Impaired hepcidin expression in alpha-1-antitrypsin deficiency associated with iron overload and progressive liver disease

Human Molecular Genetics, 2015, Vol. 24, No. 21 6254–6263 doi: 10.1093/hmg/ddv348 Advance Access Publication Date: 26 August 2015 Original Article ORIGINAL ARTICLE Impaired hepcidin expression in alpha-1-antitrypsin deficiency associated with iron overload and progressive liver disease 1 Department of Medicine II, Gastroenterology and Hepatology, 2Department of Medicine VI, Infectious Diseases, Immunology, Rheumatology, Pneumology, 3Department of Radiology, 4Department of Pediatrics I, 5Division of Human Genetics, Medical University of Innsbruck, Anichstrasse 35, A-6020 Innsbruck, Austria, 6Institute of Clinical Molecular Biology, Christian-Albrechts-University of Kiel, Schittenhelmstrasse 12, 24105 Kiel, Germany, 7 Program in Membrane Biology and Division of Nephrology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA and 8Institute of Pathology, Klinikum Bayreuth, Preuschwitzerstraße 101, 95445 Bayreuth, Germany *To whom correspondence should be addressed. Tel: +43 51250481922; Fax: +43 51250427019; Email: Abstract Liver disease due to alpha-1-antitrypsin deficiency (A1ATD) is associated with hepatic iron overload in a subgroup of patients. The underlying cause for this association is unknown. The aim of the present study was to define the genetics of this correlation and the effect of alpha-1-antitrypsin (A1AT) on the expression of the iron hormone hepcidin. Full exome and candidate gene sequencing were carried out in a family with A1ATD and hepatic iron overload. Regulation of hepcidin expression by A1AT was studied in primary murine hepatocytes. Cells co-transfected with hemojuvelin (HJV) and matriptase-2 (MT-2) were used as a model to investigate the molecular mechanism of this regulation. Observed familial clustering of hepatic iron overload with A1ATD suggests a genetic cause, but genotypes known to be associated with hemochromatosis were absent. Individuals homozygous for the A1AT Z-allele with environmental or genetic risk factors such as steatosis or heterozygosity for the HAMP non-sense mutation p.Arg59* presented with severe hepatic siderosis. In hepatocytes, A1AT induced hepcidin mRNA expression in a dose-dependent manner. Experiments in overexpressing cells show that A1AT reduces cleavage of the hepcidin inducing bone morphogenetic protein co-receptor HJV via inhibition of the membrane-bound serine protease MT-2. The acutephase protein A1AT is an inducer of hepcidin expression. Through this mechanism, A1ATD could be a trigger of hepatic iron overload in genetically predisposed individuals or patients with environmental risk factors for hepatic siderosis. † Joint first authors who contributed equally to this work. Received: July 1, 2015. Revised and Accepted: August 19, 2015 © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: 6254 Benedikt Schaefer1,†, David Haschka2,†, Armin Finkenstedt1, Britt-Sabina Petersen6, Igor Theurl2, Benjamin Henninger3, Andreas R. Janecke4,5, Chia-Yu Wang7, Herbert Y. Lin7, Lothar Veits8, Wolfgang Vogel1, Günter Weiss2, Andre Franke6 and Heinz Zoller1, * Human Molecular Genetics, 2015, Vol. 24, No. 21 Introduction Type I and Type II BMP receptors (29). Mayeur et al. (30) have shown that the BMPR1A (Alk6) is important in interleukin6-mediated induction of hepcidin expression. An orally administered BMPR1A inhibitor was found to successfully inhibit hepatic BMP signaling and may be a novel therapeutic option for patients with anemia of inflammation (31). Furthermore, BMPR1A seems to be stabilized by HFE on the cell surface by preventing proteasomal degradation of the receptor (32). In contrast, genetic defects in BMPR1B (Alk6) are reported not to be associated with apparent changes in baseline iron status in mice (30). However, this does not preclude that BMPR1B has a specific role in BMP signaling and the control of hepcidin transcription during inflammation or perturbation of iron metabolism. The apparent redundancy of the Type II BMP receptors BMPR2 and ActR2a in maintaining iron homeostasis was recently investigated. Mice lacking either BMPR2 or ActR2a showed no changes in hepcidin expression and iron status, whereas deficiency of both receptors caused iron overload and markedly reduced hepcidin mRNA expression (33). BMP-induced hepcidin expression is inhibited by the membrane-bound serine protease matriptase-2 (MT-2), which is encoded by TMPRSS6. MT-2 is thought to suppress hepcidin expression by cleaving the BMP co-receptor HJV (34). To explore a potential role of A1AT in controlling hepcidin expression by inhibition of MT-2, we have investigated the effect of A1AT on hepcidin expression and HJV cleavage. Our findings show that A1AT induces the expression of hepcidin in hepatocytes probably via inhibition of MT-2. This pathway could contribute to the association of A1ATD and iron overload. Studies in a family with A1ATD and hemochromatosis indicate a putative disease-causing effect of A1ATD in heterozygous carriers of mutations in the gene encoding hepcidin HAMP or individuals with environmental risk factors. Results Coincidence of A1ATD and non-HFE hemochromatosis The female index patient first presented at the age of 60 years for further evaluation of a reduced A1AT plasma concentration of 28 mg/dl (reference range 90–200 mg/dl), hyperferritinemia of 1033 µg/l (reference range 30–200 µg/l) and high transferrin saturation of 98% (reference range 15–45%). These abnormalities were identified during investigation for an elevated gamma-glutamyl transferase activity and a family history of liver disease. At the time of first presentation, the patient had no evidence for lung disease. A1AT phenotyping by isoelectric focusing showed an A1AT Pi Z phenotype, and subsequent genotyping revealed that the patient was homozygous for the Z-allele in rs28929474 of the SERPINA1 gene (NM_000295.4: c.1096G>A), which confirmed the diagnosis of A1ATD. Genotyping for the p.Cys282Tyr and p.His63Asp polymorphism in the hemochromatosis gene HFE showed that the patient was homozygous for the normal alleles [rs1800562 (NM_000410.3: c.845G) and rs1799945 (NM_000410.3: c.187C)]. Histological evaluation of the liver biopsy showed mild portal hepatitis with fibrosis grade 4 (Metavir) and marked accumulation of stainable iron in hepatocytes. Based on these findings, the diagnoses A1ATD Pi ZZ and non-HFE hemochromatosis were made. After 12 months of fortnightly phlebotomies, serum ferritin concentration was 59 µg/l and remained low during 3-monthly maintenance phlebotomies. Six years after initial presentation, the patient developed decompensated cirrhosis and underwent liver transplantation. Explant histology is shown in Figure 1, where microvesicular steatosis in a cirrhotic liver was found in addition to accumulation of Genetic liver diseases encompass a heterogeneous group of disorders, including alpha-1-antitrypsin deficiency (A1ATD) and hemochromatosis as the mos (...truncated)