YBCMD-01895; No. of pages: 2; 4C: Blood Cells, Molecules and Diseases xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Blood Cells, Molecules and Diseases journal homepage: www.elsevier.com/locate/bcmd
Letter to the Editor Genetic modifiers of secondary iron overload in beta thalassemia major
Dear Editor, Iron overload is a major cause of morbidity in β-thalassemia major attributed to multiple transfusions and inadequate chelation [1]. Genetic variants in iron regulating genes such as HFE, HAMP, and SLC40A1 have been identified as genetic modifiers of iron overload in thalassaemic patients [2–5]. Elucidating the role of genetic variants may explain heterogeneity of iron overload in β-thalassemia patients. In this prospective study, we measured serum ferritin, hepcidin and screened genetic variants that might influence iron status. Patients with β-thalassemia (age ≤15 years) based on clinical and molecular findings were included in this study. This study was approved by our institutional review board. Informed written consent was obtained from all patients enrolled in this study. Serum hepcidin was measured by ELISA (DRG, GmbH, Germany). Genetic variants TMPRSS6 c.2207A N G (rs855791 G N A), TFR2 c.714 C N G (rs34242818 C N G), HFE 845 G N A (rs1800562) and HFE c.187C N G (rs1799945 C N G) were screened by restriction fragment length polymorphism (RFLP). Variable number of tandem repeats (VNTRs) in TMPRSS6 c.18426_1842-2 del CACCC (rs200434923) and SLC40A1 c.-102-209_102206 in. CGG (rs371896375) were screened using genetic analyser (ABI 3130). Three SNPs in TF gene rs3811647, rs1799899 and rs1799852 [c.1330 + 278G N A, c.829.G N A, c.358 C N T respectively] and one SNP in TMPRSS6 gene rs4820268 [c.1536 C N T] were screened by high resolution melting assay (HRM). Statistical analysis was carried out using the software SPSS, version 20. One hundred and thirty patients (n = 130) with β-thalassemia major were included in the study. One hundred and two patients were receiving chelation. Patients' characteristics and other relevant details are given in Table 1. Further biochemical and molecular analyses were carried out in ninety one patients (n = 91) where all the parameters were available. Serum hepcidin was significantly higher in patients [median—26.3 (7.12–85.8) ng/ml] as compared to healthy controls [14.6 (4.82–53.6) ng/ml]; however very low hepcidin/ferritin ratio was observed in patients (Fig. 1). Serum hepcidin was significantly induced in patients but low hepcidin to ferritin ratio indicates inappropriate induction of hepcidin with respect to their iron status. As expected age and number of transfusions were positively correlated with ferritin (r = 0.343 p = 0.000 & r = 0.329 p = 0.002 respectively). Interestingly, patients with β0 mutations had significantly elevated ferritin compared to other two groups (p = 0.04); although the type of mutation did not correlate with number of transfusions (β0vs β+ median transfusion 100 vs 97.5; p = 0.2). This is consistent with the fact that severity of ineffective erythropoiesis is a strong modifier of iron absorption. Quartile analysis of transfusions with ferritin demonstrated twenty three patients with discordant ferritin levels. rs371896375 (SLC40A1)
was significantly associated with high ferritin (p = 0.017) in this sub group. HFE and HAMP genes were screened by direct sequencing in these patients and found to be negative. Two variants in TMPRSS6 (rs855791 and rs200434923) showed a significant association with low hepcidin (p = 0.01 & 0.008 respectively). It is already known that rs855791 mutant allele in TMPRSS6 suppresses hepcidin more effectively than wild type allele [6]. Another SNP rs200434923 is predicted to be an intronic enhancer region. This variant might suppress hepcidin more efficiently than wild type contributing to low hepcidin and high ferritin. None of the other nine polymorphisms were significantly associated with ferritin; rs200434923 in TMPRSS6 showed a trend towards significance (p = 0.07). Two patients were homozygous for H63D (rs1799945) polymorphism and one was heterozygous for C282Y (rs1800562). These patients had very low hepcidin/ferritin ratio. The disproportional iron overload in β-thalassemia was only partially explained by age, number of transfusions and chelation. Further studies are required to understand the mechanisms leading to iron burden in multi transfused patients [7,8]. Our study indicates that variants in genes regulating hepcidin (TMPRSS6) and iron exporter gene (SLC40A1) have a potential impact in determining body iron stores in beta thalassemia major. Authorship and disclosures R.A—carried out the sample analysis, compiled the data and wrote the manuscript. B.G, A.V,A.A, V.M, A.S—clinicians involved in patient recruitment and patient care in the study; E.S—designed the study, compiled and analysed the data, wrote, reviewed and approved the manuscript. The authors declare that they have no conflict of interest. Acknowledgements This study was supported by a grant BT/PR-13968/MED/12/465/ 2010 from the Department of Biotechnology, Government of India to ES. RA is supported by senior research fellowship from CSIR, India. Table 1 Characteristics of patients diagnosed with β thalassemia major (N = 130). Age Male/female Number of transfusions Any chelation No chelation Ferritin (ng/ml) ALT (U/l) AST (U/l) LDH (U/l) TBIL (mg%) Hepcidin (ng/ml) (n = 91)
8.78 ± 3.36 yrs 82/48 100 (7–400) 90 12 Median (range) 3117 (170.8–11,729) 54 (5–670) 51.5 (9–708) 492 (239–2056) 1.1 (0.29–9.20) 26.3 (7.12–85.8)
http://dx.doi.org/10.1016/j.bcmd.2014.12.006 1079-9796/© 2015 Elsevier Inc. All rights reserved.
