The discovery of the new haemochromatosis gene: Feder JN, Gnirke A, Thomas W, Tsuchihashi Z, Ruddy DA, Basava A, et al. A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis [Nat Genet 1996;13:399–408]

Abstract

Abstract: Hereditary haemochromatosis which affects some 1 in 400 and has an estimated carrier frequency of 1 in ten individuals of Northern European descent, results in multi-organ dysfunction caused by increased iron deposition, and is treatable if detected early. Using linkage-disequilibrium and full haplotype analysis, we have identified a 250-kilobase region more than 3 megabases telomeric of the major histocompatibility complex (MHC) that is identical-by-descent in 85% of patient chromosomes. Within this region, we have identified a gene related to the MHC class I family, termed HLA-H, containing two missense alterations. One of these is predicted to inactivate this class of proteins and was found homozygous in 83% of 178 patients. A role of this gene in haemochromatosis is supported by the frequency and nature of the major mutation and prior studies implicating MHC class I-like proteins in iron metabolism. [Abstract reproduced by permission of Nat Genet 1996;13:399–408]1. IntroductionThis discovery certainly achieved a new milestone in the history of hereditary hemochromatosis. Indeed, six names have stood out as landmarks in the field of hemochromatosis: Trousseau [[1]Trousseau A. Diabète sucré. Leçon de Clinique Médicale de l'Hôtel-Dieu. Baillère, Paris1865Google Scholar] and Troisier [[2]Troisier M. Diabète sucré.Bull Soc Anatomique Paris. 1871; 44: 231-235Google Scholar] were the first to describe the clinical and pathological entity corresponding to hemochromatosis. Von Recklinghausen in 1889 [[3]Von Recklinhausen F.D. Über hämochromatose.Tagebl Versamml Natur Ärtze Heidelberg. 1889; 62: 324-325Google Scholar] coined the term hemochromatosis. In 1935, Sheldon [[4]Sheldon J.H. Haemochromatosis. Oxford University Press, London1935Google Scholar] suggested that this disorder was an inborn error of metabolism. In 1975–1977, Simon [5Simon M. Pawlotsky Y. Bourel M. Fauchet R. Genetet B. Hémochromatose Idiopathique. Maladie associée à l'antigène tissulaire HLA-A3?.Nouv Presse Med. 1975; 4: 1432PubMed Google Scholar, 6Simon M. Bourel M. Fauchet R. Genetet B. Association of HLA-A3 and HLA-B14 antigens with idiopathic haemochromatosis.Gut. 1976; 17: 332-334Crossref PubMed Scopus (365) Google Scholar, 7Simon M. Bourel M. Genetet B. Fauchet R. Idiopathic hemochromatosis. Demonstration of recessive inheritance and early detection by family typing.N Engl J Med. 1977; 297: 1017-1021Crossref PubMed Scopus (306) Google Scholar], by demonstrating a close association between hemochromatosis and the HLA-A3 region of the short arm of chromosome 6, not only definitely proved the genetic nature and the recessive mode of transmission of the disease but showed that the implicated gene was located on the short arm of chromosome 6. ‘Finally’, in 1996, this gene was identified by Feder et al. Initially called HLA-H, the Hereditary haemochromatosis (HH) gene was later renamed HFE to avoid confusion with an HLA pseudo-gene already called HLA-H [[8]Mercier B. Mura C. Ferec C. Putting a hold on ‘HLA-H’.Nat Genet. 1997; 15: 234Crossref PubMed Scopus (51) Google Scholar]. It should be noticed, however, that there does not seem to be a clear semantic explanation for the choice of the gene symbol HFE (FE for iron?) [[9]Wain H.M. White J.A. Bruford E. Povey S. Hemochromatosis gene nomenclature.Am J Med Genet. 