Interaction between haemochromatosis and transferrin receptor genes in different neoplastic disorders

L.E. Beckman1, G.F. Van Landeghem, C. Sikström2, A. Wahlin3, B. Markevärn3, G. Hallmans4, P. Lenner1, L. Athlin5, R. Stenling6 and L. Beckman7

Department of Medical Genetics,
1 Department of Oncology,
2 Department of Clinical Microbiology,
3 Department of Medicine,
4 Department of Nutritional Research,
5 Department of Surgery and
6 Department of Pathology, Umeå University, S-901 85 Umeå, Sweden


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A number of genes are involved in iron metabolism, including the transferrin receptor (TFR) and haemochromatosis (HFE) genes. In previous investigations an increased risk for neoplastic disease has been observed in individuals homo- and heterozygous for hereditary haemochromatosis. The HFE wild-type gene product complexes with the transferrin receptor (TF) and two different HFE mutations (Cys282Tyr and His63Asp) have been found to increase the affinity of TFR for TF and increase cellular iron uptake. In a recent study we found no associations for HFE and TFR separately, but an interaction between HFE and TFR genotypes in multiple myeloma. Individuals carrying the HFE Tyr282 allele (homo- and heterozygotes) in combination with homozygosity for the TFR Ser142 allele had an increased risk. In the present study the same association was found in breast and colorectal cancer. The odds ratio for all three neoplasms combined was 2.0 (95% CI 1.0–3.8). The risk for neoplastic disease was further increased (OR 7.7, 95% CI = 1.0–59.9) when the analysis was restricted to HFE Tyr homozygotes and compound heterozygotes in combination with TFR Ser homozygosity. Thus, an interaction between HFE and TFR alleles may increase the risk for different neoplastic disorders.

Abbreviations: HFE, haemochromatosis gene; HH, hereditary haemochromatosis; TF, transferrin receptor; TFR, transferrin receptor gene; wt, wild-type.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Most neoplastic disorders have a complex and multifactorial background, which may be influenced by a number of different determinants, among which is iron. Experimental, clinical and epidemiological investigations have shown that iron can influence the process of carcinogenesis in different ways (1): by catalysing the formation of mutagenic hydroxyl radicals (2), by suppressing host defence cells (3) and by acting as an essential nutrient for proliferating tumour cells (4,5). It is therefore of interest to study the relationship between neoplastic disorders and genes involved in absorption, transport and cellular uptake of iron, particularly the transferrin (TF), transferrin receptor (TFR) and haemochromatosis (HFE) genes. TF is highly polymorphic (6,7), but no definite and reproducible differences with respect to iron binding and body iron stores have been found between common variants of TF (8,9). The proliferation of tumour cells requires adequate expression of TFR on the cell surface (1012) and blocking of the TFR with TFR antibodies has been employed to inhibit the growth of neoplastic cells by withholding iron (13). Genetic variants of TFR have recently been described (14,15), but there are so far no studies of the relationship between genetic TFR variants and neoplastic disease.

Recently it was shown that homozygosity for a point mutation (Cys282Tyr) in the HFE gene was found in 83–100% of patients with hereditary haemochromatosis (HH) (1618). Thus there is convincing evidence that HFE is the primary HH locus. Feder et al. (16) also found an additional point mutation (His63Asp) in the HFE gene and an increased frequency of compound heterozygotes (Tyr282/Asp63) was observed among the haemochromatosis patients. Homozygosity for Asp63 is associated with a 4-fold risk for haemochromatosis (19).

Significantly increased body iron stores have been found in HFE Tyr282 homozygotes and, although less pronounced, in compound heterozygotes (Tyr282/Asp63) and in heterozygotes for the HFE Tyr282 allele (20,21). In patients with HH and liver cirrhosis the risk for hepatocellular cancer has been found to be considerably increased, ~200 times higher than in normal individuals (22,23). Also, heterozygosity for HH appears to have an influence on carcinogenesis. Thus, Nelson et al. (24) observed an increased risk of colorectal neoplasia, haematological malignancy and gastric cancer in HH heterozygotes.

