Hermansky-Pudlak Syndrome and Chediak-Higashi Syndrome ......Hermansky-Pudlak syndrome (HPS) and Chediak-Higashi syndrome (CHS), share several clinical characteristics and have piqued

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© 2001 Schattauer GmbH, Stuttgart Thromb Haemost 2001; 86: 233–45

Key words

Albinism, platelet dense bodies, giant lysosomes, LYST, intracellulartrafficking

Summary

The rare autosomal recessive metabolic disorders Hermanky-Pudlaksyndrome (HPS) and Chediak-Higashi syndrome (CHS) share the clini-cal findings of oculocutaneous albinism and a platelet storage pooldeficiency. In addition, HPS exhibits ceroid lipofuscinosis and CHSis characterized by infections and an accelerated phase. The two dis-orders result from defects in vesicles of lysosomal lineage. Of the twoknown HPS-causing genes, HPS1 has no recognizable function, whileADTB3A codes for a subunit of an adaptor complex responsible for new vesicle formation from the trans-Golgi network. Other HPS-causing genes are likely to exist. The only known CHS-causing gene,LYST, codes for a large protein of unknown function. In general, HPSappears to be a disorder of vesicle formation and CHS a defect invesicle trafficking. These diseases and their variants mirror a group ofmouse hypopigmentation mutants. The gene products involved willreveal how the melanosome, platelet dense body, and lysosome areformed and trafficked within cells.

Introduction

Hermansky-Pudlak syndrome (HPS) and Chediak-Higashi syndrome(CHS), share several clinical characteristics and have piqued the inter-est of cell biologists focusing on the genesis and movement of intra-cellular vesicles (1-6). In this review, we describe these two diseasesand demonstrate why they provide insight into cell biological proces-ses. In fact, we propose that HPS and CHS represent extremes of aspectrum of disease entities that correspond to discrete steps in thegenesis and movement of vesicles of lysosomal lineage.

Hermansky-Pudlak Syndrome

HPS, first reported in 1959 by two Czechoslovakian pathologists(7), consists of a collection of genetically distinct defects having com-mon clinical and laboratory findings (3, 4). All patients have somedegree of oculocutaneous albinism and platelet storage pool deficiency.

Many also demonstrate ceroid lipofuscinosis, although the presence ofthis intracellular lipid/protein complex is no longer necessary to makethe diagnosis of HPS. In our own studies, we have also arbitrarilyrequired that dense bodies be absent from platelets for the diagnosis ofHPS to be confirmed, although future clinical/molecular correlationsmay mandate modification of this requirement. What constitutes HPSwill be discussed below, after description of the disease as we currentlyknow it.

Clinical and Laboratory Findings

The clinical manifestations of HPS are variable (3, 4) because thephysiologic processes involved are modulated by a panoply of geneticand environmental influences, and because the genetic basis of HPS isitself heterogeneous. Despite these caveats, some generalizations canbe made regarding the signs and symptoms of HPS.

Oculocutaneous albinism. In HPS, tyrosinase activity remainspresent within melanocytes (2), but melanosome function is impaired,and pigment dilution results. This affects skin, hair, and eye color, butto variable extents. Hair color ranges from nearly normal to completelywhite (Fig. 1A, B). Even among the approximately 400 Puerto Ricanpatients with exactly the same mutation in the HPS1 gene (see below),the spectrum of hair color varies enormously (8). Typically, the hair isdirty blond or tan. HPS skin also varies from pale to brown, and oftenmust be compared with that of an unaffected sibling to verify hypo-pigmentation. HPS patients are susceptible to solar damage, includingactinic keratoses, nevi, and skin malignancies (9).

Ophthalmic involvement in HPS patients (10-12) includes increasedcrossing over of nerve fibers at the optic chiasm, typical of albinism.Iris transillumination (Fig. 1C, D) occurs because cells in the iris havereduced amounts of pigment. Loss of retinal pigment epithelium causesa pale fundus and decreased visual acuity, usually 20/200 or worse(legally blind), but sometimes as high as 20/50 (8, 12). Vision in HPSis stable, uncorrectable, and accompanied by variable degrees of con-genital nystagmus.

Hematologic findings. In HPS, platelet counts are normal or ele-vated, but the platelets are dysfunctional because they lack dense bodies (Fig. 1E, F). These electron dense intracellular organelles, whichcontain ADP, ATP, serotonin, calcium, and polyphosphates (13), disgorge their contents upon activation and promote clot formation.Absence of dense bodies attenuates the secondary aggregation res-ponse and can prolong the bleeding time.

HPS patients exhibit findings typical for a platelet storage pool defi-ciency, i. e., bleeding of mucosal membranes and spontaneous softtissue bruising. Major bleeds into the joints or brain are not seen, andcoagulation factors, prothrombin times, and partial thromboplastintimes are normal (1, 8). Excess bruising usually begins at ambulation,

Correspondence to: William A. Gahl, MD, PhD, 10 Center Drive,MSC 1830, Building 10, Room 9S-241, NICHD, NIH, Bethesda, Maryland20892-1830, USA – Tel.: 301-402-2739; Fax: 301-402-2740; E-mail:[email protected]

Hermansky-Pudlak Syndrome and Chediak-HigashiSyndrome: Disorders of Vesicle Formation and Trafficking

Marjan Huizing, Yair Anikster, William A. Gahl

Section on Human Biochemical Genetics, Heritable Disorders Branch,National Institute of Child Health and Human Development,

National Institutes of Health, Bethesda, Maryland, USA

For personal or educational use only. No other uses without permission. All rights reserved.Downloaded from www.thrombosis-online.com on 2018-05-18 | ID: 1001066444 | IP: 54.70.40.11

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and epistaxis begins in childhood. Bleeding episodes can occur duringdental extractions, surgeries, acute colitis, menstrual periods, and childbirth. Fatal bleeds are rare but a significant number of patients have re-ceived transfusions of packed red cells or platelets.

Interestingly, von Willebrand factor, which is stored within plateletalpha granules, is decreased in many HPS patients (14, 15). Lympho-cyte and neutrophil function appear normal in HPS (16).

