Increased plasma transferrin, altered body iron distribution and microcytic hypochromic anemia in

30 Pages·2006·1.52 MB·English

From www.bloodjournal.org by guest on April 2, 2019. For personal use only.
Blood First Edition Paper, prepublished online September 26, 2006; DOI 10.1182/blood-2006-04-014142

Increased plasma transferrin, altered body iron distribution and microcytic
hypochromic anemia in ferrochelatase deficient mice.
*,1 *,2 3 4 5
Saïd Lyoumi , Marie Abitbol , Valérie Andrieu , Dominique Henin , Elodie Robert ,
Caroline Schmitt1, Laurent Gouya1, Hubert de Verneuil5, Jean-Charles Deybach1, Xavier
2 1 1
Montagutelli , Carole Beaumont , Hervé Puy
* These authors contributed equally to the work.
1 INSERM U773, Centre de Recherche Biomédicale Bichat Beaujon CRB3, BP 416, 75018,
Paris, France ; Université Paris 7 Denis Diderot, site Bichat, BP 416, 75018, Paris, France ; le
Centre Français des Porphyries, Hôpital Louis Mourier, Colombes, France.
2 Institut Pasteur, Unité de Génétique des Mammifères, 75015 Paris, France.
3 AP-HP, Hôpital Bichat, Laboratoire d’hématologie, 75018 Paris, France.
4 AP-HP, Hôpital Bichat, Laboratoire d’anatomo-pathologie, 75018 Paris, France.
5
INSERM E0217, 33076 Bordeaux, France ; Université V Segalen Bordeaux 2, 33076
Bordeaux, France.

Financial support : This research was supported by GIS-Maladie rares 2005-research
network on rare disorders n° A04155HS

Short Title: Ferrochelatase deficiency and iron metabolism in mouse.
Section: Red cells

Keywords: ferrochelatase, protoporphyrin IX, erythropoietic porphyria, iron, microcytic
hypochromic anemia, transferrin

Corresponding author: Carole Beaumont,
INSERM U773, Faculté Xavier Bichat, 16 rue Henri Huchard, 75018, Paris, France.
Phone: [phone] / Fax [phone]
E mail: [email protected]

Summary: 272 words
Text: 4963 words
Figures: 5
Tables: 2
References: 49

All authors participated in designing and performing the research. S.L., C.B. and H.P. wrote
the paper and all authors checked the final version of the manuscript.
1
Copyright © 2006 American Society of Hematology
From www.bloodjournal.org by guest on April 2, 2019. For personal use only.

ABSTRACT
Patients with deficiency in ferrochelatase (FECH), the last enzyme of the heme biosynthetic
pathway, present painful skin photosensitivity due to the excessive production of
protoporphyrin IX (PPIX) by erythrocytes. Controversial results have been reported on both
hematological and iron status of these patients with erythropoietic protoporphyria (EPP). We
thoroughly explored these parameters in Fechm1Pas mutant mice of three different genetic
backgrounds. FECH deficiency induces microcytic hypochromic anemia with absence of ring
sideroblasts, little or no hemolysis and no erythroid hyperplasia. Serum iron, ferritin, hepcidin
mRNA and Dcytb levels are normal. Homozygous Fechm1Pas mutant have no tissue iron
deficiency but show a clear-cut redistribution of iron stores from peripheral tissues to the
spleen, with a concomitant 2-3 fold increase in transferrin expression, both at the mRNA and
protein levels. Erythrocyte PPIX levels strongly correlated to serum transferrin levels. In
Fechm1Pas mutant mice and in contrast to iron-deficiency anemia, transferrin receptor
expression in bone marrow erythroid cells was normal, at all stages of differentiation. Based
on these observations, we propose that oral iron therapy is not the therapy of choice in EPP
patients and that the PPIX–liver transferrin pathway plays a role in the orchestration of iron
distribution between peripheral iron stores, the spleen and the bone marrow.

