Blood, 15 September 2002, Vol. 100, No. 6, pp. 2246-2248
BRIEF REPORT
Anemia and iron overload due to compound heterozygosity for novel
ceruloplasmin mutations
Sandra Bosio,
Marco De
Gobbi,
Antonella Roetto,
Gabriella Zecchina,
Eugenio Leonardo,
Mario Rizzetto,
Claudio Lucetti,
Lucia Petrozzi,
Ubaldo Bonuccelli, and
Clara Camaschella
From the Dipartimento di Scienze Cliniche e Biologiche,
and Dipartimento di Gastroenterologia, Università di Torino,
Turin, Italy; Dipartimento di Patologia, Azienda Sanitaria Ospedaliera
San Luigi-Orbassano, Turin, Italy; and Dipartimento di Neuroscienze,
Università di Pisa, Pisa, Italy.
 |
Abstract |
Aceruloplasminemia is a recessive disorder characterized by anemia,
iron overload, and neurodegeneration, caused by the absence of
ceruloplasmin (Cp), a multicopper oxidase important for iron export.
Few patients homozygous for loss of function mutations of the Cp gene
have been reported. We describe a 62-year-old white woman with heavy
liver iron overload, diabetes, anemia, and neurologic symptoms. She was
compound heterozygote for 2 novel mutations that result in the absence
of hepatocyte Cp: an adenine insertion at nucleotide 2917 causing a
truncated protein and a C-G transversion causing a glutamine
glutamic
acid substitution at position 146. Although rare in whites,
aceruloplasminemia should be considered in the differential diagnosis
of unexplained anemia associated with iron overload, because these
features anticipate progressive neurologic symptoms. We propose that
anemia, secondary to the impaired macrophage iron release, plays a
major role in hepatic iron overload through increased absorption
mediated by the erythroid regulator.
(Blood. 2002;100:2246-2248)
© 2002 by The American Society of Hematology.
| |
Introduction |
Hereditary aceruloplasminemia is a rare autosomal
recessive disease characterized by iron overload and progressive
neurodegeneration. The disease is caused by the absence of
ceruloplasmin (Cp), a copper-containing ferroxidase, which catalyses
the oxidation of ferrous to ferric iron, a change required for release
of iron to plasma transferrin.1 It is hypothesized that in
reticuloendothelial (RE) cells and hepatocytes Cp cooperates with the
iron export protein ferroportin 1 (FPN1).2 Cp deficiency
results in iron deposition in the liver, pancreas, basal ganglia, and
other organs. Patients develop diabetes mellitus, retinal degeneration,
ataxia, and dementia late in life.3,4 A mild-to-moderate
degree of anemia with low serum iron and elevated serum ferritin is a
constant feature.3-6
The Cp gene maps to chromosome 3q21-24.7 It consists of 19 exons8 and encodes a protein of 1046 amino
acids.9 Aceruloplasminemia has been described mainly in
Japanese patients3-6,10-17 and rarely in
whites.5,18
We report data on a 62-year-old Italian woman, compound
heterozygote for 2 novel mutations that hamper Cp expression in
hepatocytes. Our findings suggest that the disorder should be
considered in the differential diagnosis of anemia associated with
unexplained iron overload. Based on the severity of liver iron loading,
the mechanisms leading to iron overload in aceruloplasminemia are revisited.
| |
Study design |
Case report
The proband is a 62-year-old Italian woman. No family history of
diabetes, iron overload, anemia, or neurologic disorders was recorded.
At age 38 years the patient developed insulin-dependent diabetes
mellitus. A mild degree of anemia (hemoglobin 9.1 g/L) with
normal mean corpuscular volume (MCV; 86 fL) and low-normal values of
mean corpuscular hemoglobin (MCH; 27.2 pg) and mean corpuscular
hemoglobin concentration (MCHC; 31.7 g/dL) was documented at age 51 years. Serum iron concentration was 33 µg/dL, transferrin saturation
12%, and serum ferritin 819 µg/L. Bone marrow aspirate revealed mild
dyserythropoiesis; iron staining showed abundant iron in RE cells and
absence of iron granules in erythroblasts. Moderate anemia and abnormal
iron parameters without evidence of blood losses or inflammatory
diseases were regularly observed at follow-up. Ataxia, dystonia, mild
parkinsonism, and dementia became evident at the age of 62 and
progressed rapidly. Magnetic resonance imaging of the brain showed a
paramagnetic deposition in the basal ganglia, dentate nucleus,
thalamus, substantia nigra, and cerebral and cerebellar cortex (Figure
1). Serum Cp was undetectable. Liver
function tests were normal. No Kayser-Fleischer ring was observed, but
the retina showed a pigmentary degeneration.
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| Figure 1.
Magnetic resonance images of the proband showing iron deposition in the
brain.
Axial T2-weighted magnetic resonance images of the
proband's brain show low-signal areas (arrows) in the dentate nucleus
(A), red nucleus and substantia nigra (B), and corpus striatum
and thalamus (C), which suggest iron deposition in these regions.
|
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Two asymptomatic relatives were evaluated. The patient's son, aged 24, had normal iron parameters but reduced serum Cp concentration (10 mg/dL). The proband's sister, aged 65, had normal iron and Cp values.
Informed consent for molecular studies was obtained according to
institutional guidelines.
Methods
Biochemical determinations were performed by standard
procedures. Liver iron concentration (LIC) and copper concentrations were determined by atomic spectrophotometry on liver biopsy
specimens.19 Immunohistochemical (IHC) studies were
performed on liver biopsy specimens using a sheep anti-Cp horseradish
peroxidase-conjugated antibody (Biogenesis, Poole, United Kingdom).
