Disruption of AP3B1 by a chromosome 5 inversion: a new disease mechanism in Hermansky-Pudlak syndrome type 2

Abstract

Background: Hermansky-Pudlak syndrome 2 (HPS2; OMIM #608233) is a rare, autosomal recessive disorder caused by loss-of-function genetic variations affecting AP3B1, which encodes the β3A subunit of the adaptor-related protein complex 3 (AP3). Phenotypic characteristics include reduced pigmentation, absent platelet dense granule secretion, neutropenia and reduced cytotoxic T lymphocyte (CTL) and natural killer (NK) cell function. To date HPS2 has been associated with non-synonymous, stop-gain or deletion-insertion nucleotide variations within the coding region of AP3B1.

Case presentation: We describe a consanguineous female infant with reduced pigmentation, neutropenia and recurrent infections. Platelets displayed reduced aggregation and absent ATP secretion in response to collagen and ADP, indicating a platelet dense granule defect. There was increased basal surface expression of CD107a (lysosome-associated membrane protein 1(LAMP-1)) on NK cells and CTLs from the study subject and a smaller increase in the percentage of CD107a positive cells after stimulation compared to most healthy controls. Immunoblotting of protein extracts from EBV-transformed lymphoblasts from the index case showed absent expression of full-length AP-3 β3A subunit protein, confirming a phenotypic diagnosis of HPS2.The index case displayed a homozygous pericentric inv(5)(p15.1q14.1), which was also detected as a heterozygous defect in both parents of the index case. No loss of genetic material was demonstrated by microarray comparative genome hybridisation at 60kb resolution. Fluorescence in-situ hybridisation using the 189.6kb probe RP11-422I12, which maps to 5q14.1, demonstrated dual hybridisation to both 5q14.1 and 5p15.1 regions of the inverted Chr5. The RP11-422I12 probe maps from intron 1 to intron 16 of AP3B1, thus localising the 5q inversion breakpoint to within AP3B1. The probe RP11-211K15, which corresponds to an intergenic region on 5p also showed dual hybridisation, enabling localisation of the 5p inversion breakpoint.

Conclusion: This case report extends the phenotypic description of the very rare disorder HPS2. Our demonstration of a homozygous Chr5 inversion predicted to disrupt AP3B1 gene provides a novel pathogenic mechanism for this disorder.

Figure 1

Clinical and laboratory phenotype of the index case. Pedigree (A) showing the index case (P1; shaded) who displayed reduced skin, hair and iris pigmentation (B). Whole blood lumiaggregometry (C) was performed on EDTA-anticoagulated blood from P1 and from a healthy control (HC) using the activating agonists collagen (5 μg/ml) and ADP (10 μM). In the top panel, platelet aggregation is indicated by change in electrical impedance after addition of the agonist. In the bottom panel, platelet dense granule release is indicated by ATP secretion. Lytic granule release from lymphocytes from P1 (D) was measured by determining the increase in the percentage of CD107a positive cytotoxic T-lymphocytes CTL (top panel) and natural killer (NK) cells (bottom panel) after stimulation with phytohaemagglutinin and anti-CD3 respectively. Data are also presented from 39 healthy controls with the median control value indicated by the horizontal line. Expression of AP-3 β3A was determined in P1 by immunoblotting protein extract from EBV-immortalised B-lymphoblastoid cells using an anti-β3A subunit antibody (E). Control data are presented from a healthy control (HC) and from an unrelated individual with genetically confirmed HPS2 (HPS2). Control experiments were performed using an anti-GAPDH antibody. Migration of relevant molecular mass protein markers are indicated on each immunoblot.

Figure 2

Pericentric inversion of chromosome 5. Partial ideogram of a metaphase from the bone marrow aspirate from P2 showing both the normal and abnormal Chr5 with an inversion estimated to be between cytobands 5p15.1 and 5q13.3 (A). Schematic diagram of Chr5 showing the normal cytoband localization of the fluorescence in situ hybridisation (FISH) probes RP11-211K15 and RP11-422I12 (B). RP11-211K15 maps to an intergenic region at 5p15.1. RP11-422I12 maps to 5q14.1, and spans intron 1 to intron 16 of AP3B1. FISH was performed on metaphase chromosomes from P2, who was a heterozygote carrier of the inverted Chr5 (C and D). The images show DAPI stained chromosomes labelled with pGA-16 (green signal) which is a centromere marker of Chr5 and Chr9. The 5p15.1 probe RP11-211K15 (C; red signal) correctly localises to 5p15.1 in the normal Ch5 (solid arrow) but showed dual hybridisation to 5q14.1 and 5p15.1 in the inverted Chr5 (broken arrows). The 5q14.1 probe RP11-422I12 (D; red signal) correctly localises to 5q14.1 in the normal Ch5 (solid arrow) but showed dual hybridisation to 5p15.1 and 5q14.1 in the inverted Chr5 (broken arrows).