Iron overload in Africans and African-Americans and a common mutation in the SCL40A1 (ferroportin 1) gene☆

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Iron overload in Africans and African-Americans and a common mutation in the SCL40A1 (ferroportin 1) gene夞 Victor R. Gordeuk,a Angela Caleffi,b Elena Corradini,b Francesca Ferrara,b Russell A. Jones,c Oswaldo Castro,a Onyinye Onyekwere,a Rick Kittles,a Elisa Pignatti,b Giuliana Montosi,b Cinzia Garuti,b Innocent T. Gangaidzo,d Z.A.R. Gomo,d Victor M. Moyo,e Tracey A. Rouault,f Patrick MacPhail,g and Antonello Pietrangelob,* a

Howard University College of Medicine, Washington, DC 20059, USA Department of Internal Medicine, University of Modena, Policlinico, Via del Pozzo 71, 41100 Modena, Italy c Chattanooga Oncology & Hematology Associates, PC, 605 Glenwood Drive, Suite 200, Chattanooga, TN 37404, USA d University of Zimbabwe School of Medicine, Harare, Zimbabwe e Department of Medicine, University of Connecticut, New Haven, CT 06269, USA f Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, Bethesda, MD 20892, USA g Department of Medicine, University of the Witwatersrand, Johannesburg, South Africa b

Submitted 12 June 2003 (Communicated by E. Beutler, M.D., 19 June 2003)

Abstract The product of the SLC40A1 gene, ferroportin 1, is a main iron export protein. Pathogenic mutations in ferroportin 1 lead to an autosomal dominant hereditary iron overload syndrome characterized by high serum ferritin concentration, normal transferrin saturation, iron accumulation predominantly in macrophages, and marginal anemia. Iron overload occurs in both the African and the African-American populations, but a possible genetic basis has not been established. We analyzed the ferroportin 1 gene in 19 unrelated patients from southern Africa (N ⫽ 15) and the United States (N ⫽ 4) presenting with primary iron overload. We found a new c. 744 C3 T (Q248H) mutation in the SLC40A1 gene in 4 of these patients (3 Africans and 1 African-American). Among 22 first degree family members, 10 of whom were Q248H heterozygotes, the mutation was associated with a trend to higher serum ferritin to amino aspartate transferase ratios (means of 14.8 versus 4.3 ␮g/U; P ⫽ 0.1) and lower hemoglobin concentrations (means of 11.8 versus 13.2 g/dL; P ⫽ 0.1). The ratio corrects serum ferritin concentration for alcohol-induced hepatocellular damage. We also found heterozygosity for the Q248H mutation in 7 of 51 (14%) southern African community control participants selected because they had a serum ferritin concentration below 400 ␮g/L and in 5 of 100 (5%) anonymous African-Americans, but we did not find the change in 300 Caucasians with normal iron status and 25 Caucasians with non-HFE iron overload. The hemoglobin concentration was significantly lower in the African community controls with the Q248H mutation than in those without it. We conclude that the Q248H mutation is a common polymorphism in the ferroportin 1 gene in African populations that may be associated with mild anemia and a tendency to iron loading. © 2003 Elsevier Inc. All rights reserved.

Introduction An autosomal dominant hereditary iron overload disease characterized by marked and early iron loading of hepatic 夞 This work was supported in part by EU Grant QLK1-2001-00444, NIH Grant UH1-HL03679-05 from the National Heart, Lung, and Blood Institute and the Office of Research on Minority Health, and Howard University General Clinical Research Center Grant MO1-RR10284 from the National Center for Research Resources. * Corresponding author. Fax: ⫹39-059-4224363. E-mail address: [email protected] (A. Pietrangelo). 1079-9796/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S1079-9796(03)00164-5

Kupffer cells and elevated serum ferritin concentration, with normal transferrin saturation and slight anemia or low tolerance to iron depletion therapy has been recently described [1]. In the original pedigree the disease was associated with the A77D mutation in the SLC40A1 gene, which codes for ferroportin 1, a putative iron export protein in epithelial cells and macrophages [2]. In contrast to classical hemochromatosis due to pathogenic mutations in HFE, which almost exclusively affects populations of northern European heritage, disease due to mutations in SLC40A1 has a worldwide distribution, as evidenced by the reports of