Oligonucleotide primers were synthesized according to database
sequences (http://www.ncbi.nlm.nih.gov). Polymerase chain reaction was
carried out on genomic DNA in a thermal cycler using 12.5 pMol
primers and 0.5U Taq polymerase in a final volume of 50 µL. Hemochromatosis (HFE) mutations Cys282Tyr and
His63Asp were studied as described.20 Direct
sequencing was performed using a ThermoSequenase Cy 5.5 sequencing kit
in a Seq4 × 4 Apparatus (Amersham-Pharmacia-Biotech, Piscataway,
NJ). Single-strand conformation polymorphism was used to screen
Cp exon 3 for mutations. Fifty healthy individuals with normal iron
parameters served as controls.
| |
Results and discussion |
Liver histology, Perls staining, and IHC results for the
proband are shown in Figure 2A.
LIC was strikingly elevated (535 µmol/g dry weight [dw];
normal, 3-33) and hepatic iron index (LIC divided by years of
age = 8.6) was in the range observed in hereditary HFE.18 However, common HFE mutations were negative.
Rubeanic acid staining for copper was positive (not shown) and liver
copper concentration was remarkably increased (1260 µg/g dw; normal, 20-50 µg/g).
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| Figure 2.
Pathophysiology of aceruloplasminemia in the proband: absence of liver
Cp and causal mutations.
Panel Ai shows the histology of the proband's liver (hematoxylin and
eosin, × 250), with normal lobular architecture without evidence of
fibrosis; Aii shows heavy iron deposition in hepatocytes (Perls colors,
× 250); Aiii shows results of IHC studies with a polyclonal antibody
showing absence of Cp in proband's hepatocytes (IHC, × 250); and Aiv
shows Cp in normal hepatocytes (IHC, × 250). Insert is a high-power
view (× 400). In (B), the left panels show a sequencing chromatograph
of the proband's amplified exon 3 in the ceruloplasmin region spanning
the mutation, compared with a normal control (wild-type). The right
panels are a sequencing chromatograph of the proband's amplified exon
17 in the region encompassing the mutation, compared with a normal
control (wild-type).
|
|
Sequence analysis of the whole Cp gene revealed 2 heterozygous
nucleotide changes: a C-G transversion at nucleotide 436, causing the
glutamineglutamic acid (Gln146Glu) replacement in the protein and an
adenine (A) insertion at position 2917, causing a frameshift and a
premature stop at amino acid 983 (Figure 2B). The truncated protein resulting from A insertion causes the loss of most
copper-binding sites and affects the function of both classic and
glycosylphosphatidylinositol-anchored Cp, produced in the brain by an
alternative splicing.21 Segregation studies confirmed that
the 2 mutations were present in trans. The A insertion was
not present in the 2 relatives studied; Gln146Glu was inherited by the
proband's son, an obligate disease carrier with reduced Cp levels.
This observation and the replacement of a neutral amino acid with a
negatively charged one suggest that Gln146Glu is a causal mutation. A
common polymorphic change is excluded because Gln146Glu was not found
in healthy individuals, but we cannot formally rule out that it is in
linkage disequilibrium with other mutations in the promoter or in
intronic sequences that were not explored by our sequencing.
Moderate anemia, low serum iron, and high serum ferritin, features
shared with anemia of chronic diseases, are constant in aceruloplasminemia. Erythrocyte indices may be either reduced or still
within normal range, as in our case. As shown in Cp-deficient mice,22 a decreased efflux from the sites of iron storage
may overload RE cells and simultaneously impair iron availability for
erythropoiesis. Altered compartmentalization of iron was suggested as
the cause of increased hepatic stores in Cp
/
mouse.22 Based on the entity of hepatic stores in our
patient, we assume that such a loading cannot result only from iron
redistribution, but requires a real increase in intestinal iron
absorption. It must also be underlined that, in contrast with patients,
Cp/ mice have no anemia.22 We propose that
the hepatic iron loading is mediated by the "erythroid regulator,"
a positive determinant of iron absorption in conditions of
iron-deficient or expanded erythropoiesis.23 A similar
mechanism has been recently taken to explain iron overload in
HFE24-25 caused by mutations of FPN1,24,26 the protein that is hypothesized to cooperate with Cp to export iron in
macrophages. Although anemia is not a constant feature in
FPN1-associated HFE, a comparison of the clinical phenotype of patients
affected by these 2 "iron export" disorders may provide insights
into the role of the 2 proteins. In aceruloplasminemia the accumulation
of iron in the basal ganglia is mediated by the lack of expression of
Cp in neurons and indicates that, within the central nervous system, Cp
is essential for iron efflux from storage sites.21
Although rare, aceruloplasminemia is present in whites and should be
included in the differential diagnosis of anemia with high serum
ferritin unrelated to chronic diseases.
| |
Acknowledgments |
We thank Dr Ezio David and Prof Leonardo Lopiano for kind collaboration.
| |
Footnotes |
Submitted February 26, 2002; accepted April 16, 2002.
Prepublished
online as Blood First Edition Paper, May 17, 2002; DOI
10.1182/blood-2002-02-0584.
Partially supported by Telethon grant no. GP00255Y01, European Union
contract QLK6-1999-02237, and the Italian Ministry of the University
and Research (to C.C.).
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Reprints: Clara Camaschella, Dipartimento di Scienze
Cliniche e Biologiche, Università di Torino, Azienda Ospedaliera
San Luigi, 10043-Orbassano, Torino, Italy; e-mail:
clara.camaschella{at}unito.it.
| |
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Abstract
Introduction