2000; 93: 77Crossref PubMed Scopus (6) Google Scholar].2. The breakthrough of the 1996 Nature Genetics article by Feder et alIn their outstanding study, Feder et al. used a two-step approach. They first characterized the HH region, employing two strategies: (i) identification of the region of maximum linkage disequilibrium by point wise analysis. Forty-five markers were used to genotype 101 HH patients versus 64 controls. At each marker, the allele with the highest excess frequency in the patient set as compared to controls was defined as the ancestral allele, thereby allowing the reconstruction of the ancestral haplotype on which the common HH mutation occurred. A 600-kilobase (kb) region, limited by markers D6S2240 and D6S2233, was then considered as the most likely location of the HH gene. (ii) Haplotype analysis of patient chromosomes. In order to identify probable historic recombination events on chromosomes bearing the ancestral haplotype, the haplotypes of 46 HH chromosomes, that have been separated in somatic-cell hybrid lines, were inspected. The analysis, stratified according to the likelihood of being homozygous for the ancestral mutation, defined a 250-kb candidate region between D6S2238 and D6S2241 as the likely location of the HH gene. The second step consisted in the identification of potential genes within this 250-kb candidate region, using cDNA selection, exon trapping and genomic DNA sequencing. Fifteen genes were discovered within this region, and only two of them contained base differences predicted to result in amino acid alterations: (i) the only nucleotide change consistent with the ancestral HH mutation was found in the major histocompatibility complex (MHC) class I-like gene, cDNA 24: a G to A transition at nucleotide 845 of the open reading frame, resulting in a cysteine-to-tyrosine substitution at amino acid 282 (=mutation C282Y). This missense mutation occurred at a highly conserved residue involved in intramolecular disulphide bridging in MHC class I proteins. This C282Y mutation was detected in 85% of all HH chromosomes, 148 of them being homozygous for this mutation and nine being heterozygotes. (ii) A second missense variant in cDNA 24 was found on the non-ancestral chromosomes present in the nine C282Y heterozygotes: a C to G change was identified in exon 2 resulting in a histidine to aspartic acid substitution at position 63 (=mutation H63D). This H63D variant was present in eight of the nine non-ancestral chromosomes. Then the authors described the similarity of the HH protein with MHC class I molecules and proposed a model in which the HH protein is a single polypeptide with three extracellular domains which would be analogous to the alpha-1, -2 and -3 domains of other MHC class I proteins. In contrast, the alpha-1 and -2 domains are non-polymorphic. β2-microglobulin is a separate protein and would interact with the HH protein in a non-covalent manner in the alpha-3 domain. The HH protein contains a membrane spanning region and a short cytoplasmic tail. C282Y and H63D are located in the alpha-3 and -1 domains, respectively. Finally, the study of HH gene expression showed that one major transcript of approximately 4 kb was detected in all tissues except the brain, a tissue distribution compatible with the organs known to be affected by hemochromatosis.3. Why such a long ‘hunting’ phase between the localization of the hemochromatosis gene on chromosome 6p in 1975 and its molecular identification in 1996?It is remarkable that more than 20 years were necessary to achieve the identification of the HFE gene. Indeed, the studies by Simon et al. concluded to a genetic distance between HLA-A gene and the HFE gene of approximately 1–2 centiMorgans (cM), pointing out, however, that genetic distance did not necessary mean physical distance [10Lalouel J.M. Le Mignon L. Simon M. Fauchet R. Bourel M. Rao D.C. et al.Genetic analysis of idiopathic hemochromatosis using both qualitative (disease status) and quantitative (serum iron) information.Am J Hum Genet. 