins mediate fusion of intracellular membranes, pallidin may play a role in membrane fusion events required for melanosome biogenesis.     

Hermansky-Pudlak syndrome type 3 in Ashkenazi Jews and other non-Puerto Rican patients with hypopigmentation and platelet storage-pool deficiency

Hermansky-Pudlak syndrome (HPS), consisting of oculocutaneous albinism and a bleeding diathesis due to the absence of platelet dense granules, displays extensive locus heterogeneity. HPS1 mutations cause HPS-1 disease, and ADTB3A mutations cause HPS-2 disease, which is known to involve abnormal intracellular vesicle formation. A third HPS-causing gene, HPS3, was recently identified on the basis of homozygosity mapping of a genetic isolate of HPS in central Puerto Rico. We now describe the clinical and molecular characteristics of eight patients with HPS-3 who are of non-Puerto Rican heritage. Five are Ashkenazi Jews; three of these are homozygous for a 1303+1G-->A splice-site mutation that causes skipping of exon 5, deleting an RsaI restriction site and decreasing the amounts of mRNA found on northern blotting. The other two are heterozygous for the 1303+1G-->A mutation and for either an 1831+2T-->G or a 2621-2A-->G splicing mutation. Of 235 anonymous Ashkenazi Jewish DNA samples, one was heterozygous for the 1303+1G-->A mutation. One seven-year-old boy of German/Swiss extraction was compound heterozygous for a 2729+1G-->C mutation, causing skipping of exon 14, and resulting in a C1329T missense (R396W), with decreased mRNA production. A 15-year-old Irish/English boy was heterozygous for an 89-bp insertion between exons 16 and 17 resulting from abnormal splicing; his fibroblast HPS3 mRNA is normal in amount but is increased in size. A 12-year-old girl of Puerto Rican and Italian background has the 3,904-bp founder deletion from central Puerto Rico on one allele. All eight patients have mild symptoms of HPS; two Jewish patients had received the diagnosis of ocular, rather than oculocutaneous, albinism. These findings expand the molecular diagnosis of HPS, provide a screening method for a mutation common among Jews, and suggest that other patients with mild hypopigmentation and decreased vision should be examined for HPS.     

A germline mutation in BLOC1S3/reduced pigmentation causes a novel variant of Hermansky-Pudlak syndrome (HPS8)

Hermansky-Pudlak syndrome (HPS) is genetically heterogeneous, and mutations in seven genes have been reported to cause HPS. Autozygosity mapping studies were undertaken in a large consanguineous family with HPS. Affected individuals displayed features of incomplete oculocutaneous albinism and platelet dysfunction. Skin biopsy demonstrated abnormal aggregates of melanosomes within basal epidermal keratinocytes. A homozygous germline frameshift mutation in BLOC1S3 (p.Gln150ArgfsX75) was identified in all affected individuals. BLOC1S3 mutations have not been previously described in patients with HPS, but BLOC1S3 encodes a subunit of the biogenesis of lysosome-related organelles complex 1 (BLOC-1). Mutations in other BLOC-1 subunits have been associated with an HPS phenotype in humans and/or mouse, and a nonsense mutation in the murine orthologue of BLOC1S3 causes the reduced pigmentation (rp) model of HPS. Interestingly, eye pigment formation is reported to be normal in rp, but we found visual defects (nystagmus, iris transilluminancy, foveal hypoplasia, reduced visual acuity, and evidence of optic pathway misrouting) in affected individuals. These findings define a novel form of human HPS (HPS8) and extend genotype-phenotype correlations in HPS.     

The Drosophila pigmentation gene pink (p) encodes a homologue of human Hermansky-Pudlak syndrome 5 (HPS5)

Lysosome-related organelles comprise a group of specialized intracellular compartments that include melanosomes and platelet dense granules (in mammals) and eye pigment granules (in insects). In humans, the biogenesis of these organelles is defective in genetic disorders collectively known as Hermansky-Pudlak syndrome (HPS). Patients with HPS-2, and two murine HPS models, carry mutations in genes encoding subunits of adaptor protein (AP)-3. Other genes mutated in rodent models include those encoding VPS33A and Rab38. Orthologs of all of these genes in Drosophila melanogaster belong to the 'granule group' of eye pigmentation genes. Other genes associated with HPS encode subunits of three complexes of unknown function, named biogenesis of lysosome-related organelles complex (BLOC)-1, -2 and -3, for which the Drosophila counterparts had not been characterized. Here, we report that the gene encoding the Drosophila ortholog of the HPS5 subunit of BLOC-2 is identical to the granule group gene pink (p), which was first studied in 1910 but had not been identified at the molecular level. The phenotype of pink mutants was exacerbated by mutations in AP-3 subunits or in the orthologs of VPS33A and Rab38. These results validate D. melanogaster as a genetic model to study the function of the BLOCs.     

