Hermansky–Pudlak Syndrome and Related Disorders of Organelle Formation

Authors

  • Marjan Huizing,

    1. Section on Human Biochemical Genetics, Heritable Disorders Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA
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  • Yair Anikster,

    1. Section on Human Biochemical Genetics, Heritable Disorders Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA
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  • William A. Gahl

    1. Section on Human Biochemical Genetics, Heritable Disorders Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA
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Corresponding author: William A. Gahl, bgahl@helix.nih.gov

Abstract

Hermansky–Pudlak syndrome (HPS) consists of a group of genetically heterogeneous disorders which share the clinical findings of oculocutaneous albinism, a platelet storage pool deficiency, and some degree of ceroid lipofuscinosis. Related diseases share some of these findings and may exhibit other symptoms and signs but the underlying defect in the entire group of disorders involves defective intracellular vesicle formation, transport or fusion. Two HPS-causing genes, HPS1 and ADTB3A, have been isolated but the function of only the latter has been determined. ADTB3A codes for the β3A subunit of adaptor complex-3, responsible for vesicle formation from the trans-Golgi network (TGN). The many HPS patients who do not have HPS1 or ADTB3A mutations have their disease because of mutations in other genes. Candidates for these HPS-causing genes include those responsible for mouse models of HPS or for the ‘granule’ group of eye color genes in Drosophila. Each gene responsible for a subset of HPS or a related disorder codes for a protein which almost certainly plays a pivotal role in vesicular trafficking, inextricably linking clinical and cell biological interests in this group of diseases.

The genetic disease known collectively as Hermansky–Pudlak syndrome (OMIM #203300)1, or HPS, consists of several genetically different autosomal recessive disorders which share the clinical manifestations of hypopigmentation and a platelet storage pool deficiency. These findings reflect abnormalities of the melanocyte's melanosome and of the platelet's dense body, two intracellular organelles related to lysosomes [1]. At least 15 genetically distinct mouse strains display hypopigmentation and a storage pool deficiency [2,3], suggesting that the well-recognized locus heterogeneity of human HPS [4–6] extends far beyond the two known HPS-causing genes, HPS1 [7] and ADTB3A [8]. Every gene discovered to be responsible for HPS or a related disorder is expected to code for a protein involved in the formation, trafficking or fusion of intracellular vesicles of lysosomal lineage [1]. Consequently, these diseases and their causative genes are of immense interest to students of cellular biology.

Hermansky–Pudlak Syndrome

This disease entity was first described in 1959 by two Czechoslovakian pathologists who described patients with the unique combination of oculocutaneous albinism and a bleeding diathesis [9]. Although the accumulation of ceroid lipofuscin has been considered a hallmark of the disorder, in practice the demonstration of this amorphous, autofluorescent lipid-protein complex is not required for the diagnosis of HPS [1,10].

Clinical characteristics

The clinical manifestations of HPS are colored by the ethnic background of affected patients, as well as the causative genetic locus. Our understanding of the disease has been strongly influenced by the approximately 400 patients from northwest Puerto Rico [11] who are homozygous for exactly the same mutation in HPS1 [1,7], but at least 200 non-Puerto Rican patients are likely to exist elsewhere throughout the world. Each of the clinical areas of involvement in HPS is described below for the entire group of patients, followed by a discussion of the characteristics of the disorder unique to certain ethnic or genetic subsets.

Oculocutaneous albinism

In general, the oculocutaneous albinism of HPS is similar to other types of albinism in which tyrosinase remains present, although muted, within melanocytes [12]. However, a very wide range of severity is apparent in HPS.

The hypopigmentation of HPS affects the hair, skin, and eyes; its variability reflects the large number of genes that contribute to the color of these tissues. Hair color ranges from completely white to dark brown (Fig. 1A–F), regardless of the genetic defect [10]. Even among Puerto Rican patients with exactly the same HPS1 mutation, the spectrum of hair color is enormously broad [13]. Among all HPS patients, dirty blond or tan hair could be considered typical (Fig. 1E).

Figure 1.

Figure 1.

Varying hair colors in HPS patients. (A) A 6-year-old boy of English and Polish ancestry with no mutation in HPS1 or ADTB3A. (B) A 35-year-old woman of German, Welsh, and Irish heritage with no mutation in HPS1 or ADTB3A. Note natural streaks of silvery pigment. (C) A 52-year old Jewish woman with no mutation in HPS1 or ADTB3A. (D) A 25-year-old male of Dutch ancestry with HPS-2 and compound heterozygous mutations in ADTB3A. (E) An 11-year-old boy of English and Irish heritage with no mutation in HPS1 or ADTB3A. (F) A 12-year-old girl with Italian and Puerto Rican parents and mutations in HPS1.

The skin of HPS patients also varies from white to brown [14] and may not appear hypopigmented unless compared with that of unaffected siblings. As for individuals with other types of albinism, HPS patients are susceptible to solar damage and are at increased risk for skin malignancies (basal cell carcinoma, squamous cell carcinoma, and melanoma), nevi, actinic keratoses (Fig. 2A), generalized bruising (Fig. 2B), and other dermatologic abnormalities [14]. Sunscreen, sun avoidance, and surveillance are critical measures in managing the skin of HPS patients.