Pulmonary fibrosis. Many HPS patients suffer a progressive restric-tive lung disease beginning in their late twenties or early thirties (1, 8,17, 18), perhaps related to accumulation of ceroid in HPS alveolarmacrophages (Fig. 1G, H). Affected patients typically exhibit extensivefibrosis by the end of the fourth or fifth decade of life (Fig. 1I, J).Infection, smoking or exposure to pulmonary toxins can lead to deathwithin two or three years. The pulmonary involvement of HPS variesconsiderably, and occasional patients are over 60 years old with normallung function. Some subgroups of HPS, specifically, patients bearingmutations in the HPS1 gene (see below), are more susceptible to pul-monary fibrosis than others (8, 18). Pulmonary fibrosis has been notedin HPS patients from Czechoslovakia, Japan, Belgium, and England(3), prior to the availability of molecular genotyping.

Granulomatous colitis. This complication affects at least 15% of allHPS patients, whether or not they are of Puerto Rican heritage (8).Although the distal colon is most often involved, the entire gastro-intestinal tract may be affected, with granulomatous gingivitis occuringin rare cases (1). Patients present with blood and mucus in their stools,and the colitis resembles Crohn’s disease histologically (19, 20).

Ceroid lipofuscin. This electron dense, autofluorescent aging pig-ment is increased in some HPS patients, who accumulate it within thekidneys, urinary sediment, lung alveolar macrophages, bone marrow,spleen, liver, and large intestine (1). Smaller amounts can be found inthe heart, lymph nodes and other tissues (1). The ceroid lipofuscin ofHPS appears to reside within lysosomes. Some theories blame ceroidlipofuscin accumulation for the pulmonary fibrosis, granulomatouscolitis, and occasional renal impairment (1, 8) and cardiomyopathy (1)of HPS. However, no convincing evidence has been put forth to supportthis.

Subtypes of HPS

Two patients have been reported with mutations in the ADTB3Agene (21, 22) (see below), and we have recently identified a third.All three had persistent neutropenia and childhood infections (22).Balance problems in the two reported cases may be related to decreasedvisual acuity. In other respects, the clinical findings are typical for HPS,i. e., moderate visual impairment, nystagmus, hypopigmentation, andabsence of platelet dense bodies.

Another subtype of HPS exists in central Puerto Rico, where affect-ed individuals have absent dense bodies but mild hypopigmentationand visual acuity deficits. The HPS1 gene has been eliminated as acause for the disease in this group of patients (23). As other HPS-causing genes are identified, additional clinical subtypes should emerge.

Diagnosis

The diagnosis of HPS relies on the clinical suspicion of the physician.Dirty blond hair color, excessive bruising in toddlers, and epistaxis inmiddle childhoood provide hints, but bleeding times may remain nor-mal. Nevertheless, virtually every patient has some degree of horizon-tal nystagmus present from birth (12), and every patient has a visualacuity of 20/50 or poorer.

Fig. 1 Clinical Findings in HPS. A. Typical light brown/dirty brown haircolor in a 19-year old Puerto Rican patient. B. Extreme lack of hair pigmentin a 9-year old Puerto Rican boy with HPS. C. Mild iris transillumination ina 4-year old girl from central Puerto Rico with no mutation in HPS1. Note resid-ual, scattered pigment. D. Nearly complete iris transillumination in a 40-yearold patient from northwest Puerto Rico with the classic 16-bp duplicationin HPS1. E. Wet mount electron microscopy showing normal platelet densebodies in an unaffected individual. F. Platelet of an HPS patient showing absentdense bodies. G. Normal pulmonary alveolar macrophage. H. Alveolar macro-phage from a 34-year old northwest Puerto Rican patient showing engorgedvesicles typical of ceroid lipofuscinosis. I. Normal chest CT scan of a 53-yearold Puerto Rican HPS patient with normal pulmonary function. J. Chest CTscan from a 38-year old Puerto Rican HPS patient with severe pulmonaryfibrosis. (C, D courtesy of Dr. M. I. Kaiser-Kupfer and Mr. E. Kuehl, Natio-nal Eye Institute, NIH; E, F courtesy of Dr. James White, University of Minne-sota)


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Huizing: Hermansky-Pudlak and Chediak-Higashi Syndromes

The sine qua non of the HPS diagnosis remains demonstration ofabsent platelet dense bodies using the method of wet-mount electronmicroscopy (24). This procedure is currently performed by Dr. JamesWhite of the University of Minnesota. Studies showing reduced orabsent serotonin uptake or an absent secondary aggregation responsecan also be helpful.

Many physicians, recognizing that HPS occurs with increasedfrequency among Puerto Ricans, mistakenly discount the diagnosiswhen caring for non-Puerto Rican patients. One HPS patient carriedthe diagnosis of isolated ocular albinism until her mid-twenties, whenher excessive bleeding during menstrual periods prompted an examina-tion for platelet dense bodies, which were absent by electron microscopy.We recommend consideration of platelet dense body analysis in albinopatients without a molecular basis for their disorder.

Treatment

For HPS patients, sun avoidance and sunscreens are critical to pre-vent solar damage, and skin cancer surveillance can detect early malig-nancies (9). For the bleeding diathesis of HPS, thrombin and gelfoamcan be used for minor wounds, and some, but not all, patients respondto 1-desamino-8-D-arginine vasopressin (DDAVP), 0.2 �g/kg in 50 mlof normal saline given intravenously over 30 minutes (25). Platelets can be either on hand or transfused prior to major procedures. The mostcrucial issue, however, is recognition of the functional platelet defect,which cannot be appreciated from a simple platelet count. Hence, theuse of a medical alert bracelet can be extemely helpful.

Only supportive care is available for the pulmonary fibrosis of HPS, al-though a double-blind, placebo-controlled trial of the investigational anti-inflammatory drug, pirfenidone, is currently underway. Lung transplan-tation, although never performed on an HPS patient, should be feasiblewith close attention to coagulation, and a transplanted lung should last thenatural lifetime of a patient. The granulomatous colitis of HPS generallyresponds to steroids and other treatments appropriate for Crohn’s disease.

