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Juvenile hemochromatosis associated with pathogenic mutations of adult hemochromatosis genes

2005; Elsevier BV; Volume: 128; Issue: 2 Linguagem: Inglês

10.1053/j.gastro.2004.11.057

1528-0012

Antonello Pietrangelo, Angela Caleffi, Jean Henrion, Francesca Ferrara, Elena Corradini, Hasan Kulaksiz, Wolfgang Stremmel, Pietro Andreoné, Cinzia Garuti,

Trace Elements in Health

Background & Aims: Juvenile hemochromatosis is a severe form of hereditary iron overload that has thus far been linked to pathogenic mutations of the gene coding for hemojuvelin (HJV), on chromosome 1, or, more rarely, that coding for hepcidin (HAMP), on chromosome 19. A milder adult-onset form is due to pathogenic mutations of HFE or, rarely, serum transferrin receptor 2. Methods: We studied a pedigree with siblings affected by both juvenile and adult-onset hereditary hemochromatosis. Affected subjects underwent full clinical evaluation, as well as microsatellite and gene sequencing analysis. Results: Two siblings (male and female, aged 24 and 25 years, respectively) were hospitalized for severe endocrinopathy and cardiomyopathy. At age 18 and 17 years, they had presented with impotence and amenorrhea, respectively, and increased serum iron levels. Hypogonadotropic hypogonadism was confirmed in both, and liver biopsy showed marked hepatic iron accumulation and micronodular cirrhosis. Iron levels were normalized after 24 months (female) and 36 months (male) of weekly phlebotomies. Microsatellite analysis showed no linkage with chromosome 1 and 19, and gene sequencing showed no hemojuvelin or hepcidin gene mutations. Instead, combined mutations of HFE (C282Y/H63D compound heterozygosity) and serum transferrin receptor 2 (Q317X homozygosity) were found. A 21-year-old brother with a milder phenotype resembling classic adult-onset hereditary hemochromatosis carried only the Q317X serum transferrin receptor 2 homozygote mutation. Conclusions: Juvenile hereditary hemochromatosis is not a distinct monogenic disorder invariably due to hemojuvelin or hepcidin mutations: it may be genetically linked to the adult-onset form of hereditary hemochromatosis. Background & Aims: Juvenile hemochromatosis is a severe form of hereditary iron overload that has thus far been linked to pathogenic mutations of the gene coding for hemojuvelin (HJV), on chromosome 1, or, more rarely, that coding for hepcidin (HAMP), on chromosome 19. A milder adult-onset form is due to pathogenic mutations of HFE or, rarely, serum transferrin receptor 2. Methods: We studied a pedigree with siblings affected by both juvenile and adult-onset hereditary hemochromatosis. Affected subjects underwent full clinical evaluation, as well as microsatellite and gene sequencing analysis. Results: Two siblings (male and female, aged 24 and 25 years, respectively) were hospitalized for severe endocrinopathy and cardiomyopathy. At age 18 and 17 years, they had presented with impotence and amenorrhea, respectively, and increased serum iron levels. Hypogonadotropic hypogonadism was confirmed in both, and liver biopsy showed marked hepatic iron accumulation and micronodular cirrhosis. Iron levels were normalized after 24 months (female) and 36 months (male) of weekly phlebotomies. Microsatellite analysis showed no linkage with chromosome 1 and 19, and gene sequencing showed no hemojuvelin or hepcidin gene mutations. Instead, combined mutations of HFE (C282Y/H63D compound heterozygosity) and serum transferrin receptor 2 (Q317X homozygosity) were found. A 21-year-old brother with a milder phenotype resembling classic adult-onset hereditary hemochromatosis carried only the Q317X serum transferrin receptor 2 homozygote mutation. Conclusions: Juvenile hereditary hemochromatosis is not a distinct monogenic disorder invariably due to hemojuvelin or hepcidin mutations: it may be genetically linked to the adult-onset form of hereditary hemochromatosis. Hereditary hemochromatosis is a common genetic iron-loading disorder characterized by excessive deposition of iron in parenchymal organs that can cause tissue damage and disease. The most common form is characterized by gradual iron accumulation, which sometimes leads to organ disease, particularly liver cirrhosis, during the fourth and fifth decades of life. This adult-onset form is usually caused by pathogenic mutations in the HFE gene1Feder J.N. Gnirke A. Thomas W. et al.A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis.Nat Genet. 