The V736A TMPRSS6 polymorphism influences liver iron concentration in nontransfusion-dependent thalassemias
Authors: Milena Cau, Fabrice Danjou, Roberta Chessa, Marianna Serrenti, Maria Addis, Susanna Barella, Raffaella Origa
Journal: American Journal of Hematology (2015)

TMPRSS6 is a transmembrane serine protease expressed mainly in the liver that plays an important role during erythropoiesis. TMPRSS6 loss-of-function mutations result in the over-expression of hepcidin, impaired intestinal iron absorption and iron-refractory iron deficiency anemia (IRIDA). Tmprss6 deletion has been shown to improve iron overload and anemia in murine models of beta-thalassemia intermedia, and the siRNA- or antisense oligonucleotide-mediated suppression of Tmprss6 mRNA expression has been demonstrated to increase the hepcidin level, leading to diminished iron uptake and recycling as well as improved erythropoiesis and anemia in beta thalassemic animals 1, 2. TMPRSS6 polymorphisms have been implicated in iron metabolism in both animal and human studies. The common rs855791 polymorphism (NM_153609.3:c.2207C>T) causes a nonsynonymous valine to alanine change (p.V736A), and the T allele (encoding valine) has been associated with lower hemoglobin and ferritin concentrations as well as increased serum transferrin receptor and transferrin concentrations in all populations 3. Moreover, this polymorphism influences iron overload in hereditary hemochromatosis as well as the development of nonalcoholic fatty liver disease 4, 5 and it has been associated with iron deficiency anemia in women of reproductive ages 6. The mechanism of action of TMPRSS6 is still unclear. Some studies have indicated that TMPRSS6 polymorphisms directly affect hepcidin transcription, while others have suggested that the polymorphisms may addictionally influence iron homeostasis by mechanisms independent of hepcidin expression. The latter theory could explain why rs855791 has not been found to be significantly associated with serum hepcidin in some studies 3. In this study, we evaluated the influence of rs855791 on liver iron concentration (LIC) in patients with different levels of ineffective erythropoiesis: mild and severe thalassemia intermedia (TI) and Hemoglobin H (HbH) disease patients, which are clinically distinct forms of the so-called nontransfusion-dependent thalassemias (NTDTs), a term used to label patients who do not require regular transfusion for survival. We classified TI in agreement with the extent of imbalance between the alpha and non alpha globin chains. Accordingly, it was considered as mild in case of coinheritance of heterozygous beta thalassemia and, respectively, triple or quadruple alpha globin gene arrangement, delta/beta0 thalassemia, and mild or silent beta mutation. It was considered as severe in case of homozygosity or compound heterozygosity for beta0 mutations. In patients with NTDTs, the increased intestinal absorption of iron caused by ineffective erythropoiesis is the main cause of iron overload, which can become clinically relevant by the age of 10 years. Iron overload is associated with liver dysfunction and vascular, bone, and endocrine morbidities and is therefore an important challenge in terms of diagnosis, monitoring, and treatment. Moreover, although much more rarely than in thalassemia major, when LIC increases, labile pool iron rises quickly and may enter the heart, facilitating the occurrence of arrhythmias and heart failure. The huge variability in iron overload in NTDT patients has been hitherto attributed to age and degree of ineffective erythropoiesis, and we recently showed that ineffective erythropoiesis follows a steady increase from HbH to mild TI and severe TI patients 7. Previous studies have suggested that TMPRSS6 is a genetic modifier of the beta-thalassemia phenotype in animal models 1. Indeed, the deletion of Tmprss6 has been shown to attenuate the phenotype in a mouse model of beta-thalassemia 1, but data on its effect on iron overload in NTDT patients are not available. The study was approved by the local Ethics Committee, and all patients signed informed consent forms. Twenty-two subjects with severe TI, 7 with mild TI and 16 with HbH disease who were followed at Ospedale Regionale per le Microcitemie-Cagliari, Italy were enrolled. Their demographic, hematologic, and genetic characteristics may be found in Supporting Information. They either had no history of transfusions