Clinico‐molecular analysis of eleven patients with Hermansky–Pudlak type 5 syndrome,a mild form of HPS

Hermansky–Pudlak syndrome (HPS), first described in 1959, is a rare form of syndromic oculocutaneous albinism associated with bleeding diathesis and in some cases pulmonary fibrosis and granulomatous colitis. All 10 HPS types are caused by defects in vesicle trafficking of lysosome‐related organelles (LRO) proteins. The HPS5 protein associates with HPS3 and HPS6 to form the biogenesis of lysosome‐related organelles complex‐2 (BLOC‐2). Here, we report the clinical and genetic data of 11 patients with HPS‐5 analyzed in our laboratory. We report 11 new pathogenic variants. The 11 patients present with ocular features that are typical for albinism, with mild hypopigmentation, and with no other major complication, apart from a tendency to bleed. HPS‐5 therefore appears as a mild form of HPS, which is often clinically undistinguishable from mild oculocutaneous or ocular forms of albinism. Molecular analysis is therefore required to establish the diagnosis of this mild HPS form, which has consequences in terms of prognosis and of clinical management of the patients.     

Genetic variants and mutational spectrum of Chinese Hermansky–Pudlak syndrome patients

Hermansky–Pudlak syndrome (HPS) is a rare recessive disorder characterized by oculocutaneous albinism or ocular albinism, bleeding diathesis, and other symptoms such as colitis and pulmonary fibrosis. Eleven causative genes have been identified for HPS‐1–HPS‐11 subtypes in humans. We have identified 16 newly reported patients including the first HPS‐2 case in the Chinese population. In a total of 40 HPS patients, hypopigmentation was milder in HPS‐3, HPS‐5, and HPS‐6 patients than in HPS‐1 and HPS‐4 patients. HPS‐1 accounted for 47.5% (19 of 40) of HPS cases which is the most common subtype. Exons 11 and 19 were the hotspots of the HPS1 gene mutations. In total, 55 allelic variants were identified in HPS1–HPS6 gene, of which 17 variants were previously unreported. These results will be useful for the evaluation of the relationship between HPS genotypes and phenotypes, and for the precise intervention of HPS patients in the Chinese population.     

Instability of BLOC‐2 and BLOC‐3 in Chinese patients with Hermansky‐Pudlak syndrome

Hermansky‐Pudlak syndrome (HPS) is a rare recessive disorder characterized by oculocutaneous albinism (OCA) or ocular albinism (OA), bleeding tendency, and other symptoms due to multiple defects in tissue‐specific lysosome‐related organelles. Ten HPS subtypes have been characterized with mutations in HPS1 to HPS10, which encode the subunits of BLOC‐1, ‐2, ‐3, and AP‐3. Using next‐generation sequencing (NGS), we have screened 100 hypopigmentation genes in OCA or OA patients and identified four HPS‐1, one HPS‐3, one HPS‐4, one HPS‐5, and three HPS‐6. The HPS‐4 case is the first report in the Chinese population. Among these 20 mutational alleles, 16 were previously unreported alleles (6 in HPS1, 1 in HPS3, 2 in HPS4, 2 in HPS5, and 5 in HPS6). BLOC‐2 and BLOC‐3 were destabilized due to the mutation of these HPS genes which are so far the only reported causative genes in Chinese HPS patients, in which HPS‐1 and HPS‐6 are the most common subtypes. The mutational spectrum of Chinese HPS is population specific.     

A BLOC-1 mutation screen reveals a novel BLOC1S3 mutation in Hermansky-Pudlak Syndrome type 8

Hermansky-Pudlak Syndrome (HPS) is a genetically heterogeneous disorder of lysosome-related organelle biogenesis and is characterized by oculocutaneous albinism and a bleeding diathesis. Over the past decade, we screened 250 patients with HPS-like symptoms for mutations in the genes responsible for HPS subtypes 1-6. We identified 38 individuals with no functional mutations, and therefore, we analyzed all eight genes encoding the biogenesis of lysosome-related organelles complex-1 (BLOC-1) proteins in these individuals. Here, we describe the identification of a novel nonsense mutation in BLOC1S3 (HPS-8) in a 6-yr-old Iranian boy. This mutation caused nonsense-mediated decay of BLOC1S3 mRNA and destabilized the BLOC-1 complex. Our patient's melanocytes showed aberrant localization of TYRP1, with increased plasma membrane trafficking. These findings confirm a common cellular defect for HPS patients with defects in BLOC-1 subunits. We identified only two patients with BLOC-1 defects in our cohort, suggesting that other HPS genes remain to be identified.     