1996; 13: 399-408Crossref PubMed Scopus (3393) Google Scholar or, in rarer cases, mutations of the gene encoding the serum transferrin receptor 2, TfR2.2Camaschella C. Roetto A. Cali A. et al.The gene TFR2 is mutated in a new type of haemochromatosis mapping to 7q22.Nat Genet. 2000; 25: 14-15Crossref PubMed Scopus (745) Google Scholar The phenotypic form known as juvenile hemochromatosis shares numerous features with adult hemochromatosis, but iron loading occurs at a greatly accelerated rate.3Lamon J.M. Marynick S.P. Roseblatt R. Donnelly S. Idiopathic hemochromatosis in a young female. A case study and review of the syndrome in young people.Gastroenterology. 1979; 76: 178-183Abstract Full Text PDF PubMed Scopus (83) Google Scholar Individuals with the juvenile phenotype are more likely to present with cardiomyopathy, endocrine disease, or both, although liver cirrhosis is also part of the syndrome. Untreated patients may succumb to heart failure before the age of 30 years.4Rivard S.R. Mura C. Simard H. et al.Clinical and molecular aspects of juvenile hemochromatosis in Saguenay-Lac-Saint-Jean (Quebec, Canada).Blood Cells Mol Dis. 2000; 26: 10-14Crossref PubMed Scopus (40) Google Scholar, 5De Gobbi M. Roetto A. Piperno A. et al.Natural history of juvenile haemochromatosis.Br J Haematol. 2002; 117: 973-979Crossref PubMed Scopus (156) Google Scholar This disorder was originally linked to chromosome 1q,6Roetto A. Totaro A. Cazzola M. et al.Juvenile hemochromatosis locus maps to chromosome 1q.Am J Hum Genet. 1999; 64: 1388-1393Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar where the hemojuvelin gene, HJV (originally named HFE2), has recently been identified.7Papanikolaou G. Samuels M.E. Ludwig E.H. et al.Mutations in HFE2 cause iron overload in chromosome 1q-linked juvenile hemochromatosis.Nat Genetics. 2004; 36: 77-82Crossref PubMed Scopus (850) Google Scholar The function of hemojuvelin is currently unknown, but patients with pathogenic HJV mutations seem to produce inappropriately low levels of hepcidin, a peptide that plays a major role in human iron metabolism, suggesting that hemojuvelin is somehow involved in hepcidin synthesis. Indeed, rare cases of juvenile hemochromatosis have also been linked to homozygous mutation of the hepcidin gene (HAMP) itself.8Roetto A. Papanikolaou G. Politou M. et al.Mutant antimicrobial peptide hepcidin is associated with severe juvenile hemochromatosis.Nat Genet. 2003; 33: 21-22Crossref PubMed Scopus (778) Google Scholar In this report, we describe the pedigree of a family in which 2 siblings presented with classic clinical signs of juvenile hemochromatosis in the absence of mutations involving the known “juvenile” hemochromatosis genes. Recently completed gene studies indicate that their disease is, instead, the result of digenic mutations of TfR2 and HFE, which are usually associated with the adult form of the disease. We studied the pedigree of a southern Italian family that included 3 siblings with primary iron overload (subject IV-4, the proband, and subjects IV-2 and IV-5 in Figure 1) who had emigrated to Belgium. The study was approved by the ethics committees of the University of Modena and the Hospital de Jolimont, and all subjects provided written informed consent for collection and publication of data. In all subjects (the proband, his parents, and his 4 siblings), serum levels of iron, transferrin, and ferritin were measured by standard methods in morning specimens drawn after an overnight fast, and transferrin saturation levels were calculated. Hepatic iron concentrations (expressed as micromoles per gram of liver; dry weight) were determined by atomic absorption spectrophotometry (S2380; Perkin-Elmer, Norwalk, CT), and the hepatic iron index (hepatic iron concentration/age) was calculated. Liver biopsy was performed in all 3 affected siblings. Five-micrometer tissue sections were stained with hematoxylin and eosin and Perls’ Prussian blue for the presence of iron and with Sirius red for the presence of collagen. All family members were