Genetics

The population frequency of HPS in northwest Puerto Rico is 1in 1800, with a gene frequency of 1 in 21 (26). All 400 affected individ-uals in northwest Puerto Rico are homozygous for a specific mutation,reflecting a founder effect. However, HPS has been reported through-out the globe (1), with different mutations and different genes involved(27-30). Of the approximately 60 non-Puerto Rican HPS patientsexamined molecularly, only approximately 25-40% have their diseasedue to mutations in a known gene (28, 29). In addition to the knownHPS-causing genes HPS1 and ADTB3A, there is certainly anothergene causing the disease in central Puerto Rico (23). The many animalmodels of HPS (31) also indicate locus heterogeneity. All subtypes ofHPS appear to be inherited in an autosomal recessive fashion (1).

The HPS1 gene. The first HPS-causing gene was mapped to chromo-some 10q23.1-23.3 in 1995 using Puerto Rican and Swiss families (32).Subsequently, the gene (GenBank accession #1654350), called HPSand later HPS1, was sequenced and shown to contain an open readingframe of 2100 bp (27). The genomic structure was then determined (33).HPS1 consists of 20 exons spanning approximately 30.5 kb. Itsstandard transcript, 3.0 kb in size, is expressed in most tissues (27).Minor 3.9-kb, 4.4-kb, and 1.5-kb mRNAs also occur (34). Four alter-native splices of HPS1 have been described (27, 33). A pseudogeneof HPS1 (GenBank accession #6707403) exists on chromosome22q12.2-12.3 and contains complete exons 3, 4, and 6 (35).

The 16-bp duplication in exon 15 of HPS1 is present in the homo-zygous state in all northwest Puerto Rican patients but not in any otherpatient group (27-29). This frameshift mutation produces no mRNA,and is easily detected by PCR amplification (27). Other mutations inHPS1 include T322insC and T322delC, indicating that the region ofcodons 321-324 represents a mutation hot spot (28). Codon 396 maybe another area subject to recurrent mutation, since S396delC hasappeared in several other patients (28, 29). In all, 12 different mutations,including deletions, insertions, nonsense mutations, and splice junctionmutations, have been reported for HPS1 (3, 5, 30).

Most of the HPS1 gene mutations, as well as the frameshift mutationof the mouse homologue of HPS1, pale ear (36, 37) (see below), arepredicted to result in a protein with a truncated carboxy terminus and,presumably, no residual function. This points to the critical nature ofthe terminal portion of the HPS1 protein. To date, no missense mutationsin HPS1 have been found, although at least 24 nonpathologic DNAsequence polymorphisms have been reported (29, 30, 33).

The ADTB3A gene. ADTB3A (GenBank accession #1923267),whose coding sequence has 3281 bp, produces a 4.2-kb mRNA presentin all tissues and cell lines examined (38). The product is �3A, a subunitof adaptor complex-3 (AP3), which functions to form vesicles fromexisting membranes such as the trans-Golgi network, or TGN (39). Theonly reported cases of mutations in ADTB3A are those found in thecompound heterozygous state in two brothers affected with HPS (22).The mutations are a 21-amino acid deletion (∆390-410) and a singleamino acid substitution, L580R (21). Mutations in the pearl mouse, whichis the murine equivalent of the humans with ADTB3A mutations, inclu-de a 793-bp internal tandem duplication and a 107-bp deletion (40).

Cell Biology

HPS has long been considered a defect of vesicular membranes, inpart because the intracellular compartments causing the clinical mani-festations of the disease share integral membrane proteins. Specifically,the melanosome’s ME491 protein is the same as the platelet’s CD63or granulophysin which is the same as the lysosome’s LAMP-3 orLIMP-1 (3). In addition, two lysosomal membrane proteins, LAMP-1and LAMP-2, are also found on platelet dense body membranes (41).The hypothesis that HPS results from dysfunctional membrane traf-ficking has been strongly supported by the finding that AP3 deficiencycan cause HPS, and by the fact that several genes responsible for HPSin murine and Drosophila models are involved in vesicle formation andtrafficking (see below).

The HPS1 protein. The HPS1 gene product has 700 amino acids anda predicted molecular weight of 79.3 kDa (27), with no apparent glyco-sylation (42) and no homology to proteins with a known function.It does contain the sequence DKF(L/V)KNRG, which resembles aregion of the CHS protein (27, 43) (see below), and the carboxy termi-nus of the HPS1 protein contains a putative melanosomal localizationsignal, PLL (44). The amino acid sequence of the HPS1 protein is 81%conserved between human and mouse (36), with similar conservationbetween human and rat (5). A predicted Drosophila protein of un-known function has high sequence identity to the carboxy terminal portion of HPS1, but no homologues exist in lower species.

A role for the HPS1 protein in vesicle formation is suggested by theHis-Leu-Leu sequence near its carboxy teminus. This recognition mar-ker could serve as a sorting signal to target the protein to compartmentsof lysosomal lineage, such as the melanosome and dense granule(45, 46), although the HPS1 protein does not appear to be directly asso-ciated with lysosomes (42).


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The HPS1 protein is a component of two high molecular weightcomplexes, a cytosolic complex of approximately 200 kDa in nonmela-notic cells and a larger, 500-kDa complex in melanotic cells (42). Thelarge complex is perinuclear and associated with tubulovesicular struc-tures, small noncoated vesicles, and nascent and early-stage melanoso-mes but not with later stage melanosomes (42). Other studies place theHPS1 protein in the perinuclear region of normal melanocytes, possiblyassociated with a cisternal network outside of the Golgi zone (47). Thissuggests that the protein is a part of the premelanosome as it forms fromthe smooth endoplasmic reticulum.

