The Consequences of Failing to Obey the Traffic Rules

Vilboux T, Lev A, Malicdan MCV, et al. A congenital neutrophil defect syndrome associated with mutations in VPS45. N Engl J Med. 2013;369:54-65.

The genetic basis of the multitude of disorders associated with neutrophil function reflects the interdependence of the numerous intracellular systems required to maintain a vigilant defense against infections. Mutations interfering with granule formation, chemotaxis, migration, adhesion, differentiation, apoptosis, and intracellular/membrane trafficking can result in a spectrum of immunodeficiencies resulting from qualitative and/or quantitative neutrophil defects. Susceptibility to bacterial or fungal infections may be only one feature of these disparate syndromes whose clinical expression may reflect the penetrance of the particular mutations and whether different cell lineages or organ systems are also affected by a particular mutation.

Now, two groups of investigators have identified an immunodeficiency syndrome linked to mutation of VPS45, one of the endocytic pathway vacuolar-sorting proteins found in neutrophils. VPS45 is a member of the Sec1/Munc18 protein family that regulates the assembly of SNARE complexes that modulate membrane traffic through the endocytic-lysosomal pathway. The phenotype of VPS45-associated human disease consists of congenital neutropenia, myelofibrosis, progressive bone marrow failure, nephromegaly, thrombasthenia, and recurrent, life-threatening infections. In both case series, patients presented during infancy and were identified as the offspring of consanguineous marriages from several unrelated Palestinian families. Homozygosity for the Thr224Asn mutation (c.671 C–>A) within exon 7 of VPS45 segregated with affected individuals and followed an autosomal recessive pattern of inheritance. The mutated codon was highly conserved throughout evolution and was not found in almost 400 Palestinian controls or in a panel of other healthy individuals. In a Moroccan pedigree, affected siblings exhibited a different homozygous mutation (c.712G→A; p.Glu238Lys). Affected infants with mutated VPS45 frequently exhibited transfusion-dependent anemia, life-threatening thrombocytopenia, and G-CSF-resistant neutropenia. Outcomes were determined primarily by susceptibility to infection that resulted in death either before or as a complication of hematopoietic stem cell transplantation. Laboratory and histopathologic findings included the following: normocytic anemia with reticulocytopenia, aniso/poikylocytosis (including tear drop red cells), neutropenia with increased band forms, and thrombocytopenia with occasional giant platelets. Compared with controls, platelet aggregation was normal with ristocetin, but it was reduced in the presence of ADP, collagen, or epinephrine. In the bone marrow, myeloid hyperplasia was a consistent feature, but myelodysplasia or karyotypic abnormalities were not identified. Significant reticulin (and sometimes collagen) fibrosis was found in all patients. Kidney biopsies revealed nephromegaly that was due to extramedullary hematopoiesis.

Ultra-structural and functional studies were undertaken in selected patients to assess the biologic effects of mutated VPS45. Compared with age-matched controls, electron microscopy demonstrated  immature neutrophils with fewer azurophilic granules, increased mitochondria, more developed Golgi and endoplasmic reticulum, and less condensed chromatin and cytoplasm.

Lysosomes were depleted in cultured fibroblasts as well as in platelets, which also exhibited decreased alpha-granule content. Expression of VPS45 and its binding partners rabenosyn-5 and syntaxin-16 were decreased in circulating neutrophils and/or fibroblasts from an affected patient. Similar reduction of syntaxin-16 was observed with loss of VPS45 function in yeast. Migration of fibroblasts was impaired in an in vitro wound assay in which a scratch was introduced across a field of cells in culture. VPS45-mutant neutrophils were found to express less β1 integrin, a cell surface protein critical for motility. In addition, markers of apoptosis were significantly increased in fibroblasts and in bone marrow biopsy specimens from patients with the mutation. In zebrafish, knockdown of VPS45 resulted in a marked reduction in myeloperoxidase staining, consistent with decreased numbers of mature neutrophils. Transfection of mutant fibroblasts with non-mutated VPS45 cDNA was capable of correcting the abnormal phenotype.