questioned about previous blood transfusions, use of iron-containing medications, and daily consumption of alcohol. Serum samples were obtained for standard tests for hepatitis B virus surface antigen and hepatitis C virus antibodies and RNA. Hemoglobin electrophoresis was performed, and erythrocyte glucose-6-phosphate dehydrogenase and pyruvate kinase levels were measured. The 3 siblings with clinically expressed hemochromatosis underwent liver and bone marrow biopsies and complete cardiac (including chest radiograph, electrocardiography, and echocardiography) and endocrinology workups. The endocrinology workups included glucose tolerance, insulin response, and luteinizing hormone-releasing and thyrotropin-releasing hormone tests. For the luteinizing hormone-releasing hormone test, 100 μg of luteinizing hormone-releasing hormone was injected, and serum luteinizing hormone and follicle-stimulating hormone levels were measured 30 and 60 minutes later by radioimmunologic methods. The test was also performed in 36 healthy volunteers (12 men, 12 premenopausal women, and 12 postmenopausal women). Serum prohepcidin levels were measured with a sensitive enzyme-linked immunosorbent assay, as previously described.9Kulaksiz H. Gehrke S.G. Janetzko A. et al.Pro-hepcidin expression and cell specific localisation in the liver and its regulation in hereditary haemochromatosis, chronic renal insufficiency, and renal anaemia.Gut. 2004; 53: 735-743Crossref PubMed Scopus (217) Google Scholar Reference values determined in healthy volunteers were 51.6–153.4 ng/mL.9Kulaksiz H. Gehrke S.G. Janetzko A. et al.Pro-hepcidin expression and cell specific localisation in the liver and its regulation in hereditary haemochromatosis, chronic renal insufficiency, and renal anaemia.Gut. 2004; 53: 735-743Crossref PubMed Scopus (217) Google Scholar Assays were performed on serum samples collected in 2004 from the proband, his parents, and all 4 of his siblings. For comparison purposes, we also assayed specimens from 3 patients with anemia of chronic disease (due to rheumatic disorders; hemoglobin, 700 ng/mL; serum transferrin saturation, 13 g/L; serum ferritin, <50 ng/mL; serum transferrin saturation, <35%). Blood specimens were collected after an overnight fast, and serum was separated and maintained frozen at −20°C until assayed. Haplotype analysis was performed with 8 microsatellite markers: 4 from the juvenile 1q locus (D1S344, D1S442, D1S2347, and D1S2343)6Roetto A. Totaro A. Cazzola M. et al.Juvenile hemochromatosis locus maps to chromosome 1q.Am J Hum Genet. 1999; 64: 1388-1393Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar and 4 from the region on chromosome 19 where the HAMP gene is located (D19S931, D19S414, D19S220, and D19S420).10Delatycki M. Allen K. Gow P. et al.A homozygous HAMP mutation in a multiply consanguineous family with pseudo-dominant juvenile hemochromatosis.Clin Genet. 2004; 65: 378-383Crossref PubMed Scopus (46) Google Scholar Polymerase chain reaction (PCR) reactions with fluorescently labeled primers were performed according to standard protocols in a CEQ 2000 XL Beckman DNA sequencer (Beckman Coulter Inc, Fullerton, CA), and results were processed with CEQ Fragment Analysis software (Beckman Coulter Inc). For analysis of the HFE, HAMP, TfR2, and HJV coding regions (exons plus intron-exon boundaries), PCR-amplified fragments were purified with the QIAquick PCR purification kit (Qiagen, Valencia, CA) and sequenced by using the dye-terminator cycle-sequencing kit (Beckman Coulter Inc). Fragments were then electrophoretically separated and analyzed with a CEQ 2000 XL Beckman Coulter DNA sequencer. The pedigree studied is shown in Figure 1, and Table 1 summarizes the main characteristics of the 3 subjects with iron overload at the time of diagnosis. The study began in 1978, when the proband, subject IV-4, aged 24 years, was hospitalized with fatigue, arthralgia, orthopnea, and tachyarrhythmia. He had been diagnosed with hypogonadism and cardiomyopathy at age 18 and had been treated with digoxin and diuretics ever since. Review of the chart from that admission showed that he had also presented with increased serum levels of iron (210 μg/dL). The electrocardiogram performed during the 1978 hospitalization showed rapid atrial