or had received only sporadic transfusions during infections, pregnancy or surgery (less than 10 blood units in total, 5 years or more before the evaluation) and had undergone LIC measurements by magnetic resonance imaging (Ferriscan®-Resonance Health, Australia) (22 subjects, 6 with TI) or liver biopsy (23 subjects with TI) before receiving any iron chelation. The hemoglobin level at the time of the LIC assessment had been determined with an automated red blood cell counter (Beckman Coulter LH 750), while the serum ferritin level had been measured with an automated chemiluminescence immunoassay analyzer (IMMULITE®2000, Siemens-DPC). Plasma hepcidin-25 measurements, which were available for 20 patients with TI and 6 with HbH disease, had been performed within a year before LIC evaluation by a combination of weak cation exchange chromatography and time-of-flight mass spectrometry (WCX-TOF MS). All patients were negative for the HFE p.C282Y mutation. rs855791 was assessed by the direct sequencing of DNA isolated from peripheral mononuclear cells by previously described standard methods. The relationships between LIC and genetic parameters was assessed by linear regression using Plink software version 1.07 and assuming an additive genetic model for rs855791. Further independent variables considered were: age, gender, and levels of ineffective erythropoiesis (coded upon disease type: severe TI, mild TI and HbH disease), as well as hemoglobin and ferritin levels at the time of LIC determination. In a separate linear model, we also included the hepcidin level as an additional predictor (either continuous or categorical; for the latter undetectable levels of hepcidin were coded as 0 when <0.5 nM and 1 otherwise), only considering the 26 patients for whom hepcidin measurements were available. Here, the T allele of rs855791 was found to be significantly associated with decreased LIC (P = 9.7 x 10–03; beta = 2.6), and with magnitude of ineffective erythropoiesis (as reflected in disease type, see Figure 1 - P = 6.9 × 10−04). The T allele was also inversely associated with patient age (likely reflecting severity of disease and age at presentation - P = 0.048). Analysis including hepcidin as a covariate in the smaller subset of patients for whom hepcidin measurements were available revealed that rs855791 was still significantly associated to LIC with these variables (P = 6.0 x 10–03; beta = 4.3), while hepcidin as a continuous variable showed a borderline P-value of 0.059 (the interaction term between each showed a P-value of 0.084). Liver iron concentration upon rs855791 genotypes in Thalassemia Intermedia and Hemoglobin H diseases patients. Box-and-whisker plots of liver iron concentrations for each genotype, clustered by disease type: central band characterizes the median, box boundaries delimit interquartile range (IQR), whiskers represent 1.5 IQR, and outliers are symbolized with circles. The present sample size, together with the borderline P-values observed, does not allow for the exclusion of a mediating effect of hepcidin. However, the effect of rs855791 on LIC was clearly observed, and this effect was strong in this cohort of thalassemia patients. In conclusion, our data show that p.V736A variant (rs855791) influences the susceptibility to hepatic iron accumulation in NTDT patients, and the risk allele is 736(A). The confirmation of this finding in larger populations could be particularly relevant in terms of personalization of clinical management in NTDT patients. M.C. contributed to research design and collected patient material; F.D. performed the statistical analyses; R.C and M.S. performed genetic analysis; M.A. critically reviewed the manuscript; S.B. collected clinical information and informed consent; R.O. collected clinical information, contributed to research design and supervised the study; M.C., F.D., and R.O. interpreted the data and wrote the manuscript. All authors gave final approval for the manuscript to be published. Milena Cau1† Fabrice Danjou1† Roberta Chessa1 Marianna Serrenti1 Maria Addis1 Susanna Barella2 Raffaella Origa1* 1Dipartimento Di Sanità Pubblica, Medicina Clinica E Molecolare, Università Di Cagliari, Via Jenner s/n, Cagliari, Italy 2Ospedale Microcitemico, Via Jenner s/n Cagliari, Italy Additional Supporting Information may be found in the online version of this article. Supporting Information Table 1. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.