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Alison T Merryweather-Clarke,
Ann Atzberger,
Shamit Soneji,
Nicki Gray,
Kevin Clark,
Craig Waugh,
Simon J McGowan,
Stephen Taylor,
Asoke K Nandi,
William G Wood,
David J Roberts,
Douglas R Higgs,
Veronica J Buckle,
Kathryn J H Robson
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ABSTRACT: Understanding the pattern of gene expression during erythropoiesis is crucial for a synthesis of erythroid developmental biology. Here, we isolated 4 distinct populations at successive erythropoietin-dependent stages of erythropoiesis, including the terminal, pyknotic stage. The transcriptome was determined using Affymetrix arrays. First, we demonstrated the importance of using defined cell populations to identify lineage and temporally specific patterns of gene expression. Cells sorted by surface expression profile not only express significantly fewer genes than unsorted cells but also demonstrate significantly greater differences in the expression levels of particular genes between stages than unsorted cells. Second, using standard software, we identified more than 1000 transcripts not previously observed to be differentially expressed during erythroid maturation, 13 of which are highly significantly terminally regulated, including RFXAP and SMARCA4. Third, using matched filtering, we identified 12 transcripts not previously reported to be continuously up-regulated in maturing human primary erythroblasts. Finally, using transcription factor binding site analysis, we identified potential transcription factors that may regulate gene expression during terminal erythropoiesis. Our stringent lists of differentially regulated and continuously expressed transcripts containing many genes with undiscovered functions in erythroblasts are a resource for future functional studies of erythropoiesis. Our Human Erythroid Maturation database is available at https://cellline.molbiol.ox.ac.uk/eryth/index.html. [corrected].
Blood 01/2011; 117(13):e96-108. · 9.90 Impact Factor
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ABSTRACT: On admission to hospital Caucasian 61 year old male with jaundice was found to have unexplained increased serum iron indices. He had bilateral peripheral arthritis. On further investigation he had grade II hepatocellular iron staining and a hepatic index of 5.4 leading to a diagnosis of hereditary hemochromatosis. He lacked the common C282Y HFE mutation. We sequenced the complete HFE gene and found that he was heterozygous for a novel single nucleotide deletion (c.del478) in exon 3 of HFE. He lacks any other mutation in HFE or HJV, TFR2, HAMP and SLC40A1. The HFE mutation causes a frameshift (p.P160fs) that introduces a premature termination codon leading to mRNA degradation by nonsense-mediated decay. Haploinsufficiency of HFE may be one possible explanation for hemochromatosis in this patient.
Blood Cells Molecules and Diseases 06/2009; 43(2):194-8. · 2.35 Impact Factor
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ABSTRACT: Hereditary hemochromatosis is most frequently associated with mutations in HFE, which encodes a class Ib histocompatibility protein. HFE binds to the transferrin receptor-1 (TfR1) in competition with iron-loaded transferrin (Fe-Tf). HFE is released from TfR1 by increasing concentrations of Fe-Tf, and free HFE may then regulate iron homeostasis by binding other ligands. To search for new HFE ligands we expressed recombinant forms of HFE in the human cell line 293T. HFE protein was purified, biotinylated and made into fluorescently labelled tetramers. HFE tetramers bound to TfR1 in competition with Tf, but in addition we detected a binding activity on some cell types that was not blocked by Fe-Tf or by mutations in HFE that prevent binding to TfR1. We identified this second HFE ligand as the cation independent mannose-6-phosphate receptor (CI-MPR, also known as the insulin-like growth factor-2 receptor, IGF2R). HFE:CI-MPR binding was mediated through phosphorylated mannose residues on HFE. Recombinant murine Hfe also bound to CI-MPR. HFE bound to TfR1 was prevented from binding CI-MPR until released by increasing concentrations of Fe-Tf, a feature consistent with an iron sensing mechanism. However, it remains to be determined whether endogenous HFE in vivo also acquires the mannose-6 phosphate modification and binds to CI-MPR.
