PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of jmedgeneJournal of Medical GeneticsVisit this articleSubmit a manuscriptReceive email alertsContact usBMJ
 
J Med Genet. 2006 October; 43(10): e52.
PMCID: PMC2563163

Iron genes, iron load and risk of Alzheimer's disease

Abstract

Background

Compound heterozygotes of the haemochromatosis gene (HFE) variants, H63D and C282Y, have raised transferrin saturation compared with that in the wild type. In the cohort of the Oxford Project To Investigate Memory and Ageing (OPTIMA), bicarriers of the HFE C282Y and the transferrin C2 gene variants are at five times greater risk of developing Alzheimer's disease; the addition of HFE H63D may raise the risk still further.

Objective

To investigate transferrin saturation by HFE and transferrin genotype among people without dementia–that is, controls and those with mild cognitive impairment (MCI)—and also among those with Alzheimer's disease.

Methods

Serum iron status and genotype were examined of 177 patients with Alzheimer's disease, 69 patients with MCI and 197 controls from the OPTIMA cohort.

Results

Although each of these variants alone had relatively little effect on iron status, the combination of either HFE C282Y and HFE H63D or of HFE C282Y and transferrin C2 markedly raised transferrin saturation in those without dementia, but had little effect in those with mature Alzheimer's disease.

Conclusions

These combinations may raise the risk for Alzheimer's disease, owing to higher iron loads and therefore oxidative stress in the preclinical phase. If replicated, these findings will have implications for the prevention of Alzheimer's disease.

The effects of the haemochromatosis gene (HFE) variants, H63D and C282Y, on serum iron status of a healthy human population were first examined in 19981 and have been extensively studied since. Several large studies2,3,4,5,6 have obtained broadly consistent results. For instance, Jackson et al3 summarised their results for transferrin saturation as follows: C282Y homozygotes >compound heterozygotes (C282Y and H63D) >H63D homozygotes >C282Y heterozygotes >H63D heterozygotes >wild type. However, to our knowledge, the influence on iron status of the combination of HFE and transferrin variants has not been studied, either in healthy controls or in those with Alzheimer's disease.

We previously reported7 that in the cohort of the Oxford Project To Investigate Memory and Ageing (OPTIMA), carriers of either the HFE C282Y or transferrin C2 variants alone had no effect on risk for Alzheimer's disease; however, bicarriers of the two alleles, whether heterozygotes or homozygotes, were at five times greater risk for Alzheimer's disease. The addition of the third variant, HFE H63D, seemed to increase the risk still further.

Alzheimer's disease has a long preclinical phase.8 One of the early events in the development of the disease is thought to be oxidative stress,9,10 due crucially to the actions of redox‐active iron.11,12 The above results therefore suggested that combinations of the HFE C282Y, HFE H63D and transferrin C2 alleles might contribute to higher iron loads during the preclinical phase of Alzheimer's disease. We therefore investigated transferrin saturation by genotype among those without dementia—that is, controls and patients with mild cognitive impairment (MCI)—as well as among patients with Alzheimer's disease, in the OPTIMA cohort. We examined combinations of the HFE H63D, HFE C282Y and transferrin C2 alleles.

Key points

  • Bicarriers of the haemochromatosis gene (HFE) C282Y and the transferrin C2 variants may have higher iron loads than those of the wild type, as has previously been shown for compound heterozygotes of HFE H63D and HFE C282Y.
  • The association of the combination of HFE C282Y and transferrin C2 with increased risk for Alzheimer's disease, as previously reported, may therefore be due to oxidative stress in the preclinical phase.
  • If confirmed, these results have implications for the prevention of Alzheimer's disease in the 6% of northern Europeans who carry one or both combinations, as iron overload is a treatable condition.

