Molecular genetics of the Hermansky-Pudlak and Chediak-Higashi syndromes

Hermansky-Pudlak syndrome(HPS) and Chediak-Higashi syndrome(CHS) are similar but distinct autosomal recessive genetic diseases in which a bleeding diathesis resulting from platelet storage pool deficiency is accompanied by deficient pigmentation of the skin and hair and various systemic abnormalities associated with defective lysosomal function. The diverse multi-systemic manifestations of HPS and CHS are associated with abnormalities of a number of different cytoplasmic organelles--platelet dense granules, melanosomes, lysosomes and various cytoplasmic secretory granules. Though rare, HPS and CHS probably represent just the first of what will eventually be a novel class of genetic disorders resulting from defective biogenesis, structure or function of these organelles. The genes responsible for HPS and CHS have recently been identified and are beginning to yield insights into the molecular genetics and cellular pathophysiology of these intriguing disorders.     

The regulation of platelet-dense granules by Rab27a in the ashen mouse,a model of Hermansky-Pudlak and Griscelli syndromes,is granule-specific and dependent on genetic background

The ashen (ash) mouse, a model for Hermansky-Pudlak syndrome (HPS) and for a subset of patients with Griscelli syndrome, presents with hypopigmentation, prolonged bleeding times, and platelet storage pool deficiency due to a mutation which abrogates expression of the Rab27a protein. Platelets of mice with the ashen mutation on the C3H/HeSnJ inbred strain background have greatly reduced amounts of dense granule components such as serotonin and adenine nucleotides though near-normal numbers of dense granules as enumerated by the dense granule-specific fluorescent dye mepacrine. Thus, essentially normal numbers of platelet dense granules are produced but the granule interiors are abnormal. Collagen-mediated aggregation of mutant platelets is significantly depressed. No abnormalities in the concentrations or secretory rates of 2 other major platelet granules, lysosomes and alpha granules, were apparent. Similarly, no platelet ultrastructural alterations other than those involving dense granules were detected. Therefore, Rab27a regulates the synthesis and secretion of only one major platelet organelle, the dense granule. There were likewise no mutant effects on levels or secretion of lysosomal enzymes of several other tissues. Together with other recent analyses of the ashen mouse, these results suggest a close relationship between platelet dense granules, melanosomes of melanocytes and secretory lysosomes of cytotoxic T lymphocytes, all mediated by Rab27a. Surprisingly, the effects of the ashen mutation on platelet-dense granule components, platelet aggregation, and bleeding times were highly dependent on genetic background. This suggests that bleeding tendencies may likewise vary among patients with Griscelli syndrome and HPS with Rab27a mutations.     

The Slc35d3 gene, encoding an orphan nucleotide sugar transporter, regulates platelet-dense granules

Platelet dense granules are lysosome-related organelles which contain high concentrations of several biologically important low-molecular-weight molecules. These include calcium, serotonin, adenine nucleotides, pyrophosphate, and polyphosphate, which are necessary for normal blood hemostasis. The synthesis of dense granules and other lysosome-related organelles is defective in inherited diseases such as Hermansky-Pudlak syndrome (HPS) and Chediak-Higashi syndrome (CHS). HPS and CHS mutations in 8 human and at least 16 murine genes have been identified. Previous studies produced contradictory findings for the function of the murine ashen (Rab27a) gene in platelet-dense granules. We have used a positional cloning approach with one line of ashen mutants to establish that a new mutation in a second gene, Slc35d3, on mouse chromosome 10 is the basis of this discrepancy. The platelet-dense granule defect is rescued in BAC transgenic mice containing the normal Slc35d3 gene. Thus, Slc35d3, an orphan member of a nucleotide sugar transporter family, specifically regulates the contents of platelet-dense granules. Unlike HPS or CHS genes, it has no apparent effect on other lysosome-related organelles such as melanosomes or lysosomes. The ash-Roswell mouse mutant is an appropriate model for human congenital-isolated delta-storage pool deficiency.     