NGS‐based 100‐gene panel of hypopigmentation identifies mutations in Chinese Hermansky–Pudlak syndrome patients

Hermansky–Pudlak syndrome (HPS) is a rare recessive disorder characterized by hypopigmentation, bleeding diathesis, and other symptoms due to multiple defects in lysosome‐related organelles. Ten HPS subtypes have been identified with mutations in HPS1 to HPS10. Only four patients with HPS‐1 have been reported in Chinese population. Using next‐generation sequencing (NGS), we have screened 100 hypopigmentation genes and identified four HPS‐1, two HPS‐3, one HPS‐5, and three HPS‐6 in Chinese HPS patients with typical ocular or oculocutaneous albinism and the absence of platelet dense granules together with other variable phenotypes. All these patients except one homozygote were compound heterozygotes. Among these mutations, 14 were previously unreported alleles (four in HPS1, three in HPS3, two in HPS5, five in HPS6). Our results demonstrate the feasibility and utility of NGS‐based panel diagnostics for HPS. Genotyping of HPS subtypes is a prerequisite for intervention of subtype‐specific symptoms.     

A single base deletion in the SLC45A2 gene in a Bullmastiff with oculocutaneous albinism

Oculocutaneous albinism type 4 (OCA4) in humans and similar phenotypes in many animal species are caused by variants in the SLC45A2 gene, encoding a putative sugar transporter. In dog, two independent SLC45A2 variants are known that cause oculocutaneous albinism in Doberman Pinschers and several small dog breeds respectively. For the present study, we investigated a Bullmastiff with oculocutaneous albinism. The affected dog was highly inbred and resulted from the mating of a sire to its own grandmother. We obtained whole genome sequence data from the affected dog and searched specifically for variants in candidate genes known to cause albinism. We detected a single base deletion in exon 6 of the SLC45A2 gene (NM_001037947.1:c.1287delC) that has not been reported thus far. This deletion is predicted to result in an early premature stop codon. It was confirmed by Sanger sequencing and perfectly co‐segregated with the phenotype in the available family members. We genotyped 174 unrelated dogs from diverse breeds, all of which were homozygous wildtype. We therefore suggest that SLC45A2:c.1287delC causes the observed oculocutaneous albinism in the affected Bullmastiff.     

Characterization of melanosomes and melanin in Japanese patients with Hermansky–Pudlak syndrome types 1, 4, 6, and 9

Hermansky–Pudlak syndrome (HPS) is an autosomal recessive disorder characterized by oculocutaneous albinism (OCA), a bleeding tendency, and ceroid deposition. Most of the causative genes for HPS encode subunits of the biogenesis of lysosome‐related organelles complex (BLOC). In this study, we identified one patient each with HPS4, HPS6, and HPS9 by whole‐exome sequencing. Next, we analyzed hair samples from the three patients and representative patients with HPS1 and controls using electron microscopy and chemical methods. All HPS patients had fewer, smaller, and more immature melanosomes than healthy controls. Further, all patients showed reduced total melanin content and increased levels of benzothiazine‐type pheomelanin. The results of this study demonstrate the impact of the dysfunctions of BLOCs on the maturation of melanosomes and melanin levels and composition through analysis of their hair samples.     

