A cellular disease model for autosomal dominant ferroportin gene mutations

After HFE associated hemochromatosis mutations in SLC40A1, encoding the only known cellular iron exporter ferroportin, are the commonest cause of genetic iron overload. Patients with mainly private SLC40A1 mutations can present with distinct phenotypes. Loss of iron export function mutations have been associated with the ferroportin disease phenotype whereas the hemochromatosis type 4 phenotype is associated with gain of function mutations, where specific mutation render ferroportin constitutively active and resistant against hepcidin mediated degradation.

However, the molecular disease mechanism of ferroportin disease and hemochromatosis type 4 has only been reported for a subset of disease-associated mutations and limited information on genotype-phenotype correlation exists. Recent structural information from bacterial homologues of SLC40A1 and modelling studies suggest that the aspartate residue at position 157 is exposed at the intracellular side of of ferroportin's iron entry channel and could therefore be crucial for its export function. The aim of the present study was to determine the functional consequence of D157 mutations on the iron export function and hepcidin responsiveness of ferroportin. Residue 157 was mutated to histidine, glutamine, tyrosine or glycine using site-directed mutagenesis and the cDNA was cloned into a mammalian expression vector as N-terminal fusion protein with GFP and mCherry. The identity of the constructs was confirmed by Sanger sequencing. To create an authentic disease model of autosomal dominant SLC40A1 mutation, HEK293T cells were cotransfected with the normal and mutant expression vectors fused to GFP and mCherry. Hepcidin responsiveness and iron export were investigated in cells overexpressing wild type and mutant ferroportin using flow cytometry and ferritin ELISA.

In conclusion, this overexpression model based on a dual fluorescent protein reporter readout offers a versatile tool to study molecular disease mechanisms of autosomal dominant transporter defects including heterozygous SLC40A1 mutations, but could also be used for studying other autosomal dominant gastrointestinal channelopathies.