The mouse organellar biogenesis mutant buff results from a mutation in Vps33a,a homologue of yeast vps33 and Drosophila carnation

In the mouse, more than 16 loci are associated with mutant phenotypes that include defective pigmentation, aberrant targeting of lysosomal enzymes, prolonged bleeding, and immunodeficiency, the result of defective biogenesis of cytoplasmic organelles: melanosomes, lysosomes, and various storage granules. Many of these mouse mutants are homologous to the human Hermansky-Pudlak syndrome (HPS), Chediak-Higashi syndrome, and Griscelli syndrome. We have mapped and positionally cloned one of these mouse loci, buff (bf), which has a mutant phenotype similar to that of human HPS. Mouse bf results from a mutation in Vps33a and thus is homologous to the yeast vacuolar protein-sorting mutant vps33 and Drosophila carnation (car). This is the first found defect of the class C vacuole/prevacuole-associated target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (t-SNARE) complex in mammals and the first mammalian mutant found that is directly homologous to a vps mutation of yeast. VPS33A thus is a good candidate gene for a previously uncharacterized form of human HPS.     

Storage pool deficiency in cattle with the Chediak-Higashi syndrome results from an absence of dense granule precursors in their megakaryocytes

Platelets from cattle with the Chediak-Higashi syndrome (CHS) have a storage pool deficiency and virtual absence of platelet dense granules. Megakaryocytes (MKs) from five control (n = 135) and five CHS (n = 133) cattle were evaluated using standard transmission electron microscopy. Osmiophilic dense granules were not observed in control or CHS MKs. In MKs from normal cattle, clear vesicles of 200- to 650-nm diameter bounded by a sharp membrane were observed. They were easily differentiated from the demarcation membrane system, endoplasmic reticulum, and alpha granules. The clear vesicles were virtually absent in MKs from CHS cattle at all stages of maturation. MKs in bone marrow samples from two control (n = 91) and two CHS (n = 61) cattle that had been processed for the uranaffin reaction were also evaluated. The clear vesicles were replaced by uranaffin-positive granules in MKs from control cattle, but positive uranaffin granules were not observed in CHS MKs. These findings indicate that the platelet dense granule storage pool deficiency in CHS cattle results from an anatomic absence of dense granule precursors in maturing and mature CHS MKs.     

Quantification of a novel dense granule protein (granulophysin) in platelets of patients with dense granule storage pool deficiency.

An antigen-capture sandwich enzyme-linked immunosorbent assay (ELISA) was developed for a novel protein granulophysin, a constituent of the platelet dense granule (DG) membrane and used to characterize patients with dense granule storage pool deficiency (delta-SPD). The assay uses two monoclonal antibodies against the protein, one of which is conjugated to peroxidase. Purified DGs, an enriched source of the protein, were used for the standard curve. Granulophysin levels were only low in forms of delta-SPD associated with albinism. Granulophysin levels in platelet homogenates of 30 patients with the Hermansky-Pudlak syndrome form of delta-SPD were 1/4 to 1/5 of levels in controls or obligate heterozygotes. Two patients with the Chediak-Higashi form of delta-SPD syndrome also had markedly reduced levels of granulophysin. Patients with other forms of delta-SPD had normal levels of granulophysin. Two sisters with delta-SPD in one family had normal granulophysin present in empty dense granule membrane vesicles. Three members of another family with delta-SPD had low DG counts but normal granulophysin levels, indicating that in this group the level of granulophysin was maintained despite the reduction in granule formation. Thus, granulophysin quantitation facilitates characterization of delta-SPD patients and may provide clues to the nature of defective granules in delta-SPD subtypes.     