The HPS1 gene product appears to influence trafficking of melano-cyte-specific proteins from the TGN to preformed premelanosomes(48). Specifically, the pigment-forming proteins TRP-1 (for tyrosinaserelated protein-1) and granulophysin displayed a large granular patternof expression in HPS-1 melanocytes, consistent with localization tolarge membrane complexes visible ultrastructurally in the mutant cells(48). The appropriate location for these proteins would be within mela-nosomes.

The HPS1 protein is present on normal platelets but not on plateletsfrom patients with null mutations in HPS1. Other studies have shownthat fibroblasts do not require the HPS1 protein to grow, and that HPS1-deficient cells display a normal distribution and trafficking of the lyso-somal membrane proteins CD63 and LAMP-1. HPS1 does not appearto interact with AP3 (46).

The �3A subunit of AP3. The �3A protein is a subunit of AP3, oneof four known adaptor complexes (AP1, AP2, AP3, and AP4).Adaptors are components of cytosolic protein coats that mediate vesicleformation and incorporation of cargo proteins into the nascent vesicle’smembranes (39). AP3 apparently produces vesicles from the TGNand/or endosomes. It interacts with clathrin (49), which provides struc-ture to the newly formed vesicle. AP3 consists of a 160 kDA � subunit,a 47-kDA � subunit, a 23-kDa � subunit, and the 140 kDa �3A subunit(38, 50-52). The �3A subunit is phosphorylated on serine residues (38).The � subunit of AP3 recognizes tyrosine-based (50, 53) and dileucine-based (54) sorting signals of cargo proteins targeted for incorporationinto the membranes of new vesicles.

AP3-deficient fibroblasts display enhanced trafficking of lysosomalmembrane proteins through the plasma membrane (21), a defaultpathway employed when direct movement from the TGN to the endo-some is impaired. Similar findings were reported for fibroblasts of thepearl mouse bearing ADTB3A mutations (40) and the mocha mousecarrying mutations in the � subunit of AP3 (55). However, not allintegral membrane proteins use AP3 for targeting to lysosome-relatedorganelles. Trafficking of MHC class II molecules and the associatedinvariant chains to their intracellular compartments appeared normal inAP3-deficient cells (56).

In �3A-deficient human melanocytes, TRP-1 localization is normal,but tyrosinase expression is restricted to the perinuclear region (Fig. 2)and localized in large vesicles resembling late endosomes (57). More-over, the abnormal tyrosinase distribution is corrected by transfectionwith the normal �3A gene, supporting the concept that tyrosinase traf-ficking is regulated by AP3 (57).

Fibroblasts from patients with ADTB3A mutations show reduction ofthe �, �, and � subunits of AP3 as well; apparently the �3A subunitstabilizes the entire AP3 complex against degradation (21).

Models of Pigment Dilution in Other Organisms

The earliest and closest models for HPS consist of inbred micehaving variable degrees of pigment dilution combined with a plateletstorage pool deficiency (31) (Table 1). This group of mutants actuallyrepresents a spectrum of disease ranging from HPS to CHS and reflect-ing various human conditions (see below). Several of the model miceexhibit increased urinary excretion of lysosomal enzymes. All the HPSmice inherit their disease in an autosomal recessive fashion.

Six mouse models have had their causative genes isolated. Two ofthese, pale ear and pearl, represent the murine counterparts of humanswith mutations in HPS1 and ADTB3A, respectively. In fact, the humanADTB3A mutations were discovered because the murine pearl genewas found to code for the �3A subunit of AP3 (40). The mocha geneencodes the � subunit of AP3 (55) and pallid encodes a 25-kDa protein,pallidin (58), that interacts with syntaxin 13, part of the membrane

Table 1 Murine models of hypopigmentation due to impaired vesicle formation or trafficking

Chromosome HumanModel Mouse Human Gene Disease Comment

1. pale ear 19 10q23.1-23.3 HPS1 HPS-1 Small eumelanin granules in hair2. pearl 13 5q11-14 ADTB3A HPS-2 Night blindness in mouse; neutropenia in human3. mocha 10 19p13.3 AP3D1 – Small melanosomes in hair, hyperactivity, balance problem4. pallid 2 15q11.2-22.3 pallidin – Emphysema, balance defect, enhanced response to morphine5. gunmetal 14 14q11.2 RABGGTA – Both � and � granule defect, thrombocytopenia, large platelets6. cappuccino 5 4p15-16 – – Elevated liver lysosomal enzymes7. cocoa 3 8q13-22 – – Normal lysosomal function8. subtle grey 3 3q24-28 – – No lysosomal dysfunction, mild bleeding/pigment dilution9. light ear 5 4p16 – – Few, large melanin granules in choroid, small in hair

10. reduced 7 19q13 – – Increased anesthetic risk, small pigment granules in hairpigment

11. ruby eye 19 10q24.1-25.1 – – Reduced number of melanocytes12. ruby eye-2 7 11p15/15q11 – – –13. muted 13 6p21-23/5p15 – – Balance defect, ears lack otoliths14. sandy 13 6p21-23/5p15 – – –15. ashen 9 15q21 rab27a Griscelli Large pigment clumps on hairshafts, hemophagocytic syndrome

syndrome II16. dilute 9 15q21 myo5a Griscelli Neurologic involvement

syndrome I17. leaden 1 – – – Large pigment clumps on hairshafts18. beige 13 1q42.1-42.2 lyst CHS Giant intracellular granules


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fusion machinery (59). The gunmetal gene codes for the � subunit ofrab geranylgeranyltransferase, an enzyme that adds 20-carbon prenylgroups to cysteine residues on the carboxy termini of rab proteins tomake them membrane-bound (60). Rab proteins are key to vesicletransport and organelle dynamics (61), and the gunmetal mutationresults in decreased prenylation and decreased membrane associationof rab27a (60). The ashen mouse, which recently became a model ofHPS based upon the finding of absent platelet dense bodies, codes forrab27a itself (62). Mutations in rab27a result in Griscelli syndrome(63), which falls within the HPS-CHS spectrum of disorders (64)(see below).