A germline mutation in BLOC1S3/reduced pigmentation causes a novel variant of Hermansky-Pudlak syndrome (HPS8)

Hermansky-Pudlak syndrome (HPS) is genetically heterogeneous, and mutations in seven genes have been reported to cause HPS. Autozygosity mapping studies were undertaken in a large consanguineous family with HPS. Affected individuals displayed features of incomplete oculocutaneous albinism and platelet dysfunction. Skin biopsy demonstrated abnormal aggregates of melanosomes within basal epidermal keratinocytes. A homozygous germline frameshift mutation in BLOC1S3 (p.Gln150ArgfsX75) was identified in all affected individuals. BLOC1S3 mutations have not been previously described in patients with HPS, but BLOC1S3 encodes a subunit of the biogenesis of lysosome-related organelles complex 1 (BLOC-1). Mutations in other BLOC-1 subunits have been associated with an HPS phenotype in humans and/or mouse, and a nonsense mutation in the murine orthologue of BLOC1S3 causes the reduced pigmentation (rp) model of HPS. Interestingly, eye pigment formation is reported to be normal in rp, but we found visual defects (nystagmus, iris transilluminancy, foveal hypoplasia, reduced visual acuity, and evidence of optic pathway misrouting) in affected individuals. These findings define a novel form of human HPS (HPS8) and extend genotype-phenotype correlations in HPS.     

Hermansky–Pudlak Syndrome: Vesicle Formation from Yeast to Man

The disorders known as Hermansky–Pudlak syndrome (HPS) are a group of genetic diseases resulting from abnormal formation of intracellular vesicles. In HPS, dysfunction of melanosomes results in oculocutaneous albinism, and absence of platelet dense bodies causes a bleeding diathesis. In addition, some HPS patients suffer granulomatous colitis or fatal pulmonary fibrosis, perhaps due to mistrafficking of a subset of lysosomes. The impaired function of specific organelles indicates that the causative genes encode proteins operative in the formation of certain vesicles. Four such genes, HPS1, ADTB3A, HPS3, and HPS4, are associated with the four known subtypes of HPS, i.e. HPS‐1, HPS‐2, HPS‐3, and HPS‐4. ADTB3A codes for the β3A subunit of adaptor complex‐3, known to assist in vesicle formation from the trans‐Golgi network or late endosome. However, the functions of the HPS1, HPS3, and HPS4 gene products remain unknown. These three genes arose with the evolution of mammals and have no homologs in yeast, reflecting their specialized function. In contrast, all four known HPS‐causing genes have homologs in mice, a species with 14 different models of HPS, i.e. hypopigmentation and a platelet storage pool deficiency. Pursuit of the mechanism of mammalian vesicle formation and trafficking, impaired in HPS, relies upon investigation of these mouse models as well as studies of protein complexes involved in yeast vacuole formation.     

The CHiPS Domain--ancient traces for the Hermansky-Pudlak syndrome

Hermansky-Pudlak syndrome (HPS) is a rare disorder caused by malfunctions of lysosomes and specialized lysosome-related organelles, resulting primarily in oculocutaneous albinism and bleeding diathesis. The majority of the HPS genes have been described as novel, but herein we report the identification of a conserved protein family which includes human HPS4, as well as distant homologs for other HPS genes. Our results suggest that the cellular machinery involved in the HPS syndrome is ancient.     

A 4‐bp deletion promoter variant (rs984225803) is associated with mild OCA4 among Japanese patients

Oculocutaneous albinism (OCA) type 4 is one of the most common types of albinism among Japanese population. In some patients who were clinically diagnosed with OCA, we have found a heterozygous pathological mutation in the coding region of SLC45A2, the gene responsible for OCA4, not leading to a DNA‐based diagnosis. In this study, we evaluated pathological variants in the promoter region of SLC45A2 in these patients. The results indicated that the majority of the patients had a 4‐bp deletion in the said region (c.‐492_489delAATG; GenBank accession number: NM_016180 ; rs984225803) in the contralateral allele. These patients displayed a mild phenotype, especially regarding eye manifestations. The results of the luciferase reporter assay and electrophoretic mobility shift assay supported the pathological role of the variant. In addition, four of 220 alleles in Japanese normal control subjects also showed the deletion variant, indicating that this variant could possibly be a skin color‐associated variant.     

