Primary Iron Overload With Inappropriate Hepcidin
Expression in V162del Ferroportin Disease
Heinz Zoller,1,2Ian McFarlane,3Igor Theurl,4Sylvia Stadlmann,5Elizabeta Nemeth,6David Oxley,7Tomas Ganz,6
David J. Halsall,3Timothy M. Cox,2and Wolfgang Vogel1
Ferroportin disease (hemochromatosis type 4) is a recently recognized disorder of human
iron metabolism, characterized by iron deposition in macrophages, including Kupffer cells.
Mutations in the gene encoding ferroportin 1, a cellular iron exporter, are responsible for
this iron storage disease, inherited as an autosomal dominant trait. We present clinical,
histopathological, and radiological findings in a family with the most common ferroportin
mutation, V162del. In the index case, the disorder is characterized by abundant deposition
mucosa, and also in squamous cell carcinoma of the lung). The radiological findings indi-
cated the presence of excess iron in bone marrow and spleen. Despite a significant burden of
iron, no features of chronic liver disease were found in affected members of the family,
including individuals aged up to 80 years. Hyperferritinemia greater than 1,000 ?g/L was a
penetrant biochemical finding before the second decade in life and was associated with
significantly increased serum concentrations of pro-hepcidin that correlated positively with
urinary hepcidin concentrations. In conclusion, the systemic iron burden in ferroportin
disease is not a sufficient cause for chronic liver disease. In patients with most, but not all,
ferroportin mutations, retention of iron in macrophages of the liver and other organs may
disease is associated with elevated serum pro-hepcidin levels. (HEPATOLOGY 2005;42:466-472.)
syndrome, recently named hemochromatosis type 4 or
ferroportin disease.1-9Clinically, ferroportin disease is
rarely associated with hepatic cirrhosis, and the principal
pathological finding is iron deposition predominantly in
utations in the human gene encoding ferro-
portin 1 (FPN1/IREG-1/MTP-1/SLC40A1)
are associated with an unusual iron overload
spicuously Kupffer cells within the liver.
The most common FPN1 mutation is deletion of a
valine at position 162 and has been identified in patients
of various ethnic backgrounds.3,8,10,11Deletion of this
amino acid causes loss of function and when studied in
vitro causes accumulation of iron in cultured cells.12
FPN1 is a putative transmembrane iron channel impli-
cated in the egress of iron from duodenal enterocytes,
macrophages, hepatocytes, and placenta (reviewed in
McKie and Barlow13). Impaired iron export from macro-
phages in patients with mutations in the FPN1 gene has
been proposed as the explanation for the accumulation of
iron that occurs in organs containing abundant macro-
phages such as the liver, spleen, and bone marrow.14
The differential diagnosis of iron accumulation in
macrophages also includes the anemia of inflammation,
in which anemia is associated with inappropriate reten-
Hepcidin, which is expressed in the liver, heart,16and
kidney,17is the key mediator of anemia of inflamma-
tion,18,19and synthetic hepcidin was shown to interact
physically with ferroportin in a cellular overexpression
Abbreviations: MRI, magnetic resonance imaging; CT, computed tomography;
PCR, polymerase chain reaction.
From the Clinical Divisions of
4General Internal Medicine, Innsbruck Medical University, Innsbruck, Austria;
2Department of Medicine, University of Cambridge, Cambridge, UK;3Depart-
ment of Clinical Biochemistry, Cambridge University Hospitals NHS Trust, Cam-
bridge, UK;5Department of Pathology, Innsbruck Medical University, Innsbruck,
California, Los Angeles, CA; and7Babraham Institute, Cambridge, UK.
Received November 1, 2004; accepted May 11, 2005.
Supported by the Wellcome Trust, the Sackler Foundation, the Verein zur Fo ¨r-
derung der Forschung in Gastrenterologie und Hepatologie, and the BBSRC.
Cambridge, Addenbrooke’s Hospital, Level 5, Box 157, Hills Road, Cambridge
CB2 2QQ, United Kingdom. E-mail: firstname.lastname@example.org; fax: (44)
Copyright © 2005 by the American Association for the Study of Liver Diseases.
Published online in Wiley InterScience (www.interscience.wiley.com).
Potential conflict of interest: Nothing to report.
1Gastroenterology and Hepatology, and
system, causing internalization and degradation, and de-
creased export of iron.20Because hepcidin expression in-
feedback loop in which body iron homeostasis is con-
trolled by hepcidin has been postulated.
