title: Heterozygous Mutations in BMP6 Pro-peptide Lead to Inappropriate Hepcidin Synthesis and Moderate Iron Overload in Humans.
abstract: OBJECTIVE
  Hereditary hemochromatosis is a heterogeneous group of genetic disorders characterized by parenchymal iron overload. It is caused by defective expression of liver hepcidin, the main regulator of iron homeostasis. Iron stimulates the gene encoding hepcidin (HAMP) via the bone morphogenetic protein (BMP)6 signaling to SMAD. Although several genetic factors have been found to cause late-onset hemochromatosis, many patients have unexplained signs of iron overload. We investigated BMP6 function in these individuals.
  
  
  METHODS
  We sequenced the BMP6 gene in 70 consecutive patients with a moderate increase in serum ferritin and liver iron levels who did not carry genetic variants associated with hemochromatosis. We searched for BMP6 mutations in relatives of 5 probands and in 200 healthy individuals (controls), as well as in 2 other independent cohorts of hyperferritinemia patients. We measured serum levels of hepcidin by liquid chromatography-tandem mass spectrometry and analyzed BMP6 in liver biopsy specimens from patients by immunohistochemistry. The functions of mutant and normal BMP6 were assessed in transfected cells using immunofluorescence, real-time quantitative polymerase chain reaction, and immunoblot analyses.
  
  
  RESULTS
  We identified 3 heterozygous missense mutations in BMP6 (p.Pro95Ser, p.Leu96Pro, and p.Gln113Glu) in 6 unrelated patients with unexplained iron overload (9% of our cohort). These mutations were detected in less than 1% of controls. p.Leu96Pro also was found in 2 patients from the additional cohorts. Family studies indicated dominant transmission. Serum levels of hepcidin were inappropriately low in patients. A low level of BMP6, compared with controls, was found in a biopsy specimen from 1 patient. In cell lines, the mutated residues in the BMP6 propeptide resulted in defective secretion of BMP6; reduced signaling via SMAD1, SMAD5, and SMAD8; and loss of hepcidin production.
  
