Human Chediak-Higashi Syndrome Research Articles

The Tetrahymena bcd1 mutant implicates endosome trafficking in ciliate, cortical pattern formation.

Ciliates, such as Tetrahymena thermophila, evolved complex mechanisms to determine both the location and dimensions of cortical organelles such as the oral apparatus (OA: involved in phagocytosis) cytoproct (Cyp: for eliminating wastes) and contractile vacuole pores (CVPs: involved in water expulsion). Mutations have been recovered in Tetrahymena that affect both the localization of such organelles along anterior-posterior and circumferential body axes, and their dimensions. Here we describe BCD1, a ciliate pattern-gene that encodes a conserved beige-BEACH-domain containing protein a with possible PKA-anchoring activity. Similar proteins have been implicated in endosome trafficking, and are linked to human Chediak-Higashi syndrome and autism. Mutations in the BCD1 gene broaden cortical organelle domains as they assemble during pre-division development. The Bcd1 protein localizes to membrane pockets at the base of every cilium that are active in endocytosis. PKA activity has been shown to promote endocytosis in other organisms, so we blocked clathrin-mediated endocytosis (using 'dynasore') and inhibited PKA (using H89). In both cases, treatment produced partial phenocopies of the bcd1 pattern mutant. This study supports a model in which the dimensions of diverse cortical organelle assembly-platforms may be determined by regulated balance between constitutive exocytic delivery and PKA-regulated endocytic retrieval of organelle materials and determinants.

Drosophila rugoseIs a Functional Homolog of MammalianNeurobeachinand Affects Synaptic Architecture, Brain Morphology, and Associative Learning

Neurobeachin (Nbea) is implicated in vesicle trafficking in the regulatory secretory pathway, but details on its molecular function are currently unknown. We have used Drosophila melanogaster mutants for rugose (rg), the Drosophila homolog of Nbea, to further elucidate the function of this multidomain protein. Rg is expressed in a granular pattern reminiscent of the Golgi network in neuronal cell bodies and colocalizes with transgenic Nbea, suggesting a function in secretory regulation. In contrast to Nbea(-/-) mice, rg null mutants are viable and fertile and exhibit aberrant associative odor learning, changes in gross brain morphology, and synaptic architecture as determined at the larval neuromuscular junction. At the same time, basal synaptic transmission is essentially unaffected, suggesting that structural and functional aspects are separable. Rg phenotypes can be rescued by a Drosophila rg+ transgene, whereas a mouse Nbea transgene rescues aversive odor learning and synaptic architecture; it fails to rescue brain morphology and appetitive odor learning. This dissociation between the functional redundancy of either the mouse or the fly transgene suggests that their complex composition of numerous functional and highly conserved domains support independent functions. We propose that the detailed compendium of phenotypes exhibited by the Drosophila rg null mutant provided here will serve as a test bed for dissecting the different functional domains of BEACH (for beige and human Chediak-Higashi syndrome) proteins, such as Rugose, mouse Nbea, or Nbea orthologs in other species, such as human.

Identification of proteins in the ceroid-like autofluorescent aggregates from liver lysosomes of Beige, a mouse model for human Chediak–Higashi syndrome

Chediak–Higashi syndrome is characterized by oculocutaneous albinism, a bleeding tendency and severe recurrent infections. Age-dependent formations of autofluorescent ceroid-like substances have been noted in a variety of tissues. In this study, we isolated an autofluorescent ceroid-like aggregate from purified Beige mouse liver lysosomes and analyzed the composition of the aggregate by ion trap mass-spectrometry. In addition to lysosomal proteins, this aggregate contains proteins normally localized in the ER, mitochondria, peroxisomes, and the cytosol. Bip, a luminal ER protein was abundant in lysosomal ceroid. The ER, mitochondria, and cytosol proteins could arise in lysosomes through stimulation of autophagy, but we found no differences between normal and CHS fibroblasts in the degree of lysosomal acidity and in the level of conversion of soluble microtubular-associated protein 1 light chain 3 type I to membrane-associated type II, an accepted probe for hyper-autophagy suggesting that ceroid formation is unlikely to arise via this mechanism.

