Molecular and clinical correlates in iron overload associated with mutations in ferroportin

Abstract

Mutations in ferroportin (Fpn) result in iron overload. We correlate the behavior of three Fpn mutants in vitro with patients' phenotypes. Patients with Fpn mutations A77D or N174I showed macrophage iron loading. In cultured cells, FpnA77D did not reach the cell surface and cells did not export iron. Fpn mutant N174I showed plasma membrane and intracellular localization, and did not transport iron. Fpn mutation G80S was targeted to the cell surface and was transport competent, however patients showed macrophage iron. We suggest that FpnG80S represents a class of Fpn mutants whose behavior in vitro does not explain the patients' phenotype.

Figure 1

Human Fpn mutations affect Fpn localization and hepcidin-induced internalization. A. HEK293T cells were transiently transfected with plasmids containing wild type (WT) Fpn-GFP, FpnA77D-GFP, FpnG80S-GFP or FpnN174I-GFP. Eighteen to 24 h after transfection, localization of Fpn-GFP and response to hepcidin were assessed by epifluorescent microscopy. Cells were incubated with or without 1 μg/mL hepcidin for 4 and 24 h to assess hepcidin response. B. 18–24 h after transfection cells were incubated with or without 1 μg/mL hepcidin for 4 h, and extracts were analyzed by western blot analysis using antibody to GFP and actin as a loading control, as described in the Design and Methods.

Figure 2

Fpn mutations affect intracellular ferritin levels and ¹²⁵I-hepcidin uptake. A. HEK293T cells were transiently transfected with plasmids containing wild type (WT) Fpn-GFP, FpnA77D-GFP, FpnG80S-GFP or FpnN174I-GFP. Eighteen hours after transfection, cells were cultured with ferric ammonium citrate (FAC) (20 μM iron) for 24 h. Cells were incubated with 100 μM cycloheximide for 1 h followed by 1 μg/mL hepcidin for 4 h. Ferritin levels were determined by ELISA and normalized to total protein concentration. Error bars represent the standard error of the mean of three independent experiments. B. Eighteen hours after transfection, ¹²⁵I-hepcidin was added to HEK293T and ¹²⁵I-hepcidin uptake measured as described previously.^, Error bars represent the average of three separate experiments in triplicate (n=9).