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additional mutations with similar iron-loading syndromes in several different countries [3–7]. A distinct iron-loading disorder, termed dietary iron overload, is prevalent in Africa [8,9]. This disease is particularly frequent among Africans who drink a traditional beer brewed in non-galvanized steel drums [10]. Although the disorder was once exclusively attributed to dietary excess, severe iron overload does not develop in all beer drinkers, and not all patients with iron overload consume excessive amounts of the beer [11]. Segregation analysis of African pedigrees selected because of index subjects with iron overload has led to the conclusion that an unidentified iron-loading gene may confer susceptibility to the disease [12,13]. African iron overload is not due to the C282Y or H63D mutations in the HFE gene [14] and is not linked to the HLA locus [12]. The phenotype of African overload [15] is strikingly similar to that of ferroportin 1 disease [1].

Methods The research was approved by the committees on human investigation of The University of Modena and Reggio Emilia, The George Washington University, Washington, DC, Howard University, Washington, DC, University of the Witwatersrand, Johannesburg, South Africa, and the University of Zimbabwe, Harare, Zimbabwe. Written informed consent was obtained for all participants.

iron overload on the basis of their serum ferritin concentration being less than 400 ␮g/L. Finally, we analyzed the ferroportin 1 gene in an anonymous panel of DNA samples from 100 African-Americans. Three hundred Caucasian blood donors at the University Hospital of Modena and 25 Caucasian Italian subjects with non-HFE iron overload were also tested for the Q248H ferroportin 1 mutation. Laboratory methods Serum iron concentration was determined by a method modified from that recommended by the International Committee for Standardization in Hematology or by an automated method. Total iron-binding capacity was determined by a method modified from that recommended by the International Committee for Standardization in Hematology or was calculated after measuring the transferrin concentration by nephelometry. Serum ferritin was measured using enzyme immunoassay, complete blood counts by automated cell sorter, reticulocyte counts by light microscopy, erythrocyte sedimentation rates by the Westergren method, and liver function tests by autoanalyzer. Transferrin saturation was calculated as the serum iron divided by the total iron binding capacity times 100, with a maximum value of 100%. The ratio of serum ferritin to aspartate aminotransferase (AST) was calculated by dividing the serum ferritin concentration by the AST. Estimation of traditional beer consumption

Index subjects We first examined the ferroportin 1 gene of four AfricanAmerican patients who had the diagnosis of primary iron overload on the basis of liver biopsy, quantitative phlebotomy, and/or unexplained elevation in serum ferritin concentration. Upon finding that one of these patients was a heterozygote for a novel c.DNA 744 G3 T (Q248H) mutation in the SLC40A1 gene, we then analyzed the ferroportin 1 gene of 15 black Africans from South Africa, Zimbabwe, or Swaziland who had dietary iron overload on the basis of estimated lifetime traditional beer consumption over 1000 liters along with elevated hepatic iron as documented by diagnostic liver biospy and/or unexplained elevation in serum ferritin concentration. Three of these African patients proved to be heterozygotes for the same Q248H mutation. Family members and controls We analyzed the ferroportin 1 gene of 22 first degree family members of African-American or African index subjects identified to have the Q248H ferroportin 1 mutation. We also analyzed the ferroportin 1 gene for the Q248H change in 51 black South Africans and Swazis from the same community as the African index subjects with the Q248H ferroportin 1 mutation. These community members were selected because they were thought not to have dietary

Traditional beer was defined as a beverage that is brewed at home in non-galvanized iron drums and is known to have high iron content. Each African participant was asked to estimate his or her consumption of traditional beer: the amount ingested on a typical day, the number of days in a typical month that the beverage was consumed, the year that the subject began drinking traditional beer, and, if no longer drinking, the year he or she stopped. This estimate only provides a broad approximation of the lifetime traditional beer consumption because consumption was probably not uniform over time and information was obtained by recollection. The African-American participants were assumed to have a lifetime traditional beer consumption of 0 liters. Analysis of the ferroportin 1 gene To screen for mutations, each exon of the ferroportin 1 gene was amplified by PCR from patient and control samples and subjected to sequence analysis by a Beckman Coulter DNA sequencer. The mutation itself destroyes a restriction endonuclease site for PvuII. Therefore, to facilitate analysis of large numbers of samples, the portion of exon 6 containing the mutation was amplified by PCR with the Expand High Fidelity PCR system (Roche, Monza, Italy) and digested with PvuII (MBI Fermentas GMBH, St. Leon-Rot, Germany). The resulting DNA fragments were