Grey, a novel mutation in the murine Lyst gene, causes the beige phenotype by skipping of exon 25

The murine beige mutant phenotype and the human Chediak-Higashi syndrome are caused by mutations in the murine Lyst (lysosomal trafficking regulator) gene and the human CHS gene, respectively. In this report we have analyzed a novel murine mutant Lyst allele, called Lyst(bg-grey), that had been found in an ENU mutation screen and named grey because of the grey coat color of affected mice. The phenotype caused by the Lyst(bg-grey) mutation was inherited in a recessive fashion. Melanosomes of melanocytes associated with hair follicles and the choroid layer of the eye, as well as melanosomes in the neural tube-derived pigment epithelium of the retina, were larger and irregularly shaped in homozygous mutants compared with those of wild-type controls. Secretory vesicles in dermal mast cells of the mutant skin were enlarged as well. Test crosses with beige homozygous mutant mice (Lyst(bg)) showed that double heterozygotes (Lyst(bg)/Lyst(bg-grey)) were phenotypically indistinguishable from either homozygous parent, demonstrating that the ENU mutation was an allele of the murine Lyst gene. RT-PCR analyses revealed the skipping of exon 25 in Lyst(bg-grey) mutants, which is predicted to cause a missense D2399E mutation and the loss of the following 77 amino acids encoded by exon 25 but leave the C-terminal end of the protein intact. Analysis of the genomic Lyst locus around exon 25 showed that the splice donor at the end of exon 25 showed a T-to-C transition point mutation. Western blot analysis suggests that the Lyst(bg-grey) mutation causes instability of the LYST protein. Because the phenotype of Lyst(bg) and Lyst(bg-grey) mutants is indistinguishable, at least with respect to melanosomes and secretory granules in mast cells, the Lyst(bg-grey) mutation defines a critical region for the stability of the murine LYST protein.

Establishment of a Set of Combined Immunodeficient DA/Slc-Foxn1rnu Lystbg Congenic Rat Strains

The congenitally athymic nude rat is used for studying cancer and transplantation owing to its hairlessness and T-cell defective function caused by the Foxn1(rnu) gene. However, NK cell activity of the nude rat is markedly increased. It is known that NK cells play a major role in rejection of xenografts and in cytotoxicity against tumor cells. Thus, the athymic nude rat with impaired NK cell activity should be a useful model for extensive studies. The DA-Lyst(bg)/Lyst(bg) rat, a model for human Chediak-Higashi syndrome (CHS) is characterized by diluted-coat color and impairment of NK cell activity. We planned to establish a combined immunodeficient double mutant rat introgressed with the Foxn1(rnu) and Lyst(bg) genes and a set of congenic strains having an identical genetic backgrounds simultaneously. Based on the phenotypic and genetic characteristics of the parental rat strains, the new strains were produced using continuous backcross and diagnosis with molecular genetic techniques. Each disease gene was diagnosed with PCR-RFLP or the long-nested PCR method. Furthermore, we used a marker-assisted congenic strategy based on scanning the genetic backgrounds of the parental rats with 461 rat microsatellite markers. We think that the newly established DA/Slc-Foxn1(rnu)/Foxn1(rnu) Lyst(bg)/Lyst(bg) double mutant will be useful as a severe disease model for human CHS, and the set of DA/Slc-Foxn1(rnu) Lyst(bg) congenic strains which have impaired NK cell activity and/or defective T cell function should be useful for studying in cancer research, xenotransplantation, immune function and other wide-ranging studies.

The role of BEACH proteins in Dictyostelium.

The BEACH family of proteins is a novel group of proteins with diverse roles in eukaryotic cells. The identifying feature of these proteins is the BEACH domain named after the founding members of this family, the mouse beige and the human Chediak-Higashi syndrome proteins. Although all BEACH proteins share a similar structural organization, they appear to have very distinct cellular roles, ranging from lysosomal traffic to apoptosis and cytokinesis. Very little is currently known about the function of most of these proteins, few binding-partner proteins have been identified, and no molecular mechanism for any of these proteins has been discovered. Thus, it is important to establish good model systems for the study of these novel proteins. Dictyostelium contains six BEACH proteins that can be classified into four subclasses. Two of them, LvsA and LvsB, have clearly distinct roles in the cell. LvsA is localized on the contractile vacuole membrane and is essential for cytokinesis and osmoregulation. LvsB is most similar in sequence to the mammalian beige/Chediak-Higashi syndrome proteins and shares with them a common function in lysosomal trafficking. Structural and functional analysis of these proteins in Dictyostelium will help elucidate the function of this enigmatic novel family of proteins.

A New Beige Mutant Rat ACI/N-Lystbg-Kyo.

