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.

Lamellar bodies (LBs) are tissue-specific lysosome-related organelles in type II alveolar cells that are the main site for the synthesis, storage, and secretion of pulmonary surfactants. Defects in pulmonary surfactants lead to a variety of respiratory and immune-related disorders. LB biogenesis is closely related to their function, but the underlying regulatory mechanism is largely unclear. Here, we found that deficiency of HPS6, a subunit of BLOC-2 (biogenesis of lysosome-related organelles complex-2), led to a reduction of the steady-state concentration of vacuolar-type H+-ATPase and an increase in the luminal pH of LBs. Furthermore, we observed increased LB size, accumulated surfactant proteins, and altered lipid profiling of lung tissue and BAL fluid due to HPS6 deficiency. These findings suggest that HPS6 regulates the distribution of vacuolar-type H+-ATPase on LBs to maintain its luminal acidity and LB homeostasis. This may provide new insights into the LB pathology.

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.

The Weibel-Palade body (WPB) is one of the lysosome-related organelles (LROs) in endothelial cells, whose main content is von Willebrand factor (vWF). The biogenesis of LROs is regulated by the Hermansky-Pudlak syndrome (HPS) protein-associated complexes through transporting cargo proteins to WPBs. Our previous studies have shown that HPS6, a subunit of BLOC-2 complex, is likely involved in the maturation of WPBs. However, the underlying mechanism remains unknown. In this study, we found that the knockdown of HPS6 in human umbilical vein endothelial cells (HUVECs) resulted in misshaped WPBs, decreased WPB number, and impaired vWF tubulation, which are similar to the characteristics of HPS6-deficient mouse endothelial cells. We observed similar morphological changes of WPBs in HUVECs after the knockdown of ATP6V0D1 (a subunit of v-ATPase). Furthermore, we found that HPS6 interacted with ATP6V0D1, suggesting that HPS6 transports ATP6V0D1 to the WPB limiting membrane for the assembly of the v-ATPase complex to maintain its acidic luminal pH, which is critical for the formation of vWF tubules during WPB maturation. In conclusion, HPS6 likely regulates the biogenesis of WPBs by participating in the trafficking of v-ATPase to the WPB membrane.

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.