The rat Ruby (<Emphasis Type="Italic">R</Emphasis>) locus is <Emphasis Type="Italic">Rab38</Emphasis>: identical mutations in Fawn-hooded and Tester-Moriyama rats derived from an ancestral Long Evans rat sub-strain

Hermansky-Pudlak syndrome (HPS) is a group of rare, recessive disorders in which oculocutaneous albinism, progressive pulmonary fibrosis, bleeding diathesis, and other abnormalities result from defective biogenesis of multiple cytoplasmic organelles. Seven different HPS genes are known in humans; in mouse, at least 16 loci are associated with HPS-like mutant phenotypes. In the rat, only two HPS models are known, Fawn-hooded (FH) and Tester Moriyama (TM), non-complementing strains in which HPS-like hypopigmentation and platelet storage pool deficiency result from a mutation of the Ruby (red eyed dilution; R) locus on Chromosome (Chr) 1. We have identified the R locus as the Rab38 gene, establishing that rat R is homologous to mouse chocolate (cht). Further, we show that FH and TM rats have identical Rab38 Met1Ile mutations, occurring on an identical Chr 1 marker allele haplotype, indicating that these two strains derive from a common ancestor. This ancestor appears to have been a sub-strain of the outbred Long Evans (LE) strain, and several modern LE sub-strains carry the Rab38 Met1Ile R mutation on the same Chr 1 marker haplotype. These findings have significant implications for the many past and ongoing studies that involve the FH and LE-derivative rat strains. Hermansky-Pudlak syndrome (HPS; MIM 203300) is a group of autosomal recessive diseases in which oculocutaneous albinism (OCA), progressive and fatal pulmonary fibrosis, and bleeding diathesis due to platelet storage pool deficiency result from defects in the biogenesis of specific cytoplasmic organelles and granules: melanosomes, lysosomes, and platelet dense granules (reviewed in Spritz 1999, 2000; Spritz et al. 2003). In humans, seven different HPS genes are known (Oh et al. 1996; DellAngelica et al. 1999; Anikster et al. 2001; Suzuki et al. 2002; Li et al. 2003; Zhang et al. 2003). In the mouse, at least 16 loci associated with HPS-like mutant phenotypes are known, seven of which are homologous to the human HPS loci (Swank et al. 1998; Bennett and Lamoreux 2003). The nucleotide sequence data reported in this paper have been submitted to GenBank and have been assigned the accession number AY425759. (Naoki Oiso) Present address: Department of Dermatology, Saiseikai Tondabayashi Hospital, Tondabayashi, Osaka 584-0082, Japan.     

Association of the Hermansky-Pudlak syndrome type-3 protein with clathrin

Background  

Hermansky-Pudlak syndrome (HPS) is a disorder of lysosome-related organelle biogenesis characterized by oculocutaneous albinism and prolonged bleeding. These clinical findings reflect defects in the formation of melanosomes in melanocytes and dense bodies in platelets. HPS type-3 (HPS-3) results from mutations in the HPS3 gene, which encodes a 1004 amino acid protein of unknown function that contains a predicted clathrin-binding motif (LLDFE) at residues 172–176.     

Interaction of MC1R and PMEL alleles on solid coat colors in Highland cattle

Six solid colors occur in Highland cattle: black, dun, silver dun and red, yellow, and white. These six coat colors are explained by a non‐epistatic interaction of the genotypes at the MC1R and PMEL genes. A three base pair deletion in the PMEL gene leading to the deletion of a leucine from the signal peptide is observed in dilute‐colored Highland cattle (c.50_52delTTC, p.Leu18del). The mutant PMEL allele acts in a semi‐dominant manner. Dun Galloway cattle also have one copy of the deletion allele, and silver dun Galloway cattle have two copies. The presence of two adjacent leucine residues at the site of this deletion is highly conserved in human, horse, mouse and chicken as well as in cattle with undiluted coat colors. Highland and Galloway cattle thus exhibit a similar dose‐dependent dilution effect based on the number of PMEL :c.50_51delTTC alleles, as Charolais cattle with PMEL :c.64G>A alleles. The PMEL :c.64G>A allele was not found in Highland or Galloway cattle.     