Figure 2.

Figure 2.

Clinical findings in HPS. (A) Extensive actinic keratoses in sunexposed areas of skin in a 53-year-old Puerto Rican male homozygous for the 16-bp duplication in exon 15 of HPS1. (B) Bruise of unknown origin appearing on the left calf of a 34-year-old man of Scottish ancestry with a HPS1 mutations. (C) Iris transillmination in a 30-year-old Jewish woman with no HPS1 mutation. The orange appearance represents abnormal transmission of light through the iris. The dark areas indicate the presence of pigment, which normally occupies the entire iris. (Photograph courtesy of Ernest Kuehl and Dr Muriel I. Kaiser-Kupfer of the National Eye Institute.) (D) Fundus of same patient as in C, showing pale areas devoid of retinal pigment epithelium. (Photograph courtesy of Ernest Kuehl and Dr Muriel I. Kaiser-Kupfer of the National Eye Institute.) (E) Wet mount electron micrograph of a normal platelet showing typical dense bodies or δ granules (arrows). [Reprinted with permission from Am J Hum Genet [4].] (F) Platelet from a Puerto Rican individual without HPS1 mutations, showing absent platelet dense bodies. [Reprinted with permission from Am J Hum Genet [4].]

Several ophthalmic complications result from the dysfunction of pigmented cells in HPS [13,15–17]. Affected patients display reduced decussation (hemispheric crossover) of the optic nerve fibers, typical of albinism. This may occur because of abnormal pigmentation and migration of neural crest cells early in embryonic development. Pigmented cells are also sparse in the irides, as illustrated by the finding of iris transillumination (Fig. 2C). In this phenomenon, light shone through the pupil is transmitted back through the iris because the normal contingent of iris pigment is absent and cannot absorb the light. Finally, loss or reduction of the retinal pigment epithelium results in a pale fundus (Fig. 2D) and decreased visual acuity. Typically, HPS patients are legally blind (visual acuity of 20/200 or worse) but occasionally a bona fide patient has an acuity as high as 20/50 [13,17]. In general, the visual acuity defects of HPS are stable, cannot be corrected and result in congenital nystagmus (rapid, uncontrolled movement of the eyes), usually in a horizontal direction.

Bleeding diathesis

The hematological manifestations of HPS are those associated with a platelet abnormality. They involve apparently spontaneous soft tissue bruising (Fig. 2B) and bleeding of mucosal membranes rather than major bleeds into the joints, brain or other organs, which are common in hemophiliacs [10,13]. Coagulation factors, prothrombin times, and partial thromboplastin times are normal. The first sign of a bleeding diathesis in HPS patients usually consists of excess bruising beginning at the time of ambulation. Episodes of epistaxis (nosebleeds) are frequent but often remit during adolescence. Other events which result in excess bleeding include dental extractions, surgeries, acute colitis, menstrual periods, and childbirth. Fatalities due to bleeding are rare but a significant number of patients have received transfusions of red blood cells or platelets [10,13]. For minor bleeding, topical thrombin can be helpful and 1-desamino-8-D-arginine vasopressin (DDAVP) can be used for prophylaxis [18]. This drug helps activate von Willebrand factor, which is curiously decreased in a significant number of HPS patients [19,20]. Von Willebrand factor is stored within platelet alpha granules.

The cause of bleeding in HPS is absence of platelet dense granules, making the disease a ‘storage pool deficiency’. Dense granules are intracellular organelles which contain ADP, ATP, serotonin, calcium, and polyphosphates and disgorge their contents upon stimulation [10]. Released components, ADP in particular, cause aggregation of surrounding platelets, contributing to clot formation. HPS patients have a normal or increased platelet count but an attenuated secondary aggregation response; the bleeding time is often but not always prolonged. Absence of dense bodies (Fig. 2E,F), which confirms the diagnosis of HPS, is demonstrable by wet-mount electron microscopy [21].

Ceroid lipofuscin

This poorly defined electron dense complex autofluoresces a bright yellow color. It is considered an aging pigment because it normally accumulates progressively during life. However, its formation is exaggerated in some patients with HPS [22]. The greatest amounts occur within the kidney, urinary sediment, lung alveolar macrophages (Fig. 3A), bone marrow, spleen, liver, and large intestine. Moderate amounts are present in the heart, lymph nodes, and other tissues [10]. In HPS, ceroid lipofuscin is thought to reside within lysosomes, although it is difficult to differentiate a lysosomal membrane from the electron dense ceroid itself. The ceroid lipofuscin of HPS appears to contain excessive amounts of dolichols [22]; these isoprenoid compounds are normally enriched in the lysosomal membrane, in contrast to the cholesterol-rich plasma membrane. Animal models are available for the study of ceroid lipofuscin accumulation [23].

Figure 3.

Figure 3.