The important physiological function of hepcidin is
further demonstrated by the observation that homozy-
igrees with juvenile hemochromatosis.22-25Moreover,
targeted disruption of the hepcidin gene in mice induces
severe iron overload of the liver and pancreas, resembling
that observed in human hemochromatosis.26In the clas-
sical HFE1-associated hemochromatosis (hemochroma-
tosis type 1), as well as in rare hemochromatosis due to
homozygous mutations in genes encoding transferrin re-
ceptor 2 (hemochromatosis type 3) or hemojuvelin
(hemochromatosis type 2), liver hepcidin mRNA expres-
sion or urinary hepcidin excretion appears to be inappro-
priately low, a finding that may explain the persistently
increased absorption of iron in the face of systemic iron
excess in these diseases.27-29We present the clinical, ra-
diological, histological, and biochemical findings in a
pedigree affected by ferroportin disease (hemochromato-
sis type 4). Extrahepatic iron deposition in an adult man
led to the diagnosis and, as a result, investigations identi-
fied 4 more affected individuals. In all affected individu-
als, serum pro-hepcidin concentrations before iron
depletion therapy were found to be increased more than
threefold above the mean concentration in healthy per-
sons, and a statistically significant correlation between
serum pro-hepcidin concentrations and urinary hepcidin
excretion was found.
Patients and Methods
Informed consent was obtained from all patients in-
from venous blood samples using the QiaAMP DNA
mini Kit (Qiagen, Hilden, Germany) according to the
manufacturer’s instructions. Genetic testing for C282Y
mutation in the HFE gene was performed as previously
described.30Molecular analysis of the human ferroportin
reaction (PCR) amplified, and direct sequencing analysis
carried out on an ABI Prism 310 automated sequencer.
Primers and PCR conditions were used as described in
Cazzola et al.8Serum iron parameters were determined
using standard laboratory techniques (Abbott, Vienna,
Pro-hepcidin ELISA was purchased from DRG Diag-
nostics (Marburg, Germany) and carried out following
the maufacturer’s instructions and as previously de-
scribed.31For quantification of serum pro-hepcidin, fro-
zen serum samples were used, avoiding repeated freeze–
thaw cycles, which can cause significant pro-hepcidin
degradation. Tissues were embedded in paraffin and
stained with hematoxylin-eosin according to standard
techniques. Perls’ Prussian blue stain was carried out as
described by Perls32and Stevens.33
Measurement of urinary hepcidin concentration was
carried out as described elsewhere.34
Index Case. Patient no. 1 underwent cholecystec-
tomy for symptomatic cholelithiasis in 1986, when histo-
logical examination of a macroscopically suspicious
(Fig. 1). The 68-year-old patient was subsequently found
to have a serum ferritin of 5,265 ?g/L (reference range,
5-307 ?g/L) and a serum transferrin saturation of 37.5%
(reference range, 20%-50%). Initial genetic investiga-
tions for hereditary hemochromatosis type 1 showed the
HLA phenotype A2/A3, B7/B40, BW 6/CW 3, of which
A3 and B7 are known to be associated with this hemo-
which was performed in 1997, showed no pathological
mutations in the HFE1 gene. The patient refused liver
biopsy, but magnetic resonance imaging showed abnor-
ing tissue iron storage.
In light of these findings, secondary causes for iron
overload, such as heavy alcohol consumption or iron-
Fig. 1. (A) Mesenteric lymph node with brown hemosiderin deposits
on HE staining (arrows) (original magnification ?200). (B) Duodenal
biopsy–stainable iron in villous macrophages (Perls’ Prussian blue stain;
original magnification ?100). (C) Gastric antrum biopsy–stainable iron
in gastric mucosa (original magnification ?200). (D) Squamous cell
carcinoma of the lung–stainable iron in tissue macrophages (Perls’
Prussian blue stain; original magnification ?100).
HEPATOLOGY, Vol. 42, No. 2, 2005ZOLLER ET AL.467
loading anemias, were excluded. Neither was there any
evidence of chronic inflammation, which could cause se-
omy was started after serum iron parameters remained
unchanged over 5 years. Phlebotomy was well tolerated,
22 months (equivalent to approximately 12 g iron). Dur-
ing this treatment, serum ferritin decreased from 5,265
?g/L to 1,140 ?g/L. Although hemoglobin levels were
never below 12 g/dL, therapeutic phlebotomy was
stopped for 8 months because of non-compliance, which
resulted in an increase in serum ferritin concentration to
2,111 ?g/L. On follow-up hemoglobin, aminotrans-
ferases, bilirubin, and coagulation tests remained normal;
At the age of 77 years, the patient complained of grad-
ually worsening shortness of breath: a diagnosis of squa-
mous carcinoma of the bronchus was made by biopsy of a
tumor obstructing a main bronchus. At the age of 80
years, the patient died of complications of the carcinoma
after a course of palliative radiation therapy.