Please cite this article as: R. Athiyarath, et al., Genetic modifiers of secondary iron overload in beta thalassemia major, Blood Cells Mol. Diseases (2015), http://dx.doi.org/10.1016/j.bcmd.2014.12.006
2
Letter to the Editor
Fig. 1. A) Serum hepcidin levels in patients and controls. B) Hepcidin/ferritin ratio in patients and controls. Dot plot illustrating high hepcidin level in thalassemia patients compared to controls. Low hepcidin to ferritin ratio in patients suggests inappropriate induction of hepcidin for iron load.
References [1] L. Melchiori, S. Gardenghi, S. Rivella, β-Thalassemia: hiJAKing ineffective erythropoiesis and iron overload, Adv. Hematol. 2010 (2010), http://dx.doi.org/ 10.1155/2010/938640. [2] E. Létocart, G. Le Gac, S. Majore, C. Ka, F.C. Radio, I. Gourlaouen, et al., A novel missense mutation in SLC40A1 results in resistance to hepcidin and confirms the existence of two ferroportin-associated iron overload diseases, Br. J. Haematol. 147 (2009) 379–385, http://dx.doi.org/10.1111/j.1365-2141.2009.07834.x. [3] M. Andreani, F.C. Radio, M. Testi, C. De Bernardo, M. Troiano, S. Majore, et al., Association of hepcidin promoter c.-582 A N G variant and iron overload in thalassemia major, Haematologica 94 (2009) 1293–1296, http://dx.doi.org/ 10.3324/haematol.2009.006270. [4] L. Duca, P. Delbini, I. Nava, M.D. Cappellini, A. Meo, Hepcidin mutation in a betathalassemia major patient with persistent severe iron overload despite chelation therapy, Intern. Emerg. Med. 5 (2010) 83–85, http://dx.doi.org/10.1007/s11739009-0306-8. [5] V. Sharma, I. Panigrahi, P. Dutta, S. Tyagi, V.P. Choudhry, R. Saxena, HFE mutation H63D predicts risk of iron over load in thalassemia intermedia irrespective of blood transfusions, Indian J. Pathol. Microbiol. 50 (2007) 82–85. [6] A. Nai, A. Pagani, L. Silvestri, N. Campostrini, M. Corbella, D. Girelli, et al., TMPRSS6 rs855791 modulates hepcidin transcription in vitro and serum hepcidin levels in normal individuals, Blood 118 (2011) 4459–4462, http://dx.doi.org/10.1182/blood2011-06-364034. [7] Y. Aydinok, J.B. Porter, A. Piga, M. Elalfy, A. El-Beshlawy, Y. Kilinç, et al., Prevalence and distribution of iron overload in patients with transfusion-dependent anemias differs
across geographic regions: results from the CORDELIA study, Eur. J. Haematol. (2014), http://dx.doi.org/10.1111/ejh.12487. [8] J.B. Porter, Pathophysiology of transfusional iron overload: contrasting patterns in thalassemia major and sickle cell disease, Hemoglobin 33 (Suppl. 1) (2009) S37–S45, http://dx.doi.org/10.3109/03630260903346627.
