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Prepublished online as a Blood First Edition Paper on April 17, 2002; DOI 10.1182/blood-2002-01-0133.
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Blood, 1 August 2002, Vol. 100, No. 3, pp. 1075-1077
BRIEF REPORT
Transferrin receptor 2 (TfR2) and HFE
mutational analysis in non-C282Y iron overload: identification of a
novel TfR2 mutation
Andre Mattman,
David Huntsman,
Gillian Lockitch,
Sylvie Langlois,
Noel Buskard,
Diana Ralston,
Yaron Butterfield,
Pedro Rodrigues,
Steven Jones,
Graça Porto,
Marco Marra,
Maria De Sousa, and
Greg Vatcher
From the Genes, Elements, and Metabolism Program,
Children and Women's Hospital of British Columbia; the British
Columbia Genome Sequencing Centre, British Columbia Cancer Agency,
Vancouver, British Columbia, Canada; the Instituto de Ciências
Biomedicas Abel Salazar, Hospital Geral de Santo António, and
Instituto de Biologia Molecular e Celular, Porto, Portugal; the
Vancouver General Hospital, Vancouver, British Columbia, Canada; and
the Department of Pathology, British Columbia Cancer Agency, Vancouver,
British Columbia, Canada.
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Abstract |
Hereditary hemochromatosis (HH) is classically associated with a
Cys282Tyr (C282Y) mutation of the HFE gene.
Non-C282Y HH is a heterogeneous group accounting for 15% of HH in
Northern Europe. Pathogenic mutations of the transferrin receptor 2 (TfR2) gene have been identified in 4 Italian pedigrees
with the latter syndrome. The goal of this study was to perform a
mutational analysis of the TfR2 and HFE genes
in a cohort of non-C282Y iron overload patients of mixed ethnic
backgrounds. Several sequence variants were identified within the
TfR2 gene, including a homozygous missense change in exon
17, c2069 A C, which changes a glutamine to a proline residue
at position 690. This putative mutation was found in a severely
affected Portuguese man and 2 family members with the same genotype. In
summary, pathologic TfR2 mutations are present outside of
Italy, accounting for a small proportion of non-C282Y HH.
(Blood. 2002;100:1075-1077)
© 2002 by The American Society of Hematology.
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Introduction |
In 1996, Feder et al1 identified 85%
of hereditary hemochromatosis (HH) patients of Northern European
descent as being carriers of a C282Y mutation in the HFE
gene. Since 1996, several other mutations of the HFE
gene have been identified in association with HH2-4;
however, with rare exception, these mutations occur in tandem with
trans C282Y mutations and are infrequent causes of HH.
Mutations of the transferrin receptor 2 (TfR2) gene have
been implicated as the genetic basis of a novel autosomal recessive adult-onset HH syndrome in 4 Italian pedigrees (Hemochromatosis, Type
3, OMIM 604250).5-6 The TfR2 transcript is a homolog of the transferrin receptor (TfR1) protein with 48% identity and 66%
similarity in the extracellular portion of the protein.7 TfR2 has both similar and distinct functional properties from TfR1,
including a common affinity for diferric transferrin7 and
a lack of affinity for the HFE protein,8 respectively. Nonsense or nonfunctional mutations of TfR2 are associated
with HH5-6; however, the frequency and physiologic basis
of this association are unclear.
The goal of the current study was to identify a genetic basis for iron
overload in a cohort of patients with non-C282Y iron overload by direct
sequencing of both the TfR2 and the HFE genes.
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Study design |
The study population consisted of all patients referred to the
Molecular Diagnostic Laboratory of Children and Women's Health Centre
in Vancouver, BC, Canada, for genetic testing for HH prior to May 5, 2000. From this database, we selected all patients with a transferrin
saturation greater than or equal to 60%, an elevated serum ferritin
level, and a negative test result for the C282Y HFE mutation.
From the same laboratory, 93 control samples were obtained.
Testing for the HFE C282Y mutation was performed according to standard
methods. Polymerase chain reaction (PCR) amplification and
direct sequencing of all coding regions and all intron/exon boundaries
of the HFE and TfR2 genes were performed on each
patient at the British Columbia Genome Sequencing Centre (BCGSC).
Method details are available in the supplementary information stored on
the BCGSC website (www.bcgsc.bc.ca/fg/hemoc). The project received
approval from the ethical review boards of all participating
institutions. Investigations were undertaken with the informed consent
of all study participants.
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Results and discussion |
HFE and TfR2 sequencing was performed on DNA
from 89 patients of mixed ethnic origin (patient demographic data
available in the supplementary information). Of these patients, 15 had
risk factors for secondary iron overload. Among this cohort, one
probable pathogenic mutation of the TfR2 gene was
identified, c2069 AC, Q690P (Figure
1). This novel mutation occurred in a
single patient who was a child of a consanguineous marriage
(Figure 2). The remaining patients either
did not have a TfR2 or HFE genotype
suggestive of HH (n = 74) or had genotypes associated with normal or
mild iron overload status (HFE H63D/H63D, n = 11; HFE H63D/S65C,
n = 3).9-11 In addition, 18 TfR2
polymorphisms were identified, including 3 novel and one previously
described12 amino acid coding changes (Figure 1 and
www.bcgsc.bc.ca/fg/hemoc). Five HFE single nucleotide
polymorphisms were identified (www.bcgsc.bc.ca/fg/hemoc).
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| Figure 1.
Summary of new and previously
described coding sequence changes of the
TfR2 gene. Text box color code is as follows:
purple indicates previously described homozygous mutations, white
indicates a novel homozygous mutation, and orange indicates
heterozygous sequence changes. The asterisk indicates a previously
described heterozygous sequence change. For each sequence change
identified in this study, both the amino acid (above) and cDNA
nucleotide (below) changes are indicated.
