The diagnostic approaches for Hermansky-Pudlak Syndrome (HPS) include genetic sequencing, immunoblotting, electron microscopy (EM), and flow cytometry with mepacrine staining. However, these methods are often impractical for routine clinical use due to high cost, technical complexity, and limited availability. In this study, we evaluated dense granules (DGs) function in HPS mouse models using flow cytometry with mepacrine and FluoZin-3 staining. We then developed a standardized, practical flow cytometry-based protocol and validated it in patients with HPS and oculocutaneous albinism (OCA), which were confirmed by whole-mount EM. In HPS mouse models (BLOC-1, BLOC-2, BLOC-3, and AP-3 deficient mutants), mepacrine uptake was consistently reduced. FluoZin-3 fluorescence showed subtype-specific zinc dysregulation, with elevated levels in BLOC-1, BLOC-2, and AP-3 mutants but decreased levels in the BLOC-3 mutant. In contrast, the OCA-6 mouse mutant showed no significant changes in either mepacrine or FluoZin-3 uptake. Similar patterns were observed in HPS and non-syndromic OCA patients. Our findings indicate that the protocol can enable the precise diagnosis and preliminary subtype classification of HPS, while also facilitating differential diagnosis between HPS and OCA. This method offers a rapid, clinically accessible alternative to conventional diagnostic techniques and may also be applicable to other storage pool disorders with DG defects.

Photoprotective melanins in the skin are synthesized by epidermal melanocytes within specialized lysosome-related organelles called melanosomes. Melanosomes coexist with lysosomes; thus, melanocytes employ trafficking machineries that possess cell-type-specific functions to ensure correct cargo delivery to either the endolysosomal system or maturing melanosomes. Mutations in some of the protein complexes required for melanogenic cargo delivery, such as biogenesis of lysosome-related organelles complex 1 (BLOC-1), result in hypopigmentation due to mistrafficking of cargo to endolysosomes. We show that hypopigmented BLOC-1-deficient melanocytes retain melanogenic capacity that can be enhanced by treatment with cyclic adenosine monophosphate (cAMP)-elevating agents despite the mislocalization of melanogenic proteins. The melanin formed in BLOC-1-deficient melanocytes is not generated in melanosomes but rather within late endosomes/lysosomes to which some cargoes mislocalize. Although these organelles generally are acidic, a cohort of late endosomes/lysosomes have a sufficiently neutral pH to facilitate melanogenesis, perhaps due to mislocalized melanosomal transporters and melanogenic enzymes. Modulation of the pH of late endosomes/lysosomes by genetic manipulation or via treatment with lysosomotropic agents significantly enhances the melanin content of BLOC-1-deficient melanocytes. Our data suggest that upregulated expression of mistargeted cargoes leads to both increased tyrosinase expression and subsequent activity due to pH modulation facilitating the reprogramming of a subset of endolysosomes to replicate some functions of lysosome-related organelles.

Hermansky-Pudlak syndrome (HPS) is a heterogeneous group of autosomal recessive genetic disorders characterized by oculocutaneous albinism, bleeding diathesis, and variable presentation of immune deficiency and dysregulation. The pathogenesis of HPS involves mutations in genes responsible for biogenesis and trafficking of lysosome-related organelles, essential for the function of melanosomes, platelet granules, and immune cell granules. Eleven genes coding for proteins in the BLOC-1, BLOC-2, BLOC-3 and AP-3 complexes have been implicated in the pathogenesis of HPS. To date, the rare subtype HPS-7 associated with bi-allelic mutations in DTNBP1 (dysbindin) has only been reported in 9 patients. We report a novel DTNBP1 splicing mutation in a 15-month-old patient with HPS-7 phenotype and severe inflammatory bowel disease (IBD). This patient's leukocytes have undetectable dysbindin protein. We also identify dysregulated expression of several genes involved in activation of the adaptive immune response. This case underscores the emerging immunological consequences of dysbindin deficiency and suggests that DTNBP1 mutations may underlie some rare cases of very early onset IBD.

Hermansky-Pudlak syndrome (HPS) is a heterogeneous disorder combining oculocutaneous albinism (OCA) and a platelet function disorder of varying severity as its most prominent features. The genes associated with HPS encode for different BLOC- (biogenesis of lysosome-related organelles complex) complexes and for the AP-3 (adaptor protein-3) complex, respectively. These proteins are involved in maturation, trafficking, and the function of lysosome-related organelles (LROs) such as melanosomes and platelet δ-granules. Some patients with different types of HPS can develop additional complications and symptoms like pulmonary fibrosis, granulomatous colitis, and immunodeficiency. A new type of HPS has recently been identified associated with genetic alterations in the BLOC1S5 gene, which encodes the subunit Muted of the BLOC-1 complex. Our aim was to unravel the genetic defect in two siblings with a suspected HPS diagnosis (because of OCA and bleeding symptoms) using next generation sequencing (NGS). Platelet functional analysis revealed reduced platelet aggregation after stimulation with ADP and a severe secretion defect in platelet δ-granules. NGS identified a novel homozygous essential splice site variant in the BLOC1S5 gene present in both affected siblings who are descendants of a consanguine marriage. The patients exhibited no additional symptoms. Our study confirms that pathogenic variants of BLOC1S5 cause the recently described HPS type 11.

Lysosome-related organelles (LROs) are a category of secretory organelles enriched with ions such as calcium, which are maintained by ion transporters or channels. Homeostasis of these ions is important for LRO biogenesis and secretion. Hermansky-Pudlak syndrome (HPS) is a recessive disorder with defects in multiple LROs, typically platelet dense granules (DGs) and melanosomes. However, the underlying mechanism of DG deficiency is largely unknown. Using quantitative proteomics, we identified a previously unreported platelet zinc transporter, transmembrane protein 163 (TMEM163), which was significantly reduced in BLOC-1 (Dtnbp1sdy and Pldnpa)-, BLOC-2 (Hps6ru)-, or AP-3 (Ap3b1pe)-deficient mice and HPS patients (HPS2, HPS3, HPS5, HPS6, or HPS9). We observed similar platelet DG defects and higher intracellular zinc accumulation in platelets of mice deficient in either TMEM163 or dysbindin (a BLOC-1 subunit). In addition, we discovered that BLOC-1 was required for the trafficking of TMEM163 to perinuclear DG and late endosome marker-positive compartments (likely DG precursors) in MEG-01 cells. Our results suggest that TMEM163 is critical for DG biogenesis and that BLOC-1 is required for the trafficking of TMEM163 to putative DG precursors. These new findings suggest that loss of TMEM163 function results in disruption of intracellular zinc homeostasis and provide insights into the pathogenesis of HPS or platelet storage pool deficiency.