Identification of a homozygous deletion in the AP3B1 gene causing Hermansky-Pudlak syndrome, type 2.

We report on the molecular etiology of an unusual clinical phenotype associating congenital neutropenia, thrombocytopenia, developmental delay, and hypopigmentation. Using genetic linkage analysis and targeted gene sequencing, we defined a homozygous genomic deletion in AP3B1, the gene encoding the beta chain of the adaptor protein-3 (AP-3) complex. The mutation leads to in-frame skipping of exon 15 and thus perturbs proper assembly of the heterotetrameric AP-3 complex. Consequently, trafficking of transmembrane lysosomal proteins is aberrant, as shown for CD63. In basal keratinocytes, the incorporated immature melanosomes were rapidly degraded in large phagolysosomes. Despite distinct ultramorphologic changes suggestive of aberrant vesicular maturation, no functional aberrations were detected in neutrophil granulocytes. However, a comprehensive immunologic assessment revealed that natural killer (NK) and NKT-cell numbers were reduced in AP-3-deficient patients. Our findings extend the clinical and molecular phenotype of human AP-3 deficiency (also known as Hermansky-Pudlak syndrome, type 2) and provide further insights into the role of the AP-3 complex for the innate immune system.

Mutations in neutrophil elastase causing congenital neutropenia lead to cytoplasmic protein accumulation and induction of the unfolded protein response.

Severe congenital neutropenia (SCN) and cyclic neutropenia (CyN) are sporadic or inherited hematologic disorders of myelopoiesis. Heterozygous mutations in the gene encoding neutrophil elastase (ELA2) have been reported in both diseases. We used an inducible system to express a panel of ELA2 mutations and found for almost all mutants disruption of intracellular neutrophil elastase (HNE) protein processing at different levels. This disruption resulted in cytoplasmic accumulation of a nonfunctional protein, thereby preventing its physiologic transport to azurophil granules. Furthermore, the secretory capacity of the mutant proteins was greatly diminished, indicating alteration of the regulated and the constitutive pathways. Through analysis of primary granulocytes from SCN patients carrying ELA2 mutations, we found an identical pattern of intracellular accumulation of mutant HNE protein in the cytoplasm. Moreover, cells expressing mutant HNE protein exhibited a significant increase in apoptosis associated with up-regulation of the master ER chaperone BiP, indicating that disturbance of intracellular trafficking results in activation of the mammalian unfolded protein response.