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. 2018 Nov;103(11):1796-1805.
doi: 10.3324/haematol.2018.189845. Epub 2018 Jul 12.

The SLC40A1 R178Q Mutation Is a Recurrent Cause of Hemochromatosis and Is Associated With a Novel Pathogenic Mechanism

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Free PMC article

The SLC40A1 R178Q Mutation Is a Recurrent Cause of Hemochromatosis and Is Associated With a Novel Pathogenic Mechanism

Chandran Ka et al. Haematologica. .
Free PMC article

Abstract

Hemochromatosis type 4 is one of the most common causes of primary iron overload, after HFE-related hemochromatosis. It is an autosomal dominant disorder, primarily due to missense mutations in SLC40A1 This gene encodes ferroportin 1 (FPN1), which is the sole iron export protein reported in mammals. Not all heterozygous missense mutations in SLC40A1 are disease-causing. Due to phenocopies and an increased demand for genetic testing, rare SLC40A1 variations are fortuitously observed in patients with a secondary cause of hyperferritinemia. Structure/function analysis is the most effective way of establishing causality when clinical and segregation data are lacking. It can also provide important insights into the mechanism of iron egress and FPN1 regulation by hepcidin. The present study aimed to determine the pathogenicity of the previously reported p.Arg178Gln variant. We present the biological, clinical, histological and radiological findings of 22 patients from six independent families of French, Belgian or Iraqi decent. Despite phenotypic variability, all patients with p.Arg178Gln had elevated serum ferritin concentrations and normal to low transferrin saturation levels. In vitro experiments demonstrated that the p.Arg178Gln mutant reduces the ability of FPN1 to export iron without causing protein mislocalization. Based on a comparative model of the 3D structure of human FPN1 in an outward facing conformation, we argue that p.Arg178 is part of an interaction network modulating the conformational changes required for iron transport. We conclude that p.Arg178Gln represents a new category of loss-of-function mutations and that the study of "gating residues" is necessary in order to fully understand the action mechanism of FPN1.

Figures

Figure 1.
Figure 1.
Family studies and pedigrees with the p.Arg178Gln missense mutation. Arrows indicate the index case. Biological data of family members are presented, when available. TS: transferrin saturation; SF: serum ferritin.
Figure 2.
Figure 2.
Liver histology of the index case of family 1 at diagnosis. Perls’ stain of liver biopsy shows significant amounts of stainable iron in Kupffer macrophages, while mild iron overload is observed in hepatocytes.
Figure 3.
Figure 3.
The ferroportin p.Arg178Gln mutant shows normal cell surface expression, but substantial loss of 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. Human leukocyte antigen (HLA)-A was used as control and standard for normalization, being a cell surface protein with no known role in iron metabolism. At 24h after transfection, cell surface proteins were selectively purified and analyzed by Western blotting using a peroxidase conjugated mouse anti-V5 antibody. Densitometric scans of SLC40A1 levels (normalized to HLA-A) are shown in the lower part of the figure. The results of three independent experiments are presented. (B) HEK293T cells were grown in 20 μg/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. The 55Fe exported into the supernatant was collected at 36h. Data are presented as percentage of cellular radioactivity at time zero. Each point represents the average value (from triplicate) of five independent experiments. P values were calculated with the Student’s t-test; **P<0.01 and ****P<0.0001. WT: wild-type.
Figure 4.
Figure 4.
The ferroportin p.Arg178Gln mutant is not resistant to hepcidin. HEK293T cells were transiently co-transfected with plasmids expressing HLA(A)-V5 and either wild-type SLC40A1-V5 (WT) or SLC40A1-V5 variants. At 16h post-transfection, the cells were incubated in the presence or absence of hepcidin for 3h. Plasma membrane proteins were purified and analyzed by Western blotting and densitometry. Data are expressed as the percentage of ferroportin in cells not treated by hepcidin, according to the formula 100 × (SLC40A1 – hepcidin / SLC40A1 + hepcidin). Error bars are the standard deviation of three independent experiments.
Figure 5.
Figure 5.
Ribbon representation of a human ferroportin-1 3D structure model in an outward facing conformation, with atomic representation of amino acids involved in non-covalent bonds, likely leading to the stabilization of this conformation. The iron within the iron-binding site is represented as a red ball at the top of the figure. This figure was drawn using Chimera.
Figure 6.
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 selectively 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 μg/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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