Platelet granule disorders

The present review has cataloged the inherited and acquired disorders of platelet granules. Unfortunately, a mere listing of different conditions in which dense bodies, alpha granules, or both are decreased, absent, or fused does little to define their importance in human platelet physiology or as a causative factor in hemorrhagic disease. The inherited disorders serve as the best index of granule involvement in platelet hemostatic function. Our experience with storage pool deficiency in patients with Hermansky-Pudlak syndrome has suggested that in many individuals virtual absence of dense bodies and their contents does not present a serious threat to hemostasis. Placing HPS patients on aspirin did not cause spontaneous hemorrhage, suggesting that secretion of dense body contents and synthesis of endoperoxides and thromboxane A2 are not absolutely essential for platelet function. However, the literature strongly suggests that many patients with HPS and SPD face a serious risk from bleeding, and hemorrhage may cause death. We can only conclude that some patients with HPS have platelet defects or other hemostatic problems that render SPD a far more serious threat than in other patients who appear to have the same disease. Dense bodies of and by themselves do not appear absolutely required for platelet function. Isolated deficiency of alpha granules presents the same enigma. Only a few patients with this rare inherited disease have been reported. They are generally considered to have mild to severe hemorrhagic problems. However, the past medical history of our two patients with GPS has recently been reviewed and platelet function studies repeated. Despite the mild thrombocytopenia, they are free of any significant bleeding episodes and their platelet function appears virtually normal. Our findings do not support the concept that alpha granules are essential for platelet function. The only condition that seems to support a critical role for storage organelles in hemostasis is the combined alpha-granule, dense body deficiency in one patient reported by Weiss. This patient does have bleeding problems. However, it is difficult to draw conclusions based on a single patient, and the discovery of other patients will help to clarify the hemostatic problem of patients with dual storage organelle deficiencies. In the meantime, we have prepared platelets from normal individuals free of storage granules by sedimentation through gradients containing cytochalasin B. The function of the normal agranular platelets is compromised, but they do respond to some aggregating agents.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pseudo Chediak-Higashi Granules in Acute Lymphoblastic Leukemia: A Rare Entity

Pseudo-Chediak-Highashi granules are giant cytoplasmic inclusions commonly encountered in myeloblasts or other myeloid precursors in acute myeloid leukemia and myelodysplastic syndromes. They derive their name from the inherited Chediak-Higashi syndrome that presents with oculocutaneous albinism, chronic infections and platelet dense granule deficiency. We report possibly the third case in world literature where these granules were seen in the blast cells of acute lymphoblastic leukemia in a 15-year-old male.     

Heterogeneous abnormalities of platelet dense granule ultrastructure in 20 patients with congenital storage pool deficiency

Studies on platelet dense granule structure were carried out in 20 patients with various types of congenital storage pool deficiency (SPD), including 15 with specific deficiencies of dense granules and dense granule substances (δ-SPD), and five with combined deficiencies of dense and α-granules (αδ-SPD). Dense granules were identified by their high affinity for uranyl ions (uranaffin reaction), by their ability to accumulate the fluorescent dye mepacrine, and by their inherent electron opacity on unfixed, unstained whole mount preparations. By all these methods, dense granules were markedly decreased in seven albino patients with the Hermansky-Pudlak syndrome (HPS) variant of δ-SPD. These findings suggest that the basic defect in these patients is a specific abnormality in organelle development which prevents the formation of an intact granule structure, a quantitative abnormality which may differ from that in animals with related pigment disorders. In contrast, eight non-albino patients with δ-SPD had, on average, only a slightly reduced number of uranaffin-positive and mepacrine-positive granules, but a shift in uranaffin-granule distribution towards those lacking a dense core (‘empty granules’), suggesting a more qualitative type of dense granule defect. These results are consistent with previous evidence suggesting a decreased uptake of ATP across the granule membrane in δ-SPD. In addition, on whole mounts, these patients’platelets contained substantial numbers of electron dense chains and clusters which contained P and Ca, but with a P/Ca ratio less than that of typical dense granules, and which were retained, along with a larger amount of ATP, after thrombin treatment of the platelets. The various findings in these patients raise the possibility that these structures may represent microvesicles, derived from the Golgi apparatus, which provide a transport mechanism for concentrating adenine nucleotides and calcium in dense granules and which is impaired in some patients with SPD. Additional defects may account for the more extensive granule abnormalities observed in αδ-SPD.     

