Volume 140, Issue 7, Pages 2056-2063.e1 (June 2011) Identification of Mutations in SLC40A1 That Affect Ferroportin Function and Phenotype of Human Ferroportin Iron Overload  Roman Mayr, William J.H. Griffiths, Martin Hermann, Ian McFarlane, David J. Halsall, Armin Finkenstedt, Andrew Douds, Susan E. Davies, Andreas R. Janecke, Wolfgang Vogel, Timothy M. Cox, Heinz Zoller  Gastroenterology  Volume 140, Issue 7, Pages 2056-2063.e1 (June 2011) DOI: 10.1053/j.gastro.2011.02.064 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 (A and B) Pedigree of family A and family B. Arrows indicate index cases. SLC40A1 mutation p.W158C was identified in family A and p.H507R in family B. The index case of family A tested negative for the common HFE polymorphism C282Y. The index case of family B tested heterozygote for H63D. TS, transferrin saturation. Gastroenterology 2011 140, 2056-2063.e1DOI: (10.1053/j.gastro.2011.02.064) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 Magnetic resonance imaging (MRI) of the upper abdomen of index cases 1 and 2. (A) T2-weighted MRI of index case 1 (p.W158C) shows decreased signal intensity in liver, spleen, and bone marrow, indicating iron accumulation in these organs. (B) MRI of index case 2 (p.H507R) shows iron-related signal attenuation in the liver but not in spleen and bone marrow. Gastroenterology 2011 140, 2056-2063.e1DOI: (10.1053/j.gastro.2011.02.064) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 Liver sections of biopsy specimens from index cases. (A–C) Perls' stained liver section of index case A (p.W158C). (B) High-power view of the area indicated by the bold arrow in A shows that iron deposits are predominantly confined to Kupffer cells. (C) High-power view of the area indicated by a thin arrow in panel A shows small amounts of stainable iron in hepatocytes. (D–F) Perls' stained liver section of index case B (p.H507R). Mild steatosis siderosis (E) and siderosis of periportal hepatocytes (F) is shown. Iron overload affects predominantly hepatocytes. Gastroenterology 2011 140, 2056-2063.e1DOI: (10.1053/j.gastro.2011.02.064) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 Ferritin expression in ferroportin transfected 293T cells and its response to hepcidin. Incubation of 293T cells with transferrin induces ferritin expression, which can be reverted by cellular overexpression of normal ferroportin. Addition of hepcidin to normal ferroportin transfected 293T cells induces an increase in ferritin expression, showing hepcidin-mediated inactivation of normal ferroportin. Ferritin expression in p.C326Y transfected ferroportin cells is unresponsive to hepcidin, and low baseline ferritin level shows normal iron export. Ferritin expression in p.A77D mutant ferroportin transfected cells is increased when compared with normal ferroportin-expressing cells, showing impaired iron export. Baseline ferritin expression in p.W158C mutant ferroportin transfected cells is comparable to p.A77D ferroportin transfected cells. Baseline ferritin expression in 293T cells transfected with p.H507R mutant ferroportin and its unresponsiveness to hepcidin is comparable to p.C326Y ferroportin transfected cells. Gastroenterology 2011 140, 2056-2063.e1DOI: (10.1053/j.gastro.2011.02.064) Copyright © 2011 AGA Institute Terms and Conditions

Figure 5 Effect of hepcidin on the expression of FPN-GFP assessed by flow cytometry. Hepcidin induces a significant decrease in mean cell fluorescence of normal ferroportin overexpressing 293T cells (100% vs 78%; P < .05), whereas no such decrease can be observed in cells expressing ferroportin that harbor the p.C326Y, p.W158C, or p.H507R mutation. Apparent hepcidin resistance in p.W158C mutant cells can be explained by intracellular retention of mutant ferroportin; see Figure 6. Gastroenterology 2011 140, 2056-2063.e1DOI: (10.1053/j.gastro.2011.02.064) Copyright © 2011 AGA Institute Terms and Conditions

Figure 6 Subcellular localization of normal ferroportin and mutants studied by live cell confocal microscopy. Normal, p.C326Y, and p.H507R mutant FPN-GFP fusion protein shows prominent staining of the plasma membrane in transfected 293T cells, whereas p.W158C-FPN-GFP predominantly localizes to a perinuclear compartment with no cytoplasmic staining. p.A77D-FPN-GFP fusion protein is located at the plasma membrane and intracellularly. Gastroenterology 2011 140, 2056-2063.e1DOI: (10.1053/j.gastro.2011.02.064) Copyright © 2011 AGA Institute Terms and Conditions

Figure 7 (A) Hepcidin induces a significant decrease in FPN-GFP expression as assessed by flow cytometry. *P < .05 when compared with untreated cells. (B) The effect of hepcidin preincubated with peptide H is significantly reduced, as shown by higher normal ferroportin expression. In contrast, peptide R, which harbors the H507R mutation, has no effect on hepcidin activity (**P < .05 when compared with cells treated with hepcidin alone). (C) Amino acid sequence of peptide H and peptide R corresponding to residues 500 to 518 of normal and p.H507R mutant ferroportin, respectively. Gastroenterology 2011 140, 2056-2063.e1DOI: (10.1053/j.gastro.2011.02.064) Copyright © 2011 AGA Institute Terms and Conditions