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Loss-of-function ferroportin disease: novel mechanistic insights and unanswered questions
L. Tom Vlasveld1 and Dorine W. Swinkels2
1Department of Internal Medicine, Haaglanden Medical Center, Location Bronovo, The Hague and 2Translational Metabolic Laboratory, Department of Laboratory Medicine, Radboud University Medical Center, Nijmegen, the Netherlands
E-mail: dorine.swinkels@radboudumc.nl doi:10.3324/haematol.2018.203315
Ferroportin, a 571 amino acid cation transporter encoded by the SLC40A1 gene, is the only known human cellular iron exporter and primarily expressed in the basolateral membrane of duodenal enterocytes, macrophages, and hepa- tocytes.1,2 By regulating ferroportin-mediated iron export in these cells, the hepatocyte-derived peptide hormone hepcidin plays a central role in intestinal iron resorption, macrophage iron recycling and hepatic iron storage (Figure 1A).3 Functional studies reveal the internalization of ferroportin upon hepcidin binding with subsequent ubiquitination of the protein result- ing in the diminution of cellular iron export. The mechanism of the inhibition of cellular iron efflux is not fully elucidated.4 Genetic ferroportin variants result in autosomal dominantly inherited hereditary hemochromatosis (HH) type 4 or ferro- portin disease which is traditionally divided into two entities primarily based on the pattern of cellular iron distribution. Classical ferroportin disease (type 4A) is characterized by macrophage iron retention and decreased circulating iron avail- ability for erythropoiesis, and is clinically recognized by the presence of elevated serum ferritin concentrations with low to normal transferrin saturation (TSAT) and poor tolerance to phlebotomy. Non-classical (atypical, type 4B or ferroportin- associated HH) ferroportin disease is characterized by parenchymal (hepatocellular) iron overload with both elevated serum ferritin and TSAT and is indistinguishable from other forms of HH.5
To assess the pathogenicity of a ferroportin variant function- al studies on iron export and/or ferroportin expression are per- formed in cell-lines (mostly HEK293T, a human embryonic kidney cell line) transfected with the variant. However, inter- pretation of results may be hampered by these studies being done in non-enterocyte and non-macrophage cell lines. Moreover, comparability between reports may have short- comings, since iron loading and hepcidin exposure protocols, the mode of iron export measurements and the determination of ferroportin expression differ largely between studies. Nevertheless, the results obtained are quite consistent, in that patients with the type 4A phenotype are mostly associated with loss-of-function ferroportin variants which display decreased membrane expression and/or iron transport in func- tional assays, while patients with the type 4B phenotype have gain-of-function variants with preserved membrane expres- sion and iron export capacity even after hepcidin exposure. Notwithstanding a different pattern of cellular iron distribu- tion, a review of numerous case series of ferroportin disease reveals that in those patients who underwent magnetic reso- nance imaging (MRI) or liver biopsy, hepatic iron content is increased for both types of ferroportin disease. These observa- tions indicate that loss-of-function variants also result in body iron overload.
Variety in iron parameters reported for patients with ferro- portin variants is large and the correlation between iron export capacity in functional studies and clinical phenotype is poor,
especially on an individual level. The establishment of the pathogenicity of ferroportin variants may not only be impeded by the presence of iron homeostasis modulating co-morbidi- ties such as alcohol consumption and liver steatosis, but also by the limited applicability of in silico prediction models in the absence of a fully elucidated three-dimensional (3D) structure of ferroportin. In the most widely accepted secondary struc- ture, ferroportin comprises 12 helices located in 12 transmem- brane (TM) domains bound via six extracellular (ES) and five intracellular (IS) segments with a large intracellular loop between the 6th and 7th transmembrane helix and an intracellu- larly located N- and C-terminus. The available 3D models are based on a comparison with membrane transport proteins from a wide range of other species with only a 10 - 24% sequence homology and a maximal 40% similarity. Two stud- ies, using E. coli glycerol-3-phosphate transporter GlpT and lac- tose permease LacY or the Bdellovibrio bacteriovorus Bd2019 iron transporter as template, respectively, reveal an open inward and an open outward structure with an intra- and extracellular gate between the 6th and 7th transmembrane domain.6,7 Site mutagenetic and conformational studies revealed the residues, located at IS1, IS2 and IS5, that are important in intracellular gate interaction and the residues, located at TM1, TM12 and ES1 and ES4, that are involved in extracellular gate interaction. These studies also identified ferroportin residues that are essential for binding and ubiquitination of hepcidin and may be involved in iron binding and egress. The unequivocally hep- cidin-resistant gain-of-function ferroportin variants Cys326Ser, Tyr501Cys, Asp504Asn and Tyr64Asn and His507Arg are reported to have impaired hepcidin binding and hepcidin- dependent ubiquitination, respectively. In the open outward structure, variants causing impaired hepcidin binding were found to be localized within the extracellular gate, while vari- ants causing impaired ubiquitination were found in the periph- ery of the molecule, suggesting that these latter variants inter- fere with appropriate folding after hepcidin binding.8 These findings provide a molecular basis for the observed cellular dis- tribution pattern of the type 4B iron overload in patients and the behavior in functional tests performed for these hepcidin- resistant gain-of-function variants (Figure 1B).
The study by Ka et al., published in this issue, provides inter- esting insights into the pathophysiologic mechanisms involved in ferroportin disease caused by loss-of-function variants. They describe 22 patients from six independent families with hyper- ferritinemia, a normal TSAT and the heterozygous presence of the Arg178Gln variant. The serum hepcidin levels determined in two patients were above the reference range. The hepatic iron content, estimated by MRI methodology in three of the patients, was mildly elevated and a liver biopsy, performed in one patient, revealed predominant iron deposition in Kupffer cells. Although Arg178Gln displayed a reduced export of 55Fe out of transfected HEK293T cells, the variant was properly localized on the cellular membrane with disappearance after
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