2
From www.bloodjournal.org by guest on April 2, 2019. For personal use only.

INTRODUCTION
Erythropoietic Protoporphyria (EPP, MIM 177000) is an inherited disorder due to partial
deficiency of ferrochelatase (FECH, EC 4.99.1.1.), the last enzyme of the heme biosynthetic
pathway1. FECH is an inner membrane mitochondrial enzyme catalyzing the insertion of
ferrous iron into protoporphyrin IX (PPIX) to form heme. FECH deficiency in bone marrow
erythroid cells is responsible for a primary over production of PPIX leading to accumulation
of protoporphyrin in the bone marrow, plasma, erythrocytes, skin, bile and feces2. Because of
its hydrophobic nature, PPIX can only be removed from the body through the liver and
secreted into bile followed by fecal elimination3. Over 100 mutations in the FECH gene,
including missense, nonsense, splicing, deletions and insertions, have been identified in EPP
families (Human Gene Mutation Database,
http://archive.uwcm.ac.uk/uwcm/mg/hgmd0.html). In most cases, EPP is inherited as an
autosomal pseudo-dominant disorder and the clinical penetrance is mainly modulated by the
presence of a common intronic single-nucleotide polymorphism (SNP), IVS3 –48 C, in trans
to a dominant mutation4,5.
The commonest clinical manifestation is lifelong acute photosensitivity of sun-exposed skin
appearing in early childhood6. Although it is generally a benign disease, hepatic
complications like cholelithiasis or in rare cases (about 2%) rapid fatal liver disease with
cirrhosis may occur7-9. The chronic liver disease is associated with marked PPIX
accumulation in liver, which can begin insidiously10. The source of the excessive amounts of
PPIX in patients with EPP is, according to most authors, the bone marrow and mild anemia is
observed in 20 – 50 % of the EPP patients11,12. Pathogenesis of the hematologic symptoms is
not yet fully understood and controversial hypotheses have been reported about its origin.
Two EPP mouse models have been reported. A FECH exon 10 deletion was generated by
gene targeting, resulting in a dominant-negative effect and embryonic lethality of
homozygotes13. The best animal model is an ENU-induced point mutation named
ferrochelatase deficiency (symbol Fechm1Pas, fch thereafter). The mutation shows a fully
recessive transmission. A T to A substitution at position 293 replaces a methionine by a lysine
at residue 9814,15. In the BALB/cByJCrl genetic background, to which the mutation was
originally backcrossed, homozygote mice show 5% residual FECH activity in the liver and
spleen, and develop skin lesions, jaundice and severe hepatic dysfunction with massive PPIX
deposits. This model, which mimics the severe forms of the disease, has been used to show
that gene therapy and cellular therapy may greatly improve the condition16 and represents a
3
From www.bloodjournal.org by guest on April 2, 2019. For personal use only.

useful model for studying the pathophysiological feature of the human disease17. However,
iron metabolism has never been extensively investigated in this model. It is noteworthy that
ferrochelatase has two substrates, iron and PPIX, and only PPIX seems to accumulate in
excess.
Therefore, we decided to characterize into more details the hematological and iron phenotypes
of the Fechm1Pas mutation on three different genetic backgrounds, namely BALB/cByJCrl,
C57BL/6JCrl, and SJL/JOrlCrl. Our results show that the impact of the mutation on
erythropoiesis is mainly a microcytic hypochromic anemia without ring sideroblasts or
significant reticulocytosis. This study provides evidence for normal total body iron associated
with redistribution of iron from peripheral tissues to the spleen, which is unexplained by
hemolysis or a spleen erythropoiesis recovery. We investigated this peculiar
pathophysiological process and found that PPIX might act as a signaling molecule,
stimulating the production of transferrin by the liver to facilitate mobilization of tissue iron
stores.
4
From www.bloodjournal.org by guest on April 2, 2019. For personal use only.

MATERIAL AND METHODS

Production and maintenance of congenic strains
Wild-type BALB/cByJCrl, C57BL/6JCrl, and SJL/JOrlCrl mice (thereafter BALB/c,
C57BL/6, and SJL/J, respectively) were purchased from Charles River Laboratories
(L'Arbresle, France). The original Fechm1Pas mutation had been previously backcrossed to the
BALB/c inbred background for over ten generations. Congenic strains were developed
similarly on C57BL/6 and SJL, with ten generations of backcrossing to the recipient strain. At
each backcross generation, and in further crosses, mouse genotypes were identified by
amplifying a genomic segment encompassing the point mutation, which removes a BspHI
restriction site. PCR products were produced and digested as previously described17. The
mutant mice were maintained in the Animal Facility of Institut Pasteur. According to standard
husbandry procedure, they were maintained in filter-top cages with artificial fluorescent light,
under a 12-hr light/dark cycle. They received unlimited, autoclaved water and irradiated food
pellets (standard laboratory mouse chow, AO3, SAFE, Augy, France). Two series of mice
were bred and analyzed. The first series included 6 groups (+/+ and fch/fch for each congenic
strain) of 12-14-week-old female mice which were analyzed for hematological parameters. In
the second series, BALB/c +/+ and fch/fch mice were analyzed at 12-14 weeks of age (6
females per group) for biochemical and iron parameters. For phenylhydrazine, phlebotomy or
PPIX treatments, 12-14-week-old control female mice were purchased from Charles River
Laboratories (L’Arbresle, France). All procedures on animals were performed in compliance
with the French and European regulations on Animal Welfare and with Public Health Service
recommendations.