Methods

All 177 patients with Alzheimer's disease (100 women), 69 with MCI (30 women) and 197 controls (98 women) were Caucasians from the longitudinal, observational cohort of OPTIMA, drawn from the Oxford region. Mean age at onset of Alzheimer's disease was 70.3 (standard deviation (SD) 9.1) years, and age at death or last examination of controls was 76.2 (9.7) years (195 controls). Of the patients with Alzheimer's disease, 112 were neuropathologically confirmed by the criteria of the Consortium to Establish a Registry for Alzheimer's Disease13 (99 had “definite” and 13 had “probable” disease) and 65 were diagnosed as having “probable Alzheimer's disease” by NINCDS‐ADRDA criteria.14 Possible autosomal dominant cases were excluded on the basis of family history. Diagnosis of MCI was according to Petersen et al.15 All 197 controls were without cognitive impairment and had Cambridge Cognitive Examination scores16 >80. Informed consent was obtained in writing from all participants and the study was approved by the Central Oxford Ethics Committee (approval number 1656).

Blood samples were taken during the earlier assessments, when people with MCI were still controls—that is, between December 1989 and March 2003. Samples were then stored at −70°C until the iron assays were carried out between November 2003 and July 2004, during which period they were stored at −20°C. The final diagnoses were made in April 2004. Genotyping of HFE C282Y and HFE H63D and of transferrin C2 was as described previously.7 Serum iron and unsaturated iron‐binding capacity were determined by a microtitre plate assay17; transferrin saturation was calculated from values for serum iron and unsaturated iron‐binding capacity.17

We used analysis of covariance, followed if significant by pairwise comparisons. A Bonferroni correction factor of 6 was applied to the pairwise comparisons. We used linear regression analysis to test for interactions between variants. In all analyses, we controlled for age, sex and the apolipoprotein E ε4 allele (APOE4), removing covariates with p>0.2 in steps. All p values are after controlling for these covariates; all mean values are unadjusted.

Results

HFE C282Y and HFE H63D

Table 11 shows that with these relatively small numbers, the presence of only one variant allele—that is, HFE C282Y or HFE H63D—had little effect on transferrin saturation. On the other hand, compound heterozygotes had markedly higher transferrin saturation among controls or when combining controls and those with MCI (hereafter called the non‐AD group). We found no significant differences between subgroups of patients with Alzheimer's disease (p = 0.18). As we also found no significant differences in any analysis between the first three genetic subgroups—that is, participants negative for either or both variants—we combined these three categories. We found that among the non‐AD group, compound heterozygotes had higher transferrin saturation than all others combined: 52.1% (SD 6.0%), v 28.7% (11.4%; p<0.001), controlling for age, sex and APOE4). Also, among the non‐AD group, compound heterozygotes were more likely to have iron overload, as measured by transferrin saturation >45% in women or >50% in men, with an adjusted odds ratio (OR) of 8.4 (95% confidence interval (CI) 1.2 to 61). In linear regression analysis, controlling for age, sex and APOE4, the interaction between the presence of HFE C282Y and HFE H63D was a significant predictor of transferrin saturation (p = 0.008).

Table thumbnail
Table 1 Transferrin saturation (%) in controls, in those with mild cognitive impairment and in those with Alzheimer's disease, by haemochromatosis genotype

Among compound heterozygotes, patients with Alzheimer's disease had lower transferrin saturation than that in the non‐AD group (p = 0.01). In contrast, we found no overall reduction in transferrin saturation in patients with Alzheimer's disease.

HFE C282Y and transferrin C2

Table 22 shows the effect of the two variant alleles, HFE C282Y and transferrin C2. Again, the presence of only one variant had little effect on transferrin saturation. Only bicarriers of the two variants, whether heterozygotes or homozygotes, had higher transferrin saturation and only among the non‐AD group. We found no significant differences between subgroups of patients with Alzheimer's disease (p = 0.81). Again, we combined the first three genetic subgroups, as we found no significant differences in any analysis between them. Among the non‐AD group, bicarriers had higher transferrin saturation than all others combined: 41.4% (SD 9.7%), versus 28.8% (SD 11.6%; p = 0.007, controlling for age, sex and APOE4). Also among the non‐AD group, bicarriers, like compound heterozygotes, were more likely to have iron overload, with an adjusted OR of 8.3 (95% CI 1.8 to 39). However, in linear regression analysis, controlling for age, sex and APOE4, the interaction between HFE C282Y and transferrin C2 was not a significant predictor of transferrin saturation (p = 0.13).

Table thumbnail
Table 2 Transferrin saturation (%) in controls, in those with mild cognitive impairment and in those with Alzheimer's disease, by haemochromatosis and transferrin genotype

Among bicarriers, as among compound heterozygotes, patients with Alzheimer's disease had lower transferrin saturation than non‐AD (p = 0.004).