Very recently it has been shown that there is a functional association between the HFE and TFR gene products. The wild-type HFE protein forms a stable complex with the TFR, thereby reducing its affinity for TF. The Cys282Tyr mutation nearly completely prevents the association of the mutant HFE protein with TFR, allowing a high affinity TF binding to the uncomplexed TFR (25), which could explain the lack of control of iron absorption in HH patients. Like the wild-type HFE protein, the His63Asp mutant protein forms a stable complex with the TFR, although it fails to decrease the affinity of TFR for TF compared with the wild-type HFE protein. Thus the Cys282Tyr and His63Asp mutations appear to have similar functional consequences although through different molecular mechanisms. The finding of a functional association between the HFE and TFR gene products led us to the hypothesis that HFE and TFR alleles may interact in iron metabolism and thereby influence the risk for neoplastic disorders.

In a previous investigation we observed an interaction between HFE and TFR genotypes in multiple myeloma (26). There were no significant differences between myeloma patients and controls with respect to the genotype distributions of HFE and TFR, but individuals carrying the HFE Tyr282 allele (homo- and heterozygotes) in combination with homozygosity for the TFR Ser142 allele had a significantly higher risk for multiple myeloma compared with carriers of the HFE Tyr allele not homozygous for TFR Ser.

The aims of this investigation were: (i) to study whether the same HFE/TFR genotype combination as in multiple myeloma is associated with an increased risk for two other neoplastic disorders, where iron has been implicated, e.g. breast (12,27) and colorectal cancer (24,27); (ii) to study the influence of compound heterozygotes in the analysis of HFE–TFR interaction.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Blood samples were collected from patients with multiple myeloma (n = 92, 39 males and 53 females), breast cancer (n = 165, all females) and colorectal cancer (n = 173, 90 males and 83 females). All patients were diagnosed at the Umeå University Hospital in northern Sweden. The patients with breast and colorectal cancer have been described previously (28,29). In the patients with multiple myeloma TFR and HFE (Cys282Tyr) genotypes have been studied previously (26). The studies of patients with neoplastic disorders were approved by the local Ethics Committee.

The population controls were a series of placental samples from 201 consecutive deliveries at the Umeå University Hospital and 93 individuals from central Sweden, previously used as controls in a study of lung cancer (30). Since there were no significant differences between the population samples from northern and central Sweden they were pooled.

Extraction of DNA, PCR amplification and electrophoresis were performed essentially as described previously (31). The following primers were used, all at an annealing temperature of 62°C.

HFE, Cys282Tyr:5'-TGG CAA GGG TAA ACA GAT CC-3'

5'-CTC AGG CAC TCC TCT CAA CC-3'

HFE, His63Asp:5'-ACA TGG TTA AGG CCT GTT GC-3'

5'-GCC ACA TCT GGC TTG AAA TT-3'

TFR, Ser142Gly:5'-TGA CCT GAA GAG AAA GTT GTC GGAG-3'

5'-GTT CTA CCT TTT CCC CTA CCA GTA TAG TTT-3'

The amplified fragments were digested with RsaI for HFE Cys282Tyr, MboI for HFE His63Asp and BanI for TFR Ser142Gly.

Frequencies of the HFE and TFR alleles were calculated by gene counting. Testing of the goodness of fit to the Hardy–Weinberg equilibrium and the statistical analysis of differences between patients and controls were performed by means of the {chi}2 test.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The Cys282Tyr and His63Asp mutations do not occur on the same chromosome and are therefore allelic. Together with the most common (wt, wild-type) allele they form a three allele system. All samples showed a good agreement with the Hardy–Weinberg equilibrium. There were no significant differences between patients and controls with respect to HFE genotype and allele frequencies, although there was a somewhat increased frequency of compound heterozygotes (Tyr/Asp) in patients (3%) compared with controls (1.4%). The frequencies of the wt, Tyr and Asp alleles were respectively 0.806, 0.073 and 0.121 in the controls and 0.778, 0.074 and 0.148 in all neoplasms.

The frequency of the Gly allele was 0.395 and 0.401 in the controls and all neoplasms, respectively. As in the HFE system, there was a good agreement with the Hardy–Weinberg equilibrium and there were no significant differences between patients and controls concerning genotype and allele frequencies.