Phenylhydrazine, phlebotomy and PPIX treatments
Phenylhydrazine hydrochloride (Sigma Chemical Co., St. Louis, MO), a potent hemolytic
agent, was dissolved in phosphate-buffered saline (PBS) at 20 mg/ml and pH adjusted to 7.4
with NaOH. Wild-type BALB/c female mice, 12-14-week-old (n=10), received an intra
peritoneal injection of freshly prepared phenylhydrazine (80 µg for each). Animals were
killed two days after the final injection. Blood was collected via the vena cava, centrifuged
and serum was frozen at -20°C until assayed for haptoglobin. A 20 mM stock solution of
PPIX (Sigma) was prepared by dissolving PPIX in 0.2 M of trisodium orthophosphate-buffer
5
From www.bloodjournal.org by guest on April 2, 2019. For personal use only.

and adjusted to pH 7.6, filtered through a 2 µm bacteriological filter and stored in the dark.
12-14 week-old wild type BALB/c female mice (n=7) received an intraperitoneal injection of
500 µL of this solution daily for fourteen consecutive days and sacrificed 24 hours after the
final injection.

Cell culture and PPIX treatment
Human HepG2 hepatoma cells were grown in Dulbecco’s modified Eagle’s medium
supplemented with 10 % fetal calf serum and seeded at 106 cells/ml. After 24 hours in culture,
the cells were either scrapped directly in RNA Plus Extraction Solution Kit (Quantum
Biotechnologies, Illkrich, France) (T0) or grown for another 24 or 48 hours in the presence of
30 µM PPIX prior to RNA extraction.

Hematological and iron status parameters
Mice were anaesthetized by intra peritoneal injection of xylazine/ketamine mixture and
weighed. Blood was collected by puncture of the orbital sinus. Red blood cells (RBC),
hemoglobin (Hb), hematocrit (Ht), mean cell volume (MCV), and mean cell content in Hb
(MCCH) were measured using a SCIL Vet'ABC counter (SCIL GmbH, Viernheim,
Germany). Protoporphyrin levels in RBC and in stools were determined by a method adapted
from Poulos et al.18. FECH activities were determined by synthesis of mesoporphyrin-Zn,
adapted from Li et al.19. Final mesoporphyrin-Zn concentration was measured using a
Shimadzu RF540 (Shimadzu Co, Kyoto, Japan) spectrofluorimeter with 410 nm excitation
and 580 nm detection. Serum iron, ferritin and transferrin levels were measured on the
Olympus AU400 automate. Human reagents calibrated with commercial mouse transferrin or
recombinant mouse ferritin were used as previously described20. Serum haptoglobin was
measured using rabbit anti-haptoglobin antibodies. A direct sandwich ELISA for mouse
serum haptoglobin was developed, using affinity-purified reagents as previously described21.
The working range of the haptoglobin standard curve was 0.02-0.5 µg/mL. Tissues were
isolated from fch/fch mice and their control wild-type littermates and fixed in 3.5 %
formaldehyde for three to five hours. Fixed tissues were then subjected to routine histological
processing and the sections were stained with Perls Prussian blue for the detection of tissue
iron. Bone marrow smears were analyzed in the same conditions. Tissue iron content was
determined by acid digestion of tissue samples as described by Torrance and Bothwell22,
6
From www.bloodjournal.org by guest on April 2, 2019. For personal use only.

followed by iron quantification using an IL test (Instrumentation Laboratory, Lexington, MA)
on an Olympus AU400 automate.
Electron microscopy.
For electron microscopy, tissues were cut in 1mm3 blocks and immediately fixed in 2.5%
glutaraldehyde-buffered solution (PBS pH 7.4) for 2 hours at +4°C. After washing in PBS,
blocks were post-fixed for two hours in1% buffered osmium tetroxide solution , dehydrated in
graded series of ethanol and embedded in epoxy resin. Semi-thin sections stained with
toluidine blue were made on each block for orientation. Ultrathin stained with uranyl acetate
and lead citrate were examined with a JEOL 1010 electron microscope (Jeol, Tokyo, Japon).
In some cases, counterstaining was omitted to identify electron-dense iron-containing
granules.