HFE H63D and transferrin C2

We found no significant differences in transferrin saturation owing to these two variants occurring either singly or in combination.

HFE C282Y, HFE H63D and transferrin C2

This combination of all three variants was not examined, as we had only five patients with this combination: four with Alzheimer's disease, one with MCI and no controls.

Discussion

In much larger studies,2,3,5,6 even single copies of either HFE C282Y or HFE H63D produce small, significant differences in iron status. However, examination of our present results with those of our earlier study7 suggests that a combination of two variants is needed to produce a substantially higher iron load—that is, sufficient to have a marked effect on the risk for Alzheimer's disease.7 That combination could be either HFE C282Y and HFE H63D, or HFE C282Y and transferrin C2. It seems that HFE C282Y is an essential element in these combinations, as the other two variants together but without HFE C282Y had little effect either on the risk for Alzheimer's disease7 or on iron status. Feder et al18 suggested that, although both the HFE C282Y and the HFE H63D proteins are dysfunctional, HFE C282Y may be more so; any dysfunction associated with the transferrin C2 protein is yet to be established.19

The damaging effects of the at‐risk combinations were limited to those without dementia and were not seen in those with fully developed Alzheimer's disease. This is consistent with the view that misregulated iron and the associated oxidative stress exert their harmful influence in the preclinical phase of Alzheimer's disease. Some other risk factors for Alzheimer's disease, such as high blood pressure,20,21 are mainly or only seen in the presymptomatic phase. The lack of any raised iron status associated with the at‐risk combinations in patients with mature Alzheimer's disease could be due to iron withholding (anaemia of inflammation), as Alzheimer's disease is a chronic inflammatory condition. However, we found no evidence of iron withholding in our cohort with Alzheimer's disease (data not shown), other than among these two genetic combinations.

In our earlier study,7 we found that the presence of APOE4 further increased the risk for Alzheimer's disease associated with HFE C282Y and transferrin C2. In this study, we found no effect of APOE4 on transferrin saturation (data not shown). This suggests that APOE4 does not contribute directly to iron load, but rather aggravates the oxidative stress caused by raised iron levels.22,23

Several studies2,3,4,5,6 have shown a difference between the sexes in the effect of compound heterozygotes on iron status. Our sample size was insufficient to examine this. A general limitation of the present study was the few patients with at‐risk combinations, particularly among controls. For instance, the status of bicarriers of HFE C282Y and transferrin C2 was seen in only 4 of 197 controls, 4 of 67 patients with MCI and 15 of 172 patients with Alzheimer's disease. These figures illustrate the high risk for Alzheimer's disease associated with bicarrier status (OR 4.6; 95% CI 1.5 to 14). Owing to this shortage of at‐risk combinations, and also to capture as many preclinical cases as possible, we combined controls and MCI to form one non‐AD group (we found no significant differences in transferrin saturation between controls and those with MCI among bicarriers, among compound heterozygotes or among the other groups combined; also, virtually all those with MCI were controls when their blood was taken). Given the small numbers, it was surprising to achieve as many significant results, for instance, when comparing only eight bicarriers without Alzheimer's disease, first with the 256 others from the non‐AD group and then with 15 bicarriers with Alzheimer's disease. Further, the two different genetic combinations had a similar pattern of results ((tablestables 1 and 22).

In conclusion, although the association of HFE compound heterozygotes with higher transferrin saturation is well established,2,3,4,5,6 our study suggests two new findings: that a similar association may exist in bicarriers of HFE C282Y and transferrin C2, and that neither association may be found in patients with mature Alzheimer's disease, only in those without dementia. We may thus suggest a mechanism for the associations with risk for Alzheimer's disease reported in our earlier study7—higher iron load and therefore oxidative stress in the preclinical phase of Alzheimer's disease. However, our results should be considered provisional. Two larger studies are now needed—one to examine the associations of compound heterozygosity and of bicarrier status on risk for Alzheimer's disease, and another to study the effects of these two genetic combinations on the iron status of those without dementia, particularly elderly people. A further study should examine the genetic effects on iron levels in the brain, both in those with Alzheimer's disease and in those without dementia, using the latest scanning techniques. If replicated, these results will be relevant to the prevention of Alzheimer's disease. The allelic frequencies of our controls were 5.6% for HFE C282Y, 17.5% for HFE H63D and 24.6% for transferrin C2. Taking more conservative figures of 6%, 13%3,24 and 19%,7,25 respectively, from the literature, then 4% of northern Europeans are expected to be bicarriers of HFE C282Y and transferrin C2, and just <3% to be compound heterozygotes with both HFE C282Y and HFE H63D. That makes altogether around 6% of northern Europeans at risk. Further, iron overload is a readily treatable condition.