Platelet alpha granules in BLOC-2 and BLOC-3 subtypes of Hermansky-Pudlak syndrome

Hermansky-Pudlak syndrome (HPS) is a disorder of lysosome-related organelle biogenesis that displays genetic locus heterogeneity. The eight known HPS proteins combine in functional complexes, two of which are called BLOC-2 and BLOC-3; a BLOC is a Biogenesis of Lysosome-related Organelles Complex. Organelles affected in HPS include the melanosome, resulting in hypopigmentation, and the platelet delta (dense) granule, resulting in prolonged bleeding times. Whole mount electron microscopy (EM) detects the absence of platelet delta granules and confirms the diagnosis of HPS. To date, the status of other organelles and granules in HPS platelets has not been documented. We performed ultrastructural studies on platelets of patients with different genetic forms of HPS, specifically those comprising the BLOC-2 and BLOC-3 subtypes. No differences in distribution, size or quantity of other platelet organelles and membrane structures could be detected in our patients. Since alpha and delta granules are formed from multivesicular bodies in the megakaryocyte, and since only delta granules are defective in HPS, we conclude that HPS genes function within the portion of delta granule biogenesis that has diverged from that of alpha granules. Thus, it is unlikely that the generalized bleeding diathesis of HPS is attributed to a deficiency of alpha granules.     

Platelet alpha granules in BLOC-2 and BLOC-3 subtypes of Hermansky-Pudlak syndrome

Hermansky-Pudlak syndrome (HPS) is a disorder of lysosome-related organelle biogenesis that displays genetic locus heterogeneity. The eight known HPS proteins combine in functional complexes, two of which are called BLOC-2 and BLOC-3; a BLOC is a Biogenesis of Lysosome-related Organelles Complex. Organelles affected in HPS include the melanosome, resulting in hypopigmentation, and the platelet delta (dense) granule, resulting in prolonged bleeding times. Whole mount electron microscopy (EM) detects the absence of platelet delta granules and confirms the diagnosis of HPS. To date, the status of other organelles and granules in HPS platelets has not been documented. We performed ultrastructural studies on platelets of patients with different genetic forms of HPS, specifically those comprising the BLOC-2 and BLOC-3 subtypes. No differences in distribution, size or quantity of other platelet organelles and membrane structures could be detected in our patients. Since alpha and delta granules are formed from multivesicular bodies in the megakaryocyte, and since only delta granules are defective in HPS, we conclude that HPS genes function within the portion of delta granule biogenesis that has diverged from that of alpha granules. Thus, it is unlikely that the generalized bleeding diathesis of HPS is attributed to a deficiency of alpha granules.     

Hermansky-Pudlak syndrome subtype 5 (HPS-5) novel mutation in a 65 year-old with oculocutaneous hypopigmentation and mild bleeding diathesis: The importance of recognizing a subtle phenotype

Hermansky-Pudlak syndrome (HPS) ? characterized by the distinct clinical phenotypes of both oculocutaneous albinism and mild bleeding diathesis–is caused by mutations in genes that have crucial roles in the assembly of cellular organelles (skin melanosomes, platelet delta [dense] granules, lung lamellar bodies, and cytotoxic T-cell lymphocyte granules). Immunodeficiency, pulmonary fibrosis and granulomatous colitis are associated with some, but not all subtypes of HPS, with varying degrees of clinical severity. We describe a patient diagnosed with platelet dense granule storage pool deficiency (DG-SPD) at age 38 years after he presented with spontaneous intracranial hemorrhage. His mild oculocutaneous hypopigmentation was subtle. In the following 27 years, he did not develop severe bleeding nor pulmonary or gastrointestinal complications. A novel homozygous c.1960A>T; p.Lys654* mutation in the HPS-5 protein gene (HPS5) was identified through next generation sequencing, (NGS) which is consistent with the patient’s clinical and laboratory phenotypes. This case underscores the importance of recognizing the mild clinical phenotype of HPS-5 and utilization of both laboratory and molecular testing for diagnosis, prognostication, and surveillance for end organ damage in patients affected with HPS.     

