Evidence for an Association Between Compound Heterozygosity for Germ Line Mutations in the Hemochromatosis (HFE) Gene and Increased Risk of Colorectal Cancer

1460 Cancer Epidemiology, Biomarkers & Prevention Evidence for an Association between Compound Heterozygosity for Germ Line Mutations in the Hemochromatosis (HFE) Gene and Increased Risk of Colorectal Cancer

James P. Robinson,1 Victoria L. Johnson,1 Pauline A. Rogers,2 Richard S. Houlston,2 Earmonn R. Maher,3 D. Timothy Bishop,4 D. Gareth R. Evans,5 Huw J.W. Thomas,1 Ian P.M. Tomlinson,1,6 Andrew R.J. Silver,1 and Colorectal Cancer Gene Identification (CORGI) consortium 1Cancer Research UK Colorectal Cancer Unit, St Mark’s Hospital, Harrow, United Kingdom; 2Section of Cancer Genetics, Institute of Cancer Research, Sutton, United Kingdom; 3Section of Medical and Molecular Genetics, University of Birmingham, Birmingham, United Kingdom; 4Genetic Epidemiology Division, Cancer Research UK Clinical Centre in Leeds, St. James’s University Hospital, Leeds, United Kingdom; 5Academic Unit of Medical Genetics and Regional Genetics Service, St. Mary’s Hospital, Manchester, United Kingdom; and 6Molecular and Population Genetics Laboratory, London Research Institute, Cancer Research UK, London, United Kingdom

Abstract

Whereas a recent study reported an increased risk of contrast, individuals compound heterozygous for both colorectal cancer associated with any HFE germ line mutations (15 cases versus 5 controls) had thrice the odds mutation (C282Y or H63D), other investigators have of developing colorectal cancer (odds ratio, 3.03; 95% concluded there is no increased risk, or that any increase confidence interval, 1.06-8.61) compared with those with a is dependent on polymorphisms in HFE-interacting genes single mutation. This finding did not quite reach statistical such as the transferrin receptor (TFR). We have established significance after allowing for multiple post hoc testing the frequency of HFE mutations in colorectal cancer (Pobserved = 0.038 versus P = 0.025, with Bonferonni patients (n = 327) with a family history of the disease correction). Overall, our data indicate that individuals with and randomly selected controls (n = 322); this design a single HFE mutation, C282Y or H63D, are unlikely increases greatly the study’s power. Genotyping for the predisposed to develop colorectal cancer. However, risk of TRF S142G polymorphism was also conducted on a large colorectal cancer might be increased by compound hetero- proportion of the study group. Using PCR, restriction zygosity for the HFE mutations in the small number of enzyme mapping, sequencing followed by data analysis subjects studied. TFR gene polymorphism was not an with Fisher’s exact test and logistic regression, we show independent risk factor and did not modify the disease that the presence of any HFE mutation (Y282 or D63) was risk associated with HFE mutation. (Cancer Epidemiol not associated with colorectal cancer risk (P = 0.57). In Biomarkers Prev 2005;14(6):1460–3)