BLOC-1, a novel complex containing the pallidin and muted proteins involved in the biogenesis of melanosomes and platelet-dense granules

Recent studies have led to the identification of a group of genes required for normal biogenesis of lysosome-related organelles such as melanosomes and platelet-dense granules. Two of these genes, which are defective in the pallid and muted mutant mouse strains, encode small, coiled-coil-forming proteins that display no homology to each other or to any known protein. We report that these two proteins, pallidin and muted, are components of a novel protein complex. We raised antibodies that allow for detection of pallidin from a wide variety of mammalian cells. Endogenous pallidin was distributed in both soluble and peripheral membrane protein fractions. Size-exclusion chromatography and sedimentation velocity analyses indicated that the bulk of cytosolic pallidin is a component of an asymmetric protein complex with a molecular mass of approximately 200 kDa. We named this complex BLOC-1 (for biogenesis of lysosome-related organelles complex 1). Steady-state pallidin protein levels were reduced in fibroblasts derived from muted and reduced pigmentation mice, suggesting that the genes defective in these two mutant strains could encode components of BLOC-1 that are required for pallidin stability. Co-immunoprecipitation and immunodepletion experiments using an antibody to muted confirmed that this protein is a subunit of BLOC-1. Yeast two-hybrid analyses revealed that pallidin is capable of self-association through a region that contains its two coiled-coil forming domains. Unlike AP-3-deficient pearl fibroblasts, which display defects in intracellular zinc storage, zinc distribution was not noticeably affected in pallid or muted fibroblasts. Interestingly, immunofluorescence and in vitro binding experiments demonstrated that pallidin/BLOC-1 is able to associate with actin filaments. We propose that BLOC-1 mediates the biogenesis of lysosome-related organelles by a mechanism that may involve self-assembly and interaction with the actin cytoskeleton.     

Hermansky-Pudlak syndrome and pale ear: melanosome-making for the millennium

Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder characterized principally by oculocutaneous albinism, a bleeding tendency, and a ceroid-lipofuscin lysosomal storage disease. These clinical manifestations of HPS are associated with defects of multiple cytoplasmic organelles--melanosomes, platelet granules, and lysosomes--suggesting that the HPS gene product is involved in some shared feature of the biogenesis or functions of these diverse organelles. The HPS gene has been cloned, and a number of pathologic mutations of the gene have been identified. Functional studies indicate that the HPS protein is part of a high-molecular weight complex involved in the biogenesis of early melanosomes. Additional disorders with similarities to HPS have been identified in man, mouse, flies, and yeast, and it is rapidly becoming clear that understanding these disorders will shed new light on the mechanisms by which cells traffic newly synthesized proteins through the cytoplasm to assemble functional organelles.     

Pallidin is a component of a multi-protein complex involved in the biogenesis of lysosome-related organelles

The Hermansky–Pudlak syndrome defines a group of genetic disorders characterized by defective lysosome-related organelles such as melanosomes and platelet dense bodies. Hermansky–Pudlak syndrome can be caused by mutations of at least four genes in humans and 15 genes in mice. One of these genes is mutated in the pallid mouse strain and encodes a novel protein named pallidin (L. Huang, Y. M. Kuo and J. Gitschier, Nat Genet 1999; 23: 329–332). Pallidin has no homology to any other known protein and no recognizable functional motifs. We have conducted a biochemical characterization of human pallidin using a newly developed polyclonal antibody. We show that pallidin is a ubiquitously expressed ∼ 25 kDa protein found both in the cytosol and peripherally associated to membranes. Sedimentation velocity analyses show that native pallidin has a sedimentation coefficient of ∼ 5.1 S, much larger than expected from the molecular mass of the pallidin polypeptide. In line with this observation, cosedimentation and coprecipitation analyses reveal that pallidin is part of a hetero-oligomeric complex. One of the subunits of this complex is the product of another Hermansky–Pudlak syndrome gene, muted. Fibroblasts derived from the muted mouse strain exhibit reduced levels of pallidin, suggesting that the absence of the muted protein destabilizes pallidin. These observations indicate that pallidin is a subunit of a novel multi-protein complex involved in the biogenesis of lysosome-related organelles.     

Clinico‐molecular analysis of eleven patients with Hermansky–Pudlak type 5 syndrome,a mild form of HPS

Hermansky–Pudlak syndrome (HPS), first described in 1959, is a rare form of syndromic oculocutaneous albinism associated with bleeding diathesis and in some cases pulmonary fibrosis and granulomatous colitis. All 10 HPS types are caused by defects in vesicle trafficking of lysosome‐related organelles (LRO) proteins. The HPS5 protein associates with HPS3 and HPS6 to form the biogenesis of lysosome‐related organelles complex‐2 (BLOC‐2). Here, we report the clinical and genetic data of 11 patients with HPS‐5 analyzed in our laboratory. We report 11 new pathogenic variants. The 11 patients present with ocular features that are typical for albinism, with mild hypopigmentation, and with no other major complication, apart from a tendency to bleed. HPS‐5 therefore appears as a mild form of HPS, which is often clinically undistinguishable from mild oculocutaneous or ocular forms of albinism. Molecular analysis is therefore required to establish the diagnosis of this mild HPS form, which has consequences in terms of prognosis and of clinical management of the patients.     