Medich giant platelet disorder: a unique alpha granule deficiency I. Structural abnormalities

Human platelet granule deficiency disorders include the gray platelet syndrome (GPS), alpha delta storage pool deficiency, the Hermansky-Pudlak syndrome and the White platelet syndrome. The present study describes a patient with a lifelong history of easy bleeding, thrombocytopenia and giant platelets. Her cells were found to have normal numbers of dense bodies, but a markedly decreased number of alpha granules. Many platelets had no alpha granules and resembled the gray platelets of patients with GPS. However, the empty vacuoles without granule contents that fill the cytoplasm of GPS platelets were not present in significant numbers in her platelets. In addition to the decrease in alpha granules the patients platelets contained membranous inclusions resembling cigars or scrolls. Usually, only one scroll open at each end was present, but many platelets contained two and some as many as five. Freeze-fracture revealed an absence of intramembranous particles in many layers of the scrolls. They occur in no other human platelet disorder, but are common in platelets from the Wistar-Furth rat. Thus, the patient is a unique variant of human platelet granule deficiency disorders unlike any described previously.     

Medich giant platelet disorder: a unique α granule deficiency I. Structural abnormalities

Human platelet granule deficiency disorders include the gray platelet syndrome (GPS), α δ storage pool deficiency, the Hermansky–Pudlak syndrome and the White platelet syndrome. The present study describes a patient with a lifelong history of easy bleeding, thrombocytopenia and giant platelets. Her cells were found to have normal numbers of dense bodies, but a markedly decreased number of α granules. Many platelets had no α granules and resembled the gray platelets of patients with GPS. However, the empty vacuoles without granule contents that fill the cytoplasm of GPS platelets were not present in significant numbers in her platelets. In addition to the decrease in α granules the patients platelets contained membranous inclusions resembling cigars or scrolls. Usually, only one scroll open at each end was present, but many platelets contained two and some as many as five. Freeze-fracture revealed an absence of intramembranous particles in many layers of the scrolls. They occur in no other human platelet disorder, but are common in platelets from the Wistar–Furth rat. Thus, the patient is a unique variant of human platelet granule deficiency disorders unlike any described previously.     

The empty sack syndrome: a platelet storage pool deficiency associated with empty dense granules

Summary. Two sisters with lifelong bleeding tendencies were examined to determine whether their condition was associated with a platelet defect. Their platelet aggregation in response to epinephrine and collagen was abnormal, and the secretion of serotonin and ATP was markedly reduced. The platelet contents of serotonin, ADP, and ATP were all diminished and the ATP:ADP ratio was increased. Direct enumeration by whole-mount and quinacrine-fluorescence techniques demonstrated that the platelets from both sisters had significantly fewer dense granules than controls. These characteristics are similar to an individual with Hermansky-Pudlak syndrome and are consistent with a platelet dense granule deficiency. In contrast, immunofluorescence studies using an antibody against the dense granule membrane protein granulophysin suggested that both sisters had numbers of granules within the normal range. Evaluation by immunoblotting and ELISA indicated the presence of normal levels of granulophysin in the platelets from both sisters: FACS analysis demonstrated the surface expression of granulophysin under conditions of selective dense granule release. These results are consistent with these sisters having a form of dense granule storage pool deficiency where the granular membranes are present but the granules have reduced contents. This observation represents a novel form of storage pool disease which we have termed the empty sack syndrome.     

Platelet storage pool deficiency in Jacobsen syndrome

Jacobsen syndrome and Paris-Trousseau Syndrome share similar congenital anomalies, thrombocytopenia, giant platelet alpha granules resulting from fusion of smaller organelles, and an 11q terminal deletion at 11q23.3. Similarities in the two cohorts have suggested that the Paris-Trousseau Syndrome is a variant of Jacobsen syndrome, or the same disorder. The present study has pointed out a significant difference between the two syndromes. Platelets from six patients with Jacobsen syndrome were markedly diminished in serotonin adenine nucleotide rich dense bodies, indicating the presence of platelet storage pool deficiency. Since platelet dense bodies are reported to be normal in size, number and distribution in the Paris-Trousseau Syndrome, the presence of platelet storage pool deficiency in six patients evaluated in the present study may distinguish the two disorders.     

Platelet storage pool deficiency in Jacobsen syndrome

Jacobsen syndrome and Paris-Trousseau Syndrome share similar congenital anomalies, thrombocytopenia, giant platelet alpha granules resulting from fusion of smaller organelles, and an 11q terminal deletion at 11q23.3. Similarities in the two cohorts have suggested that the Paris-Trousseau Syndrome is a variant of Jacobsen syndrome, or the same disorder. The present study has pointed out a significant difference between the two syndromes. Platelets from six patients with Jacobsen syndrome were markedly diminished in serotonin adenine nucleotide rich dense bodies, indicating the presence of platelet storage pool deficiency. Since platelet dense bodies are reported to be normal in size, number and distribution in the Paris-Trousseau Syndrome, the presence of platelet storage pool deficiency in six patients evaluated in the present study may distinguish the two disorders.     