flutter/fibrillation, and the chest radiograph showed cardiomegaly and pleural effusion. Cardiac catheterization showed increased right-heart pressures and signs of restrictive cardiomyopathy (Table 1). The liver biopsy documented massive iron deposits and micronodular cirrhosis (Figure 2A and B). At this point, a clinical diagnosis of hereditary hemochromatosis was made.Table 1Biochemical and Clinical Characteristics of the 3 Affected Family Members at the Time of DiagnosisVariableSubject (hemochromatosis phenotype)IV-2 (adult)IV-4 (juvenile)IV-5 (juvenile)SexMMFAge at diagnosis (y)212425Symptoms and signsSkin pigmentationSkin pigmentation, fatigue, arthralgia, dyspnea and orthopnea, tachyarrhythmia, impotenceSkin pigmentation, amenorrhea, fatigue, metacarpophalangeal joint pain, dyspnea, palpitationsPhysical examinationHepatomegalyProtodiastolic gallop, apical systolic murmur; hepatomegaly (firm consistency); splenomegaly; testicular atrophyHepatomegaly (firm consistency), splenomegalyHematology workup Hb (g/L)aTo convert the values for hemoglobin to millimoles per liter, multiply by 0.6206.152137122 WBC (n/mL)590031002900 Platelets (n/mL)285,000157,000163,000 Bone marrow biopsyNormal cellularity; no blastsNormal cellularity; no blastsNormal cellularity; no blastsIron indices Serum iron (μg/dL)315225290 Serum transferrin saturation (%)10010086 Serum ferritin (ng/mL; reference range, 20–250 ng/mL)95552007200 24-h urinary iron excretion after 500 mg intramuscular deferoxamine (reference value, <1.2 mg)3.67.98.0 Hepatic iron (μmol/g of liver, dry weight; reference value, <30 μmol/g)62411429 Hepatic iron index (reference range, 0–1.0)bThe hepatic iron index is the ratio of the hepatic iron concentration to the patient’s age at the time of the evaluation.2.917.117.1 Total iron removed (g)cThe total amount of iron removed was calculated for each subject considering the total number of phlebotomies (400 mL of blood drawn per session) multiplied by the number of milligrams of iron removed per session. The latter was based on the patient’s prephlebotomy hematocrit, assuming that 1 mL of red cells contains 1 mg of iron.7.230.721.6Endocrine workup ACTH (pg/mL; reference range,14.155.76.0 10–100 pg/mL) Urinary 17-ketosteroids (mg/24 h; reference range, 9.2–22.4 mg/24 h)18.12.22.3 Urinary 17-hydroxysteroids (mg/24 h; reference range, 4.5–15.7 mg/24 h)10.36.05.4 Plasma testosterone (ng/100 mL; reference range, 150–1100 ng/100 mL)7509.5— Luteinizing hormone (ng/mL; reference range: male, 1.15–2.96 ng/mL; female, follicular phase, 1–3.5 ng/mL; luteal phase, 0.9–2.9 ng/mL)2.181.810.38 Follicle-stimulating hormone (ng/mL; reference range: male, 1.2–2.5 ng/mL; female, follicular phase, 1.8–4.9 ng/mL; luteal phase, 0.6–2.9 ng/mL)1.900.280.31 Luteinizing hormone-releasing testNormalNo change from baselineNo change from baseline Oral glucose tolerance testNormalAbnormalAbnormalHepatic workup AST (U/L)1438145 ALT (U/L)1824189 Prothrombin time (%)1005080 Laparoscopic findingsBrown/red hepatic coloration, hepatomegaly, smooth hepatic surfaceDark brownish hepatic coloration, hepatic micronodularity, ascitesDark brownish hepatic coloration, hepatic micronodularity, Mild ascites Liver histologyPeriportal siderosis, minimal periportal fibrosis with preserved lobular architectureMicronodular cirrhosis, massive siderosisMicronodular cirrhosis, massive siderosisCardiac workup ElectrocardiogramNormalAtrial flutter/fibrillationNormal Chest radiographNormalCardiomegaly, hilar enlargement, pulmonary venous congestionCardiomegaly, pulmonary venous congestion EchocardiogramNormalLeft ventricular and atrial dilatation, markedly reduced ejection fractionNormalHb, hemoglobin; WBC, white blood cell; ACTH, adrenocorticotropic hormone; AST, aspartate aminotransferase; ALT, alanine aminotransferase.a To convert the values for hemoglobin to millimoles per liter, multiply by 0.6206.b The hepatic iron index is the ratio of the hepatic iron concentration to the patient’s age at the time of the evaluation.c The total amount of iron removed was calculated for each subject considering the total number of phlebotomies (400 mL of blood drawn per session) multiplied by the number of milligrams of iron removed per session. The latter was based on the patient’s prephlebotomy