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| Figure 2.
Q690P pedigree.
Age (A, years), ferritin (F, µg/L), transferrin saturation (S, %),
and TfR2 Q690P and HFE H63D mutation status for the Portuguese
family. "+" indicates the presence of the mutant allele, and
" " indicates its absence. The mother was heterozygous for both
TfR2 Q690P and HFE H63D. Abnormal results are indicated in bold type.
Values listed are those of the initial assessment for each
patient.
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With respect to HFE mutations, a high percentage of patients
carried the HFE H63D allele (48 of 89; 54%). This value is above that
expected as compared to carrier rates for H63D ranging from 12% to
37% in European population studies.13 This
overrepresentation of HFE H63D genotypes is in keeping with the
hypothesis that HFE H63D mutations contribute to an iron overload
phenotype, even in the absence of the HFE C282Y
allele.14-15
The TfR2 Q690P mutation was identified in an individual of Portuguese
descent. He presented at 29 years of age with fatigue, hypogonadotropic
hypogonadism, hyperpigmentation, mild elevation of liver transaminases,
and idiopathic thrombocytopenic purpura. He had markedly elevated serum
iron indices (Figure 2) and has had more than 12 g of iron removed via
phlebotomy in the past 4 years. Additional hematologic abnormalities
included mild normocytic anemia and lymphopenia. He is heterozygous for
the HFE H63D mutation. The Q690P mutation was not identified in our
control group, 20 Portuguese controls, and 39 Portuguese patients with
nonclassical HH.
The family of the Q690P proband was investigated for iron overload and
the presence of the TfR2 Q690P mutation. The results of the family
study are summarized in Figure 2 (additional data available at
www.bcgsc.bc.ca/fg/hemoc). Biochemical evidence of iron overload was
detected in 2 female siblings (IV-3 and IV-4 in Figure 2), both of whom
are homozygous for the TfR2 Q690P mutation. They had no clinical
manifestations of disease aside from arthralgia, which affected sibling
IV-3. Sibling IV-3 had a transferrin saturation level measured at
greater than 100% with a normal serum ferritin and no evidence of
hepatic iron overload as assessed by magnetic resonance
imaging.16 Her hematologic profile revealed neutropenia but was otherwise normal. She has a wild-type HFE genotype.
Sibling IV-4 had a transferrin saturation value of 77% and elevations of serum ferritin and hepatic iron stores (263 µmol/g). Her
hematologic profile revealed a mild normocytic anemia and lymphopenia.
She is heterozygous for the HFE H63D mutation. Sibling IV-1 and
the mother of the proband showed no evidence of an iron overload
phenotype or hematologic abnormalities. The mother is heterozygous for
the TfR2 Q690P allele, while sibling IV-1 is wild type.
The impact of the Q690P mutation on TfR2 function can be estimated by
analysis of the corresponding position in the homologous TfR1 protein
(threonine 658). TfR1 thr658 is residue 18 of 23 amino acids forming
the helix 3 of the helical domain in the extracellular component of
the protein.17 Sixteen of the 23 amino acids are identical
or structurally conserved between TfR1 and TfR2.17 In
TfR1, this helix forms the proposed binding sites for both diferric
transferrin and HFE.17-18 Specifically, mutagenesis of
TfR1 helix 3 amino acids 643, 646-648, and 650 all yield TfR1
proteins with significantly reduced affinity for transferrin.18-19 Therefore, disruption of the helix 3 secondary structure via insertion of a proline residue at position 690 in TfR2 (position 658 in TfR1) would be expected to disrupt a key binding site for diferric transferrin. This loss of function could then
manifest as an iron overload phenotype similar to that associated with
TfR2 nonsense mutations.
The strong clinical expression of HH in the proband and family members
with the Q690P homozygous mutation suggest that the TfR2 interaction
with transferrin plays a critical role in normal iron homeostasis.
However, because TfR2 is primarily expressed in hepatocytes
and in erythroid precursors,20 much remains to be
elucidated as to how an abnormal interaction with the transferrin protein results in systemic iron overload.
In our cohort of non-C282Y HH patients of predominately mixed European
descent, only 1 of 89 was found to have a novel pathogenic mutation of
either the TfR2 or HFE genes. The isolated nature of this finding is concordant with previous studies of
TfR212,21-22 or
HFE15,23 in non-C282Y homozygous HH. However,
the identification of the TfR2 Q690P mutation helps to establish
TfR2 mutations as infrequent but important contributors to
autosomal recessive adult-onset HH. This patient's family is the first
TfR2-associated HH pedigree to be described outside Italy.
| |
Acknowledgments |
We give special thanks to Duane Smailus and the Genome
Sequencing Centre sequencing team for providing extensive
technical support in this sequencing project. We also thank Dr Doug
Horsman from the British Columbia Cancer Agency for his critical review of this manuscript. Most important, we thank the patients and family
members for agreeing to participate in this study.
| |
Footnotes |
Submitted January 17, 2002; accepted March 25, 2002.
Prepublished
online as Blood First Edition Paper, April 17, 2002; DOI
10.1182/blood-2002-01-0133.
Supported by a fellowship grant from the National Science and Energy
Research Council (NSERC) of Canada (G.V.).
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: Gillian Lockitch, Dept of Pathology and Laboratory
Medicine, Children's and Women's Health Centre of BC, 4480 Oak St,
Vancouver, British Columbia, Canada, V6H 3V4; e-mail:
glockitch{at}cw.bc.ca. In Europe: Maria De Sousa, Laboratory of Molecular
Immunology, Institute for Molecular and Cell Biology, Rua do Campo
Alegre, 823, 4150-180 Porto, Portugal; e-mail: mdesousa{at}ibmc.up.pt.
| |
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Abstract
Introduction