Assay of ferrireductase activity in duodenal brush-border membranes
A fragment of 2 cm of upper intestine was excised, thoroughly rinsed with sterile 9 g/L NaCl
and scrapped with a glass blade by the method of Kessler et al., adapted by Simpson and
Peters23. Briefly, the scraped mucosa was suspended in 50 mM mannitol, 2 mM HEPES-
NaOH pH 7.1 (30 mL/g tissue). The mucosal suspension was homogenized for 2 minutes in a
chilled Warring Blender and solid MgCl added to a final concentration of 10 mM, and the
2
homogenate stirred on ice for 20 minutes before centrifugation at 3000 g for 10 minutes. The
resulting supernatant was centrifuged at 40,000g for 40 minutes; the pellet was then
suspended in resuspension buffer (100 mM mannitol, 100 mM NaCl, 100 mM MgSO , 20
4
mM HEPES-NaOH, pH 7.4; 20 mL/g mucosa weight). The suspension was centrifuged at
6,000g for 20 minutes and the resulting supernatant centrifuged at 40,000g for 40 minutes.
The final vesicle pellet was suspended in resuspension buffer at an approximate concentration
of 10mg/ml and stored at –80°C. Protein concentration was determined in triplicate using the
BioRad protein assay and BSA as standard. Ferricyanide reducing activity was determined at
room temperature by measuring the disappearance of the chromogenic substrate ferricyanide
as optimized by Pountney et al.24. Briefly, respectively 50µL and 20µL of freshly prepared
NADH and FMN solutions were added to a 1.5mL cuvette containing 1 mL of [1.66 mM
ferricyanide, 100 mM HEPES-NaOH; pH7.1 and 1 mM lauryl maltoside (0.03% w/v)], and
the basal ferricyanide reduction rate was recorded. 50µL aliquots of brush-border membrane
vesicles were then added and using an extinction coefficient of 1020 mol -1 cm-1 for
7
From www.bloodjournal.org by guest on April 2, 2019. For personal use only.

ferricyanide, the specific activity was calculated in µmoles e- transferred x min-1 x mg protein
-1. A basal rate of reduction attributable to direct reduction of ferricyanide by NADH was
subtracted from the assay rate prior to calculation of specific activity.

RNA extraction and quantitative RT-PCR
Total RNA from liver and duodenum were isolated using RNA Plus Extraction Solution Kit
(Quantum Biotechnologies, Illkrich, France). The purity and yield of total RNA were
determined spectrophotometrically and the integrity of RNA bands (18 and 28S) was checked
on agarose gel electrophoresis. Single-stranded cDNA was synthesized using SuperScriptTM
RnaseH- Reverse Transcriptase (Invitrogene Life Technologies, Cergy-Pontoise, France).
Real-time quantification of transcripts was performed in 25 µl in ABI PRISM 7700
Sequence Detector (PE Applied Biosystems, Courtaboeuf, France) using SYBR Green PCR
master mix (PE Applied Biosystems), 5 pmoles of forward and reverse primers and 2.5 µl of
reverse transcriptase reaction mixture as previously described25. Sequences of the primers
were as follows:
Hepc1 (171 bp), 5’-CCTATCTCCATCAACAGATG-3’ (forward) and
5’-AACAGATACCACACTGGGAA-3’ (reverse);
Transferrin (Tf; 69 bp), 5’-TGTAGCCTTTGTGAAACACCAGA-3’ (forward) and
5’-TCGGCAGGGTTCTTTCCTT-3’ (reverse);
Dcytb (98 bp), 5’-GCAGCTTTCCGAGACCCCGT-3’ (forward) and
5’-CATGCCCACATCTTTGACAG-3’ (reverse);
Glyceraldehyde-3-phosphate dehydrogenase (Gapdh; 177 bp), 5’-
TGCACCACCAACTGCTTAG-3’ (forward) and 5’-GAATGCAGGGATGATGTTC-3’
(reverse).
The result was normalized arbitrary on the sample with the C value for Gapdh (named C
T T -
). For all others samples, the relative quantification was calculated using comparative
Gapdh R
C method with the following arithmetic formula:
T
(1 + E )(C - C ) / (1 + E )(C - C ), where E is the efficiency of
Gapdh T-Gapdh R T-Gapdh S X T-X R T-X S Gapdh
Gapdh target amplification and E the efficiency of amplification for the gene of interest. C
X T-
and C are the respective threshold cycles for Gapdh and for the gene of interest of
Gapdh R T-X R
8
From www.bloodjournal.org by guest on April 2, 2019. For personal use only.

the reference sample (R); C and C are the respective threshold cycles for Gapdh
T-Gapdh S T-X S
and for the gene of interest of every sample (S) but the reference sample (R). The
amplification efficiency of each target was determined using serial two-fold dilutions of
cDNA.

