Hermansky-Pudlak syndrome: the importance of molecular subtyping

The Hermansky-Pudlak syndrome (HPS) is named after the Czechoslovakian physicians Hermansky and Pudlak who in 1959 described two patients with oculocutaneous albinism, prolonged bleeding and pigmented macrophages in the bone marrow. Since then, 8 different subtypes have been identified (HPS-1 to HPS-8) [1,2]. HPS is inherited in an autosomal recessive fashion and symptoms are caused by abnormal biogenesis of lysosomes and lysosome-related organelles, such as melanosomes and platelet-dense granules. Haematologists recognize HPS because of the combination of bleeding diathesis, due to the diminished or absent dense granules causing secondary aggregation defects, oculocutaneous albinism causing congenital nystagmus, decreased visual acuity, iris transillumination and hypopigmentation of skin and hair. However, it is important to regonize that in certain types of HPS, pulmonary fibrosis, granulomatous colitis or neutropenia can develop [1,2]. HPS subtypes can be distinguished by molecular subtyping. The following cases underline the importance of mutation analysis in patients with similar initial clinical characteristics.

Four patients (three adult siblings aged 26, 28 and 30 from Libyan ancestry and a 3-year old Dutch boy) were analysed because of oculocutaneous albinism, nystagmus, subnormal vision and mild bleeding manifestations. Based on these symptoms, the three siblings (patients 1, 2, and 3) were already tentatively diagnosed with HPS in Libya by a pediatrician, without genetic testing. None of the patients were suffering pulmonary, gastro-intestinal or infectious problems, except for one adult patient who had been previously diagnosed with asthma. High resolution CT scanning of the lungs revealed no pulmonary fibrosis. All patients were enrolled in a protocol approved by the National Human Genome Research Institute (NHGRI) institutional review board. Written informed consent was obtained from the patient or the patients’ parent. Peripheral blood was collected from each patient for platelet analysis and DNA extraction.

Platelet aggregation was tested using the standard chrono-log aggregometer (Kordia, Leiden, the Netherlands). Ristocetin (1 mg/ml), ADP (2.3 µM) and collagen (0.6 µg/ml) were used as platelet agonists. For whole mount electron microscopy, platelet-rich plasma, prepared from fresh citrated blood, was placed on copper grids and treated as described [3]. Genomic DNA of each patient was screened by sequence analysis for mutations in the known Hermansky-Pudlak syndrome candidate genes HPS1, HPS3, HPS4, HPS5, and HPS6 by sequencing each genes’ exons and intronic boundaries.

Platelet aggregation tests performed in the three adult patients showed no aggregation after exposure to collagen, no secondary aggregation after exposure to ADP and the ATP/ADP ratios were increased, suggesting a dense granule defect. Indeed, electron microscopy from all 4 patients showed that platelet dense granules were absent, confirming the diagnosis of HPS (Fig 1A). Genomic DNA analysis of patients 1, 2 and 3 showed homozygosity for a novel mutation in the HPS3 gene (NM_032383) on chromosome 3q24. This splice-site mutation on the boundary of intron 3 and exon 4, likely results in skipping of exon 4 and/or other exons (Fig. 1B). Unfortunately, patients’ RNA was not available for exon-skipping experiments. Patient 4 was compound heterozygous for 2 mutations in the HPS1 gene (NM_000195) on chromosome 10q23 (Fig. 1C).

Fig. 1.

(A) Whole mount electron microscopy. Left panel: Normal platelet with dense bodies (arrows) and alpha granula. Right panel: Platelet from a patient with HPS lacking dense bodies. (B) Patient 1 exhibited a homozygous splice site mutation c.855-1G>A (IVS4-1 G>A) in the HPS3 gene at the intron 3-exon 4 boundary (right panel) compared to the normal splice consensus sequence (left panel). (C) Patient 4 was compound heterozygous for mutations in HPS1; a novel nonsense mutation (c.461G>A; p.W154X) in exon 6 (left panel) and a previously reported missense mutation (c.716T>C; p.L239P) in exon 8 (right panel).

Molecular subtyping allowed us to reassure the patients of Libyan ancestry that they had a mild form of HPS without known risk for pulmonary fibrosis [1,3]. They were instructed to consult a clinician in case of bleeding, trauma or planned surgery. The parents of the youngest patient, diagnosed with HPS-1, were informed about the diagnosis of a more severe form of HPS. This patient will need regular follow ups for lung function testing and endoscopic examinations in case of gastro-intestinal complaints [1,2].

