Association of hepcidin promoter c.-582 A>G variant and iron overload in thalassemia major

Hepcidin is a 25-amino acid peptide, derived from cleavage ofan 84 amino acid pro-peptide produced predominantly by hepatocytes.This molecule, encoded by the hepcidin antimicrobial peptide(HAMP) gene shows structural and functional properties consistentwith a role in innate immunity. Moreover, as demonstrated inmice and humans, hepcidin is a major regulator of iron metabolism,and acts by binding to ferroportin and controlling its concentrationand trafficking. In this study we investigated the influencethat mutations in HAMP and/or hemocromatosis (HFE) genes mightexert on iron metabolism in a group of poly-transfused thalassemicpatients in preparation for bone marrow transplantation. Ourresults showed that the presence of the c.-582 A>G polymorphism(rs10421768) placed in HAMP promoter (HAMP-P) might play a rolein iron metabolism, perhaps varying the transcriptional activationthat occurs through E-boxes located within the promoter.

Key words: HAMP, HFE, iron metabolism, liver iron concentration, β-thalassemia.

Introduction

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Introduction

Sophisticated mechanisms maintain body iron homeostasis andcontrol uptake of dietary iron and its mobilization from stores,in order to satisfy erythropoietic needs and to recycle previouslyused iron. Communication between cells that consume iron andcells that acquire and store iron must be strictly regulatedto provide adequate iron supply while avoiding the toxic effectsof elevated iron stores.^1,2

Hepcidin, encoded by HAMP gene, is a recently discovered 25amino acid peptide that, in addition to being involved in innateimmunity,³ appears to play a crucial role in iron homeostasisin humans, regulating both iron absorption from the intestineand its recycling by macrophages.^4–7 It has been demonstratedthat iron overload and inflammation stimulate hepcidin expressionwhile erythropoietic activity decreases its production. Hepcidinsynthesis is also suppressed by anemia and hypoxia.^8–10The HAMP role in iron metabolism has been described in thalassemicpatients.^11–14 Additionally, hepcidin deficiency is thecause of iron overload in most forms of hereditary hemochromatosis.^15–21In this study we investigated the influence of the p.H63D HFEand of the c.-582 A>G HAMP promoter (HAMP-P) gene variantsin a group of 97 β-thalassemic patients in preparationfor hematopoietic stem cell (HSC) transplant. We analyzed theiron status in patients with a wild type (WT) profile and comparedthe data to the iron status of patients with p.H63D HFE and/orthe c.-582 A>G HAMP-P variants. Estimation of iron overloadwas based on liver iron concentration (LIC) and serum ferritin(SF) levels.^22–24

Design and Methods

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Design and Methods

Patients
All patient-related procedures were approved by the EthicalReview Board. Ninety-seven patients affected by β-thalassemia,originating from an extremely wide geographical area, mainlyfrom Middle Eastern countries, proposed for HSC transplant,were included in the study, as reported in Table 1. Almost allthe patients were massively transfused: 70 patients receivedmore than 50 transfusion units (range 51–550, median 148),while 27 received less than 50 transfusion units (6–48,median 26). Thirty-eight patients had been submitted to regulartransfusion therapy since childhood and to chelation with subcutaneousdesferrioxamine 5–6 days per week, 8–12 h per day,while 59 patients received irregular iron chelation therapy.Eighteen patients were hepatitis C virus (HCV) positive andshowed a median of 43 UI/L alanine transaminase (ALT) levels(range: 23–218).

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Table 1. Patients’ characteristics.

Clinical parameters
LIC was measured on liver biopsies in 93 patients by acetyleneflame atomization and atomic absorption spectrometry (AAnalyst800, Perkin-Elmer, Waltham, MA, USA). Samples were exsiccatedat 120°C for 30' and then submitted to mineralization withnitric and sulphuric acids (100 mL each) at 120°C for 1h. Finally, samples were diluted to 10 mL with a solution ofdouble distilled water with 1% nitric acid and then analyzedby atomic absorption spectrometry. Liver biopsies were storedat –20°C until analysis. LIC was expressed as mg/gdry weight. Only liver samples with a dry weight of at least1 mg were considered for the present study. ^22,24 Mean valueof dry weight liver biopsies was 2.34 mg (range: 1.06–4.58mg). SF levels were determined by standard methods during routineblood testing prior to transplant on all the 97 patients.

Molecular analysis
A panel of known HFE gene mutations (C282Y, H63D, S65C, Q127H,V53M, V59M, H63H, P160delC, E168Q, E168X, W169X, Q283P) wasscreened. Specific regions of the HFE gene were amplified bystandard multiplex-PCR and reverse dot-blot was performed accordingto the kit protocol (Haemochromatosis StripAssay A^{^TM}-ViennaLab). HAMP coding regions, intron-exon junctions, and promoterregion were analyzed by direct sequence analysis. Primer sequencesand PCR conditions for molecular testing are available on request.

Statistical analysis
Statistical analysis was performed on MedCalc Software, (Mariakerke,Belgium) using non-parametric tests including Student’st test and Welch’s test. Correlations between variableswere evaluated by Spearman’s correlation coefficient associatedwith t test. A p value less than 0.05 was considered statisticallysignificant.