1985; 37: 700-718PubMed Google Scholar, 11Simon M. Brissot P. The genetics of haemochromatosis.J Hepatol. 1988; 6: 116-124Abstract Full Text PDF PubMed Scopus (52) Google Scholar]. Logically, a lot of efforts were subsequently devoted to analyze this putative target area and some studies suggested a 400 kb candidate region within the HLA-A class I region [12Boretto J. Jouanolle A.M. Yaouanq J. El Kahloun A. Mauvieux V. Blayau M. et al.Anonymous markers located on chromosome 6 in the HLA-A class I region: allelic distribution in genetic haemochromatosis.Hum Genet. 1992; 89: 33-36Crossref PubMed Scopus (29) Google Scholar, 13Yaouanq J. Perichon M. Chorney M. Pontarotti P. Le Treut A. el Kahloun A. et al.Anonymous marker loci within 400 kb of HLA-A generate haplotypes in linkage disequilibrium with the hemochromatosis gene (HFE).Am J Hum Genet. 1994; 54: 252-263PubMed Google Scholar]. Some reports of recombinants within families supported a gene localization centromeric to HLA-F [14Edwards C.Q. Griffen L.M. Dadone M.M. Skolnick M.H. Kushner J.P. Mapping the locus for hereditary hemochromatosis: localization between HLA-B and HLA-A.Am J Hum Genet. 1986; 38: 805-811PubMed Google Scholar, 15Gasparini P. Borgato L. Piperno A. Girelli D. Olivieri O. Gottardi E. et al.Linkage analysis of 6p21 polymorphic markers and the hereditary hemochromatosis: localization of the gene centromeric to HLA-F.Hum Mol Genet. 1993; 2: 571-576Crossref PubMed Scopus (38) Google Scholar], while several others concluded a telomeric location [16Powell L.W. Summers K.M. Board P.G. Axelsen E. Webb S. Halliday J.W. Expression of hemochromatosis in homozygous subjects.Gastroenterology. 1990; 98: 1625-1632Abstract Full Text PDF PubMed Google Scholar, 17Calandro L.M. Baer D.M. Sensabaugh G.F. Characterization of a recombinant that locates the hereditary hemochromatosis gene telomeric to HLA-F.Hum Genet. 1995; 96: 339-342Crossref PubMed Scopus (15) Google Scholar]. In the absence of definitive results, the explored domain was progressively extended so that, in the close period preceding HFE discovery, the gene was localized more than 1 megabase (Mb) telomeric to HLA-A [18Jazwinska E.C. Pyper W.R. Burt M.J. Francis J.L. Goldwurm S. Webb S.I. et al.Haplotype analysis in Australian hemochromatosis patients: evidence for a predominant ancestral haplotype exclusively associated with hemochromatosis.Am J Hum Genet. 1995; 56: 428-433Crossref PubMed Google Scholar, 19Raha-Chowdhury R. Bowen D.J. Stone C. Pointon J.J. Terwilliger J.D. Shearman J.D. et al.New polymorphic microsatellite markers place the haemochromatosis gene telomeric to D6S105.Hum Mol Genet. 1995; 4: 1869-1874Crossref PubMed Scopus (94) Google Scholar, 20Stone C. Pointon J.J. Jazwinska E.C. Halliday J.W. Powell L.W. Robson K.J. et al.Isolation of CA dinucleotide repeats close to D6105; linkage disequilibrium with haemochromatosis.Hum Mol Genet. 1994; 3: 2043-2046PubMed Google Scholar, 21Seese N.K. Venditti C.P. Chorney K.A. Gerhard G.S. Ma J. Hudson T.J. et al.Localisation of the hemochromatosis gene: linkage disequilibrium analysis using an American patient collection.Blood Cells Mol Dis. 1996; 22: 36-46Crossref PubMed Scopus (21) Google Scholar, 22Worwood M. Raha-Chowdhury R. Dorak M.T. Darke C. Bowen D.J. Burnett A.K. Alleles at D6S65 and D6S105 define a haemochromatosis specific genotype.Br J Haematol. 1994; 86: 863-866Crossref PubMed Scopus (50) Google Scholar, 23Gandon G. Jouanolle A.M. Chauvel B. Mauvieux V. Le Treut A. Feingold J. et al.Linkage disequilibrium and extended haplotypes in the HLA-A to D6105 region: implications for mapping the hemochromatosis gene (HFE).Hum Genet. 