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Fig. 1. Mutation detection. (A) Sequencing chromatographs of exon 6 spanning the c.DNA 744 G3 T (Q248H) substitution are compared for a ferroportin exon 6 sequence, a normal control, and the proband African-American. The mutation is indicated by an arrow. (B) Restriction digestion of exon 6 PCR with PvuII. The G3 T change destroys the PvuII recognition site. Normally, two fragments are obtained (302 and 123 bp): lanes 3, 7, and 8 (proband’s siblings). The amplification product from the mutant allele is not digested and three fragments are obtained including the full-length 425-bp PCR product: lane 1: proband; lanes 2, 4, 5, 6: siblings. M: molecular-weight marker.

fractionated on a 12% polyacrylamide gel and detected with ethidium bromide.

Results Case report The first participant found to be heterozygous for the Q248H mutation in the ferroportin 1 gene was a 56-year-old woman who presented with a general feeling of weakness and was found to have borderline microcytic anemia (hemoglobin 11.6 g/dL and MCV 71 fL), serum ferritin concentration ⬎1300 ␮g/L, and transferrin saturation 28%. The reticulocyte count, total bilirubin concentration, and LDH

were in the normal range and hepatitis B surface antigen and antibody to hepatitis C were negative. The hemoglobin A2 level was 3.9% (normal up to 3.5%). She had had four fullterm pregnancies and had received no blood transfusions except on one occasion to replace blood lost during hysterectomy at age 23 years. She did not have a long history of medicinal or supplemental iron use, did not abuse alcohol and was negative for C282Y and H63D mutations in the HFE gene. A diagnostic liver biopsy revealed moderate fatty change, grade 3⫹ (on a scale of 0 to 4⫹) deposition of iron in hepatocytes, the presence of iron in Kupffer cells, a hepatic iron concentration of 106 ␮mol/g dry weight (normal ⬍30), and an hepatic iron index of 1.9 ␮mol/year (normal ⬍1.0). A bone marrow aspirate and biopsy revealed orderly maturation of hematopoietic precursors, the pres-

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Table 1 Hematologic and iron measurements in 19 Africans and African-Americans with the diagnosis of primary iron overload according to whether they possessed the ferroportin 1 Q248H mutation

Hemoglobinb (g/dL; mean ⫾ SE) MCV (fL; mean ⫾ SE) Ferritinc (␮g/L; geometric mean and SE range) Ferritin/AST ratioc (␮g/U; mean ⫾ SE) Transferrin saturationd (%; mean ⫾ SE)

Ferroportin Q248H heterozygotes (N ⫽ 4)

Ferroportin wild type (N ⫽ 15)

P

13.1 ⫾ 0.6 87 ⫾ 5 1626 (982–2692) 56.5 ⫾ 19.1 62 ⫾ 12

13.4 ⫾ 0.3a 92 ⫾ 2a 2780 (2183–3540) 65.6 ⫾ 7.9 81 ⫾ 6

0.6 0.4 0.4 0.7 0.2

N ⫽ 15. ANOVA in which parameter is adjusted for sex. c ANOVA in which parameter is adjusted for age, sex, and estimated traditional beer consumption. d ANOVA in which parameter is adjusted for sex and estimated traditional beer consumption. a

b

ence of markedly increased macrophage iron stores (4⫹ to 5⫹ on a scale of 0 to 6⫹), and no ringed sideroblasts. The patient was treated with phlebotomy therapy with the removal of 25 units of blood over 10 months until her serum ferritin concentration was 81 ␮g/L, yielding an estimated iron burden of 5.5 g before the onset of phlebotomies (normal ⬍1.0 g). Five full siblings and two half-siblings of this index patient were examined. Two full sisters with normal hemoglobin A2 levels were ferroportin 1 Q248H heterozygotes and had serum ferritin concentrations of 102 and 314 ␮g/L. Two full brothers with normal hemoglobin A2 levels were ferroportin 1 Q248H heterozygotes and had serum ferritin concentrations of 23 and 719 ␮g/L. One half-sister with a normal hemoglobin A2 level was ferroportin 1 wild type and had a serum ferritin concentration of 469 ␮g/L. One full brother with a normal hemoglobin A2 level was ferroportin 1 wild type and had a serum ferritin concentration of 262 ␮g/L. One half-brother with an elevated hemoglobin A2 level was ferroportin 1 wild type and had a serum ferritin concentration of 239 ␮g/L. Iron overload index subjects One of 4 African-Americans (25%) with the diagnosis of primary iron overload and 3 of 15 black Africans (20%) with the diagnosis of dietary iron overload were heterozy-

