Hermansky-Pudlak syndrome type 2: Aberrant pre-mRNA splicing and mislocalization of granule proteins in neutrophils

Abstract

Hermansky-Pudlak syndrome type 2 (HPS2) is a syndrome caused by mutations in the beta-3A subunit of the adaptor protein (AP)-3 complex (AP3B1 gene). We describe five unreported cases with four novel mutations, one of which caused aberrant pre-mRNA splicing. A point mutation c.2702C>G in exon 23 of the AP3B1 gene caused deletion of 112 bp in the mRNA in two siblings. This mutation activates a cryptic donor splice site that overrules the wild-type donor splice site of this exon. Three other novel mutations in AP3B1 were identified, that is, a nonsense mutation c.716G>A (p.Trp239Ter), a 1-bp and a 4-bp deletion c.177delA and c.1839_1842delTAGA, respectively, both causing frameshift and premature termination of translation. Mass spectrometry in four of these HPS2 patients demonstrated the (near) absence of all AP-3 complex subunits. Immunoelectron microscopy on the neutrophils of two of these patients showed abnormal granule formation. We found clear mislocalization of myeloperoxidase in the neutrophils even though the content of this protein but not the activity seemed to be present at normal levels. In sum, HPS2 is the result of the absence of the entire AP-3 complex, which results in severe neutropenia with a defect in granule formation as the major hematological finding.

Hermansky–Pudlak syndrome: Mutation update

“…Alternatively, demonstrating an absence of a secondary aggregation response of platelets to exogenous stimuli through platelet aggregation testing also supports the HPS diagnosis (White & Witkop, 1972). Quantification of mepacrine uptake (Billio et al, 2001), and super-resolution immunofluorescence microscopy analyses (Westmoreland et al, 2016) have also been presented as potential alternatives to wholemount EM analysis for diagnosis of dense granule deficiency.…”

“… i This nucleotide change activates a cryptic donor splice and causes deletion of 112 bp within exon 23 on the mRNA level, resulting in a frameshift and a premature termination codon p.Val900Thrfs*63 (de Boer et al, 2017). …”

“…These features led to the discovery of the involvement of AP‐3 in different trafficking processes. AP‐3 is involved in neutrophil formation (Badolato & Parolini, 2007; Massullo et al, 2005); AP‐3 deficient cells mislocalize the neutrophil granule proteins myeloperoxidase and elastase and the lysosomal membrane protein CD63 (de Boer et al, 2017; Di Pietro et al, 2006; Jung et al, 2006; Meng et al, 2010). The AP‐3 immunodeficiency also involves defective AP‐3‐mediated lytic granule exocytosis in NK‐cells and cytotoxic T cells (Clark et al, 2003; Fontana et al, 2006; Gil‐Krzewska et al, 2017; Jung et al, 2006).…”

“…Immunodeficiency and/or neutropenia occurs in AP‐3‐deficient HPS, resulting in susceptibility to infections (subtypes HPS‐2 and HPS‐10; Ammann et al, 2016; de Boer et al, 2017; Fontana et al, 2006; Huizing et al, 2002; Mohammed et al, 2018). No BLOC‐2 or BLOC‐3 deficient subjects are described with immunodeficiency, but it was reported in two unrelated individuals with HPS‐9 (Badolato et al, 2012; Okamura et al, 2018) and therefore needs consideration in future BLOC‐1 deficient individuals.…”

“…i This nucleotide change activates a cryptic donor splice and causes deletion of 112 bp within exon 23 on the mRNA level, resulting in a frameshift and a premature termination codon p.Val900Thrfs*63 (de Boer et al, 2017). j This frameshift in the AP3B1 C-terminal coding region results in a prolonged altered protein, beyond the termination codon, with 42 additional C-terminal amino acids compared with the wild-type protein (Kurnik et al, 2013).…”