1996; 97: 103-113Crossref PubMed Scopus (36) Google Scholar].Several factors can explain such a delay: a limited number of available genetic markers, a small number of recombinants provided by family studies, and an especially large zone of linkage disequilibrium responsible for the fact that markers, despite being far apart, exhibited comparable levels of linkage disequilibrium. Suppression of recombination in this widespread area was supported by the mapping studies which located markers within 1 cM more than 6 Mb apart!This HH gene discovery constituted the starting point of an exceptional improvement in our understanding of the pathophysiology of iron as well as in our clinical knowledge and management of hemochromatosis. It also paved the road for the identification of new iron overload syndromes.4. Improvement in our understanding of the pathogenesis of hemochromatosis and general iron pathophysiology [[24]Fleming RE, Britton RS, Waheed A, Sly WS, Bacon BR. HFE. In: Templeton, DM, editor. Molecular and Cellular Iron Transport, New-York-Basel: Marcel Dekker, Inc., 2002. pp. 189–205.Google Scholar]4.1 The HFE geneIndirectly suggested by the model of the β2-microglobulin knock-out mouse [[25]Santos M. Schilham M.W. Rademakers L.H. Marx J.J. de Sousa M. Clevers H. Defective iron homeostasis in beta2-microglobulin knockout mice recapitulates hereditary hemochromatosis in man.J Exp Med. 1996; 184: 1975-1985Crossref PubMed Scopus (192) Google Scholar], the proof of the link between C282Y HFE mutation and altered iron metabolism was provided by the fact that HFE knockout mice [[26]Zhou X.Y. Tomatsu S. Fleming R.E. Parkilla S. Waheed A. Jiang J. et al.HFE gene knockout produces mouse model of hereditary hemochromatosis.Proc Natl Acad Sci USA. 1998; 95: 2492-2497Crossref PubMed Scopus (489) Google Scholar] as well as mice homozygous for the C282Y mutation (knockin) [[27]Levy J.E. Montross L.K. Cohen D.E. Fleming M.D. Andrews N.C. The C282Y mutation causing hemochromatosis does not produce a null allele.Blood. 1999; 94: 9-11Crossref PubMed Google Scholar] developed iron overload mimicking that observed in human hemochromatosis. The structure of the HFE gene has been refined: it consists of seven exons. The murine HFE is structurally identical to the human gene [[28]Riegert P. Gilfillan S. Nanda I. Schmid M. Bahram S. The mouse HFE gene.Immunogenetics. 1998; 47: 174-177Crossref PubMed Scopus (17) Google Scholar]. HFE gene expression is not induced by various cytokines (in contrast to other MHC molecules) and no sequences resembling iron-responsive elements have been found in the 3′ or 5′ untranslated region the HFE mRNA.4.2 The HFE proteinThe HFE protein, detected by immunohistochemistry all along the digestive tract with various intracellular localizations, exhibits a peculiar pattern in the duodenum, which is the primary site of iron absorption [[29]Waheed A. Parkkila S. Saarnio J. Fleming R.E. Zhou X.Y. Tomatsu S. et al.Association of FHE protein with transferrin receptor in crypt enterocytes of human duodenum.Proc Natl Acad Sci USA. 1999; 96: 1579-1584Crossref PubMed Scopus (180) Google Scholar]: it is highly expressed in crypt cells but not in apical cells. HFE protein is synthesized within the cytosol, then binds to the β2-microglobulin (through a disulfide bridge) and the protein couple migrates towards the baso-lateral border of the crypt cells to join the transferrin receptor1 (TFR1) molecule [30Parkkila S. Waheed A. Britton R.S. Feder J.N. Tsuchihashi Z. Schatzman R.C. et al.Immunochemistry of HLA-H, the protein defective in patients with hereditary hemochromatosis, reveals unique pattern of