A new beige-like coat color mutant was identified in the ACI/N rat colony. Other features characteristic of beige mutants, such as giant granule cells in various tissues, and prolonged bleeding time were also observed. The genetic complementation test, mating beige-like mutant with the authentic beige mutant rat, DA/Ham-Lystbg, revealed that the mutant gene is allelic to Lystbg. The new beige mutant allele was denoted Lystbg-Kyo. Molecular genetic analysis revealed deletion of exons 28, 29, and 30 of the Lyst gene owing to recombination between L1 elements in the mutant rats. Although the deletion was similar to that identified in DA/Ham-Lystbg rats, the putative deletion break points in L1 elements were different in the two strains. Further characterization of the ACI/N-Lystbg-Kyo rats should make it useful as an animal model for human Chediak-Higashi syndrome.

Localization of the locus responsible for Chediak-Higashi syndrome in cattle to bovine chromosome 28.

Chediak-Higashi syndrome in Japanese black cattle is a hereditary disease with prolonged bleeding time and partial albinism. In the present study, we mapped the locus responsible for the disease (CHS) by linkage analysis using microsatellite genotypes of paternal half-sib pedigrees obtained from commercial herds. Analysis revealed significant linkage between the CHS locus and marker loci on the proximal end of bovine chromosome 28. The CHS locus was mapped on the region incorporating the microsatellite markers BMC6020, BM2892, and RM016 with recombination fraction 0 and lod score 4.9-11.2. We also assigned the bovine CHS1/LYST, the homologue of the gene responsible for human Chediak-Higashi syndrome, to bovine chromosome 28 using a bovine/murine somatic cell hybrid panel. These findings suggest that a mutation in the CHS1/LYST gene is likely to be responsible for Chediak-Higashi syndrome in Japanese black cattle.

Clinical, Molecular, and Cell Biological Aspects of Chediak–Higashi Syndrome

Chediak–Higashi syndrome (CHS) is a rare autosomal recessive disorder characterized by variable degrees of oculocutaneous albinism, easy bruisability, and bleeding as a result of deficient platelet dense bodies, and recurrent infections, with neutropenia, impaired chemotaxis and bactericidal activity, and abnormal NK cell function. Neurologic involvement is variable, but often includes peripheral neuropathy. Most patients also undergo an “accelerated phase,” which is a nonmalignant lymphohistiocytic infiltration of multiple organs resembling lymphoma. Death often occurs in the first decade from infection, bleeding, or development of the accelerated phase. The hallmark of CHS is the presence of huge cytoplasmic granules in circulating granulocytes and many other cell types. These granules are peroxidase-positive and contain lysosomal enzymes, suggesting that they are giant lysosomes or, in the case of melanocytes, giant melanosomes. The underlying defect in CHS remains elusive, but the disorder can be considered a model for defects in vesicle formation, fusion, or trafficking. Because the beige mouse demonstrates many characteristics similar to those of human CHS patients, including dilution of coat color, recurrent infections, and the presence of giant granules, it is considered the animal homologue of CHS. The beige gene, Lyst, was mapped and sequenced in 1996, prompting identification of the human LYST gene on chromosome 1q42. Lyst and LYST show 86.5% sequence homology. LYST encodes a 429 kDa protein with a function that remains unknown, but the source of extensive speculation among students of cell biology.

Deletion in the beige gene of the beige rat owing to recombination between LINE1s.

We have determined the molecular genetic basis of the rat beige mutant, a model for human Chediak-Higashi syndrome. Deletion of a 578-bp sequence, which led to a frame shift and a presumably non-functional truncated BEIGE protein, was identified in beige cDNA. The beige rat had a deletion of about 20 kb of genomic DNA, including three exons, which constitute the deleted 578-bp cDNA fragment. LINE1s (Long Interspersed Nucleolar Element 1) were identified at the site of the deletion. Consensus recognition sequences for DNA topoisomerase I were clustered at the putative deletion junction sites in LINE1s. We conclude that the deletion in the beige gene mediated by recombination between LINE1s is the causative mutation in the beige rat. The recombination might have been induced by DNA topoisomerase I and the extensive sequence homology between LINE1s.

Genetic defects in Chediak-Higashi syndrome and the beige mouse.

Chediak-Higashi syndrome (CHS) is a rare, autosomal recessive, multisystem disorder in which severe immune deficits are accompanied by abnormalities of pigmentation, blood clotting, and neurologic function. There is no specific treatment, and without bone marrow transplantation, most patients succumb to frequent bacterial infections or to a lymphoproliferative syndrome that appears to result principally from lack of natural killer cell function. Disorders similar to human CHS occur in many mammalian species, the most important being the beige mouse, long considered a likely homologue of human CHS. This supposition has recently been confirmed by the mapping, cloning, and mutation analysis of the homologous human CHS1 and mouse beige genes. Identification of the human CHS1 gene, and the availability of a ready mouse model for human CHS, will likely facilitate investigation of the disease pathophysiology and the development of novel and specific treatments for the disorder.

