In vitro functional analysis of human ferroportin (FPN) and hemochromatosis-associated FPN mutations

Abstract

Type IV hemochromatosis is associated with dominant mutations in the SLC40A1 gene encoding ferroportin (FPN). Known as the "ferroportin disease," this condition is typically characterized by high serum ferritin, reduced transferrin saturation, and macrophage iron loading. Previously FPN expression in vitro has been shown to cause iron deficiency in human cell lines and mediate iron export from Xenopus oocytes. We confirm these findings by showing that expression of human FPN in a human cell line results in an iron deficiency because of a 3-fold increased export of iron. We show that FPN mutations A77D, V162⌬, and G490D that are associated with a typical pattern of disease in vivo cause a loss of iron export function in vitro but do not physically or functionally impede wild-type FPN. These mutants may, therefore, lead to disease by haploinsufficiency. By contrast the variants Y64N, N144D, N144H, Q248H, and C326Y, which can be associated with greater transferrin saturation and more prominent iron deposition in liver parenchyma in vivo, retained iron export function in vitro. Because FPN is a target for negative feedback in iron homeostasis, we postulate that the latter group of mutants may resist inhibition, resulting in a permanently "turned on" iron exporter. IntroductionHemochromatosis is an iron overload disease characterized by excessive iron uptake through the enterocytes of the gut and subsequent deposition in the liver, spleen, and heart, leading to tissue damage. Currently 4 subtypes of hemochromatosis are recognized. In Caucasian populations disease is predominantly associated with mutations in the HFE gene, discovered in 1996 1 ; HFE-linked hemochromatosis is designated type I. A more severe form of the disease, juvenile hemochromatosis (type II hemochromatosis), is linked to mutations in either the recently identified hemojuvelin 2 or the antimicrobial peptide hepcidin. 3,4 Hepcidin is normally up-regulated in response to high serum iron, but it is unexpectedly low in patients with hemochromatosis because of mutations in HFE, 5 hemojuvelin, 2 and transferrin receptor 2 (TfR2). 6,7 TfR2, which is expressed by hepatocytes, 8,9 is mutated in hemochromatosis type III. The iron exporter ferroportin/iron-regulated transporter 1/metal transporter protein 1 (FPN/IREG-1/MTP-1; gene symbol SLC40A1) was discovered simultaneously by 3 groups. [10][11][12] Since that time, numerous mutations in the gene have been implicated in patients from diverse ethnic origins with previously unexplained hemochromatosis. Iron overload disease because of a mutation in FPN is referred to as type IV hemochromatosis or ferroportin disease. 13 FPN is expressed on basolateral membranes of mature intestinal enterocytes and the basal membrane of the placental syncytiotrophoblast. [10][11][12] Another site of high expression of FPN is in macrophages, including Kupffer cells in the liver and in the red pulp of the spleen. 12,14 These sites of expression are consistent with a role for FPN in transport of iron from the gut to the serum, f...

Hemizygous deletion of COL3A1, COL5A2, and MSTN causes a complex phenotype with aortic dissection: a lesson for and from true haploinsufficiency

“…This lack of evidence for HFE4 is somewhat unexpected, as heterozygous loss-offunction SLC40A1 mutations have previously been associated with HFE4. 41,42 However, as the age of onset of HFE4 is up to 60 years in males and B10 years later in females (age-related penetrance), 43 it is possible that young carriers of the SLC40A1 deletion failed to present clinical symptoms of HFE4 in this study because of their age of 15 (53I, female), 18 (53J, male), and 49 (53E, male) years at examination. As, to our knowledge, no patient with complete hemizygous deletion of this gene has previously been reported at the molecular level, this is the first description of the (so far normal) clinical phenotype of individuals with true SLC40A1 haploinsufficiency, suggesting the negligible role of haploinsufficiency in the pathogenesis of HFE4.…”

The molecular genetics of haemochromatosis

“…24 It has been reported for the main V162del mutation and to a lesser extent for the older A77D mutation, which has been associated with an age-dependent rise in transferrin saturation. These loss-of-function mutations do not impede the action of wild-type ferroportin in vitro 115 and may cause the disease by haploinsufficiency, a model consistent with the autosomal dominant trait of the disease and first suggested by Montosi et al 106 The model is related to a more pronounced effect of the mutation considered on iron recycling from senescent erythrocytes by macrophages, which normally supply B20 -30 mg of iron per day, than on absorption of dietary iron by enterocytes, which normally supply B1 mg of iron per day. 122 In that case, iron retention by macrophages leads to accumulation of iron in the tissue, which explains the high-serum ferritin levels, and reduces the serum iron available for erythropoiesis, which explains the low transferrin saturation.…”

“…Further investigations may help to explain this phenotypic heterogeneity, in particular the search for modifier factors, but the very recent studies of Schimanski et al 115 have supplied some very important information. These authors first showed that the 77D, V162del and 490D ferroportin protein displayed a greatly reduced capacity to reach the Figure 5 Schematic representation of mutations in the SCL40A1 gene.…”

Measurement of serum hepcidin-25 levels as a potential test for diagnosing hemochromatosis and related disorders

“…In patients with type A disease, SLC40A1 mutations generate a protein that is defective in cell surface expression or loss of iron export function. 42,43,44 In the less prevalent type B, the mutant gene generates proteins that show normal cell surface expression but reduced sensitivity to hepcidin. 42,43 The resistance of ferroportin to hepcidin might be the key not only to the iron overload of ferroportin disease, but also to the secondary iron overload syndromes.…”