Chediak-Higashi syndrome: a review of the past, present, and future

Abstract

Since the initial description of Chediak-Higashi syndrome (CHS), over 75 years ago, several studies have been conducted to underscore the role of the lysosomal trafficking regulator (LYST) gene in the pathogenesis of disease. CHS is a rare autosomal recessive disorder, which is caused by biallelic mutations in the highly conserved LYST gene. The disease is characterized by partial oculocutaneous albinism, prolonged bleeding, immune and neurologic dysfunction, and risk for the development of hemophagocytic lympohistiocytosis (HLH). The presence of giant secretory granules in leukocytes is the classical diagnostic feature, which distinguishes CHS from closely related Griscelli and Hermansky-Pudlak syndromes. While the exact mechanism of the formation of the giant granules in CHS patients is not understood, dysregulation of LYST function in regulating lysosomal biogenesis has been proposed to play a role. In this review, we discuss the clinical characteristics of the disease and highlight the functional consequences of enlarged lysosomes and lysosome-related organelles (LROs) in CHS.

Graphical abstract

Background

Although Chediak-Higashi syndrome (CHS) bears the names of the French physician Moises Chediak and the Japanese physician Ototaka Higashi, it was Antonio Beguez-Cesar, a Cuban physician, who described the initial family with atypical enlarged granules within leukocytes¹. Additional clinical characteristics included neutropenia, albinism, and features of hemophagocytic lymphohistiocytosis (HLH). Consultation with Chediak concluded this was a novel disease. Additional cases were described by Steinbrinck in 1948 and Higashi in 1954. Today, CHS is best recognized as a primary immunodeficiency with pathognomonic giant inclusions within leukocytes, among other cell types. The disease is exceptionally rare with fewer than 500 cases reported in the literature².

Clinical characteristics

Characteristic features of CHS include partial oculocutaneous albinism, predilection for bleeding, immune dysfunction, neurodegeneration, and risk for development of HLH^{3; 4}. Pigment dilution is variable and can be subtle^{5; 6}. Within the hair, clumps of pigment are dispersed throughout the hair shaft (Figure 1a). Ocular pigmentation can also be variable. Often iris pigment is reduced with iris transillumination visible on examination (Figure 1b). Retinal pigment may also be reduced. Once thought to be an essential criterion for clinical diagnosis, it is now appreciated that there are some individuals with no evidence of oculocutaneous albinism⁷.

Figure 1.

Clinical features of Chediak-Higashi syndrome, LYST domain structure, and cellular defects

a. A hair image from a patient with CHS shows dispersal of pigment clumps throughout the hair shaft.

b. An image from a patient with CHS shows iris transillumination.

c. Peripheral blood smear from a patient with CHS shows a neutrophil with the typical giant cytoplasmic granules (arrow).

d. Peripheral blood smear from a patient with CHS shows a neutrophil with prominent cytoplasmic granules (arrow).

e. MRI of the brain of a child with CHS demonstrating the small posterior fossa and high pitched tentorium.

f. MRI of the brain of a young adult with CHS demonstrating cerebellar and cerebral atrophy.

g. A schematic of the domain structure shows the four defined domains of the human lysosomal trafficking regulator (LYST) protein. The amino-terminal ARM/HEAT domain contains a series of Armadillo (ARM) and Huntingtin (HEAT) alpha-helix repeat motifs that are predicted to mediate membrane association and vesicle transport. LYST also contains a Pleckstrin-homology (PH) domain, a Beige and Chediak-Higashi (BEACH) domain, and seven carboxy-terminal WD-40 domains containing conserved amino acids tryptophan (W) and aspartic acid (D).

h. Diagrams displaying cellular defects leading to the functional impairments in CHS. The giant azurophilic granules in neutrophils do not release their contents properly and affect bactericidal and chemotaxis activity (upper). The reduced number of dense granules in platelets leads to abnormal aggregation and prolonged bleeding time (middle). The presence of enlarged lytic granules leads to impairments of the cytotoxic activity of NK and cytotoxic T lymphocytes (lower).

Although platelet numbers are generally within the normal range, bleeding occurs due to absent or severely diminished platelet dense granules⁸. Dense granules contain calcium, ADP, ATP, and serotonin and, when released, are responsible for platelet recruitment and aggregation. In the absence of dense granules, the initial platelet plug does not develop leading to prolonged bleeding times. Platelet aggregation studies show an abnormal secondary wave of aggregation. Platelet dense bodies can be quantified using platelet electron microscopy. Generally, symptoms manifest as mucosal bleeds with gingival bleeding, epistaxis, and easy bruisability. However, in the setting of trauma or surgical intervention, platelet dysfunction may contribute to prolonged bleeding.

