Raed Daher1, Caroline Kannengiesser2, Dounia Houamel1, Thibaud Lefebvre3, Edouard Bardou-Jacquet4, Nicolas Ducrot1, Caroline de Kerguenec5, Anne-Marie Jouanolle6, Anne-Marie Robreau7, Claire Oudin7, Gerald Le Gac8, Boualem Moulouel9, Veronique Loustaud-Ratti10, Pierre Bedossa11, Dominique Valla5, Laurent Gouya3, Carole Beaumont1, Pierre Brissot4, Hervé Puy3, Zoubida Karim12, Dimitri Tchernitchko13. 1. 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. 2. 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. 3. 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. 4. Department of Liver Disease and Molecular Genetics, CHU Rennes, Rennes, France. 5. AP-HP, Département d'Hépatologie, Hôpital Beaujon, Clichy, France. 6. Department of Liver Disease and Molecular Genetics, CHU Rennes, Rennes, France; CHU Rennes, French Reference Center for Rare Iron Overload Diseases of Genetic Origin. 7. AP-HP, Centre Français des Porphyries, Hôpital Louis Mourier, Colombes, France. 8. INSERM U1078, Université de Brest, CHRU de Brest, Laboratoire de Génétique Moléculaire et d'Histocompatibilité, Bretagne, Brest, France. 9. INSERM UMR1149, Centre de Recherche sur l'Inflammation, Paris, France. 10. Service d'Hépato-Gastroentérologie, INSERM UMR1092, Limoges, France. 11. INSERM UMR1149, Centre de Recherche sur l'Inflammation, Paris, France; AP-HP, Laboratoire d'Anatomo-Pathologie, Hôpital Beaujon, Clichy, France. 12. 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. 13. AP-HP, Département de Génétique, Hôpital Bichat, Paris, France.
Abstract
BACKGROUND & AIMS: 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.
BACKGROUND & AIMS: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 hyperferritinemiapatients. 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: Susanna Canali; Kimberly B Zumbrennen-Bullough; Amanda B Core; Chia-Yu Wang; Manfred Nairz; Richard Bouley; Filip K Swirski; Jodie L Babitt Journal: Blood Date: 2016-11-18 Impact factor: 22.113