The Physical and Genetic Map Surrounding theLystGene on Mouse Chromosome 13

During the recent cloning of the mouseLystgene we developed both a high-resolution genetic map and a complete YAC and BAC contig of theLystcritical region on mouse Chromosome 13. We also report the mapping of the human homologue of the mouseLystgene (LYST) to 1q43. These data are consistent withLYSTbeing the gene for the human Chediak–Higashi Syndrome and strengthen the synteny relationship between MMU13 and human 1q43.

Comparative Mapping in thebeige–satinRegion of Mouse Chromosome 13

The proximal end of mouse chromosome (Chr) 13 contains regions conserved on human chromosomes 1q42–q44, 6p23–p21, and 7p22–p13. This region also contains mutations that may be models for human disease, includingbeige(human Chediak–Higashi syndrome). An interspecific backcross of SB/Le andMus spretusmice was used to generate a molecular genetic linkage map of mouse chromosome 13 with an emphasis on the proximal region includingbeige(bg) andsatin(sa). This map provides the gene order of the two phenotypic markersbgandsarelative to restriction fragment length polymorphisms and simple sequence length polymorphisms in 131 backcross animals. In parallel, we have created a physical map of the region usingNidogen(Nid) as a molecular starting point for cloning a YAC contig that was used to identify thebeigegene. The physical map provides the fine-structure order of genes and anonymous DNA fragments that was not resolved by the genetic linkage mapping. The results show that thebgregion of mouse Chr 13 is highly conserved on human Chr 1q42–q44 and provide a starting point for a complete functional analysis of the entirebg–sainterval.

Correction: Identification of the homologous beige and Chediak–Higashi syndrome genes

Nature 382, 262-265 (1996) THE Genbank accession number of the mouse beige gene (Lysf) was incorrect. It is L77884. The Genbank accession number of the human Chediak-Higashi syndrome gene (LYST) is L77889.

Identification and mutation analysis of the complete gene for Chediak-Higashi syndrome.

Chediak-Higashi syndrome (CHS) is a rare, autosomal recessive disorder characterized by hypopigmentation, severe immunologic deficiency with neutropenia and lack of natural killer (NK) cells, a bleeding tendency and neurologic abnormalities. Most patients die in childhood. The CHS hallmark is the occurrence of giant inclusion bodies and organelles in a variety of cell types, and protein sorting defects into these organelles. Similar abnormalities occur in the beige mouse, the proposed model for human CHS. Two groups have recently reported the identification of the beige gene, however the two cDNAs were not at all similar. Here we describe the sequence of a human cDNA homologous to mouse beige, identify pathologic mutations and clarify the discrepancies of the previous reports. Analysis of the CHS polypeptide demonstrates that its modular architecture is similar to the yeast vacuolar sorting protein, VPS15.

Identification of the homologous beige and Chediak-Higashi syndrome genes.

Vesicular transport to and from the lysosome and late endosome is defective in patients with Chediak-Higashi syndrome (CHS) and in mutant beige (bg) mice. CHS and bg cells have giant, perinuclear vesicles with characteristics of late endosomes and lysosomes that arise from dysregulated homotypic fusion. CHS and bg lysosomes also exhibit compartmental missorting of proteins, such as elastase, glucuronidase and cathepsin G. Lyst, a candidate gene for bg, was identified by direct complementary DNA selection from a yeast artificial chromosome (YAC) clone containing a 650-kilobase segment of the bg-critical region on mouse chromosome 13. Lyst is disrupted by a 5-kilobase deletion in bg mice, and Lyst messenger RNA is markedly reduced in bg homozygotes. The homologous human gene, LYST, is highly conserved with mouse Lyst, and contains a frame-shift mutation at nucleotides 117-118 of the coding domain in a CHS patient. Thus bg mice and human CHS patients have homologous disorders associated with Lyst mutations. Lyst encodes a protein with a carboxy-terminal prenylation motif and multiple potential phosphorylation sites. Lyst protein is predicted to form extended helical domains, and has a region of sequence similar to stathmin, a coiled-coil phosphoprotein thought to act as a relay integrating cellular signal response coupling.