Pulmonary involvement in HPS. (A) Centrifuged sediment of pulmonary lavage fluid from a Puerto Rican patient homozygous for the 16-bp duplication in HPS1. Within the alveolar macrophages are foamy vesicles containing membranous material (arrow). (Courtesy of Dr Mark Brantly, National Heart, Lung and Blood Institute.) (B) High resolution CT scan of the chest in a 39-year-old male homozygous for the 16-bp duplication in HPS1. The increased central markings indicate advanced fibrosis. The patient died 12 months after this image was obtained.

Pulmonary fibrosis

The pulmonary fibrosis of HPS begins as restrictive lung disease [10,13,24,25], manifest by abnormal pulmonary function tests in the late twenties or early thirties. Pulmonary function can plateau for years, and then fall dramatically and result in death within 2 or 3 years. Putative precipitating factors are infection, smoking or exposure to some other insult to the lung parenchyma. Activation of alveolar macrophages, which produce excess cytokines in HPS patients [26], suggests that inflammation precedes fibrosis [27]. Affected patients typically succumb to extensive fibrotic deterioration of the lungs (Fig. 3B) by the end of the fourth or fifth decade of life [10]. As for most manifestations of HPS, the pulmonary involvement is extremely variable, and occasional patients are over 60 years old with normal lung function. Only supportive care is available for patients with pulmonary fibrosis.

Other complications

Approximately 15% of HPS patients, whether Puerto Rican or non-Puerto Rican, suffer from a granulomatous colitis which often bleeds and requires a colostomy [13]. The distal colon is most often involved by this complication. The colitis of HPS resembles Crohn's disease [10,28,29] and responds somewhat to steroid therapy. Renal impairment has been reported in occasional HPS patients [10,13], as has granulomatous gingivitis and cardiomyopathy [10]. Lymphocyte and neutrophil function appear normal in HPS [30].

HPS in specific populations

The pulmonary fibrosis of HPS has been described in the original Czech patient [9], in a Japanese male [31], in a Belgian patient [32], in an English family [33], and in many Puerto Rican patients [10,11,13,25,27]. A large study demonstrated that Puerto Rican patients homozygous for the 16-bp duplication in HPS1 were at increased risk for developing pulmonary fibrosis [13]. Recently, it was shown that this risk extends to all patients with HPS1 mutations, not simply those with the duplication [25].

The two known HPS patients with mutations in ADTB3A, brothers in their twenties, also display characteristic clinical findings [8,34]. Both patients had persistent neutropenia, with total leucocyte counts of 2300–2800/μl and neutrophil counts of 570–1160/μl. They also had balance problems, which may be related to visual deficits, and a history of childhood infections, which could be within the range of normal. Other physical and laboratory findings, including congenital nystagmus, hypopigmentation, visual acuity defects, and absence of platelet dense bodies, were typical for HPS. Pulmonary function tests were borderline low [34].

HPS-1 disease

The disease HPS-1 represents the subset of HPS caused by mutations in the gene HPS [7] or HPS1. The number of patients is large because approximately 400 individuals in northwest Puerto Rico have HPS-1 as a result of a founder effect in this genetic isolate [10,11]. All the northwest Puerto Rican patients have their disease due to homozygosity for a 16-bp duplication in exon 15 of HPS1 [1,7,13]. Among non-Puerto Rican patients with HPS, only 25–40% have demonstrable mutations in HPS1 [1,5]; in general, these individuals appear phenotypically indistinguishable from patients who do not have HPS-1. The murine counterpart of HPS-1 is pale ear [35,36].

The HPS1 gene and its mutations

In 1995, the first HPS-causing gene was mapped to chromosome 10q23.1–23.3 by linkage disequilibrium using families from northwest Puerto Rico and a Swiss genetic isolate [37,38]. Cloning of the gene's cDNA (GenBank accession #U65676) revealed an open reading frame of 2100 bp [7] and the genomic structure of HPS1 was subsequently reported [39]. HPS1 consists of 20 exons spanning approximately 30.5 kb, with an intron 16 which is a member of the rare U12-type ‘AT-AC’ class of introns [36,39]. Recently, a pseudogene of HPS1 containing several intact exons, including exons 3, 4, and 6, was localized to chromosome 22q12.2–12.3 [40].

The human HPS1 gene has a standard transcript of 3.0 kb and is expressed in most tissues [7]. Minor 3.9- and 4.4-kb mRNAs are apparent on northern blot analysis. A 1.5-kb transcript with the same 5′ sequence as the published cDNA but with a different 3′ sequence is present in bone marrow and melanoma cells [41]. Four alternative splices of HPS1 have also been described [7,39].

The original report of the isolation of HPS1 described the 16-bp duplication in exon 15 found among northwest Puerto Rican patients [7]. This frameshift mutation produces no mRNA [4], presumably due to ‘nonsense-mediated decay’ [42,43] and has not been reported in any patient from outside of northwest Puerto Rico [1,5]. The duplication is easily detected using PCR amplification of a portion of exon 15 [7].