Rekha Athiyarath Biju George Aby Abraham Auro Viswabandya Alok Srivastava Eunice Sindhuvi Edison⁎ Department of Haematology, Christian Medical College, Vellore, India ⁎Corresponding author at: Department of Haematology, Christian Medical College, Vellore, Tamilnadu, India. Fax: +91 416 2226449. E-mail address: [email protected] (E.S. Edison). 12 December 2014 Available online xxxx
Please cite this article as: R. Athiyarath, et al., Genetic modifiers of secondary iron overload in beta thalassemia major, Blood Cells Mol. Diseases (2015), http://dx.doi.org/10.1016/j.bcmd.2014.12.006
Contents lists available at ScienceDirect
Blood Cells, Molecules and Diseases journal homepage: www.elsevier.com/locate/bcmd
Letter to the Editor Genetic modifiers of secondary iron overload in beta thalassemia major
Dear Editor, Iron overload is a major cause of morbidity in β-thalassemia major attributed to multiple transfusions and inadequate chelation [1]. Genetic variants in iron regulating genes such as HFE, HAMP, and SLC40A1 have been identified as genetic modifiers of iron overload in thalassaemic patients [2–5]. Elucidating the role of genetic variants may explain heterogeneity of iron overload in β-thalassemia patients. In this prospective study, we measured serum ferritin, hepcidin and screened genetic variants that might influence iron status. Patients with β-thalassemia (age ≤15 years) based on clinical and molecular findings were included in this study. This study was approved by our institutional review board. Informed written consent was obtained from all patients enrolled in this study. Serum hepcidin was measured by ELISA (DRG, GmbH, Germany). Genetic variants TMPRSS6 c.2207A N G (rs855791 G N A), TFR2 c.714 C N G (rs34242818 C N G), HFE 845 G N A (rs1800562) and HFE c.187C N G (rs1799945 C N G) were screened by restriction fragment length polymorphism (RFLP). Variable number of tandem repeats (VNTRs) in TMPRSS6 c.18426_1842-2 del CACCC (rs200434923) and SLC40A1 c.-102-209_102206 in. CGG (rs371896375) were screened using genetic analyser (ABI 3130). Three SNPs in TF gene rs3811647, rs1799899 and rs1799852 [c.1330 + 278G N A, c.829.G N A, c.358 C N T respectively] and one SNP in TMPRSS6 gene rs4820268 [c.1536 C N T] were screened by high resolution melting assay (HRM). Statistical analysis was carried out using the software SPSS, version 20. One hundred and thirty patients (n = 130) with β-thalassemia major were included in the study. One hundred and two patients were receiving chelation. Patients' characteristics and other relevant details are given in Table 1. Further biochemical and molecular analyses were carried out in ninety one patients (n = 91) where all the parameters were available. Serum hepcidin was significantly higher in patients [median—26.3 (7.12–85.8) ng/ml] as compared to healthy controls [14.6 (4.82–53.6) ng/ml]; however very low hepcidin/ferritin ratio was observed in patients (Fig. 1). Serum hepcidin was significantly induced in patients but low hepcidin to ferritin ratio indicates inappropriate induction of hepcidin with respect to their iron status. As expected age and number of transfusions were positively correlated with ferritin (r = 0.343 p = 0.000 & r = 0.329 p = 0.002 respectively). Interestingly, patients with β0 mutations had significantly elevated ferritin compared to other two groups (p = 0.04); although the type of mutation did not correlate with number of transfusions (β0vs β+ median transfusion 100 vs 97.5; p = 0.2). This is consistent with the fact that severity of ineffective erythropoiesis is a strong modifier of iron absorption. Quartile analysis of transfusions with ferritin demonstrated twenty three patients with discordant ferritin levels. rs371896375 (SLC40A1)
was significantly associated with high ferritin (p = 0.017) in this sub group. HFE and HAMP genes were screened by direct sequencing in these patients and found to be negative. Two variants in TMPRSS6 (rs855791 and rs200434923) showed a significant association with low hepcidin (p = 0.01 & 0.008 respectively). It is already known that rs855791 mutant allele in TMPRSS6 suppresses hepcidin more effectively than wild type allele [6]. Another SNP rs200434923 is predicted to be an intronic enhancer region. This variant might suppress hepcidin more efficiently than wild type contributing to low hepcidin and high ferritin. None of the other nine polymorphisms were significantly associated with ferritin; rs200434923 in TMPRSS6 showed a trend towards significance (p = 0.07). Two patients were homozygous for H63D (rs1799945) polymorphism and one was heterozygous for C282Y (rs1800562). These patients had very low hepcidin/ferritin ratio. The disproportional iron overload in β-thalassemia was only partially explained by age, number of transfusions and chelation. Further studies are required to understand the mechanisms leading to iron burden in multi transfused patients [7,8]. Our study indicates that variants in genes regulating hepcidin (TMPRSS6) and iron exporter gene (SLC40A1) have a potential impact in determining body iron stores in beta thalassemia major. Authorship and disclosures R.A—carried out the sample analysis, compiled the data and wrote the manuscript. B.G, A.V,A.A, V.M, A.S—clinicians involved in patient recruitment and patient care in the study; E.S—designed the study, compiled and analysed the data, wrote, reviewed and approved the manuscript. The authors declare that they have no conflict of interest. Acknowledgements This study was supported by a grant BT/PR-13968/MED/12/465/ 2010 from the Department of Biotechnology, Government of India to ES. RA is supported by senior research fellowship from CSIR, India. Table 1 Characteristics of patients diagnosed with β thalassemia major (N = 130). Age Male/female Number of transfusions Any chelation No chelation Ferritin (ng/ml) ALT (U/l) AST (U/l) LDH (U/l) TBIL (mg%) Hepcidin (ng/ml) (n = 91)
8.78 ± 3.36 yrs 82/48 100 (7–400) 90 12 Median (range) 3117 (170.8–11,729) 54 (5–670) 51.5 (9–708) 492 (239–2056) 1.1 (0.29–9.20) 26.3 (7.12–85.8)
http://dx.doi.org/10.1016/j.bcmd.2014.12.006 1079-9796/© 2015 Elsevier Inc. All rights reserved.