Results and Discussion

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Results and Discussion

The aim of this study was to verify whether HAMP and HFE genevariants affect iron metabolism in poly-transfused thalassemicpatients as measured by LIC values and serum ferritin levels.These two parameters are reported in the literature to adequatelyreflect iron load,^22,24 and in our survey reveal a strong correlation,as defined by the Spearman’s test. In fact, calculationof the coefficient of determination (R₂) showed that LIC correlatedwith SF (R₂=0.417, p<0.001). As reported in Table 1, 47/97patients showed a WT genetic profile in both genes, 25/97 hada variation in HAMP-P, 16/97 in HFE, and 9/97 in both. The classicalHFE mutation p.C282Y was not identified in any of the examinedpatients. All the subjects with an HFE variation were carriersof the p.H63D variant; 24 in the heterozygous and one in thehomozygous condition. Regarding the HAMP gene, 34/97 patientsshowed the presence of a heterozygous A/G nucleotide substitutionin position -582 where the promoter E-box 1 is located. Oneout of 97 was a homozygous carrier of the same HAMP-P variant.This transition has been already recognized as a polymorphismaffecting a conserved non-coding sequence. The HAMP-P SNP isreported in the dbSNP database (http://www.ncbi.nlm.nih.gov/projects/SNP/)with the identification number rs10421768.

A statistically significant difference was observed in the LICvalues and SF levels in the group of patients with the c.-582A>G HAMP-P polymorphism compared to WT patients. As reportedin Table 2, the mean LIC value in patients with this mutationwas 23.2±12.8 mg/g vs. 14.3±9.3 mg/g observedin HAMP-P WT patients (p=0.003), and the mean SF level was 3191±1869ng/mL vs. 2263±1373 ng/mL, in patients with the polymorphismvs. controls, respectively (p=0.034). Nine patients with polymorphismsin both HAMP-P and HFE genes showed a difference in iron overloadparameters compared to WT patients, but this difference wasnot statistically significant, likely due to the small samplesize of patients carrying double variants. No statisticallysignificant differences were observed between the groups ofpatients with or without the HFE p.H63D mutation in mean LIC(15.6±9.8 vs. 14.3±9.3 mg/g) or SF (2610±2352vs. 2263±1373 ng/mL) as reported in Table 2.

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Table 2. Liver iron concentration values (LIC) and serum ferritin levels (SF) in wild type patients (WT), patients with hemochromathosis gene variant (HFE p.H63D), and patients with hepcidin moter variant (c.-582A>G HAMP-P).

In the group of 59 patients who received irregular iron chelationtherapy, the LIC values and the SF levels were 22.1±11mg/g and 3299±1853 ng/mL, respectively. When the impactof the c.-582 A>G HAMP-P polymorphism was studied, we observedthat the mean LIC and SF levels were significantly higher inthe 18 patients carrying the mutation, compared to the 26 WTpatients (LIC: 29.7±12 vs. 18.2±9.9 mg/g; p=0.0021;SF: 3880±1716 vs. 2912±1464 ng/mL; p=0.05). SFlevels were higher, but not significantly different, in patientscarrying the HFE gene mutation compared to the WT group (3455±2814vs. 2912±1464 ng/mL), while the LIC values were comparable(18.7±8 vs. 18.2±9.9 mg/g). Among the group of38 regularly treated patients, LIC and SF values were equivalentin the 3 groups of patients characterized by the different geneticprofiles.

Although no functional studies to assess the role of the describedHAMP polymorphism on hepcidin expression have been performed,our findings suggest that the presence of this polymorphic variantmight play a role in iron metabolism of poly-transfused thalassemicpatients,^11–14 perhaps changing the response to the transcriptionalactivation by both upstream stimulatory factors 1 and 2 (USF1/USF2)and cMyc/Max heterodimers that occur through E-boxes withinthe promoter, as shown by Bayele.⁵ Alterations of these elementsmight render the promoter less responsive to USF1/USF2 or c-Myc/Max,modifying regulation of the hepcidin transcription factors andits function in iron metabolism.⁵

The c.-582 A>G HAMP-P variant did not show an impact on thelevel of iron loading in the regular chelated patients, whileit influenced the LIC values and the serum ferritin among thepatients receiving an irregular chelation treatment. These observationssuggest that this HAMP-P polymorphism may be associated withdifferent functionality of the HAMP promoter. The c.-582 A>GHAMP-P substitution might, therefore, represent a risk factorfor thalassemic patients by promoting iron overload in thesesubjects. On the other hand our data showed that this effectcan be overcome by a regular and appropriate chelation therapy.Other factors, such as age and variable duration of transfusionregimens, might account for the wide range of LIC and SF levelsobserved in both WT and HAMP-P variant patients. The reasonwhy in some HAMP-P individuals iron overload was comparableto that of wild-type patients could be due to the fact thatthey are heterozygous for the SNP and, therefore, show an incompletepenetrance or compensatory mechanisms.

Although it is well known that the presence of the p.C282Y HFEmutation may significantly alter iron absorption regulation,^15–21this is not always the case for the p.H63D mutation. This commonvariant is only occasionally associated with iron overload,usually in the homozygous state or in compound heterozygoteindividuals that also harbor the p.C282Y mutation. Thus, itis not unexpected that our data confirm the results of previousstudies showing that the p.H63D heterozygous state does notmodify iron loading in thalassemic patients.¹⁹

In conclusion, although many different factors might be involved,our observations suggest a hypothetical role of the HAMP-P -582A>G polymorphism on iron metabolism. Investigation into thiscould be useful as part of the diagnostic and prognostic evaluationof β-thalassemia.

Footnotes

Authorship and Disclosures

MA, MT and PG designed and wrote the paper; MTr organized bloodsample collection and made the statistical analysis; FCR performedexperiments and reviewed the paper; PB performed the LIC analysis;CDB, PP, RR and SM revised the paper.

The authors reported no potential conflicts of interest.

Received for publication January 21, 2009. Revision received April 2, 2009. Accepted for publication April 3, 2009.

References

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