Other mutations initially reported include T322insC, found in a Swiss isolate and in Irish patients, and A441insA, found in a Japanese patient [7]. The T322insC mutation was subsequently reported in families of Italian/German/Ukrainian, Swiss, Irish/German, French, and Scottish heritage [5,7,44]. Haplotype analysis indicates that it arose at least twice in northern Europe [5]. A T322delC mutation has been found in German and Japanese families [5,44] and S396delC has appeared in Ukrainian, Dutch/German, and Irish/English/French/Norwegian patients [5,44]. The region of codons 321–324 appears to be a mutation hot spot [5] and codon 396 may be another area subject to recurrent mutation [5,44]. In all, 12 different mutations, including deletions, insertions, nonsense mutations, and splice junction mutations have been reported [1,6,45]. The mutations E133X, T322delC, and S396del have been shown to produce decreased or undetectable mRNA on northern blot analysis [44].

Except for the single codon deletion 55Ile [5], all the HPS1 gene mutations reported to date, as well as the frameshift mutations of the pale ear mouse [35,36], are predicted to result in a truncated protein. This suggests that the carboxy terminal portion of the HPS1 protein is critical for function. Furthermore, no missense mutation in HPS1 has been reported, perhaps because single base changes in HPS1 are not pathologic and constitute polymorphisms instead. At least 23 nonpathologic DNA sequence polymorphisms have been reported [39,44,45], including four which result in amino acid substitutions (G283W, P491R, R603Q, and V630I). Recently, a V4A substitution was found to also be an HPS1 polymorphism [46].

The HPS1 protein and its cell biology

The protein HPS1 consists of 700 amino acids and has a predicted molecular weight of 79.3 kDa [7]. Although it has two potential N-glycosylation sites at residues 528 and 560, HPS1 does not appear to be glycosylated [47]. The protein has no general homology to proteins with a known function but it does contain the sequence DKF(L/V)KNRG, which resembles the Chediak–Higashi syndrome (CHS) protein [7,48] (see below). In addition, the carboxy terminus of HPS1 contains a putative melanosomal localization signal, PLL, that is present in other melanosomal proteins [49]. The HPS1 amino acid sequence is 81% conserved between human and mouse [36], with similar conservation between human and rat [6]. A predicted Drosophila protein of unknown function has high sequence identity to the carboxy terminal portion of HPS1 (GenBank accession #AAF58194) but no homologues can be found in bacteria, yeast, worm or plants.

HPS1 also contains a His-Leu-Leu sequence near the carboxy terminus. This recognition marker could serve as a sorting signal to target the protein to late endosomal/lysosomal compartments [50,51], which might include the melanosomes and dense granules of specialized cells [52]. Localization studies indicate that HPS1 is not associated with lysosomes [47], consistent with the mild and indirect lysosomal defects observed in HPS-1 patients’ cells [6].

HPS1 has also been described as a component of two distinct high molecular weight complexes [47]. In non-melanotic cells such as fibroblasts, HPS1 forms a complex of approximately 200 kDa which is widely distributed through the cytosol. In melanotic cells, HPS1 is partitioned between the cytosolic 200-kDa complex and a complex of greater than 500 kDa that appears to consist of the 200-kDa complex in transient association with membranous components. The large complex, located in the perinuclear region, is associated with tubulovesicular structures, small non-coated vesicles, and nascent and early-stage melanosomes but not with later stage melanosomes. These findings suggest that the HPS1 protein complex is involved in the biogenesis of early melanosomes [47]. Other localization studies indicate that HPS1 is present in the perinuclear region of normal melanocytes and may be associated with a cisternal network outside of the Golgi zone [53]. This would place HPS1 in association with premelanosomes as they form from the smooth endoplasmic reticulum. A fine granular staining pattern throughout the cytosol and dendrites was also observed for HPS1, supporting a melanosomal localization.

This hypothesis is bolstered by studies of the pigment-forming proteins tyrosinase related protein-1 (TRP-1) and granulophysin in melanocytes cultured from HPS-1 patients. A large granular pattern of expression appeared for these proteins throughout the cells, consistent with the large membrane complexes observed ultrastructurally [54]. Consequently, it was proposed that the HPS1 gene product regulates, in part, the trafficking of melanocyte-specific proteins from the trans-Golgi network (TGN) to preformed premelanosomes [54].

In our experience, the low abundance of HPS1 in fibroblasts has prevented definitive determination of its subcellular localization. In these cells, the protein does not appear essential, since fibroblasts from HPS-1 patients appear to thrive. In fact, such fibroblasts display normal distribution and trafficking of the lysosomal membrane proteins, CD63 and LAMP-1, in contrast to cells from patients with ADTB3A mutations, which show increased routing of these lysosomal proteins through the plasma membrane [8]. It has been shown, however, that HPS1 does not functionally interact with AP3 [52].

