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Ferroportin structure and function
Introduction
Hemochromatosis type 4 (OMIM #606069), also called ferroportin disease, is an inborn error of iron metabolism transmitted through autosomal dominant inheritance and associated with mutations in the gene encoding the solute-carrier family 40 member 1 (SLC40A1). Although rare, hemochromatosis type 4 is observed in different eth- nic groups and is considered to be the second most com- mon cause of hereditary iron overload after HFE-related hemochromatosis.1,2
SLC40A1, also known as ferroportin 1 (UniProt acces- sion number Q9NP59), is the sole iron export-protein reported in mammals. It is expressed in all types of cells that handle major iron flow, including macrophages, duo- denal enterocytes, hepatocytes and placenta syncytiotro- phoblasts.3 Expression of ferroportin 1 on the cell surface is predominantly regulated by the liver-derived peptide hepcidin, which induces internalization and degradation of ferroportin 1 and thereby decreases the delivery of iron to plasma.4 The hepcidin-ferroportin axis plays an impor- tant role in the pathogenesis of inherited and acquired iron metabolism disorders, including iron overload diseases and iron-restricted anemia.5
Mutations that alter ferroportin 1 function are expected to produce stronger effects in reticuloendothelial macrophages than in enterocytes or hepatocytes. These macrophages acquire most of their iron by recycling senescent red blood cells and account for >80% of the daily iron flux within the body.6 In line with this expecta- tion, and at variance with HFE-related hemochromatosis, patients with loss-of-function mutations usually present with mesenchymal or mixed iron overload (correspond- ing to early iron deposition within Kupffer cells) and markedly elevated serum ferritin levels, contrasting with normal or low transferrin saturation values.7,8 Aggressive phlebotomy regimens can be a problem in the early stages of the disease, when patients often display border- line anemia.9,10
Although a majority of SLC40A1 mutations reported as being causally linked to hemochromatosis type 4 are true “pathogenic variants”,11 there may be some doubt in the case of variants for which phenotypic, population, segre- gation, functional and/or computational data are lacking or not fully convincing. The problem is not specific to hemochromatosis type 4, but includes all Mendelian dis- orders associated with large allelic heterogeneity, and can be mimicked by non-genetic conditions.12,13 Assessing the pathogenicity of 18 non-synonymous SLC40A1 variants found in 44 suspected hemochromatosis 4 patients, we previously demonstrated that eight very rare missense mutations had no noticeable effects on ferroportin 1 func- tion or interaction with hepcidin.14 All these variants were identified in single cases showing moderate serum ferritin elevation and normal transferrin saturation, a biological condition that is common in clinical practice and is largely related to lifestyle and environmental factors.15
The present study provides strong evidence that the SLC40A1 p.Arg178Gln missense mutation is recurrent in the SLC40A1 gene of patients showing typical reticuloen- dothelial iron overload. We further demonstrate that the p.Arg178Gln ferroportin 1 mutant shows reduced ability to export iron out of the cell. This is likely a direct conse- quence of salt bridge disruption between Arg178 and Asp473, thereby affecting the stable formation of the
intracellular gate present in the ferroportin 1 outward fac- ing state. Such a molecular mechanism of pathogenesis has never been reported in the context of hemochromato- sis type 4.
Methods
Genetic studies
DNA was extracted from the peripheral blood of patients and unaffected family members. The complete coding sequence of SLC40A1 and intron/exon boundaries was investigated by Sanger sequencing in the probands, while family members were only assessed for exon 6 (containing codon p.Arg178). All probands were negative for genotypes known to cause hemochromatosis types 1-3 (in the HFE, HFE2, HAMP and TFR2 genes) and for mutations in the FTL and BMP6 genes (hyperferritinemia cataract syndrome: OMIM#600886; hyperferritinemia without iron over- load and cataract: OMIM#134790; BMP6-related iron overload: OMIM#112266). Polymerase chain reaction and sequencing con- ditions are available upon request.
A total of 734 DNA samples from healthy subjects, exclusively from north-western France (Brittany), were investigated to control the frequency of the SLC40A1 p.Arg178Gln variant.
Informed consent for molecular studies was obtained from all patients and family members, in accordance with the Declaration of Helsinki; in line with French ethical guidelines, the Clinical Research Ethics Committee of the University Hospital of Brest approved the study on October 25, 2010.
Hepcidin measurement in human serums
Serum hepcidin concentrations were measured using liquid chromatography coupled with tandem mass spectrometry (LC- MS/MS), as previously described.16 The 95% reference interval obtained for normal hepcidin (200 serum samples from healthy subjects) ranged from 1.0 and 20.8 ng/mL (mean: 8.2 ng/mL).
Human-25 hepcidin synthesis and secretion by T-Rex-293 cells
Human HAMP cDNA was amplified with reverse-transcription polymerase chain reaction (RT-PCR) from total ribonucleic acid (RNA) isolated from human liver hepatocellular carcinoma HepG2 cells. The PCR product was cloned into the PCR2.1 vector using the TA Cloning Kit (Thermo Fisher Scientific), subcloned into the pcDNA4TM4/TO tetracycline-regulated mammalian expression vector (Thermo Fisher Scientific), and checked by sequencing.
T-Rex-293 cells (Thermo Fisher Scientific) were stably transfect- ed with calcium phosphate, and colonies were selected in the presence of 1.5 mg/ml blasticidin and 100 mg/ml zeocin for four weeks. Tetracycline (Sigma, St. Louis, MO, USA) was used to induce expression of the 84 hepcidin pre-propeptide amino acids from 1.106 T-Rex-293 cells. After 48h the cell supernatant was col- lected, filtered through a hydrophilic nylon membrane (pore size: 0.2 mm), and measured for hepcidin-25 levels using a commercially available competitive enzyme-linked immunosorbent assay kit (ELISA; Peninsula Laboratories International, San Carlos, CA, USA). The supernatant was stored at -20°C until used.
In vitro experiments
In vitro investigations were performed as previously described14,17
and are presented in detail in the Online Supplementary Data.
3D structure modeling and analysis
Models of the 3D structure of human ferroportin 1 were built using Modeller v9.15,18 considering the sequence alignment
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