Introduction

Iron is a pro-oxidant that can catalyze the formation of nucleotide 845G ! A; cysteine ! tyrosine) and H63D (exon 2; mutagenic hydroxyl radicals leading to DNA damage. Iron is nucleotide 187C ! G; histidine ! aspartic acid). Most patients unable to move freely across cellular membranes and has to be (60-100%) satisfying criteria for hemochromatosis are homozy- transported across bound to transferrin. The cell surface gous for the C282Y mutation; a few are compound hetero- transferrin receptor (TFR) binds to transferrin allowing cellular zygotes (C282Y/wild type and H63D/wild type) or apparently uptake, and the hemochromatosis gene (HFE), in turn, C282Y/wild-type heterozygotes (9-12). Homozygosity for the regulates iron uptake by binding to the TFR decreasing the H63D mutation confers only a relatively small risk of protein’s affinity for transferrin (1-4). The transferrin, TFR, and developing hemochromatosis, and there are no reported HFE proteins have all been immunolocalized to the colonic patients who carry the C282Y and H63D mutations on the crypt (5). Protein variants of these genes may influence iron- same chromosome (11, 12). Although allele frequency varies induced free radical formation and cancer risk. widely, the two mutations are relatively common throughout Several germ line mutations in HFE lead to an increase of Caucasian populations and may be regarded as polymor- iron uptake and the development of the autosomal recessive phisms. In Europe, the C282Y and H63D allele frequencies vary disorder, hereditary hemochromatosis (6-8). The most common between 1% and 10% and 1% and 28%, respectively (11, 13). germ line mutations in the HFE gene are C282Y (exon 4; Given the high frequency of these alleles in Caucasians, the effect on the number of cancer cases may be large. The C282Y mutation has been shown to prevent functional Received 9/13/04; revised 3/8/05; accepted 3/22/05. interaction between the HFE and the TFR proteins thereby Grant support: Cancer Research UK. increasing cellular iron uptake (1, 14). The interaction is not The costs of publication of this article were defrayed in part by the payment of page charges. prevented by H63D, but the mutation causes a failure to This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. decrease the affinity of the TFR protein for transferrin, Section 1734 solely to indicate this fact. resulting in increased cellular iron intake. As the receptor Note: Collaborators from the CORGI consortium who supplied patients for this study were Shirley Hodgson (St. George’s Hospital), Louise Izatt (Guy’s Hospital), and Anneke Lucassen binds transferrin, polymorphisms in the TFR gene, such as (Wessex Regional Genetics Service). the serine/glycine variant at codon 142, may alter the level of Requests for reprints: Andrew Silver, Cancer Research UK, Colorectal Cancer Unit, St. Mark’s cellular iron uptake. One study has suggested that whereas Hospital, Harrow, HA1 3UJ, United Kingdom. Phone: 208-235-4257; Fax: 208-235-4277. E-mail: [email protected] there was no association for HFE and TFR independently, Copyright D 2005 American Association for Cancer Research. individuals heterozygous or homozygous for the C282Y