HPS-2 disease

The subtype of HPS called HPS-2 has been found to affect only two brothers, whose clinical manifestations are described above [34]. These patients were ascertained by screening a large group of HPS patients who lacked HPS1 mutations. The screening, which involved western blotting for subunits of the adaptor-3 complex or AP3 [8], was performed because the murine pearl gene had been discovered to code for the β3A subunit of AP3 [55]. This protein complex functions to form vesicles out of existing membranes of the TGN (see below). After a decrease in AP3 subunit proteins was discovered in fibroblast extracts of the two brothers, sequencing revealed in-frame mutations in the gene coding for the β3A subunit (ADTB3A) of AP3 [8]. No other HPS-2 patients have been reported to date but more severe ADTB3A mutations may broaden the phenotype of this disease in the future.

The ADTB3A gene and its mutations

ADTB3A cDNA (GenBank accession #81504) contains 3950 bases and produces a 4.2-kb message present in all tissues and cell lines examined [56]. The two brothers with HPS-2 displayed compound heterozygous mutations in ADTB3A consisting of a 21 amino acid deletion (Δ390–410) and a single amino acid substitution, L580R, due to a T→G substitution [8]. Both mutations are in the ‘trunk’ region of the β3A molecule. Mutations detected in the pearl mouse include a 793-bp internal tandem duplication starting at nucleotide 2135 and a 107-bp deletion of nucleotides 2504–2610 [55].

The β3A protein and its cell biology

The β3A protein is a subunit of AP3, one of four known adaptor complexes. Adaptors are components of cytosolic protein coats that mediate vesicle formation and incorporation of ‘cargo’ proteins into the nascent vesicle's membranes [57]. Of the four known adaptor complexes, AP1, AP2, AP3, and AP4, AP2 operates at the plasma membrane, while the other AP complexes apparently function to produce vesicles from the TGN and/or endosome. Like the other heterotetrameric adaptor complexes, AP3 interacts in vitro with the vesicle-forming protein clathrin [58] and with both tyrosine-based [59,60] and dileucine-based sorting signals [61]. AP3 consists of a 160-kDa δ subunit, a 47-kDa μ subunit, a 23-kDa σ subunit, and the 140-kDa β3A subunit [56,59,62,63]. The β3A subunit is phosphorylated on serine residues [56]. The μ subunit interacts with the targeting signals of cargo proteins [59].

Consistent with a role for AP3 in protein trafficking, AP3-deficient cells displayed enhanced trafficking of lysosomal membrane proteins through the plasma membrane [8,64]. Similar findings were reported for fibroblasts of the pearl mouse bearing ADTB3A mutations [55] and the mocha mouse carrying mutations in the δ subunit of AP-3 [65]. Therefore, AP3 may mediate the trafficking of a subset of integral membrane proteins from an intracellular site to lysosomes and, presumably, to melanosomes and platelet-dense granules. However, not all integral membrane proteins use AP3 for targeting to lysosome-related organelles, since trafficking of MHC class II molecules and the associated invariant chains to their intracellular compartments appears normal in AP3-deficient cells [66].

Fibroblasts from patients with ADTB3A mutations show reduction of AP3 subunits other than β3A, i.e. μ, σ, δ. Apparently, the trunk region of the β3A subunit interacts with the other AP-3 subunits and stabilizes the complex against degradation [8].

Our understanding of the role of AP3 in pigment formation has been advanced by recent investigations. First, it was demonstrated using a surface plasmon resonance biosensor system that the μ subunit of AP-3 interacts with the pigment-forming enzyme tyrosinase [61]. Next, in β3A-deficient human melanocytes, tyrosinase expression was shown to be reduced, restricted to the perinuclear region, and localized in large vesicles resembling late endosomes, supporting the concept that tyrosinase trafficking is regulated by AP3 [67].

HPS-3 disease

At the present time, there is no HPS-3 disease. However, several additional subtypes of HPS are certain to exist as a reflection of the extensive locus heterogeneity already documented. For example, within central Puerto Rico, there resides an enclave of patients with a mild form of HPS and no mutation in HPS1 [4]. Moreover, 60–75% of HPS patients outside of Puerto Rico have a mutation in neither HPS1 nor ADTB3A [1,5]. Finally, the many distinct murine loci causing an HPS-like phenotype strongly supports the existence of several HPS-causing genes in humans.

How can those genes be identified? The sporadic occurrence of HPS in most populations, along with the heterogeneity of disease-causing loci, mitigate against the use of linkage analysis. Rather, we have employed screening of candidate genes to identify those that may cause HPS in patients who lack HPS1 and ADTB3A mutations. The technique was previously successful in ascertaining our HPS-2 patients. Potential candidate genes include any HPS-causing genes isolated in mice, genes responsible for the ‘granule’ group of eye color mutants in Drosophila, certain vesicle proteins for sorting (VPS) mutants in yeast, and selected genes found to code for other proteins involved in vesicle formation and trafficking.

