hepcidin levels in normal individuals rs855791 ...

From bloodjournal.hematologylibrary.org at FONDAZIONE CENTRO S RAFFAELE on August 31, 2011. For personal use only.

Prepublished online August 26, 2011; doi:10.1182/blood-2011-06-364034

TMPRSS6 rs855791 modulates hepcidin transcription in vitro and serum hepcidin levels in normal individuals Antonella Nai, Alessia Pagani, Laura Silvestri, Natascia Campostrini, Michela Corbella, Domenico Girelli, Michela Traglia, Daniela Toniolo and Clara Camaschella

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TMPRSS6 rs855791 MODULATES HEPCIDIN TRANSCRIPTION IN VITRO AND SERUM HEPCIDIN LEVELS IN NORMAL INDIVIDUALS Antonella Nai1,2, Alessia Pagani1,2, Laura Silvestri,1,2 Natascia Campostrini3, Michela Corbella3, Domenico Girelli3, Michela Traglia1,2, Daniela Toniolo1,4 and Clara Camaschella1,2

1

Division of Genetics and Cell Biology, San Raffaele Scientific Institute and 2Vita-Salute

University, Milan, Italy 3 4

Department of Medicine, University of Verona, Verona, Italy. Institute of Molecular Genetics-CNR, Pavia Italy

Key words: Iron, TMPRSS6, Matriptase-2, Hepcidin, GWAS

Running title: Tmprss6 genetic variants and hepcidin

Corresponding author: Clara Camaschella Vita-Salute San Raffaele University Via Olgettina, 60 20132 Milan-Italy Phone: +39-0226437782 Fax: +39-0226432640 e-mail: [email protected]

1 Copyright © 2011 American Society of Hematology


ABSTRACT The iron hormone hepcidin is inhibited by matriptase-2, a liver serine-protease encoded by TMPRSS6 gene. Cleaving the BMP-coreceptor hemojuvelin, matriptase-2 impairs the BMP/SMAD signaling pathway, downregulates hepcidin and facilitates iron absorption. TMPRSS6 inactivation causes iron-deficiency-anemia refractory to iron administration both in humans and mice. Genome wide association studies have shown that the SNP rs855791, which causes the matriptase-2 V736A amino acid substitution, is associated with variations of serum iron, transferrin saturation, hemoglobin and erythrocyte traits. Here we show that in vitro matriptase-2 736A inhibits hepcidin more efficiently than 736V . Moreover, in a genotyped population, after exclusion of samples with iron deficiency and inflammation, hepcidin, hepcidin/transferrin saturation and hepcidin/ferritin ratios were significantly lower and iron parameters were consistently higher in homozygotes 736A than in 736V. Our results indicate that rs855791 is a TMPRSS6 functional variant and strengthen that even a partial inability to modulate hepcidin influences iron parameters and indirectly erythropoiesis.

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INTRODUCTION Hepcidin is the key regulator of iron homeostasis, controlling surface expression of the iron exporter ferroportin on enterocytes and macrophages1. Inactivation of hepcidin causes severe iron overload in mice and humans, whereas hepcidin overexpression causes iron deficiency anemia2. Hepcidin expression is up-regulated in response to increased body iron, through the Bone Morphogenetic Protein (BMP)-hemojuvelin (HJV)-Son of Mothers Against Decapentaplegic (SMAD) pathway3 and inhibited by matriptase-2 (MT2), a type II transmembrane serine protease, encoded by the TMPRSS6 gene4,5 that in vitro cleaves the BMP-coreceptor HJV6. In vivo “Mask” mice, which have a deleted serine protease domain4, and Tmprss6 null mice7 show microcytic anemia due to high hepcidin levels. TMPRSS6 deleterious mutations in humans cause ironrefractory iron-deficiency anemia (IRIDA), unresponsive to oral iron administration5. The same mutations show partial inhibition of the hepcidin promoter activity when overexpressed with HJV in vitro in hepatoma cells6,8.

Recently genome-wide association studies (GWAS) reported the association of common genetic variants of TMPRSS6 (rs855791 and rs4820268) with serum iron and transferrin saturation9-11, hemoglobin (Hb), MCV and MCH12,13, highlighting a role for MT2 in the control of iron and erythrocyte parameters. The SNP rs855791 (2321G->A) causes a non-synonymous alanine to valine change (A736V) in the catalytic domain, whereas the SNP rs4820268 leads to a synonymous change at 521 and is in linkage disequilibrium with rs855791. Since rs855791 affects the MT2 catalytic domain, a common speculation was that its effects were hepcidin-mediated9,14. We tested this hypothesis using an in vitro assay, based on luciferase reporter gene driven by the hepcidin promoter and showed that the MT2736A inhibits hepcidin more efficiently than MT2736V. We also demonstrated that this variant affects hepcidin levels of normal individuals.

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MATERIAL AND METHODS In vitro studies The in vitro analyses (western blot, hepcidin promoter activity assay and binding assay) were previously reported6 and are detailed in supplemental material. The TMPRSS6 variant, encoding 736A (MT2736A) was obtained by mutagenesis of MT2736V plasmid by using the QuikChange sitedirected mutagenesis kit (Stratagene, La Jolla, CA) Human studies The population of the genetic isolate “Val Borbera” (VB) was previously described15. The study was approved by San Raffaele Ethical Committee. Serum hepcidin levels were measured by SELDI-TOF-Mass Spectrometry16 and detailed results are reported elsewhere11. Statistical analysis Association of TMPRSS6 rs855791 was first analyzed in 655 unrelated (pairwise kinship coefficient