expression in gastrointestinal tract.Proc Natl Acad Sci USA. 1997; 94: 2534-2539Crossref PubMed Scopus (243) Google Scholar, 31Waheed A. Parkkila S. Saarnio J. Fleming R.E. Zhou X.Y. Tomatsu S. et al.Association of HFE protein with transferrin receptor in crypt enterocytes of human duodenum.Proc Natl Acad Sci USA. 1999; 96: 1579-1584Crossref PubMed Scopus (212) Google Scholar, 32Gross C.N. Irrinki A. Feder J.N. Enns C.A. Co-trafficking of HFE, a non-classical major histocompatibility complex class I protein, with the transferrin receptor implies a role in intracellular iron regulation.J Biol Chem. 1998; 273: 22068-22074Crossref PubMed Scopus (198) Google Scholar]. The major structural work by Lebron et al. [[33]Lebron J.A. Bennett M.J. Vaughn D.E. Chirino A.J. Snow P.M. Mintier G.A. et al.Crystal structure of the hemochromatosis protein HFE and characterization of its interaction with transferrin receptor.Cell. 1998; 93: 111-123Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar] and Bennett et al. [[34]Bennett J.M. Lebron J.A. Bjorkman P.J. Crystal structure of the hereditary haemochromatosis protein HFE complexed with transferrin receptor.Nature. 2000; 403: 46-53Crossref PubMed Scopus (293) Google Scholar] described the 2.8 Å crystal structure of a complex between the extracellular portions of HFE and TFR1. The complex has twofold symmetry with a 2/2 stoichiometry, so that HFE contacts both polypeptide chains of the TFR1 homodimer. The relative orientations of both molecules indicate that HFE and TFR1 associate on the same membrane, rather than between opposite membranes. Due to the physical association between HFE and TFR1, the effect of HFE expression on TR1-mediated iron uptake has been investigated, yielding conflicting results. Studies have shown that the normal (=wild type) HFE protein decreased the affinity of serum transferrin for the TFR or decreased the number of TFR sites, resulting in a diminished entry of iron into the crypt cell [35Feder J.N. Penny D.M. Irrinki A. Lee V.K. Lebron J.A. Watson N. et al.The hemochromatosis gene product complexes with transferrin receptor and lowers its affinity for ligand binding.Proc Natl Acad Sci USA. 1998; 95: 1472-1477Crossref PubMed Scopus (722) Google Scholar, 36Salter-Cid L. Brunmark A. Li Y. Leturcq D. Peterson P.A. Jackson M.R. et al.Transferrin receptor is negatively modulated by the hemochromatosis protein HFE: implications for cellular homeostasis.Proc Natl Acad Sci USA. 1999; 96: 5434-5439Crossref PubMed Scopus (144) Google Scholar]. Therefore, in case of mutated HFE, one should expect an increased iron uptake, which does not fit with the iron deficient phenotype of duodenal crypt cells in hemochromatosis. More recent studies showed an opposite effect of HFE on TFR1 iron uptake. In transfected Chinese hamster ovary cells the overexpression of both HFE and β2-microglobulin resulted in an increase in TFR1-dependent iron uptake and increased iron levels in the cells [[37]Waheed A. Grubb J.H. Zhou X.Y. Tomatsu S. Fleming R.E. Costaldi M.E. et al.Regulation of transferrin-mediated iron uptake by HFE, the protein defective in hereditary hemochromatosis.Proc Natl Acad Sci USA. 2002; 99: 3117-3122Crossref PubMed Scopus (127) Google Scholar]. Trinder et al. showed that iron uptake from plasma transferrin by the duodenum was impaired in the HFE knockout mouse [[38]Trinder D. Olynyk J.K. Sly W.S. Morgan E.H. Iron uptake from plamsa transferrin by the duodenum is impaired in the Hfe knockout mouse.Proc Natl Acad Sci USA. 2002; 99: 5622-5626Crossref PubMed Scopus (86) Google Scholar]. The effect of HFE itself on the expression of the DMT1 iron transporter within apical duodenocytes remains unclear. An increased expression