The 15 murine models of HPS, with their absent platelet dense granules, hypopigmentation, and lysosomal defects [2,3], provide the most obvious candidate genes. Two of these, pale ear and pearl, represent the murine counterparts of HPS-1 and HPS-2, respectively. Only four other murine models of HPS have had their causative genes isolated. The mocha gene encodes the δ subunit of AP3 and the mocha mouse exhibits increased trafficking of lysosomal membrane proteins via the cell surface, similar to ADTB3A deficient human cells [52]. The murine pallid gene [68] encodes a 25-kDa protein, pallidin, that shows no homology to any other known proteins. Pallidin interacts with syntaxin13, which is part of the membrane fusion machinery [69]. This suggests a role for pallidin in vesicle trafficking. The gunmetal mouse carries mutations in the α subunit of rab geranylgeranyl transferase, an enzyme that adds 20-carbon prenyl groups to cysteine residues on the carboxy termini of rab proteins [70]. The mutation results in decreased prenylation and decreased membrane association of rab27a [70]. Rab proteins are recognized as key players in vesicular transport and organelle dynamics [71], so these events could result in impaired transport of proteins to lysosome-related organelles. Most recently, the ashen mouse was shown to exhibit mutations in rab27a [3]. Ashen was also shown, for the first time, to have an increased bleeding time and a reduction in platelet dense granules, making it a bone fide model for HPS.

At least 11 Drosophila melanogaster models with mutations at eye color loci are members of the ‘granule group’ of mutants [72]. In Drosophila, pigment granules are related to the lysosomes of other cell types. The 7 granule group genes recognized so far are identical to genes involved in lysosomal trafficking in other organisms. For example, all four subunits of AP3 have a Drosophila mutant model. The garnet fly has mutations in the δ subunit of AP3 [63,73], while the ruby fly is mutated in β3 [74], the carmine fly in μ3 [75], and the orange fly in σ3 [76]. In addition, three other genes of the granule group encode Drosophila homologues of the yeast VPS genes. The light fly has mutations in VPS41, whose gene product interacts with the δ subunit of AP3 and is required for the formation of AP3-coated carrier vesicles [77]. The deep orange and carnation flies are mutated in VPS18 and VPS33, respectively [78,79]. In yeast, the VPS18 and VPS33 proteins form a multisubunit complex with VPS11 and VPS16 [80]. In Drosophila, VPS33 and VPS18 interact in a large complex and mutations in deep orange result in accumulation of multivesicular bodies [79].

Finally, yeast contains more than 40 different VPS genes which influence trafficking of proteins destined for the lysosome-like vacuole [81]. Many human homologues of these genes are legitimate candidates for genes causing HPS or related disorders.

Related Disorders

Several extremely rare genetic conditions resemble HPS. Eventually, all of these disorders will be defined on molecular grounds but currently most are recognized only by their phenotypes, which have certain features in common with HPS. CHS, Griscelli syndrome (GS), Elejalde Syndrome (ES) and Cross syndrome are characterized by silvery hair, a finding seen in some patients with HPS (Fig. 1B). In addition, individuals with CHS or GS may have a syndrome of hemophagocytosis. Patients with CHS, and probably one subset of patients with GS, have absent platelet dense bodies. Choroideremia patients share visual defects with HPS patients and individuals with the Gray Platelet syndrome (GPS) have abnormalities in platelet vesicles called alpha granules rather than delta or dense granules.

Chediak–Higashi syndrome

As described in this issue [82], CHS (OMIM 214500) is characterized by variable hypopigmentation of skin, hair, and eyes, a bleeding diathesis, progressive neurological dysfunction, and severe immunological deficiency [82–84]. CHS cells show giant lysosomes, melanosomes, lytic granules, and azurophil granules [82,85,86]. Platelet dense granules are absent or reduced in number [87]. The gene responsible for the defect, LYST, encodes a protein of approximately 430 kDa [88,89], has no known function, and is localized to the cytoplasm. Expression studies of LYST suggested a role in membrane fusion/fission events [90] but the large size of LYST and its cytoplasmic location argue for a role in vesicular transport. The beige mouse is the murine counterpart of human CHS [88,91].

Griscelli syndrome

This syndrome provides an example of a composite disease which molecular genetics has retrieved from ‘catch-all’ status and separated into two distinct entities. In composite, GS is a rare autosomal recessive disorder (OMIM 214450) characterized by pigmentary dilution of the skin, silvery gray hair due to pigment clumping, severe neurologic impairment with or without immune abnormalities, and hemophagocytic syndrome, an uncontrolled T lymphocyte and macrophage activation syndrome leading to death in the absence of bone marrow transplantation [92,93].

Now that GS has been somewhat molecularly defined, it is recognized to consist of at least two different disorders. One occurs due to mutations in the myosin Va gene, myoVa, as determined by linkage to chromosome 15q21 [94] and resemblance to the dilute mouse, known to have myoVa mutations [95]. As a group, Class V myosins perform multiple functions within the cell. Myosin Va is an actin-binding motor that moves in large steps approximating the 36-nm pseudo-repeat of the actin filament [96]. It associates with the centrosome at all stages of the cell cycle, and melanocytes isolated from dilute lethal mice divide at half the rate of melanocytes isolated from wildtype mice [97]. Dilute has lightening of coat color, caused by an abnormal adendritic melanocyte morphology that results in an uneven release of pigment granules into the developing hair shaft. Most dilute mutations also produce a tendency toward convulsions and opisthotonus (rigidity) [98,99] and dilute transcripts are abundant in neurons of the central nervous system, cephalic ganglia, and spinal ganglia. The dilute gene product, myoxin, probably plays a role in the elaboration, maintenance or function of cellular processes of melanocytes and neurons [95].