The combinations of TFR and HFE genotypes are given in Table IGo together with the numbers of compound heterozygotes. Individuals who carried the HFE Tyr allele (homo- and heterozygotes) in combination with the TFR Ser/Ser genotype showed an increased frequency in the three different neoplasms (Table IIGo). There was no significant heterogeneity between the three cancer groups with respect to the frequency of this TFR/HFE combination ({chi}2 = 0.71, 2 d.f., P = 0.70), hence they may be pooled for comparison with the controls. The OR for all three neoplasms pooled was 2.0 (95% CI 1.0–3.8). However, when the analysis was restricted to HFE Tyr homozygotes and compound heterozygotes in combination with TFR Ser homozygosity (Table IIGo) a 3- to 4-fold additional risk was found (OR = 7.7, 95% CI 1.0–59.9). Finally, there was no sex difference with respect to the TFR/HFE combination at risk ({chi}2 = 2.37, 1 d.f., P = 0.12).


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Table I. Combinations of TFR (Ser/Gly) and HFE (Cys/Tyr) genotypes in population controls and in patients with multiple myeloma, breast cancer and colorectal cancer
 

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Table II. Frequencies (F), odds ratios (OR) and 95% confidence intervals (CI) for (A) TFR Ser/Ser–HFE Tyr homo- and heterozygotes and (B) TFR Ser/Ser–HFE Tyr homozygotes and compound (Tyr/Asp) heterozygotes
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Most neoplastic disorders have a multifactorial background with an interaction between genetic and environmental factors. Among these factors is the body iron level, which in turn has a complex multifactorial background, and iron metabolism is known to be influenced by a number of genes, e.g. TF, TFR, HFE, B2M (32), FT (33), IREBP (34) and Nramp 2 (35). The products of three of these genes (TFR, HFE and B2M) interact physically to form a molecular complex that influences the affinity of TFR for TF (16,25). Mutations that affect the structure of either of these gene products can be expected to interfere with the function of the molecular complex and thereby receptor function. It is therefore conceivable that mutations in TFR itself may also influence the TFR–HFE–B2M complex and the affinity for TF.

Since increased body iron stores caused by homo- and heterozygosity for HH have been found to be associated with an increased risk for neoplastic disease, we tested whether genetic variants of HFE and TFR separately and combined may influence the risk for multiple myeloma, breast cancer and colorectal cancer. The HFE and TFR genotypes tested separately were not associated with any of these neoplastic disorders, but there was a significant difference between patients and controls with respect to HFE/TFR genotype combinations. Thus the previous association found in multiple myeloma (26) was largely replicated in breast and colorectal cancer. In the present study the frequency of the HFE Tyr allele showed a significant heterogeneity between the three TFR types ({chi}2 = 11.67, 2 d.f., P = 0.0029). No such association was found in the controls ({chi}2 = 2.48, 2 d.f., P = 0.29).

Furthermore, it is of interest that the risk for neoplastic disease was considerably increased when the analysis was restricted to HFE Tyr homozygotes and compound (Tyr/Asp) heterozygotes in combination with TFR Ser homozygosity. This indicates that the carcinogenic effect may somehow be dependent on increased iron uptake. The TFR Ser allele alone does not seem to influence body iron stores as in a recent study no significant association was found between the TFR Ser/Gly polymorphism and haemochromatosis without HFE mutation (36).

Thus, based on statistical evidence it seems that an interaction between HFE and TFR alleles may increase the risk for a number of different neoplastic disorders, whereas heterozygosity for the HFE Tyr allele on its own appears to be of less importance. The mechanism behind this interaction, particularly the role of the TFR variants, remains to be elucidated.


    Acknowledgments
 
We thank Mrs S.Linghult and Mrs I.Svanholm for expert technical assistance. This study was supported by the Research Foundation of the Department of Oncology, Umeå University Hospital.


    Notes
 
7 To whom correspondence should be addressed. Email: lars.beckman{at}medgenet.umu.se Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received November 20, 1998; revised March 5, 1999; accepted March 12, 1999.