gous for a c.DNA 744 G3 T substitution in exon 6 of the ferroportin 1 gene (Fig. 1) that results in replacement of glutamine, an uncharged amino acid, with histidine, a positively charged amino acid (Q248H). Q248 lies in the predicted cytoplasmic domain and is conserved across mammals. Hemoglobin concentrations, mean corpuscular volumes, and indirect measures of iron status did not differ significantly between index subjects who were heterozygotes for the Q248H mutation in ferroportin 1 and those who were ferroportin 1 wild type (Table 1). Family members Heterozygosity for the Q248H substitution was found in 10 of 22 (45%) first degree family members of the four African and African-American patients with the diagnosis of primary iron overload and the Q248H mutation in ferroportin 1. Serum ferritin concentrations and transferrin saturations did not differ significantly between the Q248H heterozygotes and the wild type family members, but there was a trend to higher serum ferritin to amino aspartate transferase ratios (means of 14.8 versus 4.3 ␮g/U; P ⫽ 0.1) and lower hemoglobin concentrations (means of 11.8 versus 13.2 g/dL; P ⫽ 0.1). (Table 2). The ratio corrects serum ferritin concentration for alcohol-induced hepatocellular damage [16].

Table 2 Hematologic and iron measurements in first degree family members of four African and African-American Q248H heterozygote index subjects

Hemoglobina (g/dL; mean ⫾ SE) MCV (fL; mean ⫾ SE) Ferritinb (␮g/L; geometric mean and SE range) Ferritin/AST ratiob (␮g/U; mean ⫾ SE) Transferrin saturationc (%; mean ⫾ SE) a

Ferroportin Q248H heterozygotes (N ⫽ 10)

Ferroportin wild type (N ⫽ 12)

P

11.9 ⫾ 0.5 83 ⫾ 3 69 (46–102) 14.8 ⫾ 4.5 33 ⫾ 5

12.9 ⫾ 0.5 88 ⫾ 2 66 (46–94) 5.3 ⫾ 3.9 28 ⫾ 5

0.1 0.3 0.9 0.1 0.5

ANOVA in which parameter is adjusted for sex. ANOVA in which parameter is adjusted for age, sex, and estimated traditional beer consumption. c ANOVA in which parameter is adjusted for sex and estimated traditional beer consumption. b

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Table 3 Hematologic and iron measurements in African community control participants selected because their serum ferritin concentration was less than 400 ␮g/L

Hemoglobina (g/dL; mean ⫾ SE) MCV (fL; mean ⫾ SE) Ferritinb (␮g/L; geometric mean and SE range) Transferrin saturationc (%; mean ⫾ SE)

Ferroportin Q248H heterozygotes (N ⫽ 7)

Ferroportin wild type (N ⫽ 44)

P

12.5 ⫾ 0.5 85 ⫾ 3 63 (40–100) 24 ⫾ 5

13.7 ⫾ 0.2 87 ⫾ 1 35 (29–42) 26 ⫾ 2

0.026 0.5 0.2 0.7

a

ANOVA in which parameter is adjusted for sex. ANOVA in which parameter is adjusted for age, sex, and estimated traditional beer consumption. c ANOVA in which parameter is adjusted for sex and estimated traditional beer consumption. b

Controls We found ferroportin 1 Q248H heterozygosity in 7 of 51 (14%) African control subjects selected for having iron parameters within the normal range and being from the same community as the index subjects with the Q248H mutation. Among the 51 controls, transferrin saturation did not differ significantly according to Q248H genotype, but the Q248H heterozygotes showed a trend toward higher ferritin (although it was not significant) whereas they had significantly lower hemoglobin concentrations than the wild type participants (12.5 versus 13.7 g/dL; P ⫽ 0.026) (Table 3). Five of 100 (5%) anonymous African Americans were heterozygotes for the ferroportin 1 Q248H mutation. We did not find the Q248H change in 25 Caucasians with hyperferritinemia and iron overload without pathogenic mutations in other iron-loading genes (HFE, hepcidin, TfR2) or in 300 healthy Caucasian blood donors.