Cancer Epidemiol Biomarkers Prev 2005;14(6). June 2005 Downloaded from cebp.aacrjournals.org on September 25, 2021. © 2005 American Association for Cancer Research. Cancer Epidemiology, Biomarkers & Prevention 1461 mutation and homozygous for the S142 allele had an Table 1. Frequency of HFE gene genotypes in study subjects increased risk of multiple myeloma, breast cancer, and colorectal cancer (15, 16). The lack of association between Genotype Case patients, Control subjects, %(n = 327) %(n = 322) HFE mutations, when considered in isolation, and colorectal cancer has been supported by two other investigations (17, C282/C282 84.1 (275) 86.6 (279) 18), although another study has found a modestly increased C282/Y282 15.3 (50) 12.1 (39) risk of colon cancer for carriers of any HFE mutation Y282/Y282 0.6 (2) 1.2 (4) compared with subjects with no mutation (adjusted odds H63/H63 72.2 (236) 74.8 (241) H63/D63 25.4 (83) 22.7 (73) ratio [OR], 1.40; 95% confidence interval [95% CI], 1.07-1.87; D63/D63 2.4 (8) 2.5 (8) ref. 19). Any HFE mutation 39.1 (128) 37.0 (119) To determine whether there is an increased risk of colorectal Allele frequency, Y282 0.083 (54/654) 0.073 (47/644) cancer in individuals carrying mutant HFE and TFR alleles, Allele frequency, D63 0.151 (99/654) 0.138 (89/644) independently or as a combination, we established genotype frequencies of mutant alleles in a well-defined population with a family history of colorectal cancer and in a randomly selected control group. The use of cases with a family history increases Results the statistical power of the study compared with a set of unselected cases (20). The frequencies of HFE gene mutations were established in 327 cases (191 females and 136 males) and 322 controls (168 females and 154 males). There was no significant difference in Materials and Methods the distribution of males and females between cases and controls (C 2 = 2.55, P = 0.11). The allele frequencies of each Patients and HFE Genotyping. The study involved a total 1 HFE mutation were very similar in the cases and the controls of 327 unrelated individuals (191 females and 136 males) who (C282Y, 0.082 versus 0.073; H63D, 0.151 versus 0.138; P = 0.536 had been diagnosed with colorectal cancer; were known to be and 0.528, respectively, Fisher’s exact test; Table 1). The negative for predisposing mismatch repair, APC and MYH number of individuals heterozygous for the C282Y mutation germ line mutations; and had a family history of colon cancer was higher in the case population (50 of 327, 15.3%) than with a minimum of two affected family members but no controls (39 of 322, 12.1%), although this difference was not reported hemochromatosis. Cases were recruited from Re- significant (P = 0.255, Fisher’s exact test; Table 1). Heterozy- gional Genetic Centres in the South of England and either the gosity for H63D occurred at a higher frequency in the case case or a relative was affected at <70 years of age. The cases in (83 of 327, 25.4%) than the control (73 of 322, 22.7%) this study had an age range of 30 to 70 years. Controls populations (Table 1), although the difference was not comprised 322 individuals (168 females and 154 males) who significant (P = 0.463, Fisher’s exact test). The number of were selected and recruited from households in the South of homozygotes for each of the two HFE mutations was very England and randomized by using a random number similar in cases and controls (2 and 4 and 8 and 8, respectively; generator. Controls had no previous diagnosis of colorectal Table 1); no compound homozygotes were identified. In both neoplasia or family history of disease, had not been casesandcontrols,theHFE genotypes were in Hardy- investigated for colonic and/or rectal disease within 2 years Weinberg equilibrium (C282Y: cases, P > 0.5; controls, P > before recruitment, and had an age range of 50 to 75 years. 