On family screening for iron overload, 2 sons of the
index case were found to have iron overload (Fig. 2), one
aged 29 (patient no. 5) with a serum ferritin of 4,935
ferritin of 7,239 ?g/L. The latter finding may have been
exacerbated by heavy consumption of alcohol before pre-
ferritin and the other was not available for a medical in-
vestigation. In the third generation of this family, the
17-year-old twin sons of patient no. 3 were found to have
serum ferritin concentrations of 1,150 ?g/L and 1,324
?g/L. The serum iron parameters of all family members
available for investigation are displayed in Table 1.
In all affected family members, phlebotomy was well
tolerated, and in patient no. 5 fortnightly therapeutic
phlebotomy for 23 months resulted in a decline of serum
ferritin concentrations from 4,935 ?g/L to 345 ?g/L.
Complete hematological and biochemical parameters re-
mained within the normal range during follow-up. In all
other cases, the diagnosis was established only recently,
and hence data on the effects of prolonged phlebotomy
are not yet available.
Histopathology. Histological examination of the
macroscopically suspicious mesenteric lymph node,
which was removed during the cholecystectomy, showed
hemosiderin deposits (Fig. 1A).
The patient refused liver biopsy, which was offered
after identification of stainable iron in the lymph node
that led to the detection of hyperferritinemia. However,
during follow-up, gastroduodenoscopy was performed at
examination of the duodenal biopsy showed stainable
iron in the villous macrophages. Furthermore, histologi-
Fig. 2. (A-C) Liver histopathology of patient no. 5. There were hemo-
siderin deposits in the liver sinusoids, but no hemosiderin was found in
hepatocytes. (D) Perls’ Prussian blue stain of the liver showing consid-
erably less stainable iron in hepatocytes than in Kupffer cells. Original
magnification ?50 (A), ?200 (B), ?400 (C), ?500 (D).
Table 1. Serum Iron Parameters of All Family Members Available for Detailed Investigation
NOTE. Affected individuals are listed in italics.
468ZOLLER ET AL.HEPATOLOGY, August 2005
(epidermoid cellular) also reflected increased stainable
iron in tumor-associated macrophages (Fig. 1D).
The liver biopsy of patient no. 2 at the age of 29 years
is shown in Fig. 2, where excessive brown deposits (he-
mosiderin) within the liver were found. Unlike patients
with classical HFE1-associated hemochromatosis, no he-
mosiderin and little stainable iron were detected in hepa-
tocytes; hemosiderin was mainly found in the sinusoids.
accumulation, predominantly in Kupffer cells (Fig.
2B,D). No signs of inflammatory liver disease or fibrosis
were found. Taken together, these findings demonstrate
that ferroportin disease (hemochromatosis type 4) caused
by the V162del mutation is a systemic disorder in which
the macrophage is the principal cell affected.
Radiology. To exclude hepatocellular carcinoma at
the time of presentation with severe iron overload and to
further assess the severity of hepatic iron overload, mag-
netic resonance imaging (MRI) was performed. The sig-
nal intensity of T2-weighted MRI scans of the liver was
significantly reduced in patient 1 and patient 5, which
accords with the histological findings that demonstrated
heavy iron deposits in the liver. Furthermore, T2 signal
intensities in spleen and bone marrow were also reduced
in both patients, suggestive of iron deposition in these
macrophage-rich organs (Fig. 3). Computed tomography
scanning of the abdomen was carried out during surveil-
radiological changes, compatible with iron deposition in
liver, spleen, and lymph nodes, were found (not shown).
the liver veins and signal hyperintensity of the spleen are
known to be nonspecific CT findings, which can be due
to iron deposition.
Genetics. The pedigree is displayed in Fig. 4A. The
inant trait, with affected individuals represented in all 3
generations. Sequencing of the ferroportin gene revealed
the most common ferroportin mutation (a deletion of 3
base pairs in exon 5), which results in loss of a valine in
heterozygous family members. Unaffected family mem-
ferritin concentrations. Mutations in all other exons of
the ferroportin gene were excluded by complete sequenc-
ing of the coding region. In summary, the mutation was
fully penetrant and co-segregated with the phenotype of
hyperferritinemia and normal serum transferrin satura-
tion in this family.