Among humans with GS, one nonsense and one missense mutation in myoVa have been reported [94] but the missense was later shown to be a polymorphism [100]. The single Turkish patient with a mutation in myoVa exhibited primarily neurologic impairment, without immune defects [94].

A second type of GS was discovered because a large number of patients did not show a mutation in myoVa. Linkage analysis indicated the existence of a second locus for GS in the 15q21 region [101]. Physical mapping and mutation analysis identified the rab27a gene, less than 1.6 cM from myoVa, as responsible for GS in a large group of patients [92]. The rab27a gene encodes a 221-amino acid human protein which is expressed in most human tissues and cell lines [102]. Recombinant rab27a exhibits GTP-binding activity in vitro. Rab proteins function in the processes that underlie the targeting and fusion of transport vesicles with their appropriate acceptor membranes [71,103] Rab proteins require geranylgeranylation of the two consensus C-terminal cysteines of the protein [104]. Truncation of the carboxy terminal part of rab27a should thus leave it in an inactive state.

All GS patients with mutations in rab27A developed the hemophagocytic syndrome and rab27a-deficient T cells exhibited reduced cytotoxicity and cytolytic granule exocytosis required for immune homeostasis [92]. Rab27a mutations, like myoVa defects, lead to clustering of melanin pigment in the hair shaft [94] and defective melanosome transport in the melanocytes [93]. Rab27a is not expressed in brain tissue [102], in accordance with the absence of neurological features from the rab27a subset of GS patients. A direct interaction between a rab protein and a molecular motor protein has been described [105]. Although it has not been reported, interaction between the rab27A and myoVa gene products might very well occur, and might regulate melanosome transport.

The mouse ashen has mutated rab27a and represents the murine counterpart of the largest subset of GS patients [3]. In addition to pigment defects, ashen mice have reduced numbers of platelet dense granules [3], leading to prolonged bleeding times. This finding places rab27A within the melanocyte/platelet subfamily of rab proteins [102]. It also suggests that GS due to mutation of rab27A is really a type of HPS.

The three mouse models, dilute (myoVa), ashen (rab27a), and leaden (gene not yet determined), provide a unique system for studying vesicle transport in mammals. All three produce a lightened coat color because of defects in pigment granule transport [2]. All three mutations are also suppressed by the semidominant dilute-suppressor (dsu) [106], providing genetic evidence that these mutations function in the same or overlapping transport pathways. The characterization of the leaden mouse will aid in understanding the small differences among these related disorders.

Disorders related to GS

Two human syndromes resemble the myosin Va subtype of GS in having silvery hair and neurological disorders. These are neuroectodermal melanolysosomal disease or Elejalde syndrome (OMIM 256710) [107,108] and Cross syndrome or Kramer syndrome (OMIM 257800), also called oculocerebral syndrome with hypopigmentation [109]. Mutation analysis of the myoVa gene in these groups of patients would be extremely interesting.

The rab27A subtype of GS also resembles a disorder called partial albinism and immunodeficiency syndrome (PAID) (OMIM 604228) [110]. Gene mapping evidence suggested that PAID syndrome results from mutations at the myoVa locus on 15q21 and is thus allelic to GS [111]. However, since rab27A is localized to the same chromosomal locus, rab27A mutations might very well be the underlying cause of PAID syndrome.

Choroideremia

Choroideremia (CHM) (OMIM 303100) is an X-linked form of retinal degeneration that results from defects in the rab escort protein-1 (REP1) gene [112,113]. The protein REP1 assists in the attachment of geranylgeranyl groups to rab proteins, a modification that is essential for rab proteins to function as molecular switches regulating intracellular vesicular transport. Seabra et al. [114] observed a selectively unprenylated rab, rab27A in patients with choroideremia and speculated that the loss of rab27A function is the ultimate cause of choroideremia. However, in GS patients with mutations in rab27A, no retinal degeneration has been reported [92] and in choroideremia patients, the immunological problems typical of GS have not been recognized. In addition, recent research on the retina of ash mice revealed normal appearance [3]. Thus, another rab defect, caused by REP1 mutations, probably accounts for the pathophysiology of choroideremia.

Gray Platelet syndrome

GPS (OMIM139090), or α-storage pool deficiency, is a rare platelet disorder characterized by a deficiency in the number and content of alpha-granules. In this disorder, the platelets are large and contain few granules, giving them a gray appearance on light microscopy of Wright-stained blood smears [115,116]. GPS patients also show prolonged bleeding times and thrombocytopenia, so the mouse, gunmetal, could be the murine model for GPS. However, the gunmetal mouse contains a normal contingent of alpha granules. A rat model, the Wistar Furth (WF) rat, more closely resembles GPS [117,118]. The WF rat is characterized by hypopigmentation and hereditary macrothrombocytopenia, with both large mean platelet volume and deficiency of platelet alpha granule protein. This parallels GPS in humans. The WF rat demonstrates abnormal cytoskeletal protein function in megakaryocytes; this may contribute to the lack of alpha granules in the platelets of the WF rat [119]. The combined data suggest that the WF rat and the GPS human are not variants of HPS but closely related disorders.