hematocrit, assuming that 1 mL of red cells contains 1 mg of iron. Open table in a new tab Hb, hemoglobin; WBC, white blood cell; ACTH, adrenocorticotropic hormone; AST, aspartate aminotransferase; ALT, alanine aminotransferase. Investigation of the family history showed that the proband’s sister (subject IV-5; aged 25 years) also had fatigue and palpitations. She had been receiving gonadotropin treatment since age 17, when amenorrhea was first diagnosed, and the records from that period indicate that her serum iron level was already increased (225 μg/dL). At age 22, she experienced a third-trimester abortion. A second pregnancy at age 24 ended with a live birth despite late complications (preeclampsia and listeria infection), and gonadotropin was discontinued. The inpatient workup prompted by her brother’s diagnosis showed cardiomegaly, hepatomegaly, and splenomegaly. Micronodular cirrhosis with massive iron deposition was documented by liver biopsy (Figure 2C and D). Family screening also led to the identification of subject IV-2, aged 21 years, who was apparently healthy. He presented with dark skin and moderate hepatomegaly, but liver function was normal, and there were no signs of cardiomyopathy or endocrine disease (Table 1). Liver biopsy showed grade 2 iron deposition, particularly in periportal hepatocytes, and minimal signs of periportal and sinusoidal fibrosis (Figure 2E and F). None of the 3 siblings had thalassemia, hemolytic conditions, or serological evidence of infection with the hepatitis B or C virus. Their bone marrow biopsies were characterized by normal cellularity and the absence of sideroblasts. All 3 reported alcohol consumption of <10 g/day. The endocrine workup showed abnormal oral glucose tolerance and hypogonadotropic hypogonadism in subjects IV-4 and IV-5 (Table 1). In all 3, thyroid function and responses to insulin- and thyrotropin-releasing hormone tests were normal compared with age- and sex-paired control values. The 3 siblings were enrolled in a weekly phlebotomy program, and their iron stores normalized (i.e., serum ferritin, <50 ng/mL; serum transferrin saturation, <30%) after 9 months (subject IV-2), 48 months (subject IV-4), and 36 months (subject IV-5). All have continued maintenance treatment, which currently consists of 6 to 7 phlebotomies a year for subjects IV-4 and IV-5, as opposed to 2 per year for their younger brother. None of the 3 has developed diabetes. Subject IV-4’s heart disease has stabilized, and he has had no signs of heart failure or serious arrhythmias since 1978. His hypogonadism has not been reversed by treatment. The same is true of his sister, who has remained amenorrheic since gonadotropin was discontinued in 1978. In 1997, the 3 siblings with iron overload were tested for C282Y and H63D mutations of the HFE gene. Identical compound heterozygous mutations (C282Y/H63D) were found in the proband and his similarly affected sister, but no HFE mutations were found in subject IV-2. In 2003, subject IV-2 was tested for TfR2 mutations, and a novel homozygous change (C to T) was found in exon 7. The mutation, which replaces glutamine (CAG) with a stop codon (TAG) at residue 317 (Q317X), produces a truncated TfR2 molecule without the transmembrane domain. The entire HFE gene was also sequenced and confirmed to be normal. Because subjects IV-4 and IV-5 presented an iron-overload phenotype much more severe than that usually found in C282Y/H63D compound heterozygotes, we suspected that they might have additional genetic defects. Therefore, in 2004, after the 2 “juvenile hemochromatosis genes,” HJV and HAMP, had been identified, we embarked on haplotype analysis and sequencing of the exons and intron-exon boundaries of these genes. Haplotypes for all family members were first constructed by using 4 microsatellites from the juvenile 1q locus, but these showed no segregation of the iron overload state (Figure 3). Similar results emerged when microsatellite markers of the HAMP locus on chromosome 19 were analyzed (data not shown). We then directly sequenced the coding regions and exon-intron boundaries of HJV and HAMP. No mutations were identified in either gene. Although the phenotype of subjects IV-4 and IV-5 was unusually severe for classic TfR2-related hemochromatosis, we