Hemoglobin, 36(1):85–92, (2012)Copyright © Informa Healthcare USA, Inc.ISSN: 0363-0269 print/1532-432X onlineDOI: 10.3109/03630269.2011.637148

SHORT COMMUNICATION

EFFECT OF CO-INHERITANCE OF β-THALASSEMIA AND

HEMOCHROMATOSIS MUTATIONS ON IRON OVERLOAD

Herminio López-Escribano,1 Joana F. Ferragut,2 Maria M. Parera,1

Pilar Guix,1 José A. Castro,2 M. Misericòrdia Ramon,2 and

Antònia Picornell2

1Servicio de Análisis Clínicos, Hospital Universitario Son Dureta, Palma de Mallorca, Illes Balears,España2Institut Universitari d’Investigació en Ciències de la Salut (IUNICS) i Laboratori de Genètica,Departament de Biologia, Universitat de les Illes Balears, Palma de Mallorca, Illes Balears, España

� Co-inheritance of mutations in the HFE gene underlying hereditary hemocromatosis (HH) mayplay a role in the variability of iron status in patients with β-thalassemia (β-thal) minor. Differentstudies have yielded conflicting results: some suggest iron overload might arise from the interactionof the β-thal trait with homozygosity or even heterozygosity for HFE mutations and others that itwas unrelated to the HFE genotype. Because of the high frequency of HFE mutations in the BalearicIslands, where the β-thal trait is also moderately common, it is of interest to evaluate the effect of theco-inheritance of mutations in both genes on the severity of iron loading. A retrospective analysis of142 individuals heterozygous for β-thal was performed to investigate the effect of HFE mutationson iron status of these patients. No significant differences were detected between β-thal carriers withand without HFE mutations. These results suggest that in the Balearic population the β-thal traitdoes not tend to be aggravated by the co-inheritance of HFE mutations.

Keywords β-Thalassemia (β-thal), Hereditary hemochromatosis (HH), HFE mutations,C282Y, H63D, Iron overload

Many diseases have been related to defects in iron metabolism leadingto complications of iron overload that is associated with different chronicdegenerative disorders. Thalassemia syndromes represent the main cause of

Received 21 March 2011; Accepted 11 June 2011.Address correspondence to Dr. Antònia Picornell, Laboratori de Genètica, Departament de Biolo-

gia, Universitat de les Illes Balears, Cra. Valldemossa, km 7.5, 07122-Palma de Mallorca, Illes Balears,España; Tel.: +34-971-172050; Fax: +34-971-173184; E-mail: apicornell@uib.es

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86 H. López-Escribano et al.

iron overload in Mediterranean countries. These patients are known to havepathologically increased iron stores due to greater iron absorption, regulartransfusions, anemia and increased erythropoiesis. However, only a minorityof individuals heterozygous for β-thalassemia (β-thal) develops iron over-load, despite excess iron absorption induced by ineffective erythropoiesis,indicating other hereditary and environmental variables are involved in ironloading (1,2). Among genetic factors, the most common cause of iron over-load is the presence of two main mutations (C282Y and H63D) in the HFEgene, mainly responsible for hereditary hemochromatosis (HH). Althoughthe role of the H63D mutation is not as clear as the role of the C282Y muta-tion, different studies claim H63D confers an increased risk of iron overloadand, therefore, genetic susceptibility to developing HH or aggravating otherdiseases (3–5). Therefore, the presence of mutations in the HFE gene mayadversely affect iron loading in β-thal carriers and explain the variability iniron overload observed in these patients.

The role of the HFE mutation on iron status in β-thal trait is controver-sial. The homozygous state for the C282Y mutation has been described asan aggravating factor (6,7); yet the consequences of co-inheritance of β-thaland H63D homozygosity have not been clearly elucidated, some authorsclaiming this genotype shows no effect on iron loading (8,9), while othersclaim it could induce iron overload in β-thal minor (6,10,11).

Because of the high prevalence of HFE mutations (especially H63D)in some Mediterranean countries, where the β-thal trait is also common,co-segregation of both inherited conditions in this region should not beconsidered exceptional. It is important for possible therapeutical implica-tions to investigate their possible synergistic effect on iron absorption. Thus,in this study we aimed to investigate the effect of co-inheritance of HFE andβ-thal in the Balearic Islands (where H63D carrier frequency is >31%) (12),to establish whether a fortuitous association is common and if it really affectsthe severity of iron overload.

This study was performed on 142 anonymous, unrelated β-thal carriersfrom the Balearic Islands, detected during a pilot β-thal screening. Theywere diagnosed by hematological studies based on a full blood count andquantification of Hb A2 and Hb F by high performance liquid chromatog-raphy (HPLC), iron deficiency being excluded in all cases. An automatedfull blood count (ADVIA 120 Hematology System; Siemens HealthcareDiagnostics, Deerfield, IL, USA) was performed on all samples. Microcy-tosis indicator was the mean corpuscular volume (MCV) (<78 fL) andfor hypochromia, the mean corpuscular hemoglobin (Hb) (MCH) (<27pg). To discard microcytosis by ferropenic anemia, we evaluated the ironstatus in microcytosis samples in a Hitachi Modular 917 analyzer (RocheDiagnostic, Mannheim, Germany). The iron deficiency was defined as atransferrin saturation index (IST) <15% or ferritin levels <14 ng/mL. Hb

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HFE Mutations in β-Thalassemia Minor 87

A2 and Hb F levels were performed by HPLC (HA-8160, Menarini Diagnos-tic, Florence, Italy).The C282Y and H63D mutations in the HFE gene wereassessed on genomic DNA from peripheral leukocytes (QIAamp Blood kit;Qiagen, Crawley, West Sussex, UK), using the standard polymerase chainreaction-restriction fragment length polymorphism (PCR-RFLP) method.