SLC35D3 delivery from megakaryocyte early endosomes is required for platelet dense granule biogenesis and is differentially defective in Hermansky-Pudlak syndrome models

Platelet dense granules are members of a family of tissue-specific, lysosome-related organelles that also includes melanosomes in melanocytes. Contents released from dense granules after platelet activation promote coagulation and hemostasis, and dense granule defects such as those seen in Hermansky-Pudlak syndrome (HPS) cause excessive bleeding, but little is known about how dense granules form in megakaryocytes (MKs). In the present study, we used SLC35D3, mutation of which causes a dense granule defect in mice, to show that early endosomes play a direct role in dense granule biogenesis. We show that SLC35D3 expression is up-regulated during mouse MK differentiation and is enriched in platelets. Using immunofluorescence and immunoelectron microscopy and subcellular fractionation in megakaryocytoid cells, we show that epitope-tagged and endogenous SLC35D3 localize predominantly to early endosomes but not to dense granule precursors. Nevertheless, SLC35D3 is depleted in mouse platelets from 2 of 3 HPS models and, when expressed ectopically in melanocytes, SLC35D3 localizes to melanosomes in a manner requiring a HPS-associated protein complex that functions from early endosomal transport intermediates. We conclude that SLC35D3 is either delivered to nascent dense granules from contiguous early endosomes as MKs mature or functions in dense granule biogenesis directly from early endosomes, suggesting that dense granules originate from early endosomes in MKs.     

Alpha-delta platelet storage pool deficiency in three generations

Alpha-Delta platelet storage pool deficiency (alphadelta SPD) is a rare inherited bleeding disorder affecting both males and females, occurring in families, as well as sporadically. Patient platelets in most cases are moderately deficient in both alpha granules and dense bodies. Only one patient has been severely deficient in both organelles. The present study is the first to document a severe decrease in both platelet alpha granules and dense bodies in four members in three generations of the same family. Efforts to differentiate this disorder from other hypogranular platelets syndromes in the present investigation suggested that the alpha granules and dense bodies become connected to channels of the open canalicular system (OCS) and lose their contents to the exterior without prior activation of the cells. In contrast, alpha granule formation in the white platelet syndrome is too slow, and cells leave the bone marrow still in the process of producing organelles. Gray platelet syndrome platelets can make alpha granules, but their enclosing membranes are unable to retain stored products. As a result, the organelles lose their contents to surrounding cytoplasm in megakaryocytes and platelets, not selectively through the demarcation system channels and OCS channels. Thus, the pathogenesis of alphadelta SPD is unique.     

Correction of symptoms of platelet storage pool deficiency in animal models for Chediak-Higashi syndrome and Hermansky-Pudlak syndrome

Two human diseases of platelet storage pool deficiency (SPD), Hermansky- Pudlak syndrome and Chediak-Higashi syndrome, are recessively inherited disorders characterized by hypopigmentation, prolonged bleeding, and normal platelet counts accompanied by a reduction in dense granule number. We have recently described seven independent recessive mutations in the mouse regulated by separate genes which are likely animal models for human SPD. Reciprocal bone marrow transplants were carried out between normal C57BL/6J mice and two of these mutants, beige and pallid, in order to test whether the platelet defects are due to a defect in platelet progenitor cells or to humoral factors. Normal and congenic mutant mice were transplanted with marrow after 950 rad whole body radiation. The long bleeding times and low serotonin concentrations of the two mutants were converted to normal values after transplantation with normal marrow. Likewise, normal mice displayed symptoms of SPD when transplanted with mutant marrow. These studies demonstrate that with each of the two mutations, platelet SPD results from a defect in bone marrow precursor cells. Also, the studies suggest that in severe cases, platelet SPD may be successfully treated by bone marrow transplantation.