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Association of Mutations in the Hemochromatosis Gene With Shorter Life Expectancy
Lise Bathum, MD, PhD;
Lene Christiansen, PhD;
Hanne Nybo, MD;
Karen Andersen Ranberg, MD;
David Gaist, MD, PhD;
Bernard Jeune, MD;
Niels Erik Petersen, MD, PhD;
James Vaupel, PhD;
Kaare Christensen, MD, PhD
Arch Intern Med. 2001;161:2441-2444.
ABSTRACT
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Background To investigate whether the frequency of carriers of mutations in the HFE gene associated with hereditary hemochromatosis diminishes
with age as an indication that HFE mutations are
associated with increased mortality. It is of value in the debate concerning
screening for hereditary hemochromatosis to determine the significance of
heterozygosity.
Methods Genotyping for mutations in exons 2 and 4 of the HFE gene using denaturing gradient gel electrophoresis in 1784 participants
aged 45 to 100 years from 4 population-based studies: all 183 centenarians
from the Danish Centenarian Study, 601 people aged 92 to 93 years from the
Danish 1905 Cohort, 400 aged 70 to 94 years from the Longitudinal Study of
Aging Danish Twins, and 600 aged 45 to 67 years from a study of middle-aged
Danish twins.
Results All participants (N=1784) were screened for mutations in exon 4, and
a trend toward fewer heterozygotes for the C282Y mutation—the mutation
most often associated with hereditary hemochromatosis—was found. This
was significant for the whole population (P=.005)
and for women (P=.004) but not for men (P=.26). A group of 599 participants was screened for mutations in exon
2, and there was no variation in the distribution of mutations in exon 2 in
the different age groups.
Conclusions In a high–carrier frequency population like Denmark, mutations
in HFE show an age-related reduction in the frequency
of heterozygotes for C282Y, which suggests that carrier status is associated
with shorter life expectancy.
INTRODUCTION
HEREDITARY hemachromatosis is the most common inherited disease in Europeans.
It occurs in as many as 5 in every 1000 individuals of northern European heritage,1 with 10% to 15% of the population being carriers of
mutations in the HFE gene associated with hereditary
hemochromatosis.
Hereditary hemochromatosis is due to an abnormal absorption of iron
from the intestine. As iron loading progresses, it leads to irreversible damage
of many organs and tissues, resulting in hepatic fibrosis and cirrhosis, endocrine
dysfunction, cardiomyopathy, or arthropathy.2
The classic "bronzed diabetes" (skin pigmentation, diabetes mellitus, and
cirrhosis) represents only a small fraction of affected individuals, usually
those in whom the diagnosis has been undetected for many years. Hereditary
hemochromatosis more often presents with nonspecific complaints, such as joint
pain, fatigue, and abdominal pain.3
In 1996, the candidate gene for hereditary hemochromatosis, HFE, was identified.4 This gene codes
for a transmembrane protein that is presumed to be involved in the regulation
of the intracellular iron level. Three common mutations have been found: C282Y,
H63D, and S65C. Studies1-8
in different countries have shown that approximately 80% to 100% of patients
with clinically diagnosed hemochromatosis are homozygotes for the C282Y mutation,
whereas the impact of H63D and S65C is more uncertain. It seems likely that
the compound heterozygotes C282Y/H63D and C282Y/S65C are at increased risk
of developing hemochromatosis.5-8
The distribution of these mutations differs in different populations. The
frequency of heterozygosity for C282Y is 9.6% in white people in the United
States,9 17.3% in northern Ireland,10 13.2% in New Zealand,11
9.6% in northern Germany,12 and 13.3% in Denmark.13 This mutation is absent in populations of African,
Asian, or Australian descent.14
The importance of hereditary hemochromatosis is based on its prevalence,
its remarkably diverse clinical spectrum, and the fact that early treatment
(ie, venesection) is effective in preventing its clinical manifestations.
The clinical diagnosis of hereditary hemochromatosis is established much more
rarely than expected, and strong association between C282Y homozygosity and
hereditary hemochromatosis makes it tempting to suggest population-based genetic
screening for this mutation.1, 15
However, several issues need to be clarified before the conditions for screening
are fulfilled. What proportion of individuals with hereditary hemochromatosis
on a molecular level will develop serious clinical manifestations, and what
is the impact on heterozygotes? Examinations of the frequency of C282Y homozygotes
in healthy populations and in other diseases have shown that the clinical
penetrance of this mutation is low. Some studies16-17
have identified individuals who are homozygous for C282Y but do not fulfill
the clinical and biochemical criteria for hemochromatosis.
Heterozygotes for mutations in HFE have significantly
higher serum iron and transferrin saturation11
and a lower frequency of iron deficiency anemia.9
Furthermore, new studies strongly suggest a relation between the storage of
iron and cardiovascular diseases. Two studies18-19
have shown that heterozygosity for mutations in HFE
confers a significant increase in risk for cardiovascular events in men and
women. However, the evidence for adverse effects of being a carrier is still
sparse.
Iron is a potent promotor of generation of free radicals—strong
reactive ions that react with cell membranes and cell organelles. Free radicals
are believed to play a role in the development of cancer and cardiovascular
diseases and in aging, although there is no good experimental evidence.20-21 HFE mutations
thus represent a group of common mutations that may affect the aging processes,
morbidity, and mortality possibly through the accumulation of iron. Except
for apolipoprotein E,22 no longevity-associated
genes have been firmly established, and the HFE mutations
are of particular interest because of the potential for prevention of harmful
effects through venesection.