Cells preparation and flow cytometry analysis
Bone marrow cells were extracted from the femurs and tibias of BALB/c fch/fch and their
wild-type littermates (n=6 for each group), and a single-cell suspension was made by gentle
passage of the bone marrow cells through an 18-gauge needle. The cells were pelleted by
centrifugation, washed, resuspended at 106 cells/ml in 37°C DMEM containing 2% FBS, 1
mM HEPES and incubated for 90 min at 37°C. Cells were pelleted and maintained at 4°C
before Fluoresence-activated cell sorter analysis (FACS). FACS analysis for transferrin
receptor (CD71) of bone marrow erythroid cells was performed on a FACS-Calibur (BD
Biosciences, Le Pont de Claix, France). Bone marrow cells were labeled with PE-conjugated
anti-Ter119 (BD Biosciences) and FITC-conjugated anti-CD71 (Serotec, Oxford, UK)
antibodies. RBC (small cells), intermediate normoblats (cells of intermediate size) and early
normoblasts (large cells) were gated based on their FCS and SSC profiles. A ratio of the mean
fluorescence intensity (MFI) of fch/fch cells and their wild-type littermates was used to
normalize Ter-119 expression by FACS. Comparative analyses of fluorescence intensity were
performed on the same FACS machine with regular calibration standards and constant voltage
for each cell genotype as previously described26.

Statistical analysis
Statistical significance was evaluated using the unpaired, two-tailed Student’s t-test for
comparison between two means. Correlations were performed by linear regression. GraphPad
Prism software (GraphPad Software, San Diego, CA) was used for statistical evaluation.

9
From www.bloodjournal.org by guest on April 2, 2019. For personal use only.

RESULTS
Congenic strains production, clinical features and porphyrin studies.
Congenic strains were produced by a minimum of ten generations of backcrossing to the
recipient strain as previously described17. In the BALB/c and C57BL/6 backgrounds, fch/fch
homozygotes showed growth retardation and reduction of fertility in adults of both sexes,
milder in C57BL/6 than in BALB/c (data not shown). These features were only slightly
noticed on the SJL background. Serum and urine were icteric in fch/fch homozygotes of the
three backgrounds but much more intensely in BALB/c. Jaundice was visible on ears in young
albino BALB/c and SJL homozygotes. No spontaneous photosensitivity lesions were
observed under our husbandry conditions. Clinical observations suggested that the mutation
had a more severe impact on the general condition in BALB/c and C57BL/6, than in SJL/J
mice.
FECH activity was measured in the liver of 12-14-week-old, +/+, and fch/fch female mice
(Figure 1A). Wild-type mice showed identical liver FECH activity across strains at 12-14-
weeks of age. The original description of the Fechm1Pas mutation reported ~5% residual
activity in homozygotes. These results were confirmed in the liver at 12-14 weeks in BALB/c
and SJL/J. C57BL/6 homozygous mice showed slightly higher activity than expected
compared to +/+. The erythrocyte free PPIX levels were very similar in the three strains and
much higher in fch/fch than in +/+ mice (Figure 1B).

Hematological studies.
The results of hematological studies are shown in Tables 1 and Figure 2. All homozygous
fch/fch animals exhibited a mild microcytic hypochromic anemia at 12-14 weeks of age with
some strain-specific variability. Hemoglobin concentration (Hb), hematocrit (Ht), mean cell
volume (MCV) and red blood cell counts (RBC) were reduced in fch/fch mice as compared to
the wild-type in the three strains, although the severity of the anemia was more pronounced in
the BALB/c strain.
There was a modest increase in reticulocyte count only in the BALB/c genetic background
(2.5 ± 0.6% in +/+ mice as compared to 8.8 ± 3.4% in fch/fch, p<0.001), suggesting that this
anemia is poorly regenerative. However, bone marrow smears did not reveal major
abnormalities in the erythroid precursors nor an increased proportion of erythroblasts (Figure
2). Perls staining was negative in the three genetic backgrounds, with no iron-loaded
10