  
  CONCLUSIONS
  We identified 3 heterozygous missense mutations in BMP6 in patients with unexplained iron overload. These mutations lead to loss of signaling to SMAD proteins and reduced hepcidin production. These mutations might increase susceptibility to mild-to-moderate late-onset iron overload.
authors.affiliation: INSERM UMR1149, Centre de Recherche sur l'Inflammation, Paris, France; Université Paris Diderot, Site Bichat, Sorbonne Paris Cité, Paris, France; Laboratory of Excellence, GR-Ex, Paris, France.
authors.forename: Raed
authors.initials: R
authors.lastname: Daher
authors.affiliation: INSERM UMR1149, Centre de Recherche sur l'Inflammation, Paris, France; Université Paris Diderot, Site Bichat, Sorbonne Paris Cité, Paris, France; Laboratory of Excellence, GR-Ex, Paris, France; AP-HP, Département de Génétique, Hôpital Bichat, Paris, France.
authors.forename: Caroline
authors.initials: C
authors.lastname: Kannengiesser
authors.affiliation: INSERM UMR1149, Centre de Recherche sur l'Inflammation, Paris, France; Université Paris Diderot, Site Bichat, Sorbonne Paris Cité, Paris, France; Laboratory of Excellence, GR-Ex, Paris, France.
authors.forename: Dounia
authors.initials: D
authors.lastname: Houamel
authors.affiliation: INSERM UMR1149, Centre de Recherche sur l'Inflammation, Paris, France; Université Paris Diderot, Site Bichat, Sorbonne Paris Cité, Paris, France; Laboratory of Excellence, GR-Ex, Paris, France; AP-HP, Centre Français des Porphyries, Hôpital Louis Mourier, Colombes, France.
authors.forename: Thibaud
authors.initials: T
authors.lastname: Lefebvre
authors.affiliation: Department of Liver Disease and Molecular Genetics, CHU Rennes, Rennes, France.
authors.forename: Edouard
authors.initials: E
authors.lastname: Bardou-Jacquet
authors.affiliation: INSERM UMR1149, Centre de Recherche sur l'Inflammation, Paris, France; Université Paris Diderot, Site Bichat, Sorbonne Paris Cité, Paris, France; Laboratory of Excellence, GR-Ex, Paris, France.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Ducrot
authors.affiliation: AP-HP, Département d'Hépatologie, Hôpital Beaujon, Clichy, France.
authors.forename: Caroline
authors.initials: C
authors.lastname: de Kerguenec
authors.affiliation: Department of Liver Disease and Molecular Genetics, CHU Rennes, Rennes, France; CHU Rennes, French Reference Center for Rare Iron Overload Diseases of Genetic Origin.
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Jouanolle
authors.affiliation: AP-HP, Centre Français des Porphyries, Hôpital Louis Mourier, Colombes, France.
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Robreau
authors.affiliation: AP-HP, Centre Français des Porphyries, Hôpital Louis Mourier, Colombes, France.
authors.forename: Claire
authors.initials: C
authors.lastname: Oudin
authors.affiliation: INSERM U1078, Université de Brest, CHRU de Brest, Laboratoire de Génétique Moléculaire et d'Histocompatibilité, Bretagne, Brest, France.
authors.forename: Gerald
authors.initials: G
authors.lastname: Le Gac
authors.affiliation: INSERM UMR1149, Centre de Recherche sur l'Inflammation, Paris, France.
authors.forename: Boualem
authors.initials: B
authors.lastname: Moulouel
authors.affiliation: Service d'Hépato-Gastroentérologie, INSERM UMR1092, Limoges, France.
authors.forename: Veronique
authors.initials: V
authors.lastname: Loustaud-Ratti
authors.affiliation: INSERM UMR1149, Centre de Recherche sur l'Inflammation, Paris, France; AP-HP, Laboratoire d'Anatomo-Pathologie, Hôpital Beaujon, Clichy, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Bedossa
authors.affiliation: AP-HP, Département d'Hépatologie, Hôpital Beaujon, Clichy, France.
authors.forename: Dominique
authors.initials: D
authors.lastname: Valla
authors.affiliation: INSERM UMR1149, Centre de Recherche sur l'Inflammation, Paris, France; Université Paris Diderot, Site Bichat, Sorbonne Paris Cité, Paris, France; Laboratory of Excellence, GR-Ex, Paris, France; AP-HP, Centre Français des Porphyries, Hôpital Louis Mourier, Colombes, France.
authors.forename: Laurent
authors.initials: L
authors.lastname: Gouya
authors.affiliation: INSERM UMR1149, Centre de Recherche sur l'Inflammation, Paris, France; Université Paris Diderot, Site Bichat, Sorbonne Paris Cité, Paris, France; Laboratory of Excellence, GR-Ex, Paris, France.
authors.forename: Carole
authors.initials: C
authors.lastname: Beaumont
authors.affiliation: Department of Liver Disease and Molecular Genetics, CHU Rennes, Rennes, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Brissot
authors.affiliation: INSERM UMR1149, Centre de Recherche sur l'Inflammation, Paris, France; Université Paris Diderot, Site Bichat, Sorbonne Paris Cité, Paris, France; Laboratory of Excellence, GR-Ex, Paris, France; AP-HP, Centre Français des Porphyries, Hôpital Louis Mourier, Colombes, France.
authors.forename: Hervé
authors.initials: H
authors.lastname: Puy
authors.affiliation: INSERM UMR1149, Centre de Recherche sur l'Inflammation, Paris, France; Université Paris Diderot, Site Bichat, Sorbonne Paris Cité, Paris, France; Laboratory of Excellence, GR-Ex, Paris, France. Electronic address: zoubida.karim@inserm.fr.
authors.forename: Zoubida
authors.initials: Z
authors.lastname: Karim
authors.affiliation: AP-HP, Département de Génétique, Hôpital Bichat, Paris, France.
authors.forename: Dimitri
authors.initials: D
authors.lastname: Tchernitchko
mesh_terms: D000368:Aged; D000818:Animals; D001706:Biopsy; D055418:Bone Morphogenetic Protein 6 / Q000235:genetics* / Q000378:metabolism; D016022:Case-Control Studies; D002460:Cell Line; D002853:Chromatography, Liquid; D004252:DNA Mutational Analysis; D005260:Female; D005293:Ferritins / Q000097:blood; D056726:Genetic Association Studies; D020022:Genetic Predisposition to Disease; D006432:Hemochromatosis / Q000097:blood / Q000235:genetics* / Q000378:metabolism*; D064451:Hepcidins / Q000096:biosynthesis* / Q000097:blood; D006579:Heterozygote; D006801:Humans; D007150:Immunohistochemistry; D007501:Iron / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D020125:Mutation, Missense; D009893:Opossums; D010641:Phenotype; D051896:Smad Proteins, Receptor-Regulated / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D014162:Transfection
chemical_list: C525816:BMP6 protein, human; D055418:Bone Morphogenetic Protein 6; C578702:HAMP protein, human; D064451:Hepcidins; D051896:Smad Proteins, Receptor-Regulated; D005293:Ferritins; D007501:Iron
keywords: Bone Morphogenetic Protein; Genetic Analysis; HH; Signal Transduction