Conclusions

Several general principles can be put forth concerning the various genetic disorders known collectively as HPS. First, the definition of HPS is arbitrary and there exists a good deal of clinical overlap with other eponymous syndromes. We currently require albinism and absent platelet dense bodies to make the diagnosis of HPS but mild decrements in expression of an HPS-causing gene could allow for the presence of dense bodies. Molecular identification of the genetic defects involved in HPS may prompt modification of our present categorizations, so it appears appropriate to consider our nomenclature to be tentative at present.

Second, a variety of different intracellular vesicle defects could cause HPS or related disorders but only a few have been identified. These can arguably be divided into disorders of vesicle formation, movement or fusion (Table 1), with variable degrees of certainty. We consider HPS-1 and pale ear to be disorders of vesicle formation because the expression of HPS1 protein occurs in the perinuclear, non-Golgi region of melanocytes [53]. HPS-2 should also involve vesicle formation, since AP3 mediates the creation of vesicles from the TGN. The same holds for mice and flies with AP3 subunit defects, as well as light, a Drosophila mutant of VPS41, whose gene product interacts with the δ subunit of AP3 [77]. The alpha granules of GPS may be absent because of a failure of their genesis from extant membranes but that is only speculation.

Table 1.  Functions and relationships of known human, mouse, and Drosophila genes causing HPS and related disordersa
Vesicle process involvedDisorder (Gene)
 HumanMouseDrosophila
  1. a Categorization is speculative. Disorders on the same line are homologous in the different species. “?” means the causative gene has not been isolated.

FormationHPS-1 (HPS1)Pale ear (HPS1)
 HPS-2 (ADTB3A)Pearl (AP-3β1)Ruby (AP-3β)
 Mocha (AP-3δ)Garnet (AP-3δ)
 Orange (AP-3σ)
 Carmine (AP-3μ)
 Light (VPS41)
 GPS (?)
    
MovementCHS (LYST)Beige (lyst)
 GS1 (myoVa)Dilute (myoVa)
 GS2 (rab27a)Ashen (rab27a)
 Leaden (?)
 PAID (?), Elejalde (?), Cross (?)
    
Tethering-Choroideremia (REP1)
Docking-Gunmetal (RGGTA)
FusionPallid (pallidin)
 Deep orange (VPS18)
 Carnation (VPS33)

The perinuclear distribution of giant lysosomes and melanosomes in CHS humans and beige mice suggests a failure of vesicle transport to the periphery, with proximal accumulation and secondary fusion. The GS also appears to involve vesicle transport abnormalities. GS1 lacks myosin Va, an actin binding motor responsible for peripheral movement of melanosomes. Rab27a, deficient in GS2, may well power this process. The murine counterparts of these diseases are dilute and ashen. Along with leaden, these models exhibit impaired transport of melanosomes to the dendritic periphery of melanocytes and their pigment phenotypes are suppressed by dilute suppressor. We place PAID, Elejalde, and Cross syndromes within the movement group because of their resemblance to the two GS disorders.

Choroideremia in humans and gunmetal in mice provide examples of putative tethering or docking defects. Both result in impaired geranylgeranylation of rab proteins, which then cannot anchor themselves in membranes to participate in vSNARE–tSNARE interactions. The mouse protein pallidin apparently interacts with syntaxin 13, supporting a role in vesicle docking. Mutations in the Drosophila VPS18 and VPS33 genes result in failure of directed fusion of multivesicular bodies with late endosomes, causing accumulation of giant intermediate vesicles [79].

The complete group of HPS-related disorders can also be categorized according to their importance for survival. One subset of HPS represents partial or leaky defects in essential genes; organisms with complete absence of the gene products are not viable. The other subset consists of genes that are not essential for survival. In this situation, the gene products are required for normal functioning of specialized organelles such as the melanosome and platelet dense body but they are not absolutely required for the function of generalized organelles such as lysosomes. In these generic vesicles, other, redundant proteins subsume the function of the defective proteins. This probably explains why the symptoms of choroideremia are limited to the eye, although the gene is expressed ubiquitously.

Finally, HPS and related disorders stand at the crossroads of cell biology and yeast, Drosophila, mouse, and human genetics. A journey down one thoroughfare yields a map to another sector and within any region a passel of pathways requires elucidation. Blocks in these pathways produce the human disorders we call trafficking defects.

Acknowledgement

Dr Yair Anikster is a Howard Hughes Medical Institute Physician Postdoctoral Fellow.

Footnotes

  • 1

    The OMIM, Online Mendelian Inheritance in Man, database is a catalogue of human genes and genetic disorders edited by Dr VA McKusick and developed for the world wide web by the National Center for Biotechnology Information, NCBI. It can be accessed at http://www.ncbi.nlm.nih.gov:80/omim/.

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