The β-thal genotypes were characterized by the LightCycler PCRmethod. Amplification and melting analysis was carried out with the Light-Cycler instrument (Roche Diagnostic). This screening was performed forthe following mutations, β0 codon 39 (C>T), β0 codon 37 (G>A), β+ IVS-I-110 (G>A), β0 codon 6 (–A), β0 IVS-I-1 (G>A) and β+ IVS-I-6 (T>C),as these are the most frequent β-globin mutations in the Mediterraneanarea. In those individuals who did not carry any of the former mutations,the β-globin gene was sequenced with the primers described in Clark andThein (13) by using the Big Dye® Terminator Cycle Sequencing kit v.3.1 (Applied Biosystems, Foster City, CA, USA) and an ABI PRISM® 3100Genetic Analyzer (Applied Biosystems).

Twenty-eight of the 142 β-thal carriers (19.7%) presented with β+ muta-tions, mostly IVS-I-110 (14.8%) and IVS-I-6 (3.5%). Other β+ gene defectswere found in frequencies <1% [IVS-II-745 (C>G) and IVS-II-705 (T>G)].The β0 mutations were found in 80.3% of the β-thal minor patients. Themost common mutation, codon 39, accounted for 69.0% of the β0 alle-les. The IVS-I-1, codons 8/9 (+G), codon 6 and –32 (C>T) mutationsaccounted for 9.8%. The codon 44 (–C) and codon 37 (G>A) mutationswere only detected in one individual, respectively.

Of the β-thal carriers, 39% had mutations in the HFE gene: 49 H63Dheterozygotes, four H63D homozygotes, one subject was heterozygous forC282Y and one was a compound heterozygote for the C282Y/H63D muta-tions. The allelic frequencies of C282Y and H63D were 0.007 ± 0.005 and0.204 ± 0.024, respectively. As expected, no significant differences in allelicfrequencies were found between β-thal carriers and the general Balearicpopulation (12).

Concerning iron status, there is a statistically significant difference(p <0.01) between ferritin values detected in β-thal carriers and controls(Table 1, top), although a considerable inter patient variation was observed.In order to evaluate the influence of different β-globin mutations, the β-thal carriers were separated according to the type of mutation (β0 and β+)(Table 1, bottom). There was no significant difference in the iron profilebetween β0 and β+ minor thalassemic subjects, except in transferrin satura-tion in women; although, as expected, those with β0 had more iron loadingthan individuals with β+.

However, no statistical differences were detected between β-thal carriersubjects with and without HFE mutations (Figure 1). When data were ana-lyzed separately according to the type of mutation (β0 and β+) (Table 2),

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HFE Mutations in β-Thalassemia Minor 89

N= 34 23

N HFE HFE mutations

800-

700-

600-

500-

400-

300-

200-

100-

0-

P = 0.86

32

N HFE HFE mutations

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N = 34 N = 53 32

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FIGURE 1 (a) Ferritin (ng/mL) and (b) IST (%) in β-thal carriers without (N) and with HFE mutations(white: males; grey: females).

the frequency of individuals with high ferritin and/or IST values was verysimilar between those with or without HFE gene mutations. Therefore, theinter patient variation in iron loading in our thalassemia minor patientsdoes not seem to depend on the type of inherited mutation in the β-globingene or the presence of mutations on the HFE gene.

Taking into account that most (49 out of 55) patients were heterozygousfor the H63D mutation, the results suggest iron overload in β-thal carriersis not accentuated by H63D heterozygosity; even the H63D homozygotesdid not have elevated ferritin and/or IST levels. Only the single compoundheterozygote (H63D/C282Y) individual had an unusual iron overload. Alarge number of subjects carrying different HFE mutated genotypes wouldbe necessary to conclude their effect on iron status.

Our results are not in agreement with those obtained in some stud-ies (2,14,15), suggesting iron overload in β-thal trait tends to be aggra-vated by co-inheritance of the H63D mutation, even when present in the

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HFE Mutations in β-Thalassemia Minor 91

heterozygous state; but are in agreement with those found in Thai andIranian populations (8,16) for β-thal minor and even β-thal intermedia inGreeks (17). An earlier study from Italy (7) also concluded that their find-ings do not support the hypothesis that the association of the β-thal traitwith a single C282Y or H63D allele might lead to iron overload. Diversityof results are probably due to the fact that iron status depends on com-plex interactions and is influenced by factors like sex, age, other mutationsin the HFE or other genes related to iron metabolism and environmentalpopulational variables such as diet or alcohol intake.

In conclusion, in the Balearic population we found no significantchange in ferritin or transferrin saturation levels in β-thal minor patientswith HFE mutations compared to patients without them. Therefore, system-atic screening of HFE mutations in β-thal carriers does not seem warrantedunless they develop evidence of iron overload. Large studies would beneeded to further assess the possible effect of less frequent and morecommitted HFE genotypes than H63D heterozygosity.

ACKNOWLEDGMENTS

This study was partially supported by grants PRDIB-2006-687872 andPROGECIB-10A from the Direcció General de R+D+I (Comunitat Autònoma deles Illes Balears), Palma, Balearic Islands, España.

Declaration of Interest: The authors report no conflicts of interest. The authorsalone are responsible for the content and writing of this article.

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