0.3; and H63D: cases, and controls, P > 0.5). Both cases and controls were of Caucasian origin. Without The frequency of individuals with any HFE gene mutation taking into account the increased statistical power provided by was similar in cases (128 of 327, 39.1%) and controls (119 of the use of family history cases (20), a study of this size using a 322, 37.0%). However, the frequency of the compound random selection of both cases and controls would have 80% heterozygous HFE genotype (C282Y/wild type and H63D/ power to detect a 4-fold increase in the frequency of a variant wild type) was significantly higher in cases (15 of 327, 4.6%) between controls (2%) and cases (8%). All individuals than controls (5 of 322, 1.6%; P = 0.038, Fisher’s exact test; provided peripheral blood and DNA was extracted using Table 2). We therefore identified three mutually exclusive standard protocols. PCR-RFLP analysis was used to screen for groups of individuals: compound heterozygotes (both C282Y/ the HFE germ line mutations and for the TFR polymorphism, wild type and H63D/wild type), ‘‘single mutation carriers’’ G142S as described previously (6, 15, 19). Primers for the (C282Y/wild type or C282Y/C282Y or H63D/wild type or H63D region were 5V-ATGGTTAAGGCCTGTTGCTCTGTC- H63D/H63D), and those who were wild type for both 3Vand 5V-AGACTGTTGGAGTACTTCACTCC-3V. All digests mutations (Table 2). Single variable logistic regression analysis were conducted alongside control PCR products of known showed that overall, the indicator variable for these three genotype. To ensure accuracy of the HFE genotyping, we also groups only reached borderline significance (P =0.07). directly sequenced almost all the samples for C282Y and a However, Wald Tests showed that single mutation carriers random 20% of samples for H63D. (individuals with mutation at C282 or H63 but not both) to be Statistical Analysis. STATA statistical software version 8.0 at no increased risk of colorectal cancer compared with wild- (Stata Corp., College Station, TX) was used. The data were type subjects (OR, 1.01; 95% CI, 0.73-1.40; P = 0.95) and analyzed as a case-control study using single and multivar- compound heterozygotes to be possibly at increased risk iable logistic regression. The outcome variable was case or compared with individuals with a single mutation (OR, 3.03; control, and the explanatory variables were genotypes or CI, 1.06-8.61; P = 0.038). However, allowing for multiple post combinations of genotypes. Wald tests were used to compare hoc testing using the Bonferroni correction for two post hoc a single characteristic of a variable with the baseline and the tests (i.e., no mutation against a single mutation and likelihood ratio test was used to test the significance of an compound mutation against single mutation), overall statisti- overall variable. The study has been analyzed with adjust- cal significance at the 5% level would be attained only at P = ment for multiple post hoc testing using Bonferroni proce- 0.025. dure to adjust for two post hoc tests (5% level of statistical We selected at random 171 cases and 184 control samples for significance attained at P = 0.025). Controlling for gender did TRF genotyping and found that the allelic frequencies for S142 not alter the results obtained from logistic regression and and G142 were the same in both cases and controls (0.51 and Wald tests, and this was not used in the results reported 0.49; P = 0.940, Fisher’s exact test; Table 3). As allele below. frequencies are known to vary among ethnic groups (21),