NGS‐based targeted resequencing identified rare subtypes of albinism: Providing accurate molecular diagnosis for Japanese patients with albinism

Albinism, which is commonly inherited as an autosomal recessive trait, is characterized by a reduction or absence of melanin in the eyes, skin, and hair. To date, more than 20 causal genes for albinism have been identified; thus, the accurate diagnosis of albinism requires next‐generation sequencing (NGS). In this study, we analyzed 46 patients who tested negative for oculocutaneous albinism (OCA)1–4 and Hermansky‐Pudlak syndrome (HPS)1 based on conventional analysis, in addition to 28 new Japanese patients, using NGS‐based targeted resequencing. We identified a genetic background for albinism in 18 of the 46 patients (39%), who were previously tested negative according to the conventional analysis. In addition, we unveiled a genetic predisposition toward albinism in 23 of the 28 new patients (82%). We identified six patients with rare subtypes of albinism, including HPS3, HPS4, and HPS6, and found 12 novel pathological mutations in albinism‐related genes. Furthermore, most patients who were not diagnosed with albinism by the NGS analysis showed mild manifestations of albinism without apparent eye symptoms and harbored only one heterozygous mutation, occasionally in combination with skin‐color associated gene variants.     

Early Origin of Genes Encoding Subunits of Biogenesis of Lysosome‐related Organelles Complex‐1, ‐2 and ‐3

Biogenesis of lysosome‐related organelles complex (BLOC)‐1, ‐2 and ‐3 are three multi‐subunit protein complexes that are deficient in various forms of Hermansky‐Pudlak syndrome, a human disease characterized by abnormal formation of lysosome‐related organelles. Contrasting views have arisen on the evolutionary origin of these protein complexes. One view is that the BLOCs represent a recent evolutionary ‘acquisition’ unique to metazoans. However, the yeast proteins Mon1, Ccz1 and She3 have been reported to display homology to the HPS1 and HPS4 subunits of BLOC‐3 and the BLOS2 subunit of BLOC‐1, respectively. In this work, we have systematically searched for orthologs of BLOC subunits in the annotated genomes of over 160 species of eukaryotes, including metazoans and fungi in the Opisthokonta group as well as highly divergent organisms. We have found orthologs of six of the eight BLOC‐1 subunits, two of the three BLOC‐2 subunits, and the two BLOC‐3 subunits, in some non‐opisthokonts such as Dictyostelium discoideum, suggesting an early evolutionary origin for these complexes. On the other hand, we have obtained no evidence in support of the notion that yeast She3 would be an ortholog of BLOS2, and found that yeast Mon1 and Ccz1, despite displaying restricted homology to portions of HPS1 and HPS4, are unlikely to represent the orthologs of these BLOC‐3 subunits. Potential orthologs of Mon1 and Ccz1 were found in humans and several other eukaryotes.     

Hermansky–Pudlak Syndrome and Pale Ear: Melanosome‐Making for the Millennium

Hermansky–Pudlak syndrome (HPS) is a rare autosomal recessive disorder characterized principally by oculocutaneous albinism, a bleeding tendency, and a ceroid‐lipofuscin lysosomal storage disease. These clinical manifestations of HPS are associated with defects of multiple cytoplasmic organelles – melanosomes, platelet granules, and lysosomes – suggesting that the HPS gene product is involved in some shared feature of the biogenesis or functions of these diverse organelles. The HPS gene has been cloned, and a number of pathologic mutations of the gene have been identified. Functional studies indicate that the HPS protein is part of a high‐molecular weight complex involved in the biogenesis of early melanosomes. Additional disorders with similarities to HPS have been identified in man, mouse, flies, and yeast, and it is rapidly becoming clear that understanding these disorders will shed new light on the mechanisms by which cells traffic newly synthesized proteins through the cytoplasm to assemble functional organelles.     