The hallmark of CHS is the giant granules within leukocytes that are visualized on routine peripheral blood smear (Figure 1c). The granules are azurophilic, peroxidase positive, yet variable in size (Figure 1d). The large granules impair neutrophil chemotaxis, and intracellular bactericidal activity is also reduced, likely from impaired degranulation⁸. Neutropenia may be present. Natural Killer (NK) cells are also reduced in number and in function.

Neurologic disease includes both developmental and degenerative components. Children may manifest learning difficulties and behavioral abnormalities during early school age⁷. Interestingly, on MRI imaging, the posterior fossa shows structural variability with a high-pitched tentorium supporting the theory that development of the posterior fossa is impacted (Figure 1e). In late adolescence and early adulthood, patients begin to display progressive degeneration including absence of deep tendon reflexes, signs of cerebellar dysfunction, peripheral neuropathy, length dependent neuropathy and neuronopathy, weakness, spasticity, or parkinsonian symptoms^{5; 7; 9}. Brain imaging is unrevealing initially, though over time, cerebellar and/or cerebral atrophy may develop (Figure 1e, and 1f).

The accelerated phase, or HLH, is the primary cause of mortality in CHS. HLH can occur at any age and presents with unexplained fevers, lymphadenopathy, hepatosplenomegaly, and cytopenias of at least two of the three cell lineages in peripheral blood. Criteria for the diagnosis of HLH are well described, and management is the same as for HLH of other etiologies¹⁰.

Differential diagnosis

CHS most closely resembles Griscelli syndrome (GS) and Hermansky-Pudlak syndrome (HPS). Both GS and HPS have pigment reduction. There are 10 subtypes of HPS with abnormal bleeding due to absent dense granules. HPS-2, is also characterized by neutropenia, recurrent infections, and risk of HLH, although less risk of HLH than CHS or GS¹¹. There are 3 subtypes of GS. GS1 has a predominant neurologic phenotype, while GS2 presents with immunodeficiency and risk for HLH¹². GS3 manifests with only pigment dilution. CHS distinguishes itself from GS and HPS by identifying the giant granules within leukocytes as GS and HPS do not have enlarged granules on peripheral smear.

Genetic cause of CHS and genotype-phenotype correlation

CHS is an autosomal recessive disorder due to mutations in the highly conserved gene, lysosomal trafficking regulator (LYST)^{2; 13-15}. The open reading frame of the LYST mRNA (NM_000081) has 53 exons that encodes a protein (NP_000072) of 3801 amino acids. LYST is widely expressed in human tissues with higher levels of expression found in bone marrow, cerebellum, spleen and thymus. The carboxy-terminal of LYST contains three domains: a Pleckstrin-homology (PH) domain, a Beige and Chediak-Higashi (BEACH) domain, and WD-40 (tryptophan-aspartic acid) repeats (Figure 1g)¹⁴. Studies of proteins with BEACH domains revealed some of the functional significance of these domains and suggest their primary involvement in vesicular trafficking^16-18. The amino terminal of LYST contains a series of Armadillo (ARM) and Huntingtin (HEAT) α-helix repeat motifs, that may mediate membrane association and vesicle transport¹⁹.

Prior to the discovery of LYST, CHS diagnosis was based on clinical criteria, and likely only identified children with classic features of the disease. However, the clinical phenotype is better represented as a spectrum of disease with some individuals having a more classic presentation and some individuals with atypical features. While not absolute, a genotype-phenotype correlation has emerged with individuals with biallelic truncating mutations leading to more severe clinical manifestations and individuals with at least one missense mutation having a milder presentation⁷. In NK cells, it has been shown that in the classic presentation, granules are fewer in number, but extremely large in size. However, in atypical cases, the granules may be more numerous, and smaller, yet still prominent compared to normal⁷.

Unlike other clinical features, the neurologic disease does not distinguish the classic from the more atypical phenotypes. For individuals with atypical phenotypes, the neurologic features may dominate the clinical picture while the hematologic and immunologic features are more muted. Patients with classic presentation appear to be at greater risk of developing HLH, although there are exceptions reported with genotypically milder individuals accelerating²⁰.

It is not surprising that numerous different variants have been reported in LYST given its large size. Most individuals have private mutations. Mutations include nonsense, missense, frameshift, and splice site variants that have been identified throughout the gene, particularly in the ARM/HEAT, BEACH and WD-40 domains. Challenges arise when trying to determine pathogenicity of new variants. Bioinformatic predictive tools offer some guidance for determining pathogenicity, but clinical phenotype is also important for the interpretation of the molecular variations.