also analyzed the DNA sequence of this gene. Homozygous Q317X TfR2 mutations, the same mutation found in subject IV-2, were thus identified in both of the siblings with juvenile hereditary hemochromatosis. Serum levels of prohepcidin measured in 2004 were as follows: subject IV-2, 59.2 ng/mL; subject IV-4, 53.3 ng/mL; subject IV-5, 60.1 ng/mL; subject IV-1, 105.2 ng/mL; subject IV-3, 144 ng/mL; subject III-1, 140 ng/mL; and subject III-2, 71.8 ng/mL. The 3 patients with anemia of chronic disease had levels of 87.8, 111, and 340 ng/mL, and those found in the 3 patients receiving maintenance phlebotomy therapy for HFE-related hemochromatosis were 94.2, 40.1, and 70.1 ng/mL. The term juvenile hemochromatosis refers to a severe form of hereditary hemochromatosis in which iron loading and symptomatic organ disease occur in both sexes as early as the first or second decade of life.3Lamon J.M. Marynick S.P. Roseblatt R. Donnelly S. Idiopathic hemochromatosis in a young female. A case study and review of the syndrome in young people.Gastroenterology. 1979; 76: 178-183Abstract Full Text PDF PubMed Scopus (83) Google Scholar The clinical presentation is generally dominated by endocrine or cardiac manifestations, but clinically silent liver disease is also part of the syndrome. Rare cases of juvenile hemochromatosis have been attributed to pathogenic mutations of HAMP,8Roetto A. Papanikolaou G. Politou M. et al.Mutant antimicrobial peptide hepcidin is associated with severe juvenile hemochromatosis.Nat Genet. 2003; 33: 21-22Crossref PubMed Scopus (778) Google Scholar the gene encoding hepcidin, which is a putative down-regulator of iron efflux from enterocytes and macrophages. However, most cases are associated with pathogenic mutations of the recently identified HJV gene.7Papanikolaou G. Samuels M.E. Ludwig E.H. et al.Mutations in HFE2 cause iron overload in chromosome 1q-linked juvenile hemochromatosis.Nat Genetics. 2004; 36: 77-82Crossref PubMed Scopus (850) Google Scholar Deleterious HJV mutations were first documented in 12 families from Greece, Canada, and France with 1q-linked juvenile hemochromatosis. Roughly two thirds of these mutations caused a G320V substitution in the HJV gene product, hemojuvelin, whose function is unknown. However, because inappropriately low urine levels of hepcidin were found in these patients, it was suggested that hemojuvelin might be an upstream regulator of hepcidin.7Papanikolaou G. Samuels M.E. Ludwig E.H. et al.Mutations in HFE2 cause iron overload in chromosome 1q-linked juvenile hemochromatosis.Nat Genetics. 2004; 36: 77-82Crossref PubMed Scopus (850) Google Scholar More recent studies in various areas of the world have confirmed that the G320V mutation is the most common HJV mutation associated with juvenile hemochromatosis.11Lanzara C. Roetto A. Daraio F. et al.The spectrum of hemojuvelin gene mutations in 1q-linked juvenile hemochromatosis.Blood. 2004; 103: 4317-4321Crossref PubMed Scopus (167) Google Scholar, 12Lee P.L. Beutler E. Rao S.V. Barton J.C. Genetic abnormalities and juvenile hemochromatosis mutations of the HJV gene encoding hemojuvelin.Blood. 2004; 103: 4669-4671Crossref PubMed Scopus (130) Google Scholar In the southern Italian family we studied, 3 siblings were found to have iron overload. Two developed hypogonadotropic hypogonadism and cardiomyopathy during adolescence; these are classic features of the juvenile form of hereditary hemochromatosis. Their younger brother presented a mild form of hemochromatosis characterized by hepatic iron overload, with no signs of endocrine or cardiac disease. To date, no signs of iron overload have been detected in any other members of the family. All 3 of the affected siblings carry identical homozygous mutations (Q317X) in TfR2. Published reports on TfR2-related hemochromatosis are limited and clinical descriptions scanty, but it seems that homozygous stop-codon mutations of TfR2 (the type described in this report) lead to organ disease that is generally diagnosed during the fourth decade of life.13Girelli D. Bozzini C. Roetto A. et al.Clinical and pathologic findings in hemochromatosis type 3 due to a novel mutation in transferrin receptor 2 gene.Gastroenterology. 