Laboratory Assessment of Patients with Systemic Mastocytosis

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GATA proteins are lineage-restricted transcription factors that regulate tissue-specific patterns of gene expression, principally during development. GATA-2 appears to regulate early mast cell gene expression and has been shown to positively regulate mast cell identity in model systems. GATA-2 deficiency is a protean disorder in human subjects with markedly variable clinical expressivity. Relevant to the mast cell compartment, many GATA-2deficient subjects have myeloid lineage abnormalities, including marrow hypocellularity, cytopenias, and myeloid neoplasms. Given that the data available on the effects of GATA-2 disruption in mast cells are limited to nonhuman or in vitro studies, we examined the effects of heterozygous loss-of-function GATA2 mutations on clinical allergy, cultured primary mast cell growth, morphology, and activation in patients with GATA2 deficiency. Retrospective investigation revealed that the overall prevalence of documented clinical symptoms consistent with IgE-mediated immediate hypersensitivity to foods, environmental allergens, medications, and/or stinging insects was low (21%; 6/28) in GATA-2deficient subjects. Consistent with this finding, none of the GATA-2deficient subjects had been prescribed epinephrine autoinjectors, and only 4 (14%) had been prescribed second-generation antihistamines, leukotriene modifiers, or mast cell stabilizers. All GATA-2deficient subjects and 27 unselected volunteers (control) underwent skin prick tests (SPTs) to a panel of common environmental allergens and food allergens. Control subjects demonstrated a greater rate of sensitization and a greater number were polysensitized relative to GATA-2deficient subjects. SPT responses in GATA-2deficient subjects were discordant with specific IgE levels in serum, where despite low total IgE levels (median IgE, 5.9 kU/L), 4 subjects had detectable antigen-specific IgE, 3 of whom were polysensitized to 3 or 4 allergens. GATA-2deficient subjects did not have impaired responses to the mast cell mediator histamine, as quantified by wheal area, suggesting the reduced SPT responses and clinical allergy observed were both mast cell intrinsic and antigen dependent. We next set out to identify mechanisms leading to impairment of antigen-dependent degranulation in GATA-2deficient mast cells and identified KIT and FcRI as candidates; these molecules both contribute to mast cell activation and survival in human subjects and have been shown to depend on intact GATA-2 activity in a number of systems. Flow cytometric analysis revealed that FcRI expression was significantly reduced in basophils (P < .05) and plasmacytoid dendritic cells (P < .05) from GATA-2deficient subjects. KIT expression was likewise reduced in GATA-2deficient mast cells in skin biopsy specimens, whereas mast cell numbers were paradoxically comparable with those of control subjects. Although bone marrow mast cells identified in aspirates as CD45+KIT+CD34SSChigh cells from patients with GATA-2 deficiency had comparable KIT expression when compared with those from control subjects with disease, these cells were actually increased (P = .01) in number compared with those in subjects with idiopathic anaphylaxis and comparable in number to subjects with indolent systemic mastocytosis. Like indolent systemic mastocytosis, increased bone marrow mast cell counts were associated with increased basal serum tryptase (BST) levels (>11.4 ng/mL) in 3 of 11 subjects. However, examining the remaining KIT+ myeloid cells in bone marrow, expression was found to be reduced. Likewise, significantly fewer CD34+ progenitor cells, which require KIT for survival, were identified in the periphery of GATA-2deficient patients. Further studies revealed that FcRI and KIT expression were reduced in successfully cultured GATA-2deficient mast cells. Successfully cultured primary mast cells (from 5 GATA-2deficient subjects and 6 paired healthy volunteers) were sensitized with biotinylated IgE, and FcRI was cross-linked using streptavidin. Mast cell degranulation, as measured by -hexosaminidase release, was reduced relative to control values. Stem cell factor (SCF) addition enhanced IgE-mediated mast cell degranulation in control subjects; however, it did not significantly alter GATA-2deficient mast cell degranulation. Intracellular Ca2+ flux was likewise impaired in GATA-2deficient mast cells in response to IgE cross-linking, as well as after the addition of SCF. These data are consistent with functional impairment in degranulation of primary GATA-2deficient mast cells in response to both IgE and SCF, which was associated with the reduced FcRI and KIT expression we have identified. In summary, GATA-2deficient subjects have evidence of impaired IgE-mediated mast cell activation and reduced IgE-mediated clinical allergic disease. These findings are antigen dependent and associated with reduced KIT and FcRI expression and are not attributable to a numeric problem in mast cells or an impaired response to the mast cell mediator histamine.