Serum Pro-hepcidin. In the 3 affected individuals
from whom pre-treatment serum samples were available
ng/mL, and, 649 ng/mL, all of which are above the re-
ported range in healthy individuals (52-153 ng/mL).31
Furthermore, these values are even higher than the re-
ported median for chronic renal insufficiency (148 ng/
mL).31In the 2 affected family members (patient 1 and
patient 2) from whom serum samples were available only
after initiation of treatment, serum pro-hepcidin concen-
trations were determined at 68 ng/mL and 159 ng/mL,
respectively. During venesection therapy, the concentra-
tion of serum pro-hepcidin gradually decreased.
As shown in Fig. 5B, a statisticaly significant correla-
tion between serum pro-hepcidin concentration and uri-
nary hepcidin concentration was found (Deming
level,with hyperferritinemiapresent inall
Hyperferritinemia is a common finding in clinical
ical conditions such as chronic liver disease, iron-loading
anemias, inflammation, and malignant diseases, in all of
which serum iron and transferrin saturation are usually
decreased. HFE1-associated hemochromatosis is a fre-
quent cause of hyperferritinemia but usually is associated
with elevated serum iron concentrations and transferrin
saturation. Recent advances in the study of hereditary
iron overload syndromes have shown that hereditary
hemochromatosis can be caused by homozygous muta-
tions in several other genes (TfR2, HJV, HAMP)35and
Fig. 3. Images of a T2-weighted MRI scan of the
abdomen of patient no. 5. Note the low signal intensity
in liver, spleen, and bone marrow with normal signal
intensities in pancreas and kidneys. (A) Abdomen
transversal section showing iron accumulation in the
liver and spleen. (B) Abdomen transversal section
showing no iron accumulation in pancreas and kid-
HEPATOLOGY, Vol. 42, No. 2, 2005 ZOLLER ET AL.469
may be associated with more complex compound geno-
the most common ferroportin mutation, V162del. On
histological examination of various tissues, iron deposi-
tion was found not only in Kupffer cells but also in many
other types of macrophages. Stainable iron was detected
in duodenal villous macrophages, lymph node macro-
mous cell carcinoma of the lung. Systemic macrophage
CT and MRI scans, where the radiological abnormalities
were strongly indicative of increased tissue iron in spleen
and bone marrow.
The total burden of iron in the body was apparently
suggested by high serum ferritin concentrations and by
estimation of mobilizable iron during therapeutic phle-
botomy. The high iron burden is notable, because no
clinical, histological, biochemical, or radiological signs of
liver disease or extrahepatic manifestations of hemochro-
matosis could be found; cardiomyopathy, diabetes melli-
tus, arthropathy, hypogonadism, or endocrinopathy
(reported clinical features in patients with the N144H
mutation in the ferroportin gene9), were significant by
ferroportin disease (hemochromatosis type 4)9,10in pa-
tients with the V162del mutation, therapeutic phlebot-
omy was well tolerated and iron could be readily
mobilized without induction of anemia.
The absence of any clinical manifestation of iron-stor-
age disease in this pedigree even in individuals of ad-
vanced age, however, raises the question as to whether
phlebotomy should have been started at all. Liver fibrosis
hemochromatosis, with iron deposition in hepatocytes
and increased transferrin saturation.3-10,37,38The severity
Fig. 4. (A) Pedigree of a family with iron
storage associated with a mutation in the hu-
man ferroportin gene. Affected individuals, het-
erozygous for the V162del mutation, are
represented by solid symbols, which are la-
beled with age at diagnosis (above) and serum
ferritin values (below). (B) DNA Sequence elec-
tropherograms of an unaffected (above) and an
affected individual (below). Note that GTT
(484-486) is missing, resulting in an in-frame
deletion on one allele and loss of a valine
residue at position 162 of the mature protein.
Fig. 5. (A) Serum pro-hepcidin concentrations in healthy individuals,
untreated ferroportin disease (hemochromatosis type 4 [HH type 4]),
treated ferroprotin disease, and treated adult hemochromatosis type 1.
Broken line indicates the highest concentration reported in healthy
individuals’ normal (153 ng/mL).30(B) Correlation between serum pro-
hepcidin and urinary hepcidin concentrations.