In the last decade various mutations in the different human HPS genes have been identified by molecular analysis. The human HPS genes encode proteins that are components of multi-protein complexes; they are either subunits of the adaptor protein complex-3 (AP3) or of the complexes named biogenesis of lysosome-related organelles (BLOC-1, -2 or -3) [1]. AP3 is the only complex with a known function. It recruits and transports cargo proteins through the endosomal system to the newly-synthesized organelles. Mutations in the ADTB3A gene, coding for the β3A subunit of AP3, cause HPS-2 disease [1,2]. The cellular functions of the BLOCs are not clearly established and remain putative, although they are considered to play a role in protein and membrane trafficking from endosomes to lysosomes and to lysosome-related organelles. BLOC-1, -2 and -3 are affected in HPS subtypes other then HPS-2 [1].

All HPS subtypes present with some degree of oculocutaneous albinism and variable bleeding manifestations. However, some subtypes have additional clinical features, such as pulmonary fibrosis, granulomatous colitis and neutropenia [2]. HPS-1, HPS-2 and HPS-4 subtypes are associated with increased risk for developing pulmonary fibrosis, although sporadic cases have been described in other HPS subtypes [2]. Pulmonary fibrosis is estimated to occur in more than 80% of HPS-1 patients and leads to death in the third to sixth decade [1]. HPS-1 and HPS-4 are also associated with granulomatous colitis, resembling Crohn’s disease. Up to one-third is affected, but in contrast with pulmonary fibrosis, rarely results in death [1]. HPS-1 disease is the most common subtype of HPS with the largest population in north-west Puerto Rico. More than 20 HPS1 gene mutations have been described, with the 16-bp frameshift duplication mutation in exon 15 (c.1472_1487dup16; p.H497QfsX90) being the most prevalent [2]. The HPS1 and HPS4 proteins are both subunits of the BLOC-3 protein complex, suggesting a role for BLOC-3 in the development of pulmonary fibrosis and granulomatous colitis [1].

HPS-2 is distinguished from other forms by the presence of neutropenia and immunodeficiency with concomitant recurrent infections, especially of the respiratory tract. Pulmonary fibrosis has been detected on CT scanning in some HPS-2 patients [2,4]. The pathogenesis of neutropenia is currently unknown. In contrast, it is known that immunodeficiency in HPS-2 is characterised by a decrease in T lymphocyte-mediated cytotoxicity [5].

HPS-3 disease is the second most prevalent HPS subtype. Most patients are found in central Puerto Rico, due to a 3,904-bp deletion in the HPS3 gene [6]. Other mutations in the HPS3 gene have been reported in non-Puerto Rican patients. HPS-3 has a remarkably milder bleeding course compared to HPS-1 [2,3]. The pulmonary tract is affected only mildly or not at all, with slight reductions in forced vital capacity (FVC) [3]. Granulomatous colitis has been described in only two patients with HPS-3 [6]. Only a few patients with HPS-5, HPS-6, HPS-7 and HPS-8 have been diagnosed worldwide. They lack pulmonary fibrosis and granulomatous colitis [1,2]. The HPS5 and HPS6 proteins act together with HPS3 in BLOC-2 while HPS7 and HPS8 proteins are components of BLOC1 [1].

There is no curative treatment for HPS, so prevention of bleeding and supportive care are important aspects to decrease morbidity and mortality. Determination of the molecular subtype of a HPS patient is recommended as this will not only confirm the clinical diagnosis, but may also allow interventions such as carrier detection on relatives or even antenatal diagnosis. Moreover, molecular subtyping may assist both physicians and patients in anticipation of clinical symptoms and potential interventions. In cases of HPS-1 and HPS-4, precautions include avoidance of exposure to cigarette smoke and other lung toxins, prompt therapy of respiratory infections, and administration of influenza and pneumococcal vaccine [2]. Patients at risk for lung fibrosis need regular follow up with pulmonary function tests and high resolution CT-scanning. Pirfenidone, an anti-fibrotic agent, can slow down the progression of lung disease [7]. In addition, awareness of the possibility of developing granulomatous colitis will prevent a delay in diagnosis and proper treatment with either steroids or anti-TNF-α agents. In HPS-2 patients, symptomatic neutropenia might require the administration of G-CSF [1].

In conclusion, molecular subtyping in HPS has important prognostic and treatment implications and should always be performed. Secondly, finding novel mutations should help to unravel the biology and pathophysiology of HPS.