The patient provided written informed consent to protocols 95-HG-0193 (clinicaltrials.gov NCT00001456, “Clinical and Basic Investigations into Hermansky-Pudlak Syndrome”) and 04-HG-0211 (clinicaltrials.gov NCT00084305, “Procurement and Analysis of Specimens from Individuals with Pulmonary Fibrosis”). The research was conducted prospectively; the clinical studies were approved by the institutional review board of the National Human Genome Research Institute.

Clinical testing was performed at Regional Cancer Center, Johnson City, Tennessee and the National Institutes of Health Clinical Center, Bethesda, Maryland. Platelet aggregation was measured by the absorbance method using the AggRam system (Helena Laboratories, Beaumont, TX). High-resolution computed tomography scans of the chest, pulmonary function tests, and platelet electron microscopy were performed as described (Rouhani, 2009; Ferreira, 2017).

Next Generation Sequencing (NGS) was performed in a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory using a targeted panel (Agilent Technologies, Santa Clara, CA) encompassing 9 HPS genes (i.e., HPS1, AP3B1, HPS3, HPS4, HPS5, HPS6, DTNBP1, BLOC1S3, BLOC1S6). Large scale single-nucleotide polymorphism (SNP) analysis was performed on genomic DNA using HumanOmniExpress DNA Analysis BeadChip (Illumina, San Diego, CA) and GenomeStudio software (Illumina) as described (Bryan, 2017). PCR of HPS1 using genomic DNA was performed using Platinum^™ Pfx DNA Polymerase according to manufacturer’s instructions (Invitrogen, Carlsbad, CA).

Fibroblasts were cultured from a forearm skin biopsy as described (Stephen, 2017). Dermal fibroblasts from two healthy volunteers served as normal controls; dermal fibroblasts from 3 adults with HPS-1 served as BLOC-3 complex controls. Experiments were performed in triplicate.

Total RNA and protein were isolated (Stephen, 2017). Quantitative real-time PCR (qPCR) was performed using TaqMan primers specific for HPS6 (Thermo Fisher, Waltham, MA), and cDNA from the patient and controls was amplified using TaqMan universal PCR master mix (Thermo Fisher) as described (Stephen, 2017). Results were normalized with expression of RFLP13a (Thermo Fisher) and analyzed. Western blot analysis was performed as described (Stephen, 2017). Membranes were incubated overnight at 4°C with rabbit monoclonal anti-HPS1 antibody (Abcam, Cambridge, UK), rabbit polyclonal anti-HPS4 antibody (Santa Cruz Biotechnology Inc., Dallas TX), rabbit polyclonal anti-HPS6 antibody (Origene Technologies Inc., Rockville, MD), or mouse monoclonal anti-β-actin antibody (Sigma-Aldrich, St. Louis, MO). Densitometry analysis was performed using the Odyssey^® imaging system; results were normalized to mean HPS6 protein expression in fibroblasts from two normal controls.

Data shown are mean ± standard error of the mean. Student’s t-test was used to analyze significance of difference between means (GraphPad Prism, San Diego, CA).

The patient is a 58-year old Caucasian woman with a long history for excessive bleeding and no known oculocutaneous albinism. Her pigmentation darkens with exposure to sunlight. She reported easy bruising and prolonged epistaxis. She was treated with blood product transfusions for severe menorrhagia, hemorrhage following vaginal delivery requiring admission to an intensive care unit, and prolonged gastrointestinal bleeding with hemodynamic instability after gastric polypectomy via esophagogastroduodenoscopy. She also experienced intermittent episodes of lower gastrointestinal bleeding of unknown etiology. Colonoscopy showed no evidence of inflammatory bowel disease and incidental findings of 2 small polyps. Polypectomies caused prolonged lower gastrointestinal bleeding. Initially considered to have von Willebrand's disease, the patient’s von Willebrand factor antigen was normal, and she was subsequently diagnosed with a platelet storage pool defect.

Family history is notable for consanguinity. The patient’s parents are second and third cousins (Figure 1A). One brother had easy bruising; her other relatives, including her child, did not have albinism or excessive bleeding.

Physical examination was notable for light-colored hair, eyes, and skin. Best corrected visual acuity measured 20/25 and 20/40 in the right and left eyes, respectively. Although she did not have nystagmus, she had mild iris transillumination defects, minimal pigmentation of the peripheral retina, and a poorly developed fovea (Figures 1B-C). Optical coherence tomography demonstrated loss of the foveal depression, no outer segment lengthening relative to the parafoveal outer nuclear layer, and no extrusion of the plexifom layers, consistent with grade 3 foveal hypoplasia (Figure 1D)(Thomas, 2011). Cardiovascular, pulmonary, abdominal, and neurological findings were normal. There were no detectable petechiae, ecchymoses, or hematomas.

Laboratory testing revealed normal white blood count, hemoglobin, prothrombin time, partial thromboplastin time, factor VIII activity, and von Willebrand factor activity and antigen. Platelet count was 158 × 10³/ul [173-369 × 10³/uL]. Platelet function assay showed prolonged closure time in response to epinephrine (>300 sec [86-154 sec]), but not to adenosine diphosphate (70 sec [73-129 sec]). Platelet aggregation responses to collagen, arachidonic acid, adenosine diphosphate, and ristocetin were normal; rate of aggregation and final amplitude to epinephrine were decreased. Whole mount platelet electron microscopy testing showed virtually no delta granules (Figure 1E).

Pulmonary function and six-minute walk test were normal. High-resolution computed tomography scan of the chest showed bibasilar juxta-pleural linear and nodular atelectasis without clear evidence of interstitial lung disease (Figure 1F).

Given her findings of mild oculocutaneous albinism and history of excessive bleeding with absent platelet delta granules, the patient was diagnosed with HPS. To determine her HPS subtype, SNP analysis and exome sequencing were performed. Consistent with a history of consanguinity, SNP analysis showed regions of homozygosity, one of which included HPS1 and HPS6. PCR showed normal full length HPS1 transcript (data not shown). A targeted NGS panel of 9 HPS genes using the patient’s genomic DNA identified a homozygous mutation in HPS6 (NM_024747.5:c.383T>C; p.V128A). This variant is not found in published reports of HPS6 mutations (Table 1), and it is not reported in the large population genetics databases 1000 Genomes, NHLBI GO Exome Sequencing Project (ESP), or Genome Aggregation Database (gnomAD). Furthermore, this HPS6 mutation is predicted to be probably damaging by PolyPhen-2 (v2.2.2r398) Hum Var (score 0.921), deleterious by Sorting Intolerant from Tolerant (SIFT, score: 0.01), and disease causing by MutationTaster (prob value = 0.964).

To determine whether the patient’s mutation is associated with altered HPS6 expression, mRNA and protein levels were measured in her cells. We found that HPS6 mRNA levels in this patient’s dermal fibroblasts were 37% of that in normal cells (p<0.001) and 36% of that in HPS-1 BLOC-3 complex controls (p<0.001)(Figure 1G). Western blot analysis showed that HPS6 protein expression in the patient’s fibroblasts were 60% of that of normal controls (p<0.001) and 29% of that of HPS-1 BLOC-3 complex controls (p=0.008)(Figure 1H). HPS1 and HPS4 proteins were expressed in this HPS-6 patient’s cells; these results are consistent with this patient’s defect in BLOC-2, and not BLOC-3.