NGS‐based targeted resequencing identified rare subtypes of albinism: Providing accurate molecular diagnosis for Japanese patients with albinism

Albinism, which is commonly inherited as an autosomal recessive trait, is characterized by a reduction or absence of melanin in the eyes, skin, and hair. To date, more than 20 causal genes for albinism have been identified; thus, the accurate diagnosis of albinism requires next‐generation sequencing (NGS). In this study, we analyzed 46 patients who tested negative for oculocutaneous albinism (OCA)1–4 and Hermansky‐Pudlak syndrome (HPS)1 based on conventional analysis, in addition to 28 new Japanese patients, using NGS‐based targeted resequencing. We identified a genetic background for albinism in 18 of the 46 patients (39%), who were previously tested negative according to the conventional analysis. In addition, we unveiled a genetic predisposition toward albinism in 23 of the 28 new patients (82%). We identified six patients with rare subtypes of albinism, including HPS3, HPS4, and HPS6, and found 12 novel pathological mutations in albinism‐related genes. Furthermore, most patients who were not diagnosed with albinism by the NGS analysis showed mild manifestations of albinism without apparent eye symptoms and harbored only one heterozygous mutation, occasionally in combination with skin‐color associated gene variants.     

Hermansky–Pudlak Syndrome and Pale Ear: Melanosome‐Making for the Millennium

Hermansky–Pudlak syndrome (HPS) is a rare autosomal recessive disorder characterized principally by oculocutaneous albinism, a bleeding tendency, and a ceroid‐lipofuscin lysosomal storage disease. These clinical manifestations of HPS are associated with defects of multiple cytoplasmic organelles – melanosomes, platelet granules, and lysosomes – suggesting that the HPS gene product is involved in some shared feature of the biogenesis or functions of these diverse organelles. The HPS gene has been cloned, and a number of pathologic mutations of the gene have been identified. Functional studies indicate that the HPS protein is part of a high‐molecular weight complex involved in the biogenesis of early melanosomes. Additional disorders with similarities to HPS have been identified in man, mouse, flies, and yeast, and it is rapidly becoming clear that understanding these disorders will shed new light on the mechanisms by which cells traffic newly synthesized proteins through the cytoplasm to assemble functional organelles.     

Characterization of BLOC-2, a complex containing the Hermansky-Pudlak syndrome proteins HPS3, HPS5 and HPS6

Hermansky–Pudlak syndrome (HPS) defines a group of at least seven autosomal recessive disorders characterized by albinism and prolonged bleeding due to defects in the lysosome-related organelles, melanosomes and platelet-dense granules, respectively. Most HPS genes, including HPS3, HPS5 and HPS6 , encode ubiquitously expressed novel proteins of unknown function. Here, we report the biochemical characterization of a stable protein complex named Biogenesis of Lysosome-related Organelles Complex-2 (BLOC-2), which contains the HPS3, HPS5 and HPS6 proteins as subunits. The endogenous HPS3, HPS5 and HPS6 proteins from human HeLa cells coimmunoprecipitated with each other from crude extracts as well as from fractions resulting from size-exclusion chromatography and density gradient centrifugation. The native molecular mass of BLOC-2 was estimated to be 340 ± 64 kDa. As inferred from the biochemical properties of the HPS6 subunit, BLOC-2 exists in a soluble pool and associates to membranes as a peripheral membrane protein. Fibroblasts deficient in the BLOC-2 subunits HPS3 or HPS6 displayed normal basal secretion of the lysosomal enzyme β-hexosaminidase. Our results suggest a common biological basis underlying the pathogenesis of HPS-3, -5 and -6 disease.     

Genetic variants and mutational spectrum of Chinese Hermansky–Pudlak syndrome patients

Hermansky–Pudlak syndrome (HPS) is a rare recessive disorder characterized by oculocutaneous albinism or ocular albinism, bleeding diathesis, and other symptoms such as colitis and pulmonary fibrosis. Eleven causative genes have been identified for HPS‐1–HPS‐11 subtypes in humans. We have identified 16 newly reported patients including the first HPS‐2 case in the Chinese population. In a total of 40 HPS patients, hypopigmentation was milder in HPS‐3, HPS‐5, and HPS‐6 patients than in HPS‐1 and HPS‐4 patients. HPS‐1 accounted for 47.5% (19 of 40) of HPS cases which is the most common subtype. Exons 11 and 19 were the hotspots of the HPS1 gene mutations. In total, 55 allelic variants were identified in HPS1–HPS6 gene, of which 17 variants were previously unreported. These results will be useful for the evaluation of the relationship between HPS genotypes and phenotypes, and for the precise intervention of HPS patients in the Chinese population.