Please cite this article as: R. Athiyarath, et al., Genetic modifiers of secondary iron overload in beta thalassemia major, Blood Cells Mol. Diseases (2015), http://dx.doi.org/10.1016/j.bcmd.2014.12.006
2
Letter to the Editor
Fig. 1. A) Serum hepcidin levels in patients and controls. B) Hepcidin/ferritin ratio in patients and controls. Dot plot illustrating high hepcidin level in thalassemia patients compared to controls. Low hepcidin to ferritin ratio in patients suggests inappropriate induction of hepcidin for iron load.
References [1] L. Melchiori, S. Gardenghi, S. Rivella, β-Thalassemia: hiJAKing ineffective erythropoiesis and iron overload, Adv. Hematol. 2010 (2010), http://dx.doi.org/ 10.1155/2010/938640. [2] E. Létocart, G. Le Gac, S. Majore, C. Ka, F.C. Radio, I. Gourlaouen, et al., A novel missense mutation in SLC40A1 results in resistance to hepcidin and confirms the existence of two ferroportin-associated iron overload diseases, Br. J. Haematol. 147 (2009) 379–385, http://dx.doi.org/10.1111/j.1365-2141.2009.07834.x. [3] M. Andreani, F.C. Radio, M. Testi, C. De Bernardo, M. Troiano, S. Majore, et al., Association of hepcidin promoter c.-582 A N G variant and iron overload in thalassemia major, Haematologica 94 (2009) 1293–1296, http://dx.doi.org/ 10.3324/haematol.2009.006270. [4] L. Duca, P. Delbini, I. Nava, M.D. Cappellini, A. Meo, Hepcidin mutation in a betathalassemia major patient with persistent severe iron overload despite chelation therapy, Intern. Emerg. Med. 5 (2010) 83–85, http://dx.doi.org/10.1007/s11739009-0306-8. [5] V. Sharma, I. Panigrahi, P. Dutta, S. Tyagi, V.P. Choudhry, R. Saxena, HFE mutation H63D predicts risk of iron over load in thalassemia intermedia irrespective of blood transfusions, Indian J. Pathol. Microbiol. 50 (2007) 82–85. [6] A. Nai, A. Pagani, L. Silvestri, N. Campostrini, M. Corbella, D. Girelli, et al., TMPRSS6 rs855791 modulates hepcidin transcription in vitro and serum hepcidin levels in normal individuals, Blood 118 (2011) 4459–4462, http://dx.doi.org/10.1182/blood2011-06-364034. [7] Y. Aydinok, J.B. Porter, A. Piga, M. Elalfy, A. El-Beshlawy, Y. Kilinç, et al., Prevalence and distribution of iron overload in patients with transfusion-dependent anemias differs
across geographic regions: results from the CORDELIA study, Eur. J. Haematol. (2014), http://dx.doi.org/10.1111/ejh.12487. [8] J.B. Porter, Pathophysiology of transfusional iron overload: contrasting patterns in thalassemia major and sickle cell disease, Hemoglobin 33 (Suppl. 1) (2009) S37–S45, http://dx.doi.org/10.3109/03630260903346627.
Rekha Athiyarath Biju George Aby Abraham Auro Viswabandya Alok Srivastava Eunice Sindhuvi Edison⁎ Department of Haematology, Christian Medical College, Vellore, India ⁎Corresponding author at: Department of Haematology, Christian Medical College, Vellore, Tamilnadu, India. Fax: +91 416 2226449. E-mail address: [email protected] (E.S. Edison). 12 December 2014 Available online xxxx
Please cite this article as: R. Athiyarath, et al., Genetic modifiers of secondary iron overload in beta thalassemia major, Blood Cells Mol. Diseases (2015), http://dx.doi.org/10.1016/j.bcmd.2014.12.006