There also exist at least 11 Drosophila melanogaster models of HPSand related disorders. These flies have mutations at eye color loci andare members of the “granule group” of mutants (65). The 7 granulegroup genes isolated to date are identical to genes involved in lyso-somal trafficking in other organisms. For example, all four subunits ofAP3 have a Drosophila mutant model. The garnet gene produces a �subunit of AP3 (52, 66), while ruby codes for a �3 subunit (67), car-mine codes for �3 (68), and orange for �3 (69). Three other genes of thegranule group encode Drosophila homologues of the yeast VPS(Vesicle Proteins for Sorting) genes. The light fly has mutations inVPS41, whose protein product interacts with the � subunit of AP3 (70).Deep orange and carnation have mutations in VPS18 and VPS33,respectively (71, 72). The products of these genes interact in a largecomplex, and mutations in deep orange cause accumulation of multi-vesicular bodies (72).

In yeast, the VPS18 and VPS33 proteins form a multisubunit com-plex with VPS11 and VPS16 (73). Moreover, VPS33 interacts withthe t-SNARE VAM3, indicating that these proteins might act togetherto direct docking and/or fusion of transport intermediates with the yeastvacuole (74). VAM3 is transported via an AP3-dependent sorting

pathway (75). There are more than 40 different yeast VPS genes whichinfluence trafficking of proteins destined for the lysosome-like vacuole(76). Mutants in any of these genes could be primitive models forhuman HPS.

Chediak-Higashi Syndrome

CHS (1, 2, 77, 78) was first described by Beguez-Cesar in 1943 (79),but acquired its eponym a decade later from Moises Chediak (80) andOtotaka Higashi (81). It is characterized by an infectious diathesis,giant intracellular granules, variable degrees of oculocutaneousalbinism, and a platelet storage pool deficiency. The infections aregenerally fatal in the first decade of life, but CHS patients can alsosuccumb to a chronic lymphohistiocytic infiltration known as the accel-erated phase during the second or third decades of life.

Clinical and Laboratory Findings

As for HPS, the clinical findings in CHS are variable but includeseveral characteristic manifestations. Heterozygotes for CHS are al-ways completely normal.

Oculocutaneous albinism. Hair color in CHS patients can be blondto light brown, but the truly characteristic color is metallic silver. Gianthypomelanized melanosomes in CHS melanocytes cannot be trans-ferred to surrounding keratinocytes (82), resulting in skin color thatvaries from white to gray. Sun-exposed areas may pigment, and neviand lentigines do arise with some frequency. The eyes of CHS patientshave reduced pigment in the retina, iris, choroid and ciliary epithelium.On electron microscopy, giant aggregates of melanin appear in themelanosomes of these tissues (83). Irides can be gray, blue, or evenbrown. Patients can also have nystagmus, photophobia, and reduced

Fig. 2 Distribution of �3A, LAMP-3, TRP-1 (tyro-sinase-related protein-1) and tyrosinase in normal andHPS-2 melanocytes, fixed in formaldehyde and stainedwith monoclonal antibodies. A. In both normal andHPS-2 melanocytes, LAMP-3 (green) showed a similarmelanosomal distribution pattern. In normal melano-cytes, �3A (red) was expressed throughout the cell, par-tially colocalizing with LAMP-3. In HPS-2 melano-cytes, the �3A signal was dramatically decreased, asexpected. B. In normal melanocytes, TRP-1 (green) andtyrosinase (red) exhibited a similar distribution through-out the cells. In HPS-2 cells, TRP-1 had a normal distri-bution, but tyrosinase was localized mainly in theperinuclear area and was absent from the cell periphery.This finding indicates that tyrosinase, but not TRP-1,is carried by an AP3-dependent pathway


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visual acuity. Electroretinograms and visual evoked potentials showprogressive abnormalities (84).

Bleeding. CHS patients have the same diathesis toward easy bruis-ability, mucosal bleeding, and epistaxis that HPS patients exhibit, butsome CHS patients have reduced or irregular platelet dense bodiesrather than total absence of these intracellular organelles. CHS plateletsdo have low levels of ATP, ADP, calcium, and serotonin, and secreteddense body constituents (i. e., nucleotides and calcium) are lacking aswell (85). Aggregation studies performed on CHS patients (85, 86)reveal an absent or minimal second wave of aggregation followingstimulation with epinephrine or other agonists. Alpha granules havebeen normal in number and morphology in CHS patients.

Infections. Children with CHS suffer from recurrent infections be-ginning in infancy. Typical organs affected include the skin andrespiratory systems. Periorbital cellulitis, otitis media, pneumonias,pyoderma, abscesses, sinus infections, and dental caries are prominenttypes of infections, and Staphylococcus aureus and �-hemolyticStreptococcus are the primary organisms, although gram negative orga-nisms, Candida, and Aspergillus are also important (87). Response toantibiotics is slower than expected.

Although the bone marrow appears normal to hypercellular in CHS,neutropenia is common. CHS neutrophils, as well as lymphocytes,eosinophils, and platelets, contain giant granules, up to 4 �m in diam-eter (Fig. 3). The granules contain acid hydrolases, myeloperoxidase,and other proteins necessary for bactericidal activity (88), and existalongside normal-sized primary lysosomes (89). The giant granulesimpair leucocyte migration, perhaps by inhibiting cell deformability.Phagocytosis appears normal in CHS cells (90), with intracellularbactericidal acivity delayed but improving to near normal over time.This suggests that the primary defect in bacterial killing resides in theearly stage of the process, while later functions remain intact. In CHSpatients, natural killer (NK) cells are present in normal or slightlyelevated numbers, but their function is impaired (91), contributing tothe susceptibility to defective cellular immunity and to development ofthe accelerated phase.

A number of parameters are normal in CHS, including immuno-globulins, antibody production, delayed hypersensitivity, reticuloendo-thelial clearance, monocyte number and phagocytic function (90),levels of complement (92), suppressor cells, response to interferon,target cell recognition, and antibody-dependent, cell-mediated cyto-lysis (93, 94).

