ORIGINAL ARTICLE Association of HFE and TMPRSS6 genetic variants with iron and erythrocyte parameters is only in part dependent on serum hepcidin concentrations Michela Traglia, 1 Domenico Girelli, 2 Ginevra Biino, 3,4 Natascia Campostrini, 2 Michela Corbella, 2 Cinzia Sala, 1 Corrado Masciullo, 1 Fiammetta Vigano `, 1 Iwan Buetti, 1 Giorgio Pistis, 1 Massimiliano Cocca, 1 Clara Camaschella, 1,5 Daniela Toniolo 1,3 ABSTRACT Background Hepcidin is the main regulator of iron homeostasis: inappropriate production of hepcidin results in iron overload or iron deficiency and anaemia. Aims To study variation of serum hepcidin concentration in a normal population. Results Hepcidin showed age and sex dependent variations that correlated with ferritin but not with serum iron and transferrin saturation. The size of the study population was underpowered to find genome wide significant associations with hepcidin concentrations but it allowed to show that association with serum iron, transferrin saturation and erythrocyte traits of common DNA variants in HFE (rs1800562) and TMPRSS6 (rs855791) genes is not exclusively dependent on hepcidin values. When multiple interactions between environmental factors, the iron parameters and hepcidin were taken into account, the HFE variant, and to lesser extent the TMPRSS6 variant, were associated with ferritin and with hepcidin normalised to ferritin (the hepcidin/ferritin ratio). Conclusions The results suggest a mutual control of serum hepcidin and ferritin concentrations, a mechanism relevant to the pathophysiology of HFE haemochromatosis, and demonstrate that the HFE rs1800562 C282Y variant exerts a direct pleiotropic effect on the iron parameters, in part independent of hepcidin. INTRODUCTION Iron is essential for multiple biological functions in all tissues, but especially for haemoglobin synthesis, as shown by anaemia that results from iron deficiency. Excess iron is toxic, because it favours oxidative stress and cell damage. 1 For this reason, the amount of plasma iron is maintained within narrow limits and is tightly regulated by the liver peptide hepcidin according to the body ’s needs. 2 Hepcidin controls the surface expression of the iron exporter ferroportin on enterocytes and iron recycling macrophages. 3 Genetic disorders of the hepcidineferroportin pathway lead to opposite conditions. Haemochromatosis is caused by muta- tions in genes which encode upstream hepcidin activating proteins (HFE, TFR2, hemojuvelin) or mutations in hepcidin itself. All these forms of haemochromatosis are characterised by inappro- priately high iron absorption, elevated transferrin saturation and serum ferritin, and inappropriately low/undetectable hepcidin expression. 4 More rarely mutations affect ferroportin, the downstream target of hepcidin; as a consequence, either iron is not recycled and remains sequestered in macro- phages or the mutant is not internalised because is hepcidin resistant. 5 Iron refractory, iron deficiency anaemia (IRIDA) is caused by mutations of TMPRSS6, which encodes the liver expressed hepcidin inhibitor serine protease matriptase-2. Mask mice homozygous for a truncated matriptase-2 lacking the serine protease domain, 6 Tmprss6 null mice, 7 and patients with IRIDA 8 do not efficiently absorb oral iron because they are unable to fully suppress hepcidin activation. 9 10 They display very low transferrin saturations but moderately decreased serum ferritin because of iron retention in macrophage stores. 11 Genetic variants of two of the hepcidin regula- tory genes, TMPRSS6 and HFE, affect serum iron concentration 12 and transferrin saturation 13 14 in normal populations. Furthermore, single nucleotide polymorphisms (SNPs) at TMPRSS6 and HFE loci were found to be associated with quantitative variations of haemoglobin (Hb) concentrations and erythrocyte traits. 15e18 However, it remains uncertain whether the association is iron mediated or dependent on a direct effect of the variants on erythropoiesis. In addition, the effect of the ‘iron gene’ variants was ascribed to variations in hepcidin concentrations, 15 16 18 but serum hepcidin was not measured. We report here the analysis of serum hepcidin concentrations, measured by a mass spectrometry based method, 19 in 1657 normal individuals from the Val Borbera (VB) genetic isolate in Northern Italy. We explored relationships between hepcidin and a set of anthropometric, haematologic, and iron parameters and tested the association of two common variants rs1800562 and rs855791 in the HFE and TMPRSS6 genes, respectively, with iron, erythrocyte parameters and hepcidin values in 1545 genotyped individuals. We demonstrate a reciprocal control of serum hepcidin and ferritin concentra- tions that may be relevant to the pathophysiology of HFE haemochromatosis, and demonstrate that the HFE C282Y variant exerts a direct pleiotropic effect on several of the iron parameters, partly independent of hepcidin. < Additional tables and figures are published online only. To view these files please visit the journal online (http://jmg.bmj. com). 1 Division of Genetics and Cell Biology, San Raffaele Research Institute, Milano, Italy 2 Department of Medicine, University of Verona, Verona, Italy 3 Institute of Molecular Genetics, CNR, Pavia, Italy 4 Institute of Population Genetics, CNR, Sassari, Italy 5 Vita Salute University, San Raffaele Scientific Institute, Milano, Italy Correspondence to Daniela Toniolo, Division of Genetics and Cell Biology, San Raffaele Research Institute, Via Olgettina 58, Milano 20132, Italy; daniela.toniolo@hsr.it Received 21 March 2011 Revised 20 May 2011 Accepted 22 June 2011 Published Online First 23 July 2011 J Med Genet 2011;48:629e634. doi:10.1136/jmedgenet-2011-100061 629 Complex traits group.bmj.com on August 31, 2011 - Published by jmg.bmj.com Downloaded from