Blood Cells Molecules and Diseases 06/2009; 43(2):180-93. · 2.35 Impact Factor
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Chun Yu Lok, Alison T Merryweather-Clarke,
Vip Viprakasit,
Yingyong Chinthammitr,
Somdet Srichairatanakool,
Chanin Limwongse,
David Oleesky,
Anthony J Robins,
John Hudson,
Phyu Wai, [......],
Carole McKeown,
Maurice Jackson,
Rousseau Gama,
Nasaim Khan,
William Newman,
Gurvinder Banait,
Andrew Chilton,
Isaac Wilson-Morkeh,
David J Weatherall,
Kathryn J H Robson
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ABSTRACT: Hereditary hemochromatosis is an iron overload disorder that can lead to the impairment of multiple organs and is caused by mutations in one or more different genes. Type 1 hemochromatosis is the most common form of the disease and results from mutations in the HFE gene. Juvenile hemochromatosis (JH) is the most severe form, usually caused by mutations in hemojuvelin (HJV) or hepcidin (HAMP). The autosomal dominant form of the disease, type 4, is due to mutations in the SLC40A1 gene, which encodes for ferroportin (FPN). Hereditary hemochromatosis is commonly found in populations of European origin. By contrast, hemochromatosis in Asia is rare and less well understood and can be masked by the presence of iron deficiency and secondary iron overload from thalassemia. Here, we provide a comprehensive report of hemochromatosis in a group of patients of Asian origin. We have identified novel mutations in HJV, HAMP, and SLC40A1 in countries not normally associated with hereditary hemochromatosis (Pakistan, Bangladesh, Sri Lanka, and Thailand). Our family studies show a high degree of consanguinity, highlighting the increased risk of iron overload in many countries of the developing world and in countries in which there are large immigrant populations from these regions.
Blood 05/2009; 114(1):20-5. · 9.90 Impact Factor
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Human Genetics 01/2006; 118(3-4):547. · 5.07 Impact Factor
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Human Genetics 01/2006; 118(3-4):546. · 5.07 Impact Factor
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Hal Drakesmith,
Lisa M Schimanski,
Emma Ormerod, Alison T Merryweather-Clarke,
Vip Viprakasit,
Jon P Edwards,
Emma Sweetland,
Judy M Bastin,
Diana Cowley,
Yingyong Chinthammitr,
Kathryn J H Robson,
Alain R M Townsend
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ABSTRACT: Ferroportin (FPN) mediates iron export from cells; FPN mutations are associated with the iron overloading disorder hemochromatosis. Previously, we found that the A77D, V162del, and G490D mutations inhibited FPN activity, but that other disease-associated FPN variants retained full iron export capability. The peptide hormone hepcidin inhibits FPN as part of a homeostatic negative feedback loop. We measured surface expression and function of wild-type FPN and fully active FPN mutants in the presence of hepcidin. We found that the Y64N and C326Y mutants of FPN are completely resistant to hepcidin inhibition and that N144D and N144H are partially resistant. Hemochromatosis-associated FPN mutations, therefore, either reduce iron export ability or produce an FPN variant that is insensitive to hepcidin. The former mutation type is associated with Kupffer-cell iron deposition and normal transferrin saturation in vivo, whereas patients with the latter category of FPN mutation have high transferrin saturation and tend to deposit iron throughout the liver parenchyma. FPN-linked hemochromatosis may have a variable pathogenesis depending on the causative FPN mutant.
Blood 09/2005; 106(3):1092-7. · 9.90 Impact Factor
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Lisa M Schimanski,
Hal Drakesmith, Alison T Merryweather-Clarke,
Vip Viprakasit,
Jon P Edwards,
Emma Sweetland,
Judy M Bastin,
Diana Cowley,
Yingyong Chinthammitr,
Kathryn J H Robson,
Alain R M Townsend
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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, V162delta, 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.
Blood 06/2005; 105(10):4096-102. · 9.90 Impact Factor
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ABSTRACT: Extensive investigation into the molecular basis of iron overload disorders has provided new insights into the complexity of iron metabolism and related cellular pathways. The possible involvement of genes affecting iron homeostasis, including HFE, SLC40A1, HAMP and CYBRD1, was investigated in individuals who were referred for confirmation or exclusion of a diagnosis of haemochromatosis, but who tested negative or were heterozygous for the causative HFE mutation, C282Y. Denaturing high performance liquid chromatography analysis of these genes revealed a unique spectrum of mutations in the South African study population, including 67 unrelated patients and 70 population-matched controls. Two novel CYBRD1 gene mutations, R226H and IVS1-4C-->G, were identified in 11% of South African Caucasian patient referrals. We identified a novel D270V mutation in the SLC40A1 gene in a Black South African female with iron overload. These mutations were absent in the control population. In Africans with iron overload not related to the HFE gene, the possible involvement of the SLC40A1 and CYBRD1 genes was demonstrated for the first time. This study confirms the genetic heterogeneity of haemochromatosis and highlights the significance of CYBRD1 mutations in relation to iron overload.