Animal models: Prenatal diagnosis of Chediak‐Higashi syndrome in the cat by evaluation of cultured chorionic cells

The autosomal recessive disease Chediak-Higashi syndrome (CHS) is a progressive and generally fatal disease of humans. The underlying genetic defect in CHS is unknown and prenatal diagnostic methods have not been applied to this disease. The purpose of this study was to determine if CHS chorionic cells expressed a characteristic of CHS--enlarged lysosomes--that would permit the prenatal diagnosis of the disease. Cats with CHS, which have been shown to be homologous with human CHS, were used as the model system in this study. Chorionic tissue samples were obtained from CHS and control cat fetuses and cultures of cells were established. Acid phosphatase was utilized as a marker of lysosomes and cultures of chorionic fibroblasts from CHS and control fetuses were stained histochemically for acid phosphatase. The diameter of the largest lysosomes in 150 cells of each fetus was determined. The mean (+/- SD) diameter (in microns) of the largest lysosomes of normal fetuses was 0.9 +/- 0.13 (range 0.5-7.0 microns), whereas the mean diameter of lysosomes in CHS chorionic cells was 3.9 +/- 0.65 microns (range 0.5-25 microns). These means were significantly different (P less than 0.0001). These data suggest that it should be possible to diagnose human CHS in the first trimester by chorionic villus sampling.

Chediak-Higashi syndrome in the cat: Prenatal diagnosis by evaluation of amniotic fluid cells

Chediak-Higashi syndrome (CHS) is an autosomal recessive disease in humans, cats, and 8 other species. The homology of CHS in humans and cats has been demonstrated. Since human CHS is a progressive, serious, and eventually fatal disease, a method for prenatal diagnosis would be desirable. This study was designed to determine whether CHS could be diagnosed prenatally by examination of amniotic fluid cells. The amniotic fluid samples were obtained from CHS and control cat fetuses on the 45th day of gestation and cultures of cells were established. Because the underlying enzyme deficiency in CHS has not been identified, it was necessary to use a secondary manifestation of the syndrome in these studies. The secondary manifestation used was the characteristic enlargement of lysosomes associated with the disease. The lysosomes of these cells were stained by acid phosphatase histochemistry and the diameter of the largest lysosome in each cell was measured by light microscopy with a calibrated ocular micrometer. The diameters of the largest lysosomes in cells of normal fetuses ranged from 0.5 to 7.0 micron (means ranged from 0.9 to 1.8 micron), whereas the diameter of the largest lysosomes in the cells of CHS fetuses ranged from 0.5 to 30 microns (means ranged from 6.4 to 12.8 microns). The approximate t-test for independent samples with unequal variances disclosed that the largest acid phosphatase-positive lysosomes in amniotic fluid cells of CHS cat fetuses were significantly larger than the lysosomes in the cells of normal cat fetuses (P less than 0.0001). This information should, by extrapolation, provide the basis for the prenatal diagnosis of human CHS by amniocentesis.

Impaired antibody response of C57BL/6 beige mutant mice to a thymus-independent type 2 antigen

The murine equivalent of the human Chediak-Higashi Syndrome is the beige (bg) mutant in the C57BL/6 (B6) background. Besides the well-known lack of natural killer (NK) activity in bg-homozygous mice, functional abnormalities of T cells, macrophages and various granulocytes have been reported. With the exception of one study indicating a decreased in vitro response to lipopolysaccharide, there is no report concerning the B cell compartment of the beige mutant. The in vivo anti-trinitrophenyl antibody response to a TI-2 antigen (TNP-Ficoll) was found here to be significantly lower in B6 beige than in B6 wild mice, although both strains responded similarly to an analogous TD antigen (TNP-ovalbumin). Since the marginal zone macrophages of the spleen were previously shown to be essential for the initiation of antibody responses to TI-2 antigens, they might be another target of the beige mutation.

A Case Report of Chediak-Higashi Syndrome Complicated with Systemic Amyloidosis and Olivo-Cerebellar Degeneration

The histopathological observations in the case of a 24 year old woman with Chediak-Higashi syndrome are described. There were characteristic features of cytoplasmic giant granules in various cells and lymphohistiocytic infiltration in various tissues. Amyloid deposits, which have not been reported previously in human Chediak-Higashi syndrome, were systemically noted and were immunohistochemically revealed to be AA type protein. Another rare complication, olivo-cerebellar degeneration, was observed in the central nervous system not associated with lymphohistiocytic infiltration. These complications may develop in long surviving patients with Chediak-Higashi syndrome.