Murine models

Genetic linkage studies have shown that LYST maps within a linkage group conserved between human chromosome 1q42-q43 and the Beige region on mouse chromosome 13²¹. The Beige (bg) mutant allele was the first mutation of the murine Lyst gene that appeared spontaneously as a consequence of LINE1 element insertion within an intron of the murine Beige gene producing a premature stop codon¹⁵. The Beige mice on a C57BL/6J background have enlarged lysosomes and lysosome-related organelles (LROs), hypopigmentation, prolonged bleeding times, and immunodeficiency due to defective cytotoxicity of T and NK cells.^{22; 23}.

Another CHS model, the Lyst^bg-grey, consists of a point mutation of the splice donor site of the intron between exon 25 and exon 26, which causes an in-frame skipping of exon 25 and unstable LYST protein, and leads to a phenotype similar to the Beige mice²⁴.

The Lyst^Ing3618 mice on the C57BL/6J background display hypopigmentation and prolonged bleeding times but with no abnormality in hematopoietic lineage, impaired lysosomal morphology and function, or susceptibility to infections or tumors. Interestingly, these mice apparently present with a neurodegenerative phenotype from 5 months of age²⁵.

Recently, it has been shown that Lyst mutant mice exhibit more overt tremor on the DBA/2J genetic background when compared to C57BL/6J²⁶, suggesting the presence of genetic modifiers²⁷. This mouse strain may be useful in studying the neurodegenerative phenotype in more detail and provides an avenue for future drug testing.

Cellular defects in CHS and proposed function of LYST

The hallmark of the subcellular morphology associated with CHS is enlarged lysosomes and abnormal LROs including melanosomes, lytic granules, major histocompatibility complex (MHC) class II compartments and platelet dense bodies in various cell types^{2; 8; 28}; the mechanism underlying the characteristic subcellular morphology remains largely elusive. In a normal cell, the fusion of lysosomes with late endosomes results in intermixing of vesicular contents with lysosomal hydrolases and an increase in lysosomal surface area due to transient endosomal membrane influx. Once the endosomal contents are delivered to lysosomes, endosomes fission off, a process commonly known as “kiss-and-run”. These constant fusion and fission events result in the remodeling of organelles and the formation of mature lysosomes while keeping the steady-state median size and the number of lysosomes constant.

Two different models are presented to explain the dysregulation of lysosomal size. One model suggests that LYST is required for proper fusion events. This model is supported by the studies in human cells revealing the interaction of LYST, with proteins involved in tethering, docking, and fusion machinery, is altered in CHS²⁹, and also observed in other models showing increased fusion in granule precursors³⁰ and increased phagosome fusion³¹. The other model suggests that LYST may contribute to lysosomal membrane fission events instead of fusion. This was first supported by Lyst overexpression studies in mice^32-34. These studies showed that overexpression of Beige causes fragmentation and peripheral dispersal of lysosomes, leading to the reduction in lysosome size. These observations led the authors to conclude the rate of lysosomal fission is positively regulated by LYST. siRNA-mediated depletion of LYST in human HeLa and U2OS cell lines leads to fewer and larger lysosomes, supporting the notion that the lysosomes observed in CHS patient cells are caused by loss-of-function mutations in LYST ³⁵. Interestingly, LYST-depletion in human cells had no effect on the fusion of lysosomes with endosomes and autophagosomes, their degradative capacity or trafficking of cargo via autophagy, endocytosis or retrograde transport³⁵.

Another cellular defect observed in fibroblasts from CHS patients and the Beige mouse is the impairment of plasma membrane resealing following a wounding³⁶. Plasma membrane wounds are repaired by a Ca²⁺-dependent exocytosis mechanism, involving elevation in intracellular Ca²⁺ that triggers the fusion of small peripheral lysosomes with the plasma membrane³⁷. The abnormal enlargement of lysosomes in CHS/Beige fibroblasts is a limiting factor for normal lysosomal exocytosis and their fusion with the plasma membrane. This suggests that lysosomal enlargement may deplete the small peripheral lysosomes that are preferentially involved in Ca²⁺-triggered exocytosis in fibroblasts.