Acknowledgements

We thank all patients and volunteers, members of OPTIMA and the Department of Neuropathology, Radcliffe Infirmary, Oxford, UK.

Abbreviations

APOE4 - apolipoprotein E ε4 allele

HFE - haemochromatosis gene

MCI - mild cognitive impairment

OPTIMA - Oxford Project To Investigate Memory and Ageing

Footnotes

Funding: We thank the Norman Collisson Foundation for financial support.

Competing interests: None.

Informed consent was obtained in writing from all participants and the study was approved by the Central Oxford Ethics Committee (approval number 1656).

References

1. Merryweather‐Clarke A T, Worwood M, Parkinson L, Mattock C, Pointon J J, Shearman J D, Robson K J. The effect of HFE mutations on serum ferritin and transferrin saturation in the Jersey population. Br J Haematol 1998. 101369–373.373. [PubMed]
2. Beutler E, Felitti V, Gelbert T, Ho N. The effect of HFE genotypes on measurements of iron overload in patients attending a health appraisal clinic. Ann Intern Med 2000. 133329–337.337. [PubMed]
3. Jackson H A, Carter K, Darke C, Guttridge M G, Ravine D, Hutton R D, Napier J A, Worwood M. HFE mutations, iron deficiency and overload in 10,500 blood donors. Br J Haematol 2001. 114474–484.484. [PubMed]
4. Rossi E, Bulsara M K, Olynyk J K, Cullen D J, Summerville L, Powell L W. Effect of hemochromatosis genotype and lifestyle factors on iron and red cell indices in a community population. Clin Chem 2001. 47202–208.208. [PubMed]
5. Beutler E, Felitti V, Gelbart T, Waalen J. Haematological effects of the C282Y HFE mutation in homozygous and heterozygous states among subjects of northern and southern European ancestry. Br J Haematol 2003. 120887–893.893. [PubMed]
6. Adams P C, Reboussin D M, Barton J C, Barton J C, McLaren C E, Eckfeldt J H, McLaren G D, Dawkins F W, Acton R T, Harris E L, Gordeuk V R, Leiendecker‐Foster C, Speechley M, Snively B M, Holup J L, Thomson E , Sholinsky P. Hemochromatosis and iron‐overload screening in a racially diverse population. N Engl J Med 2005. 3521769–1778.1778. [PubMed]
7. Robson K J H, Lehmann D J, Wimhurst V L C, Livesey K J, Combrinck M, Merryweather‐Clarke A T, Warden D R, Smith A D. Synergy between the C2 allele of transferrin and the C282Y allele of the haemochromatosis gene (HFE) as risk factors for developing Alzheimer's disease. J Med Genet 2004. 41261–265.265. [PMC free article] [PubMed]
8. Ohm T G, Müller H, Braak H, Bohl J. Close‐meshed prevalence rates of different stages as a tool to uncover the rate of Alzheimer's disease‐related neurofibrillary changes. Neuroscience 1995. 64209–217.217. [PubMed]
9. Nunomura A, Perry G, Aliev G, Hirai K, Takeda A, Balraj E K, Jones P K, Ghanbari H, Wataya T, Shimohama S, Chiba S, Atwood C S, Petersen R B, Smith M A. Oxidative damage is the earliest event in Alzheimer disease. J Neuropathol Exp Neurol 2001. 60759–767.767. [PubMed]
10. Praticò D. Alzheimer's disease and oxygen radicals: new insights. Biochem Pharmacol 2002. 63563–567.567. [PubMed]
11. Smith M A, Harris P L, Sayre L M, Perry G. Iron accumulation in Alzheimer disease is a source of redox‐generated free radicals. Proc Natl Acad Sci USA 1997. 949866–9868.9868. [PubMed]