2002; 122: 1295-1302Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar This is consistent with the phenotype of subject IV-2, who had only mild asymptomatic liver changes at age 20 years. The fact that, at the same age, his affected siblings had already presented severe endocrine and cardiac disease was much more suggestive of alterations in one of the known juvenile hemochromatosis genes (HAMP and HJV). However, in both of these subjects, the coding regions and intron-exon boundaries of both of these genes were completely normal, and the presence of pathogenic mutations in regulatory sequences of the “juvenile” genes was excluded by the results of our haplotype analysis of the family. In fact, hemochromatosis did not segregate with any single haplotype constructed by microsatellite analysis of the probable HJV and HAMP enhancer-promoter regions located, respectively, on chromosomes 1q and 19q. The only genotypic difference that distinguished the proband and his sister from their younger affected brother was the presence in the older siblings of a second mutation involving the HFE gene. In HFE-related hemochromatosis, the risk of disease expression (ranging from the biochemical stigmata of iron overload to clinically evident organ damage/disease) is almost always associated with C282Y homozygosity,1Feder J.N. Gnirke A. Thomas W. et al.A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis.Nat Genet. 1996; 13: 399-408Crossref PubMed Scopus (3393) Google Scholar although a small percentage of C282Y/H63D compound heterozygotes also seem to be at risk.14Gochee P.A. Powell L.W. Cullen D.J. Du Sart D. Rossi E. Olynyk J.K. A population-based study of the biochemical and clinical expression of the H63D hemochromatosis mutation.Gastroenterology. 2002; 122: 646-651Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 15Rochette J. Pointon J.J. Fisher C.A. et al.Multicentric origin of hemochromatosis gene (HFE) mutations.Am J Hum Genet. 1999; 64 (published erratum appears in Am J Hum Genet 1999;64:1491): 1056-1062Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar Our hypothesis is the following: whereas either of the 2 mutations found in subjects IV-4 and IV-5—considered singly—would be expected to produce no more than the adult-onset form of the disease, their combined effects may be sufficient to produce the much more severe juvenile hemochromatosis phenotype. The basic metabolic abnormality in all forms of hereditary hemochromatosis is a progressive expansion of the plasma iron compartment. This expansion (which is reflected in a high saturation of circulating transferrin with iron) is caused by the uncontrolled transfer/release of iron into the bloodstream by intestinal cells and probably also macrophages. It may occur rapidly (as in juvenile hemochromatosis) or more gradually (as in adult hemochromatosis). Excess plasma iron is normally transferred to storage depots represented by the liver and other organ tissues, but when the storage capacity of these tissues is exceeded, the risk of organ damage increases, and the visceral patterns of iron overload (i.e., predominance of hepatic involvement vs. that of the endocrine glands/heart) may depend, at least in part, on the rate of plasma iron loading. Recent data indicate that hepcidin is one of the main down-regulators of iron release from enterocytes and macrophages. Assays of hepcidin have shown that this peptide is expressed at inappropriately low levels7Papanikolaou G. Samuels M.E. Ludwig E.H. et al.Mutations in HFE2 cause iron overload in chromosome 1q-linked juvenile hemochromatosis.Nat Genetics. 2004; 36: 77-82Crossref PubMed Scopus (850) Google Scholar in patients with juvenile forms of hereditary hemochromatosis. Relative hepcidin deficiency has also been associated with the adult variant related to HFE mutation.16Gehrke S.G. Kulaksiz H. Herrmann T. et al.Expression of hepcidin in hereditary hemochromatosis evidence for a regulation in response to serum transferrin saturation and non-transferrin-bound iron.Blood. 2003; 102: 371-376Crossref PubMed Scopus (221) Google Scholar, 17Nemeth E. Valore E.V. Territo M. Schiller G. Lichtenstein A. Ganz T. Hepcidin, a putative mediator of anemia of inflammation, is a type II acute-phase protein.Blood. 2003; 101: 2461-2463Crossref PubMed Scopus (1213) Google Scholar, 18Bridle K.R. Frazer D.