Accelerated phase. Approximately 85% of CHS patients experiencethe accelerated phase sometime between a few months and severalyears of age. The accelerated phase involves fever, anemia, neutro-penia, and occasionally thrombocytopenia, hepatosplenomegaly,lymphadenopathy, and jaundice (87). Liver function tests are elevated,cellular immunity is decreased, and pancytopenia presents due tosplenomegaly and hemolysis. A coagulopathy may also develop due toliver dysfunction and thrombocytopenia (87). The accelerated phaserepresents a reactive process with perivascular lymphohistiocytic in-filtrates that are benign histologically (87, 95, 96) and resemble infec-tious mononucleosis (95). Lack of NK cell function has been theorizedto allow the accelerated phase to become fulminant.

Neurologic manifestations. CHS patients can experience a peri-pheral or cranial neuropathy, autonomic dysfunction, weakness andsensory deficits, loss of deep tendon reflexes, clumsiness, a wide-basedgait, seizures, abnormal electromyograms, and decreased motor nerveconduction velocites or spinocerebellar degeneration (77, 97). Onpathological examination of four CHS patients, lymphohistiocytic infil-trates were evident throughout the nervous system (98). Cytoplasmic

inclusions, resembling lysosomes or large, irregular lipofuscin granu-les, were present in astrocytes, choroid plexus epithelial cells, Schwanncells, and satellite cells of the dorsal spinal ganglia.

Diagnosis

The hallmark of CHS is the presence of giant peroxidase-positivegranules in polymorphonuclear leucocytes, megakaryocytes, neurons,conjunctival fibroblasts, and cultured lymphoblasts (1, 2, 77, 78). Otherclinical characteristics, such as mild oculocutaneous albinism, silveryhair, bleeding problems, provide clues to seek a definitive diagnosis.Prenatal diagnosis has been achieved by examination for large, acidphosphatase-positive lysosomes in amniocytes and chorionic villuscells (99). The molecular diagnosis of CHS remains difficult because ofthe large size of the LYST gene and because several different mutationshave been identified. Molecular diagnosis is not commercially available.

Treatment

The only curative therapy for CHS is bone marrow transplantation,which has also reversed the leucocyte defect in the mouse model ofCHS, beige (100). Seven of 10 children from two separate institutionswho received related donor bone marrows had a successful transplanta-tion (101). None of the children had recurrence of the acceleratedphase, and all had improved NK cell activity. However, this therapydoes not prevent the progressive neuropathy of CHS.

Other therapeutic modalities for CHS are symptomatic (77). Child-hood immunizations are well-tolerated and should be provided (87).Antibiotics will treat the infections, although with a slow response.Aspirin-containing products should be avoided. Desmopressin and�-amino-caproic acid can be effective for treatment and prophylaxis,while platelet transfusions may be needed for major procedures orbleeds. Corticosteroids, chemotherapeutics, intravenous immunoglobu-lin, and splenectomy may occasionally induce a temporary remission ofthe accelerated phase. Ascorbic acid treatment has not proven clinicallyeffective in preventing either infections or the accelerated phase in CHSpatients (102, 103).

Genetics

CHS is an extremely rare, autosomal recessively inherited disorderthat has been found in countries all over the world. Although more than200 cases have been reported over time (1, 77), many fewer are likelyto be alive today.

Unlike HPS, CHS does not display locus heterogeneity. However, itremains possible that some CHS patients, particularly clinical variants,might have their disease due to mutations in genes not yet discovered.The clinical variability observed in the animal models of CHS, i. e., theAleutian mink, cattle, cats, killer whales, and rats (77), supportsthe possibility of locus heterogeneity. Presently, the one gene provento cause CHS is LYST, which was discovered with the assistance ofextensive investigations into the best animal model of CHS, the beigemouse (77).

The LYST gene. The lyst gene in the beige mouse, on chromo-some 13, is homologous to the human LYST (for lysosome traffickingregulator), on chromosome 1q42.1-42.2 (104-106). LYST (107-109) has a coding region of 11,403 bp and is expressed in all cell types.The 13449-bp mRNA (GenBank accession #4502838) is present inlow levels and is difficult to detect by Northern blot analysis. A large,11.4 kb transcript appears to be responsible for gene function, while the


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function of a smaller, 5.8 kb transcript derived from the 5’ terminus ofLYST is unknown (107, 108). The human LYST gene has homologues inall mammalian species (109). The human and mouse proteins are 82%identical and 88% homologous. High degrees of identity are also seenbetween the human and the rat and cow CHS genes.

To date, LYST mutations have been identified in 13 CHS patients(107-110). All human mutations, as well as those in the mouse (111)and rat (112) beige models, have been nonsense or frameshift mutationswith premature termination. There is no obvious “hot spot”, and nocorrelation between severity of disease and residual length of the trun-cated LYST protein.

The LYST protein. The LYST gene encodes a large cytosolic proteinwith 3801 amino acids and a molecular mass of 429 kDa (109). TheLYST protein contains several phosphorylation sites and myristylationsites, indicating a capacity for regulation and interaction with otherproteins or membranes. In addition, the amino-terminus of the LYSTprotein contains a series of hydrophobic helices resembling HEAT andARM domains (109). HEAT repeat proteins are involved in vesicletransport, and ARM domains are thought to mediate membrane asso-ciations (113). The N-terminus of LYST also shows low homologyto stathmin, a phosphoprotein involved in regulation of microtubulepolymerization (109, 114).

The central region of LYST contains two leucine zippers and an8-amino-acid domain homologous to a sequence in the HPS1 protein(27). HPS1 and LYST may interact through this homologous domain,possibly via another linker protein (115, 116).

The carboxy-terminus of LYST contains seven consecutive WD40motifs that form beta sheets (109). WD40 motifs are considered tomediate protein-protein interactions (117). Another motif in the C-terminus, conserved among LYST homologues in other species, is a345-amino-acid domain called BEACH, for “beige” and “CHS” (109).The BEACH domain contains a consensus “WIDL” amino acid stretchas well as other conserved amino acids. The combination of BEACH

and WD-40 domains are found in several mammalian proteins includ-ing FAN, CDC4L, and neurobeachin (78, 118). FAN links TNF� toneutral sphingomyelinase (119), and neurobeachin anchors proteinkinase A in the trafficking of neuronal post-Golgi membranes (118).The function of CDC4L (120) is unknown. The exact function of LYSTcannot be predicted from homology searches, but these structuralstudies do suggest that it functions in vesicule trafficking.