Functional consequences of enlarged lysosomes and LROs in CHS

The enlarged lysosomes and abnormal LROs could have several functional consequences particularly in the cells that perform lysosomal secretion (Figure 1h). In melanocytes, enlarged melanosomes are not properly transferred to epithelial cells, which results in hypopigmentation³⁸. The giant azurophilic granules present in the neutrophils of CHS patients do not appropriately release their contents following bacterial and viral infection, leading to impaired bactericidal activity^{39; 40}. The chemotaxis function of neutrophils is also affected in CHS⁴¹. The presence of enlarged lysosomes and abnormal LROs lead to the impaired cytotoxic activity of T and NK cells, leading to the development of life-threatening HLH. The cytotoxicity of lymphocytes is a highly regulated, stepwise process, requiring the formation of the immunological synapse (IS) between lymphocyte and target cell followed by a reorganization of the lymphocyte cytoskeleton to translocate the microtubule-organizing center (MTOC) toward the IS. Docking of the MTOC beneath the IS ensures microtubule-assisted directional transport of specialized secretory lysosomes (lytic granules) containing the soluble cytolytic proteins including perforin and granzymes. Exocytosis of secretory lysosomes at the IS leads to the release of cytolytic proteins (degranulation) into the synaptic cleft and promotes the killing of the target cell. In CHS patients, the cytotoxic granules in cytotoxic T lymphocytes (CTL) exhibited limited mobility and failed to degranulate at the IS. The secretory ability of cytotoxic granules was restored by increasing the expression of effectors of the exocytosis machinery⁴² suggesting that LYST may regulate the trafficking of effectors required for the terminal maturation of perforin containing lytic granules into exocytosis-competent secretory granules.

Knocking-out LYST in a human NK cell line resulted in enlarged lytic granules, impaired integrity of endolysosomal compartments, defective exocytosis and inhibition of NK cytotoxicity; however, the activity of lysosomal enzymes (granzyme B and cathepsin), levels of major lysosome-associated proteins (LAMP1 and LAMP2), lytic proteins (perforin and granzyme B) and proteins critical for lytic granule polarization (Dynein), secretion (myosin IIA), docking (Rab27a), priming (Munc13-4), and fusion with the plasma membrane (VAMP7) were not altered. Disruption of Rab14 expression or decreasing the density of cortical actin meshwork restored the degranulation and killing abilities of LYST- knockout NK cells⁴³. These findings could imply that LYST function may be necessary for lysosomal biogenesis but not required during the early activation stage of NK cells leading to the formation of IS for target cell killing; also, these data suggest that an enlarged lytic granule may present a physical hindrance to degranulate at the IS and kill target cell, leading to impaired cytotoxicity.

Mutations in LYST are distributed throughout the length of protein, suggesting that the position of mutations may be irrelevant for the final functional outcome. Comparing the cytotoxic function of NK among CHS patients with mutations found in the ARM/HEAT or BEACH domains of LYST, did not reveal any correlation between degree of NK cytotoxicity and position of mutations, but the position of CHS mutations correlates positively with the size and number of lytic granules in NK cells⁴⁴.

An analysis of 21 CHS patients showed a similar degree of impairment in granule exocytosis from both CTLs and NK cells despite the differences between number and size of granules in these two cell types⁴⁵. Interestingly, the cytotoxic function was more severely affected in NK cells compared to CTLs, which suggest that differences in regulation of cytotoxic granule biogenesis in CTL versus NK could account for the more severe defect both in granule morphology and cytotoxic function in NK cells. Taken together, these findings provide evidence for the unique role of LYST in lysosomal biogenesis rather than affecting the process leading to the cytotoxic activity. It should be noted that the cytotoxic function of NK cells is uniformly compromised among CHS patients, however, there are variabilities in the development of HLH, which are likely caused by the differences in the cytotoxic capacity of CTLs⁴⁶.

Current management and prospects for therapy

The large size of the LYST coding sequence (approximately 11kb) presents a significant technical hurdle in developing a gene therapy application for CHS. Even with the recombinant adeno-associated virus (AAV) vector that has recently become an attractive approach for many genes, the insert size limit of 5 kb poses a challenge. Moreover, developing a unifying minigene approach may not be feasible given the distribution of pathogenic mutations throughout the length of the coding region. The current management of CHS is limited to symptomatic care. For HLH, the initial management is the same as for primary forms of HLH and establishes remission until definitive treatment can be achieved using allogenic hematopoietic stem cell transplantation (HSCT). HSCT may have a better outcome when performed prior to presentation of HLH, which has been shown to correct the immunologic and hematologic manifestations of the disease. Unfortunately, HSCT does not alter the neurodegenerative process^{7; 47; 48}.

Neurological disease is variable and treated on a case by case basis. Levodopa therapy may be valuable for some individuals with parkinsonism⁷. Other patients will benefit from ongoing physical therapy or adaptive equipment. Other monogenic forms of Parkinson Disease (PD) have impaired LRO function, thus defining pathways and functions of LYST can provide clues about potential therapeutic targets for CHS patients.

Conclusions and future prospects

Disruption of LYST results in the rare autosomal recessive Chediak-Higashi syndrome, which presents with clinical features that are probably associated with defects in the biogenesis of lysosomes and LROs. Further studies will be needed to determine the molecular interplay between LYST and other proteins governing the regulation of size and number of lysosomes in various cells types. Understanding the cellular functions of LYST may provide therapeutic targets for CHS.