of this transporter has been reported by Fleming et al. in HFE-knockout mice [[39]Fleming R.E. Migas M.C. Zhou X.Y. Jiang J. Britton R.S. Brunt E.M. et al.Mechanism of increased iron absorption in murine model of hereditary hemochromatosis.Proc Natl Acad Sci USA. 1999; 96: 3143-3148Crossref PubMed Scopus (257) Google Scholar] and by Zoller et al. in patients with genetic hemochromatosis [[40]Zoller H. Koch R.O. Theurl I. Obrist P. Pietrangello A. Montosi G. et al.Expression of the duodenal iron transporters divalent-metal transporter 1 and ferroportin1 in iron deficiency and iron overload.Gastroenterology. 2001; 120: 1412-1419Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar] but not by Cannone-Hergaux et al. [[41]Cannone-Hergaux F. Levy J.E. Fleming M.D. Montross L.K. Andrews N.C. Gros P. Expression of the DMT1 (NRAMP2/DCT1) iron transporter in mice with genetic iron overload disorders.Blood. 2001; 97: 1138-1140Crossref PubMed Scopus (87) Google Scholar] in other strains of HFE-knockout mice. Moreover, the role of HFE in the regulation of iron absorption may be limited because HFE mutant mice retain the ability to regulate iron absorption [[42]Ajioka R.S. Levy J.E. Andrews N.C. Kushner J.P. Regulation of iron absorption in Hfe mutant mice.Blood. 2002; 100: 1465-1469Crossref PubMed Scopus (72) Google Scholar]. It is therefore highly likely that modifier genes modulate the regulatory capacity of HFE, as indicated by the data obtained with compound mutant mice [[43]Levy J.E. Montross L.K. Andrews N.C. Genes that modify the hemochromatosis phenotype in mice.J Clin Invest. 2000; 105: 1209-1216Crossref PubMed Scopus (195) Google Scholar] and with different strains of HFE knockout mice [[44]Dupic F. Fruchon S. Bensaid M. Borot N. Radosavljevic M. Loréal O. et al.Inactivation of the hemochromatosis gene differentially regulates duodenal expression of iron-related mRNAs between mouse strains.Gastroenterology. 2002; 122: 745-751Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar]. Classical MHC class I molecules could be involved [[45]Cardoso E.M. Macedo M.G. Rohrlich P. Ribeiro E. Silva M.T. Lemonnier F.A. et al.Increased hepatic iron in mice lacking classical MHC-class I molecules.Blood. 2002; 100: 4239-4241Crossref PubMed Scopus (28) Google Scholar]. Hepcidin has been reported to play a key role in iron metabolism [46Pigeon C. Ilyin G. Courselaud B. Leroyer P. Turlin B. Brissot P. et al.A new mouse liver-specific gene, encoding a protein homologous to human antimicrobial peptide hepcidin, is overexpressed during iron overload.J Biol Chem. 2001; 276: 7811-7819Crossref PubMed Scopus (1404) Google Scholar, 47Nicolas G. Bennoun M. Devaux I. Beaumont C. Grandchamp B. Kahn A. et al.Lack of hepcidin gene expression and severe tissue iron overload in upstream stimulatory factor 2 (USF2) knockout mice.Proc Natl Acad Sci USA. 2001; 98: 8780-8785Crossref PubMed Scopus (1075) Google Scholar, 48Nicolas G. Bennoun M. Porteu A. Mativet S. Beaumont C. Grandchamp B. et al.Severe iron deficiency anemia in transgenic mice expressing liver hepcidin.Proc Natl Acad Sci USA. 2002; 99: 4596-4601Crossref PubMed Scopus (758) Google Scholar, 49Courselaud B. Pigeon C. Inoue Y. Inoue J. Gonzalez F.J. Leroyer P. et al.C/EBP alpha regulates hepatic transcription of hepcidin, an antimicrobial peptide and regulator of iron metabolish. Cross-talk between C/EBP pathway and iron metabolism.J Biol Chem. 2002; 277: 41163-41170Crossref PubMed Scopus (225) Google Scholar]. Its relationship with HFE and its molecular partners remain to be determined. Macrophages might also play an important role as suggested by the fact that wild-type HFE protein normalizes transferrin iron accumulation in macrophages from patients with hemochromatosis [[50]Montosi G. Paglia P. Garuti C. Guzman C.A. Bastin J.M. Colombo M.P. et al.Wild-type HFE protein normalizes transferrin iron accumulation in macrophages from subjects with hereditary hemochromatosis.Blood. 