Discussion Ferroportin 1 is a multiple transmembrane domain protein that exports iron from cells and is conserved across vertebrate species. It has been postulated to function in release of iron from cells, particularly from macrophages where it is highly abundant (reviewed in Ref. [17]). Disease caused by ferroportin 1 mutations is likely due to defective iron exit from reticulendothelial cells, leading to iron accumulation in the liver, spleen and bone marrow, and early rise in serum ferritin levels without an accompanying elevation of transferrin saturation. Defective release of iron from macrophages may be responsible for inadequate iron supply to erythroid precursors in the bone marrow, leading to latent anemia and reduced tolerance to iron depletion [1,2]. In this report, we describe a new ferroportin 1 mutation, Q248H, that was discovered by examining the ferroportin 1 gene of Africans and African-Americans with iron overload. The mutation proved to be not only common among individuals with primary iron overload, but also prevalent in both the African and the African-American general popu-

lation samples we have studied so for. In contrast, the mutation is absent from all Caucasians we have analyzed, with or without iron overload. Although the Q248H mutation was discovered by studying patients with iron overload, standing alone it does not seem to be associated with a marked tendency to accumulate excess body iron, for among both family members and population controls we did not find serum ferritin concentrations to be significantly higher in the Q248H heterozygotes than the unaffected participants. However, among family members with the mutation there was a trend to a higher ratio of serum ferritin to aspartate amino transferase and among African controls with the mutation there was a tendency to higher serum ferritin concentration. It is possible that in combination with other genetic or environmental influences, the Q248H mutation in ferroportin 1 does lead to substantial iron loading. In this regard, it is intriguing that the African-American index subject with primary iron overload and Q248H heterozygosity has the beta-thalassemia trait but with unexpectedly high serum ferritin concentration (⬎1300 ␮g/L), hepatic non-heme iron concentration (106 ␮mol/g dry weight; normal ⬍30), and macrophage iron stores (4 –5⫹; normal ⬍3⫹) on bone marrow aspirate. It seems possible that the combination of a mild thalassemic condition and ferroportin 1 Q248H heterozygosity could have led to substantial iron loading. Also, it may be of note that the African iron overload subjects with the ferroportin 1 Q248H mutation also had increased dietary iron in the form of traditional beer. It is therefore possible that the combination of ferroportin 1 Q248H heterozygosity and high dietary iron also could have led to substantial iron loading. Determining the exact role of the ferroportin 1 Q248H mutation in the development of increased body iron stores will require the study of larger numbers of heterozygotes and controls with and without potential environmental and genetic modifying factors. Our data do suggest that the Q248H mutation may be associated with a tendency to anemia. Mean hemoglobin concentrations averaged 1 to 1.2 g/dL lower in Q248H heterozygote family members and control subjects than in wild type participants. As indicated in the original description of ferroportin 1 disease [1], patients present with mild

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anemia or low tolerance to phlebotomy. This may be due to impaired iron export from macrophages and reduced iron availability for bone marrow [1,2]. Clinically significant iron overload occurs in Americans of African descent and the possibility exists that black Americans have the same disorder as patients with African iron overload. The fact that we have found an AfricanAmerican patient with iron overload associated with the same mutation found in southern African iron overload patients supports the hypothesis. Since African-Americans are generally of West African origin and since the ferroportin 1 Q248H mutation does not appear to be present in Caucasians, the occurrence of this mutation in AfricanAmericans and black southern Africans suggests the concept of a “founder” effect and that the mutation may have originated from a single individual. This finding also implies that African populations from different geographic areas may share a common genetic background and is in keeping with our previous proposal that the finding of iron overload in both West Africa and southern Africa suggests a possible common founder effect for both populations [18]. Tracking the Q248H mutation in different African populations may offer additional information on the origin and migrations of Africans.

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