CHS Cell Biology

Mutations in LYST result in giant granular inclusions (78, 88) inmany different cell types, including platelets (Fig. 3), which are alsodeficient in dense bodies (121). To understand why giant granules formand some normal vesicles do not, several aspects of CHS cell biologymust be addressed.

Endosomal transport in CHS. Secretory lysosomes are synthesizednormally in CHS cells (122), and both early (recycling) and late (multi-vesicular) endosomes display normal morphology and accessibility toendocytic tracers (123). However, some downstream functions areimpaired. For example, peptide loading and antigen presentation, partsof the immunologic process, are delayed in CHS B cells (123), andmelanosome, secretory granule, and lysosome release are functionallyimpaired. Other endocytic functions, such as protein degradation andrecycling of transferrin receptors, are not affected.

The characteristic macrolysosomes of CHS cells are acidic, acquireendocytic tracers such as bovine serum albumin-conjugated colloidalgold with normal kinetics (123, 124), and occasionally fuse with theplasma membrane. The lysosomal membrane markers HLA-DM,LAMP-1, LAMP-2, CD63, CD82, and �-hexosaminidase all accumu-late in the macrolysosomes, as expected, but these proteins also accu-mulate at the cell surface in CHS cells (123, 125). The mannose-6-phosphate receptor (MPR), normally excluded from lysosomes, is veryabundant in the macrolysosomes in CHS. In contrast, late endosomes

Fig. 3 Giant granules (arrows) in CHS cells. A. Neu-trophil with several giant lysosomes. (�3300) B. Lym-phocyte with a single giant granule. (�3300) C. Eosino-phil with several large granules of different electrondensity. (�2600). D. Platelet showing many normalsized lysosomes and one giant lysosome. (�5500).All micrographs courtesy of Dr. James White, Univer-sity of Minnesota


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Fig. 4 Hypothetical model of LYST protein function in protein sorting and vesicle formation and trafficking. A. Schematic drawing of the motifs found in theLYST protein. B. Late Golgi processes. Proteins destined to be transported to lysosomes bind to receptor molecules in the trans-Golgi membrane (1). This resultsin activation of the “VPS15 like” C-terminal portion of LYST (2). VPS15 activation provides a binding site for the VPS34-like PI3K (3),which then translocatesto the membrane (4). Since the PI3K can be replaced by another specific PI-kinases in this LYST-mediated process, it is termed PI(x)K. Inset: PI(x)K generatesPtdIns(x)Pn (phosphatidylinositol [x]-phosphate) from PtdIns and free phosphate. PtdIns(x)Pn is a source for the supply of DAG (diacylglycerol) to the Golgi com-plex. DAG is formed by phospholipase C, and activates PKC. C. Early vesicle formation. The C-terminus of LYST activates the specific local phosphorylation ofmembrane phospholipids, resulting in recruitment of coat proteins (5) and specific guanine nucleotide exchange factors (GEF) required for vesicle formation andtransport. The GEFs stimulate activation of members of the rab family of small GTPases (6). Meanwhile, the N-terminal portion of LYST connects to microtubu-le structures (7). D. Early vesicle movement. The N-terminal portion of LYST “pulls” the vesicle out of the late Golgi membrane towards the microtubule struc-tures (8). Once the vesicle is formed and starts moving along the microtubule, the LYST protein is released from the vesicle (9), probably recycling to the late Golgi for a new round of vesicle formation


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contain a paucity of the normally abundant membrane markers HLA-DR,CD63, and CD82 (116).

Faigle et al. (116) concluded that defective lysosomal fusion withthe plasma membrane is not the primary defect in CHS cells. Further-more, the lack of T-cell and NK cell toxicity in CHS occurs becausecytolytic enzymes are missorted and thus absent from the granules,which release these enzymes upon fusion with the plasma membrane.Finally, the giant granules of CHS result from endosomal missorting ofproteins involved in the regulation of lysosomal homeostasis.

Association of LYST with microtubules. The macrolysosomes ofCHS cells are located in the perinuclear region, consistent with a defectin the microtubular network. Furthermore, the LYST protein hashomology to the microtubule polymerization protein, stathmin (119, 114),although CHS cells have normal numbers and distribution of micro-tubules themselves (126-128). We propose that the N-terminus ofLYST interacts with microtubules, but truncation of the C-terminus ofthe LYST protein, which occurs in all human LYST mutations, causesmalfunctioning of the microtubular moving system. In this case, themicrotubule-associated portion of LYST will remain present in amicrotubular distribution.

PKC activity and granule formation. Evidence indicates a correla-tion between decreased protein kinase C (PKC) activity and increasedlysosome size. Specific PKC inhibitors such as chelerythrin and cal-phostin C induce giant granule formation in normal fibroblasts (129),and inhibition of calpain, which inactivates PKC, prevents giant granuleformation in beige fibroblasts (129). These findings are important be-cause PKC activity is down-regulated in CHS cells (129-131). Moreover,decreased PKC activity in polymorphonuclear leucocytes (PMNs) andnatural killer (NK) cells from beige mice appears responsible for theimpaired immune functions in CHS. The down-regulation of PKC ac-tivity in CHS cells can be corrected by calpain inhibition (129-130),which also corrects the NK cell dysfunction of beige mice (131).

Phosphoinositol kinases. Phosphoinositol-3 kinase (PI3K) plays animportant role in the regulation of protein sorting (132). The localizedgeneration of PI-lipids by PI kinases (PI[x]K) acts to target proteinscritical for cellular trafficking, such as FYVE proteins (133, 134) tothe appropriate location on a membrane that will become a vesicle.PI-lipids also relocate specific guanine nucleotide exchange factors tothe vicinity of new vesicle formation. These exchange factors areneeded to activate members of the rab family of small GTPases whichfacilitate the docking of vesicles with target membranes. Inhibition ofPI3K with wortmannin impairs transport of membrane proteins fromendosomes to lysosomes (135), and causes accumulation of mannose-phosphate receptors in swollen lysosomal compartments of normalfibroblasts (136), an effect similar to that seen in CHS cells.