2000; 96: 1125-1129Crossref PubMed Google Scholar].5. Improvement in our clinical knowledge and management of hemochromatosis [51Brissot P, Deugnier Y. In: Bircher J, Benhamou JP, McIntyre N, Rizzetto M, Rodes J, editors. Genetic haemochromatosis, Oxford Textbook of Clinical Hepatology, 2nd ed., vol. 1. Oxford-New York-Tokyo: Oxford University Press, 1999, pp. 409–413.Google Scholar, 52Barton J.C. Edwards C.Q. Hemochromatosis. Genetics, pathophysiology, diagnosis, and treatment. Cambridge University Press, Cambridge2000Crossref Google Scholar]The ‘classical’ form of hemochromatosis, which was at the basis of the HFE discovery, can be named HFE-associated hemochromatosis (or just hemochromatosis). The term of HFE-1 hemochromatosis has also been proposed.5.1 Frequency and penetrance of HFE mutations5.1.1 C282Y homozygosity (C282Y/C282Y)The vast majority of patients presenting with the strict phenotypic criteria of hemochromatosis are homozygotes for C282Y. However, there is incomplete penetrance of C282Y homozygosity as shown by: (i) the initial reports of C282Y/C282Y adult individuals without biochemical disturbance in iron metabolism [[53]Brissot P. Moirand R. Jouanolle A.M. Guyader D. Le Gall J.Y. Deugnier Y. et al.A genotypic study of 217 unrelated probands diagnosed as ‘genetic hemochromatosis’ on ‘classical’ phenotypic criteria.J Hepatol. 1999; 30: 588-593Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar]. (ii) The data provided by systematic screening of unselected populations [54Olynyk J.K. Cullen D.J. Aouilia S. Rossi E. Summerville L. Powell L.W. A population-based study of the clinical expression of the hemochromatosis gene.N Engl J Med. 1999; 341: 718-724Crossref PubMed Scopus (642) Google Scholar, 55Burt M.J. George P.M. Upton J.D. Collett J.A. Frampton C.M. Chapman T.M. et al.The significance of haemochromatosis gene mutations in the general population: implications for screening.Gut. 1998; 43: 830-836Crossref PubMed Scopus (207) Google Scholar]. (iii) The recent report of a clinical penetrance of less than 1% in a large series of 41,038 subjects [[56]Beutler E. Felittic V.J. Koziolb J.A. Hoa N.J. Penetrance of 845G-A (C282Y) HFE hereditary haemochromatosis mutation in the USA.Lancet. 2002; 359: 211-218Abstract Full Text Full Text PDF PubMed Scopus (736) Google Scholar]. Besides criticisms related to the likely population bias and to the clinical data collection, the definition of penetrance is essential to consider. Indeed, when a biochemical definition is adopted (increased serum transferrin saturation and/or serum ferritin) 70% of men and 56% of women would have been considered as expressing their homozygosity.5.1.2 Compound heterozygosity (C282Y/H63D)Observed in 1.8–8.2% of hemochromatosis series (based on rigorous phenotypic criteria), compound heterozygosity, when it is expressed, usually corresponds to mild forms of hemochromatosis with cofactors of expression (especially alcohol). The low penetrance of this genotype has been shown by studies of unselected populations [54Olynyk J.K. Cullen D.J. Aouilia S. Rossi E. Summerville L. Powell L.W. A population-based study of the clinical expression of the hemochromatosis gene.N Engl J Med. 1999; 341: 718-724Crossref PubMed Scopus (642) Google Scholar, 55Burt M.J. George P.M. Upton J.D. Collett J.A. Frampton C.M. Chapman T.M. et al.The significance of haemochromatosis gene mutations in the general population: implications for screening.Gut. 1998; 43: 830-836Crossref PubMed Scopus (207) Google Scholar] as well as of the expression of family members [[57]Moirand R. Guyader D. Mendler M.H. Jouanolle A.M. Le Gall J.Y. David V. et al.HFE based reevaluation of heterozygous hemochromatosis.Am J Med Genet. 