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Ferroportin structure and function
transferrin saturation,25 and then observed with incom- plete penetrance in members of an independent Greek family. Serum ferritin was found to be markedly elevated in the proband (a 25-year-old female), but only slightly in her 53-year-old mother and within the normal range in her 87-year-old grandfather.26,27 We observed the p.Arg178Gln missense mutation in 11 adult males with high serum fer- ritin concentrations (>970 mg/L) and normal plasma iron levels (transferrin saturation: 26-43%). Three adult females from the larger pedigrees (3.III.4, 4.II.1 and 4.II.6) displayed a mild phenotype, with serum ferritin concen- trations ranging between 200 and 400 mg/L at diagnosis, while two males presented an intermediate phenotype (584 and 613 mg/L) in their third decade (3.III.6 and 4.II.3). Liver biopsy was available for the index case of family 1, and showed tissue iron overload, with iron deposits pri- marily observed in non-parenchymal cells. The index case
had two children who exhibited increased iron stores at 11 and seven years of age, respectively. Similar pheno- types were observed in two children from family 3 (3.III.2 and 3.III.3). None of the patients were reported to have developed significant fibrosis or cirrhosis. Taken together, these data indicate that the p.Arg178Gln substitution is responsible for the classic form of ferroportin disease, or hemochromatosis type 4A. The expressivity of the SLC40A1 p.[Arg178Gln];[=] genotype is variable, with milder phenotypes observed in women. High serum fer- ritin levels can be observed in young patients, highlighting the fact that tissue iron overload may appear early in life and that, in contrast to HFE hemochromatosis, diagnosis of ferroportin disease should not be restricted to adults.28 This recapitulates some previous observations on the rela- tionship between ferroportin loss-of-function mutations and mild to severe reticuloendothelial iron overload.2,11,29
A
B
Figure 6. Effect of p.Arg178Ala and p.Asp473Ala ferroportin variants on cell surface expression and iron export. (A) HEK293T cells were transiently co-transfected with plasmids encoding either a V5-tagged ferroportin protein (WT or variant) or a V5-tagged HLA-A protein. At 24h after transfection, cell surface proteins were selec- tively purified and analyzed by Western blotting using peroxidase-conjugated mouse anti-V5 antibody. Densitometric scans of SLC40A1 levels (normalized to HLA-A) are shown. The error bars represent the standard deviation of three independent experiments. (B) HEK293T cells were grown in 20 mg/mL 55Fe-transferrin for 24h before being washed and transiently transfected with WT or mutated SLC40A1-V5 expression plasmids. After 15h, cells were washed and then serum-starved for up to 36h. 55Fe exported into the supernatant was collected at various time points. Data are presented as a percentage of cellular radioactivity at time zero. Each point represents the mean standard deviation; n=3 in each group. The data are representative of three separate experiments. WT: wild-type.
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