Structural characteristics of the LYST protein. By virtue of itsHEAT repeats, ARM repeats, and WD40 motifs, the LYST proteinresembles a p150 yeast serine/threonine protein kinase, VPS15 (109).The VPS15 yeast mutant presents as a defect in sorting to the vacuole(137), causing secretion of vacuolar hydrolases. VPS15 regulatesVPS34, a PI3 kinase, and in yeast and mammalian cells, VPS34/PI3Kis involved in endosomal transport and sorting into late endosomesand lysosomes (138, 139). The human LYST sequence does not containa bona fide kinase domain (140), but its carboxy terminus does resembleVPS15.

The Function and Dysfunction of LYST

We hypothesize that the LYST protein acts as an intermediate thataffects lipid-related protein trafficking. In this model, which is based

upon the various domains found in LYST (Fig. 4A), the C-terminus fun-ctions at the TGN to sort specific proteins pertinent for endosomes orlysosomes (Fig. 4B). The protein sorting is supported by lipid phos-phorylation carried out by a VPS34-like PI kinase activated by theVPS15-like domain of the C-terminal region of LYST. The PI kinasestimulates PKC activity indirectly, i. e., via DAG (Fig. 4 inset). Phos-phorylation of specific phospholipids in the TGN results in localrecruitment of coat proteins required for vesicle formation and trans-port. The localized generation of PI lipids by PI(x)K also attractsexchange factors which activate members of the rab family of smallGTPases (Fig. 4C). The GTP/GDP exchange factors interact with theleucine zippers and the HPS1 homology domain of the central part ofthe LYST protein. Rab GTP hydrolysis energizes vesicle formation orvesicle attachment to LYST.

Meanwhile, the N-terminal portion of LYST binds to microtubulesvia its stathmin-like domain (Fig. 4C). Once vesicles form, they contin-ue to be transported peripherally along the microtubules, but the verylarge and sterically hindering LYST protein is released for recyclingwell before the dendrite is reached (Fig. 4D).

When LYST is mutated, PI(x)K is not activated, and decreased DAGproduction leads to down regulation of PKC. Pigment forming vesiclesand dense bodies fail to form and do not move to the periphery.

The model is supported by the finding of a microtubule associationof LYST, by the down regulation of PKC activity, and by the knownstructural domains of LYST. There is also evidence that rabs play arole in the process. Antisense expression of rab7 or rab9 in HeLa cellsresults in the formation of large vacuoles, resembling those of CHSfibroblasts (141, 142), and rab7 interacts with VPS34 (143). Finally,the large size of LYST makes it a good candidate to bind to both theTGN and microtubules.

The model does not explicitly explain the formation of giant vesiclesin CHS cells. It may be that, absent the controlled, directed formationand peripheral movement of nascent vesicles, proteins accumulatewithin the TGN until a critical, massive size is reached, whereupon agiant vesicle breaks away. Its size would preclude movement from theperinuclear region. Alternatively, the giant vesicles may representendosomes formed from the plasma membrane which were intendedto fuse with LYST-dependent vesicles derived from the TGN. In theabsence of LYST, vital proteins, crucial for maintaining the size andcourse of the fused vesicles, never reach the endosomes, resulting intheir growth to giant granules.

Where Does HPS Stop and CHS Begin?

Studies of the clinical spectrum of HPS and the mouse models ofcombined hypopigmentation and storage pool deficiency provide in-sight into the relationship between HPS and CHS. HPS-2 disease,which clearly fits into the category of HPS based upon absence of pla-telet dense bodies and other clinical findings, manifests with neutro-penia and a diathesis toward infections. The ashen mouse, now con-sidered a model for HPS because of the recent finding of absent plate-let dense bodies (62), remains a model also for Griscelli syndrome, adisorder of pigment dilution and silvery hair plus other clinical findings(64). Griscelli syndrome patients who manifest immunological defectsand hemophagocytic syndrome, an uncontrolled T-lymphocyte andmacrophage activation syndrome, have their disease due to deficiencyof rab27a (63), as for ashen (62). Griscelli syndrome patients whoexhibit neurologic impairment instead, have mutations in myosin 5a(144), the gene affected in the mouse dilute (145). (Leaden resembleashen and dilute but its molecular defect has not been resolved.)


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Clearly, the neurologic and immunological symptoms of thesepatients and mice bring them closer to CHS, and confirm the existenceof a continuum of disease. The concept of a gradation of pathologyapplies not only to clinical manifestations, but to cell biological pheno-mena as well. HPS defects appear more involved with vesicle forma-tion, while CHS appears due to vesicle transport defects. The Griscellisyndromes fall in between, with rab27a defects (vesicle formation/fusion)resembling HPS and myosin 5a defects (microtubular transport) resem-bling CHS.

The complete spectrum of disease between HPS and CHS requiressome shading. This will be accomplished by the molecular descriptionof HPS patients whose mutations have not yet been defined, and by theisolation of genes responsible for clinical variants. These genes arebeing sought among those responsible for animal models of theHPS/CHS spectrum, as well as genes involved in the pathways ofvesicle formation and trafficking. From our model of the mechanismof LYST action, we predict that specific rabs, exchange factors, andvesicle docking and fusion proteins are involved. As the gamut ofhuman disease becomes defined, so will the pathways for the formationof vesicles of lysosomal lineage.

Acknowledgement

Dr. Yair Anikster is a Howard Hughes Medical Institute Physician Post-doctoral Fellow.

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Hermansky-Pudlak Syndrome and Chediak-Higashi Syndrome ......Hermansky-Pudlak syndrome (HPS) and Chediak-Higashi syndrome (CHS), share several clinical characteristics and have piqued