470 ZOLLER ET AL. HEPATOLOGY, August 2005
of iron deposition in Kupffer cells and the absence of
no. 5 suggests that iron loading of macrophages is well
tolerated, but equivalent loading of hepatocytes may be
the cause of fibrosis and cirrhosis in classical hemochro-
Relative hepcidin deficiency has been reported in
HFE1-associated hemochromatosis, juvenile hemochro-
matosis, and TfR2-associated hemochromatosis.29,31,34
Here we provide a report of serum pro-hepcidin expres-
sion in ferroportin syndrome (hemochromatosis type 4)
in which we demonstrate a strong correlation between
serum pro-hepcidin concentration and urinary hepcidin
concentrations in individuals with elevated hepcidin ex-
pression. In contrast to other types of hemochromatosis,
serum pro-hepcidin concentrations in patients heterozy-
gous for the V162del mutations in the ferroportin gene
were found to be markedly increased, which is in accor-
dance with a recent study demonstrating increased uri-
nary hepcidin concentrations34in other patients with this
mutation. The increase, however, appears to be dynamic
so that hepcidin remains responsive to changes in iron
status, because phlebotomy resulted in a gradual decrease
in serum pro-hepcidin in at least 2 patients. The process-
ing of pro-hepcidin to hepcidin, however, has not yet
pro-hepcidin and urinary hepcidin also correlate in other
disorders of iron homeostasis.
One explanation for the coexistence of systemic iron
overload with high hepcidin concentrations, which
should reduce iron absorption, is that the hepcidin signal
can be overridden by another signal that induces iron
absorption. In our youngest patients, aged 17 years, se-
and serum pro-hepcidin concentrations also were in-
creased. Because we observed an age-dependent increase
in serum ferritin and pro-hepcidin, iron absorption in
these patients was still inappropriately high, even though
serum (pro-)hepcidin concentrations were elevated. In
the light of this observation, we question whether there is
indeed an unidentified “erythroid regulator” inducing
If so , such an entity appears to override the effect of high
(pro)-hepcidin concentrations.40However, the severe
iron overload phenotype in patients with hepcidin defi-
ciency22,23and the severe iron deficiency that develops in
gests that this peptide is the principal determinant of iron
absorption in mammals.
Recently, Nemeth et al.20found that synthetic hepci-
dins bind to ferroportin expressed as a fusion protein in
cultured cells, thereby inducing ferroportin internaliza-
tion and its subsequent degradation.20As shown in vitro,
if the V162del ferroportin variant is overexpressed in cell
lines, no such internalization can be induced by synthetic
hepcidin.43However, because hemochromatosis type 4
has only been recognized in individuals heterozygous for
mutations in the human ferroportin gene, the contribu-
tion of ferroportin expressed from the coexistent wild-
type allele to the disease phenotype cannot readily be
There are indications that type 4 hemochromatosis it-
on the nature of the mutation affecting the ferroportin
molecule. Thus, at least some patients with mutations in
amino acid 144 have been reported to show increased
transferrin saturation and evidence of liver disease, with-
out any other known causes for systemic iron overload or
hepatocyte iron loading. Ferroportin is highly expressed
in duodenal enterocytes, hepatocytes, and macrophages
(as well as in the placenta). It is conceivable that certain
mutations do not induce a major bottleneck to iron re-
lease from macrophages but still display resistance to the
inhibitory effects of hepcidin on iron export. In such pa-
tients, the phenotype could be dominated by excessive
intestinal iron absorption unresponsive to increased hep-
boring V162del mutations, may be primarily affected by
the consequences of functional deficiency of ferroportin
in macrophages. Paradoxically, retention of iron in mac-
rophages may protect these patients against injury to pa-
renchymal cells in the liver and other organs, even when
intestinal absorption and systemic iron load are also in-
In summary, we report here detailed studies in a
pedigree with an unusual presentation of hemochro-
matosis type 4 that highlight the systemic nature of the
disorder. Despite a greatly increased burden of iron,
chronic liver disease is not the clinical hallmark of the
disorder. Finally, we report high serum pro-hepcidin
concentrations that were apparent in the early stages of
hemochromatosis type 4—an unexpected finding, be-
cause aquisition of iron by absorption at this stage of
the disorder is increased.
family members for participation in the study. We also
thank Dr. M. Schocke for provision of MR images, Dr.
Peter Obrist and Prof. Gregor Mikuz for initial review of
Christian Hallbrucker for collection of clinical data. The
Ms. Gisela Egg is acknowledged.
The authors thank all patients and
HEPATOLOGY, Vol. 42, No. 2, 2005 ZOLLER ET AL.471
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