Human Genetics 11/2004; 115(5):409-17. · 5.07 Impact Factor
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ABSTRACT: Co-inheritance of HFE mutations has a substantial role in iron overload in beta-thalassaemia carriers in north European populations where two HFE mutations, C282Y and H63D, are prevalent. In Thailand, there was little information about the allele frequency of HFE mutations. It is of interest to determine whether such determinants represent a potential risk in developing iron overload as nearly 40% of the Thai population carry either one of thalassaemia or haemoglobinpathy alleles. A total of 380 normal controls from five different regions including Bangkok were screened for the HFE C282Y, H63D and IVS5+1 G-->A alleles. In addition, 70 individuals with homozygous haemoglobin E (Hb EE) were also tested and their genotypes were correlated with levels of serum ferritin. H63D is the major HFE mutation found in the Thai population with an average allele frequency of 3% (range 1-5%). One individual was heterozygous for the splice site mutation IVS5 + 1 G --> A, and the C282Y allele was not detected. In the Hb EE group, five individuals had iron deficiency (ferritin <12 microg/L) and the remaining 65 individuals had a wide range of serum ferritin levels of 16-700 microg/L. Four individuals with Hb EE were heterozygous for the H63D allele. No significant difference in serum ferritin level was detected in this group with or without the HFE mutation (137.2 +/- 78 vs. 116.3 +/- 128 microg/L). HFE mutations are relatively uncommon among the Thai population, and the average allele frequency of the ancient H63D mutation is similar to that of other countries in this region. Because of their paucity, it appears that these alleles are less likely to be responsible for high ferritin levels and iron loading in individuals with Hb E related disorders.
European Journal Of Haematology 07/2004; 73(1):43-9. · 2.61 Impact Factor
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Alison T Merryweather-Clarke,
Estelle Cadet,
Adrian Bomford,
Dominique Capron,
Vip Viprakasit,
Anne Miller,
Paddy J McHugh,
Roger W Chapman,
Jennifer J Pointon,
Victoria L C Wimhurst,
Karen J Livesey,
Voravarn Tanphaichitr,
Jacques Rochette,
Kathryn J H Robson
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ABSTRACT: Haemochromatosis (HH) is a clinically and genetically heterogeneous disease caused by inappropriate iron absorption. Most HH patients are homozygous for the C282Y mutation in the HFE gene. However, penetrance of the C282Y mutation is incomplete, and other genetic factors may well affect the HH phenotype. Ferroportin and TFR2 mutations also cause HH, and two HAMP mutations have recently been reported that causes juvenile haemochromatosis (JH) in the homozygous state. Here, we report evidence for digenic inheritance of HH. We have detected two new HAMP mutations in two different families, in which there is concordance between severity of iron overload and heterozygosity for HAMP mutations when present with the HFE C282Y mutation. In family A, the proband has a JH phenotype and is heterozygous for C282Y and a novel HAMP mutation Met50del IVS2+1(-G). This is a four nucleotide ATGG deletion which causes a frameshift. The proband's unaffected mother is also heterozygous for Met50del IVS2+1(-G), but lacks the C282Y mutation and is heterozygous for the HFE H63D mutation. Met50del IVS2+1(-G) was absent from 642 control chromosomes. In family B, a second novel, less severe HAMP mutation, G71D, was identified. This was detected in the general population at an allele frequency of 0.3%. We propose that the phenotype of C282Y heterozygotes and homozygotes may be modified by heterozygosity for mutations which disrupt the function of hepcidin in iron homeostasis, with the severity of iron overload corresponding to the severity of the HAMP mutation.
Human Molecular Genetics 10/2003; 12(17):2241-7. · 7.64 Impact Factor
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Blood 08/2002; 100(2):733-4. · 9.90 Impact Factor
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ABSTRACT: High frequencies of the haemochromatosis-related HFE C282Y mutation have been reported in North European populations, in which a high proportion of patients with the disease are homozygotes. However, the degree of penetrance of this genotype is unknown. We determined the HFE C282Y and H63D genotypes of 411 consenting volunteer blood donors on Jersey, and the serum ferritin and transferrin saturation levels of 204 of these volunteers. The C282Y allele frequency was found to be 8.3% in 822 chromosomes, indicating a homozygote frequency of 1/145. Consistent with this, four C282Y homozygotes were detected in 411 volunteers. As there are only 18 patients presently receiving treatment for haemochromatosis on Jersey, out of a total population of about 85 000, there is a large discrepancy between the number of haemochromatosis patients and the number of C282Y homozygotes in this population. In a preliminary study of 204 consenting volunteers we found a correlation between transferrin saturation and HFE H63D/C282Y genotype (P = 0.017) and between serum ferritin and genotype (P = 0.056). We also observed elevated values of transferrin saturation in the two C282Y homozygotes assayed. These results suggest that a large proportion of the many undetected C282Y homozygotes on Jersey and in similar populations could be in the preclinical stages of haemochromatosis, and warrant investigation. However, there may be a wide variation in the expression of the condition, and a more extensive study of the level of disease penetrance encompassing a large number of hitherto undetected C282Y homozygotes is therefore imperative.
