Severe Congenital Neutropenia-Type 5: Impaired T Cell Proliferation, Aberrant Th1 Cytokine Production, Abnormal Megakaryocytes, and Impaired Platelet Granule Formation in a Patient with VPS45 Deficiency Caused By Uniparental Isodisomy

Introduction

VPS45 is essential in early endosome formation with rabenosyn-5 and syntaxin 16. VPS45 deficiency is one of the inborn errors of immunity characterized by severe congenital neutropenia and myelofibrosis. Impaired production and function of neutrophils, increased cell death, and abnormal hematopoiesis have been reported, whereas there are several undetermined pathophysiologies, such as T and/or NK lymphopenia or myelofibrosis. Here, we experienced a girl with VPS45 deficiency presenting with neutropenia at birth, progressive T and NK lymphopenia, hepatosplenomegaly, and myelofibrosis in infancy. We examined the pathophysiology of VPS45 deficiency focusing on hematologic disorder, T cell function, and myelofibrosis.

Methods

We sequentially evaluated peripheral blood and bone marrow cell counts and morphologic features. Genetic analysis was performed using DNA from peripheral blood. We evaluated T cell proliferation using the CFDA-SE cell tracer kit with the stimulation of PHA-L or anti-CD3/CD28 beads. Serum cytokine analysis was performed by beads array. Protein expression was examined in platelets from the patient and the healthy control by western blot analysis.

Results

Case: The patient was born to nonconsanguineous Japanese parents. She received a blood examination for mild respiratory distress at birth. This examination demonstrated neutropenia, the emergence of myeloid cells, and nucleated red blood cells in the peripheral blood, whereas mature neutrophils were confirmed in the bone marrow. Genetic analysis demonstrated homozygous pathogenic variants of p.E238K in VPS45. The same heterozygous variant was confirmed in her father, but not in her mother. SNP array analysis demonstrated that she inherited chromosome 1 only from her father and developed VPS45 deficiency by paternal uniparental isodisomy.

At 7 months, she demonstrated T and NK lymphocytopenia, macrothrombocytopenia, and hepatosplenomegaly. Bone marrow examination demonstrated the presence of blast cells, micro or separated megakaryocytes, and pseudo-Pelger-Huet anomaly. Electronic microscopy of peripheral blood demonstrated abnormal granules in neutrophils and macro-thrombocytes with reduced alpha granules and open channel enlargement. Elevated interferon-gamma, IL-12, and IL-23 levels, representing T helper 1 (Th1) activation were found. We initiated ruxolitinib at 7 months to suppress aberrant Th1 activation and extra-medullar hematopoiesis which could be associated with massive hepatosplenomegaly. Ruxolitinib was effective for hepatosplenomegaly and reduced Th1 cytokine levels without significant complications. She received hematopoietic cell transplantation with bone marrow from an HLA-matched unrelated donor 30 days after the initiation of ruxolitinib.

T cell function: With anti-CD3/CD28 beads stimulation, the patient's CD8-positive cells were significantly less proliferative. Either CD4-positive or CD8-positive cells did not proliferate with PHA-L stimulation.

Platelet analysis: Western blot analysis of platelet demonstrated a reduced amount of alpha granule proteins (von Willebrand factor and PF4), not of p-selectin.

Discussion

VPS45 deficiency was characterized by severe congenital neutropenia and myelofibrosis. Furthermore, T cell deficiency, especially CD8-positive T lymphopenia was observed in some patients with VPS45 deficiency. Our study suggested that T cell lymphopenia would be associated with impaired TCR signaling and mitogen response, although the detailed mechanism remains to be elucidated. The impaired platelet alpha granule formation confirmed by electron microscopy and western blot analysis in our patients was compatible with the previous VPS45 deficiency patient. A similar manifestation is observed in gray platelet syndrome which is also associated with affected endosomal transport. Affected endosomal transport would be responsible for myelofibrosis.

We hypothesized that massive hepatosplenomegaly was attributable to the hyper-activated Th1 cells and extra-medullar hematopoiesis associated with JAK1 and JAK2 signaling. Indeed, JAK1/ JAK2 inhibitor, ruxolitinib improved hepatosplenomegaly in this patient.

Conclusion

VPS45 deficiency could broadly affect not only neutropenia and myelofibrosis but T cell, NK cell, megakaryocyte, and platelet function.