Germline variants in oculocutaneous albinism genes and predisposition to familial cutaneous melanoma

Approximately 1%–2% of cutaneous melanoma (CM) is classified as strongly familial. We sought to investigate unexplained CM predisposition in families negative for the known susceptibility genes using next‐generation sequencing of affected individuals. Segregation of germline variants of interest within families was assessed by Sanger sequencing. Several heterozygous variants in oculocutaneous albinism (OCA) genes: TYR, OCA2, TYRP1 and SLC45A2, were present in our CM cohort. OCA is a group of autosomal recessive genetic disorders, resulting in pigmentation defects of the eyes, hair and skin. Missense variants classified as pathogenic for OCA were present in multiple families and some fully segregated with CM. The functionally compromised TYR p.T373K variant was present in three unrelated families. In OCA2, known pathogenic variants: p.V443I and p.N489D, were present in three families and one family, respectively. We identified a likely pathogenic SLC45A2 frameshift variant that fully segregated with CM in a family of four cases. Another four‐case family harboured cosegregating variants (p.A24T and p.R153C) of uncertain functional significance in TYRP1. We conclude that rare, heterozygous variants in OCA genes confer moderate risk for CM.     

Hermansky-Pudlak syndrome type 3 in Ashkenazi Jews and other non-Puerto Rican patients with hypopigmentation and platelet storage-pool deficiency

Hermansky-Pudlak syndrome (HPS), consisting of oculocutaneous albinism and a bleeding diathesis due to the absence of platelet dense granules, displays extensive locus heterogeneity. HPS1 mutations cause HPS-1 disease, and ADTB3A mutations cause HPS-2 disease, which is known to involve abnormal intracellular vesicle formation. A third HPS-causing gene, HPS3, was recently identified on the basis of homozygosity mapping of a genetic isolate of HPS in central Puerto Rico. We now describe the clinical and molecular characteristics of eight patients with HPS-3 who are of non-Puerto Rican heritage. Five are Ashkenazi Jews; three of these are homozygous for a 1303+1G-->A splice-site mutation that causes skipping of exon 5, deleting an RsaI restriction site and decreasing the amounts of mRNA found on northern blotting. The other two are heterozygous for the 1303+1G-->A mutation and for either an 1831+2T-->G or a 2621-2A-->G splicing mutation. Of 235 anonymous Ashkenazi Jewish DNA samples, one was heterozygous for the 1303+1G-->A mutation. One seven-year-old boy of German/Swiss extraction was compound heterozygous for a 2729+1G-->C mutation, causing skipping of exon 14, and resulting in a C1329T missense (R396W), with decreased mRNA production. A 15-year-old Irish/English boy was heterozygous for an 89-bp insertion between exons 16 and 17 resulting from abnormal splicing; his fibroblast HPS3 mRNA is normal in amount but is increased in size. A 12-year-old girl of Puerto Rican and Italian background has the 3,904-bp founder deletion from central Puerto Rico on one allele. All eight patients have mild symptoms of HPS; two Jewish patients had received the diagnosis of ocular, rather than oculocutaneous, albinism. These findings expand the molecular diagnosis of HPS, provide a screening method for a mutation common among Jews, and suggest that other patients with mild hypopigmentation and decreased vision should be examined for HPS.     

Hermansky–Pudlak syndrome: a disease of protein trafficking and organelle function

The Hermansky–Pudlak syndrome (HPS) is a collection of related autosomal recessive disorders which are genetically heterogeneous. There are eight human HPS subtypes, characterized by oculocutaneous albinism and platelet storage disease; prolonged bleeding, congenital neutropenia, pulmonary fibrosis, and granulomatous colitis can also occur. HPS is caused primarily by defects in intracellular protein trafficking that result in the dysfunction of intracellular organelles known as lysosome‐related organelles. HPS gene products are all ubiquitously expressed and all associate in various multi‐protein complexes, yet HPS has cell type‐specific disease expression. Impairment of specialized secretory cells such as melanocytes, platelets, lung alveolar type II epithelial cells and cytotoxic T cells are observed in HPS. This review summarizes recent molecular, biochemical and cell biological analyses together with clinical studies that have led to the correlation of molecular pathology with clinical manifestations and led to insights into such diverse disease processes such as albinism, fibrosis, hemorrhage, and congenital neutropenia.