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 thatmight influence iron status. Patients with β-thalassemia (age ≤15 years) based on clinical and molecularfindingswere included in this study. This studywas approved byour institutional review board. Informedwritten consentwas 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 andmolecular analyses were carried out in ninety onepatients (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 β 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 (βvs 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)

Most hereditary haemochromatosis patients are homozygous for the C282Y mutation of the HFE gene. However, the phenotypic expression and clinical aggressiveness of the disease differs considerably from patient to patient. The main objective of this work was to study the role of variants in the SLC40A1 gene in the severity of iron overload and his clinical consequences in 100 Spanish probands homozygous for the C282Y mutation of the HFE gene. We performed automated sequencing of the coding regions, including intron-exon junctions of the SLC40A1 gene. We studied the association between polymorphisms in the SLC40A1 gene and median values of iron removed, taking into account statistical corrections for multiple comparisons. No pathogenic mutations in the SLC40A1 were detected. Five known single nucleotide polymorphisms (SNPs) were identified, and two of them were associated with phenotypic characteristics. IVS1-24 C>G was associated with the amount of iron removed and presence of liver disease: Of the 83 patients finally studied for this SNP, the amount of iron removed was above the median in 36 of 56 (64.3%) for C/C, in nine of 23(39.1%) for C/G and in zero of four (0%) for G/G patients (P=0.01). Liver damage was observed in 34 of 56 patients (60.7%) for C/C, in eight of 23 (34.8%) for C/G and in zero of four (0%) for G/G (P=0.01). Both associations remained significant at multivariate analysis (P=0.011 and P=0.023, respectively). IVS1-24 C>G on the ferroportin gene seems to be a genetic modifier for clinical aggressiveness of HFE1 haemochromatosis.