British Journal of Haematology 12/2001; 101(2):369 - 373. · 4.94 Impact Factor
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ABSTRACT: We have generated a detailed physical map of the 6p21.3/p22.1 boundary, using a combination of yeast artificial chromosome
(YAC) fragmentation and high-resolution sequence tagged site (STS) content mapping. YACs from the CEPH, St. Louis, and ICRF
libraries have been used to construct a 4.5-Mb contig spanning the markers D6S306 to D6S1571. YAC insert sizes were determined by pulsed field gel electrophoresis (PFGE). Chimerism of YACs was determined by fluorescent
in situ hybridization (FISH), and their integrity was determined by fingerprinting with Alu-PCR. We have identified 10 new
CA repeat loci in this region as well as over 50 novel STSs, several tRNA genes, a new histone H2B gene and the phospholipase D gene. Using these new markers, we have rapidly generated a bacterial clone contig of over 250 kb, spanning the markers D6S1260 to D6S1918 (WI-3111) with STSs spaced on average every 6 kb.
Mammalian Genome 04/1998; 9(3):220-225. · 2.89 Impact Factor
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ABSTRACT: Hereditary hemochromatosis is most frequently associated with mutations in HFE, which encodes a class Ib histocompatibility protein. HFE binds to the transferrin receptor-1 (TfR1) in competition with iron-loaded transferrin (Fe–Tf). HFE is released from TfR1 by increasing concentrations of Fe–Tf, and free HFE may then regulate iron homeostasis by binding other ligands. To search for new HFE ligands we expressed recombinant forms of HFE in the human cell line 293T. HFE protein was purified, biotinylated and made into fluorescently labelled tetramers. HFE tetramers bound to TfR1 in competition with Tf, but in addition we detected a binding activity on some cell types that was not blocked by Fe–Tf or by mutations in HFE that prevent binding to TfR1. We identified this second HFE ligand as the cation independent mannose-6-phosphate receptor (CI-MPR, also known as the insulin-like growth factor-2 receptor, IGF2R). HFE:CI-MPR binding was mediated through phosphorylated mannose residues on HFE. Recombinant murine Hfe also bound to CI-MPR. HFE bound to TfR1 was prevented from binding CI-MPR until released by increasing concentrations of Fe–Tf, a feature consistent with an iron sensing mechanism. However, it remains to be determined whether endogenous HFE in vivo also acquires the mannose-6 phosphate modification and binds to CI-MPR.
Blood Cells, Molecules, and Diseases.
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Alison T Merryweather-Clarke,
Jennifer J Pointon,
Jeremy D. Shearman,
Kathryn J H Robson,
Anne-Marie Jouanolle,
Annick Mosser,
Véronique David,
Jean-Yves Le Gall,
David J Halsall,
Terence S. Elsey, [......],
Angela Totaro,
Paolo Gasparini,
Antonella Roetto,
Clara Camaschella,
Chris Darke,
Daniel F Wallace,
Kasra Saeb-Parsy,
James S Dooley,
Mark Worwood,
Ann P Walker
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ABSTRACT: Hereditary hemochromatosis (HH) is an autosomal recessive disease caused by mutations in the HFE gene that mainly affects populations of European descent. Recently a novel mutation (IVS5+1 G-->A) has been described in a Vietnamese patient with HH that was not detected in a European control population. We have developed a novel method to screen for this mutation based on restriction enzyme digestion of a PCR product using a modified forward primer. We have screened 314 Vietnamese people from several ethnic groups and 154 people from Thailand for this mutation and have detected two heterozygotes in the Vietnamese subjects (allele frequency 0.003). Analysis of these heterozygotes indicates that the mutation is on the same haplotype as that found in the original proband. Screening for the widely distributed HFE mutation, H63D, gave an allele frequency of 0.049 in the Vietnamese subjects and 0.032 in the subjects from Thailand. This is the first report of H63D allele frequencies in these populations. We suggest that the presence of the IVS5+1 G-->A and H63D mutations should be considered when investigating iron overload in Vietnamese patients and those of mixed origin as co-inheritance of both mutations is likely to be a risk factor for iron overload.
Blood Cells Molecules and Diseases 30(3):302-6. · 2.35 Impact Factor