12. Perry G, Taddeo M A, Petersen R B, Castellani R J, Harris P L, Siedlak S L, Cash A D, Liu Q, Nunomura A, Atwood C S, Smith M A. Adventitiously‐bound redox active iron and copper are at the center of oxidative damage in Alzheimer disease. Biometals 2003. 1677–81.81. [PubMed]
13. Mirra S S, Heyman A, McKeel D, Sumi S M, Crain B J, Brownlee L M, Vogel F S, Hughes J P, van Belle G, Berg L. The Consortium to Establish a Registry for Alzheimer's Disease (CERAD). Part II. Standardization of the neuropathologic assessment of Alzheimer's disease. Neurology 1991. 41479–486.486. [PubMed]
14. McKhann G, Drachman D, Folstein M, Katzman R, Price D, Stadlan E M. Clinical diagnosis of Alzheimer's disease: report of the NINCDS‐ADRDA work group under the auspices of Department of Health and Human Services task force on Alzheimer's disease. Neurology 1984. 34939–944.944. [PubMed]
15. Petersen R C, Smith G E, Waring S C, Ivnik R J, Tangalos E G, Kokmen E. Mild cognitive impairment: clinical characterization and outcome. Arch Neurol 1999. 56303–308.308. [PubMed]
16. Roth M, Huppert F A, Tym E, Mountjoy C Q. CAMDEX: the Cambridge examination for mental disorders of the elderly. Cambridge: Cambridge University Press, 1988.
17. Worwood M. Iron deficiency anaemia and iron overload. In: Lewes SM, Bain BJ, Bates I, eds. Dacie & Lewis practical haematology. London: Churchill Livingstone, 2001. 115–128.128.
18. Feder J N, Penny D M, Irrinki A, Lee V K, Lebrón J A, Watson N, Tsuchihashi Z, Sigal E, Bjorkman P J, Schatzman R C. The hemochromatosis gene product complexes with the transferrin receptor and lowers its affinity for ligand binding. Proc Natl Acad Sci USA 1998. 951472–1477.1477. [PubMed]
19. Zatta P, Messori L, Mauri P, van Rensburg S J, van Zyl J, Gabrielli S, Gabbiani C. The C2 variant of human serum transferrin retains the iron binding properties of the native protein. Biochim Biophys Acta 2005. 1741264–270.270. [PubMed]
20. Skoog I, Lernfelt B, Landahl S, Palmertz B, Andreasson L A, Nilsson L, Persson G, Odén A, Svanborg A. 15‐year longitudinal study of blood pressure and dementia. Lancet 1996. 3471141–1145.1145. [PubMed]
21. Kivipelto M, Helkala E L, Laakso M P, Hanninen T, Hallikainen M, Alhainen K, Soininen H, Tuomilehto J, Nissinen A. Midlife vascular risk factors and Alzheimer's disease in later life: longitudinal, population based study. BMJ 2001. 3221447–1451.1451. [PMC free article] [PubMed]
22. Miyata M, Smith J D. Apolipoprotein E allele‐specific antioxidant activity and effects on cytotoxicity by oxidative insults and beta‐amyloid peptides. Nat Genet 1996. 1455–61.61. [PubMed]
23. Ramassamy C, Averill D, Beffert U, Bastianetto S, Theroux L, Lussier‐Cacan S, Cohn J S, Christen Y, Davignon J, Quirion R, Poirier J. Oxidative damage and protection by antioxidants in the frontal cortex of Alzheimer's disease is related to the apolipoprotein E genotype. Free Radic Biol Med 1999. 27544–553.553. [PubMed]
24. Merryweather‐Clarke A T, Pointon J J, Shearman J D, Robson K J H. Global prevalence of putative haemochromatosis mutations. J Med Genet 1997. 34275–278.278. [PMC free article] [PubMed]
25. Van Landeghem G F, Sikström C, Beckman L E, Adolfsson R, Beckman L. Transferrin C2, metal binding and Alzheimer's disease. Neuroreport 1998. 9177–179.179. [PubMed]

Articles from Journal of Medical Genetics are provided here courtesy of BMJ Group