these data provided good evidence of matching of the cases Table 3. Frequency of TRF polymophisms in study subjects and controls used in this study. The frequencies of S142 heterozygotes and S142 and G142 homozygotes were also Genotype Case patients, Control subjects, %(n = 171) %(n = 184) similar in cases and controls (Table 3). Statistical analysis failed to identify any interaction, as proposed by Beckman et al. (15), S142/S142 23.0 (40) 25.0 (47) between TRF genotypes and either HFE mutation or com- S142/G142 55.0 (94) 50.0 (92) pound HFE heterozygozity (Ps > 0.06). G142/G142 22.0 (37) 25.0 (45) Allele frequency, S142 0.51 (174/342) 0.51 (186/368) Allele frequency, G142 0.49 (168/342) 0.49 (182/368) Discussion

Recently, the presence of any germ line HFE mutation (C282Y or H63D) was shown associated with a modest but significant both C282Y and H63D compound heterozygous mutations. 1.4-fold increased risk (95% CI, 1.07-1.87) of colon cancer The data presented showed that there was evidence of a compared with those individuals with no HFE mutation; the possible increased risk of colorectal cancer (3.03-fold) only level of increased risk was the same for those with or without when the two different HFE mutations were present in the a family history of colon cancer (19). Curiously, heterozygosity same individual. for either mutation conferred almost the same increase in The study of Shaheen et al. did not show an increased colorectal cancer risk, although heterozygosity for the C282Y frequency of HFE compound heterozygotes in colorectal mutation has been reported to lead to more severe iron cancer cases (1.1%) compared with controls (1.3%; ref. 19). overload in carriers than the H63D mutation. The cases and The disparity between this result and our own findings of an controls in that study were made up of 43% African association between HFE compound heterozygosity and Americans and 57% Caucasian Americans, although the colorectal cancer risk may relate to differences in case and frequencies of the C282Y and H63D alleles in the two ethnic control selection between the two studies. In support of our groups are very different (African Americans, 0.0067 and findings, an increased frequency of compound heterozygotes 0.0263; Caucasian Americans, 0.0507 and 0.1512, respectively; has been reported in randomly selected sets of Swedish ref. 22). Despite the much lower mutant allele frequency in Caucasian patients diagnosed with either multiple myeloma African Americans, those who did possess an HFE mutation (2.2%), breast cancer (3.6%), or colorectal cancer (2.9%) were at significantly increased risk of colorectal cancer (OR, compared with controls (1.4%; ref. 15). Although HFE 2.1; 95% CI 1.1 to 3.9), whereas Caucasians with a similar compound heterozygote mutants are relatively uncommon genotype were not (OR, 1.2; 95% CI 0.8-1.6; ref. 19). By and hence detection of true associations may not be straight- contrast, a study conducted in the Netherlands of postmen- forward, the consistency between the Swedish study and our opausal women screened for C282Y mutations and followed own work suggests that this genotype may truly be associated for a period of 20 years showed no significant increase in with increased risk of colorectal cancer. colorectal cancer risk for women who were C282Y hetero- We found no evidence to suggest that subjects homozygous zygotes (23). Other studies have found no link between any for either HFE mutation were at increased risk of disease, HFE mutation and risk of colorectal cancer. For example, although the numbers reported in this study and by others (15, Beckman et al. (15) reported that genetic variants of HFE were 19) were very small. It has been shown that C282Y/C282Y not associated with increased risk of this disease or of multiple homozygotes have the highest iron load (6-9) and if the myeloma and breast cancer; two other studies found that association between HFE variants and colorectal cancer is heterozygosity for the C282Y mutation did not increase risk of mediated directly through iron excess, these individuals might colorectal cancer (17, 18). Hence, it would seem that the higher be expected to have the highest risk of colorectal cancer. overall statistically significant risk reported by Shaheen et al. Owing to the very low frequency of this genotype, it is not (19) may be a consequence of inclusion of the African possible to exclude an association with colorectal cancer. American subjects in their study. The reported increased risk An earlier investigation identified a 7.7-fold increase in of colorectal cancer in African Americans relative to Cauca- colorectal cancer risk in HFE Y282 homozygotes and C282Y/ sians (24, 25) may well relate to genetic factors other than the H63D heterozygotes who were also homozygous for TFR S142 HFE C282Y and H63D mutations and/or environmental suggesting that an interaction between HFE and TRF increased influences. risk for neoplastic disease (15). Using a similar number of case In view of these apparent contradictions, we have evaluated and control samples to this earlier study, we have found no whether individuals with a family history of colorectal cancer evidence to support the earlier observation; our data indicate tend to carry mutant HFE alleles, independently or in that homozygosity for the S142 does not modify the influence combination. When HFE mutations were considered separate- of the C282Y mutation on colorectal cancer risk. ly, there was only a small, nonsignificant increased risk of In summary, we have found evidence to suggest that colorectal cancer; in this respect therefore, our data support the individuals with a single HFE C282Y or H63D mutation are earlier, negative findings (15, 17, 18). Our study also unlikely predisposed to develop colorectal cancer and that considered the colorectal cancer risk to an individual carrying TRF genotype in combination with C282Y mutation does not influence the likelihood of disease. In contrast, compound heterozygosity for the HFE mutations might increase the risk of colorectal cancer. Table 2. Frequency of single and compound HFE gene mutations among cases and controls

Variable* Case Control OR Acknowledgments patients, subjects, (95% CI) We thank the patients, their doctors, and other staff from UK Genetics %(n = 327) %(n = 322) Departments. Wild type 60.9 (199) 63.0 (203) Single mutation 34.6 (113) 35.4 (114) 1.01 (0.73-1.40) Compound mutation 4.5 (15) 1.6 (5) 3.06 (1.09-8.58) References 1. Feder JN, Penny DM, Irrinki A, et al. The hemochromatosis gene product *Single mutation, C282Y or H63D as homozygotes or heterozygotes. Compound complexes with the transferrin receptor and lowers its affinity for ligand mutation, all were heterozygous for C282Y/wild type and H63D/wild type. binding. Proc Natl Acad Sci U S A 1998;95:1472 – 7.

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Cancer Epidemiol Biomarkers Prev 2005;14(6). June 2005 Downloaded from cebp.aacrjournals.org on September 25, 2021. © 2005 American Association for Cancer Research. Evidence for an Association between Compound Heterozygosity for Germ Line Mutations in the Hemochromatosis (HFE) Gene and Increased Risk of Colorectal Cancer

James P. Robinson, Victoria L. Johnson, Pauline A. Rogers, et al.

Cancer Epidemiol Biomarkers Prev 2005;14:1460-1463.

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