2002; 111: 356-361Crossref PubMed Scopus (28) Google Scholar] with often a slight elevation of transferrin saturation and sometimes mild hyperferritinemia. The percentage of individuals presenting, in unselected populations, an increase in their serum iron parameters varies between 21 and 26% for transferrin saturation and between 16 and 34% for ferritin [54Olynyk J.K. Cullen D.J. Aouilia S. Rossi E. Summerville L. Powell L.W. A population-based study of the clinical expression of the hemochromatosis gene.N Engl J Med. 1999; 341: 718-724Crossref PubMed Scopus (642) Google Scholar, 55Burt M.J. George P.M. Upton J.D. Collett J.A. Frampton C.M. Chapman T.M. et al.The significance of haemochromatosis gene mutations in the general population: implications for screening.Gut. 1998; 43: 830-836Crossref PubMed Scopus (207) Google Scholar]. A simultaneous increase of transferrin saturation and ferritin is observed in 4.8% of compound heterozygotes belonging to the first 20,130 cases of the North American HEIRS study [[58]Adams P.C. Acton R. Barton J.C. Dawkins F. Eckfeldt J. Gordeuk V.R. et al.Hemochromatosis and iron overload screening study (HEIRS): an interim analysis of 20,130 primary care persons.Gastroenterology. 2002; 122: A634Google Scholar]. Mutations other than H63D have been reported in compound heterozygosity such as S65C [59Mura C. Raguenes O. Férec C. HFE mutations analysis in 711 hemochromatosis probands: evidence for S65C implication in mild form of hemochromatosis.Blood. 1999; 93: 2502-2505PubMed Google Scholar, 60Wallace D.F. Walker A.P. Pietrangelo A. Clare M. Bomford A.B. Dixon J.L. et al.Frequency of the S65C mutation of HFE and iron overload in 309 subjects heterozygous for C282Y.J Hepatol. 2002; 36: 474-479Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 61Asberg A. Thorstensen K. Hveem K. Bjerve K.S. Hereditary hemochromatosis: the clinical significance of the S65C mutation.Genet Test. 2002; 6: 59-62Crossref PubMed Scopus (36) Google Scholar] or some exceptional mutations [62Wallace D.F. Dooley J.S. Walker A.P. A novel mutation of HFE explains the classical phenotype of genetic hemochromatosis in a C282Y heterozygote.Gastroenterology. 1999; 116: 1409-1412Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 63Barton J.C. Sawada-Hirai R. Rothenberg B.E. Acton R.T. Two novel missense mutations of the HFE gene (I105T and G93R) and identification of the S65C mutation in Alabama hemochromatosis probands.Blood Cell Mol Dis. 1999; 25: 147-155Crossref PubMed Scopus (170) Google Scholar, 64Piperno A. Arosio C. Fossati L. Vigano M. Trombini P. Vergani A. et al.Two novel nonsense mutations of HFE gene in five unrelated Italian patients with hemochromatosis.Gastroenterology. 2000; 119: 441-445Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar].5.1.3 Penetrance of other genotypic profilesIt is very low for simplex C282Y or H63D heterozygosity (C282Y/WT or H63D/WT) and remains debated for H63D/H63D subjects [65Gochee 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 (214) Google Scholar, 66Aguilar-Martinez P. Bismuth M. Picot M.C. Thelcide C. Pageaux G.P. Blanc F. et al.Variable phenotypic presentation of iron overload in H63D homozygotes: are genetic modifiers the cause?.Gut. 2001; 48: 836-842Crossref PubMed Scopus (71) Google Scholar].5.2 The diagnostic impact of the HFE discovery5.2.1 Individual diagnosisThe speed with which HFE testing transformed the diagnostic management for hemochromatosis is unique for genetic disea