In this study, we genotyped 1784 individuals aged 45 to 100 years from
population-based studies for mutations in HFE to
investigate whether the frequency of heterozygotes for mutations in HFE diminishes by age. If so, this would indicate that
heterozygotes for the mutations in HFE have increased
mortality rates starting at midlife.
SUBJECTS, MATERIALS, AND METHODS
We conducted 4 major surveys and collected biological material from
1995-1999: the Danish 1905 Cohort, all Danes born in 1905 (1632 DNA samples)23; the Longitudinal Study of Aging Danish Twins, all
Danish twins 70 years and older (2265 DNA samples)24;
a study of middle-aged Danish twins, a random sample of twin pairs born between
1931 and 1952 (4171 DNA samples)25; and the
Danish Centenarian Study, all persons living in Denmark who celebrated their
100th birthday between April 1, 1995, and May 31, 1996, and centenarians from
the island of Funen, who participated in the pilot study in 1994 (183 DNA
samples).22, 26 These studies comprised
a home-based 2-hour multidimensional interview and sampling of DNA by means
of a finger prick or a cheek swab, except in the Danish Centenarian Study,
in which full blood samples were collected. We randomly selected DNA samples
from 600 individuals in the middle-aged twins study and from 400 in the Longitudinal
Study of Aging Danish Twins, but only 1 participant from each twin pair was
included. From the Danish 1905 Cohort, we selected 300 men and 301 women.
From the Danish Centenarian Study, DNA samples from all participants were
included. This selection scheme aimed at getting precise estimates at middle
ages and among the oldest old.
DNA samples were isolated from cheek swabs and blood spots using the
QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). Exons 2 and 4 were screened
using denaturing gradient gel electrophoresis27
after previous amplification using an external set of primers. Exons 2 and
4 were analyzed in 599 samples (200 each from the Longitudinal Study of Aging
Danish Twins and middle-aged twins cohort and 199 from the Danish 1905 Cohort)
and only exon 4 was analyzed in the remaining samples. In case of abnormal
patterns, the relevant exons were sequenced using a Thermo Sequenase Fluorescent–labeled
primer cycle sequencing kit (Amersham, Pharmacia Biotech Inc, Piscataway,
NJ) and Alf Express (Amersham, Pharmacia Biotech AB, Uppsala, Sweden).
Hardy-Weinberg equilibrium was tested using the  2 test.
The 95% confidence intervals for the proportion of heterozygotes for C282Y
in an age group were calculated using the normal distribution. The 2 test for trend was used for comparing the frequencies among the different
age groups, and the uncorrected 2 test was used for comparing
proportions.28 EpiCalc 2000 (version 1.02;
Gilmann and Myatt, Brixton, England) was used for statistical analysis.
RESULTS
A total of 599 samples were screened for mutations in exon 2. The H63D
and S65C mutations were found with the same frequency across the age groups
(Table 1). Exon 4, the clinically
most important, was screened in all samples. The C282Y carrier frequencies
and 95% confidence intervals are given in Table 2. The overall distribution was in Hardy-Weinberg equilibrium
(P = .4). All samples showing variant band patterns
were sequenced (2 samples), and the following mutations were found in exon
4: C917T (this participant was also heterozygous for the H63D mutation in
exon 2) and T1105C. The reduction in heterozygotes for C282Y is significant
in the population as a whole (P = .005) and in women
(P = .004) but not in men (P
= .26). The frequency of heterozygotes for C282Y in the youngest age group
was 12.4% in men and 19.5% in women, but this difference was not significant
(P = .09).
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Table 1. Frequency of Mutations in Exons 2 and 4 in 599 Individuals
Screened in Exons 2 and 4 of the HFE Gene*
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Table 2. C282Y Carrier Frequency (and 95% Confidence Intervals [CIs])
in the Different Age Groups in Men, Women, and the Total Population
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COMMENT
In this study, we investigated the frequency of carriers of 3 common HFE gene mutations—C282Y, H63D, and S65C—in
different age groups. No apparent difference was found for H63D and S65C (Table 1). However, the results indicate
an age-related reduction in the carrier frequency of the hemochromatosis-related
C282Y mutation (Table 2). These
findings are consistent with the hypothesis that there may be a survival difference
from middle age onward, especially for women, among carriers and noncarriers
of the C282Y allele. The age-related reduction is only minor after age 65
years—a trend that has also been shown for obesity.29
Our data thus imply that there is selection against carriers of C282Y and
that this manifests before age 65 years.
The observed reduction in C282Y carrier frequency persisted until age
95 years; however, the frequency in the centenarian group is the same as that
in the youngest group for both men and women. There are several possible explanations
for this. Although 183 centenarians is a large sample, including most of the
centenarians in Denmark who reached age 100 years in the study year, it is
not large enough to give a reliable carrier frequency. This can be seen in
the broad 95% confidence intervals in Table
2; therefore, it may be a chance finding. Another possible explanation
is that heterozygosity for C282Y has a higher mortality rate in the younger
groups but becomes beneficial in the oldest old (antagonistic pleiotropy).
Furthermore, the fact that some become octogenarians or nonagenarians with
a "bad" gene could be balanced by a good composition of other relevant genes
or lifestyle, making it more likely that they become centenarians.
There are several limitations to our study. The major limitation is
the inability to study directly the effect of the C282Y allele on survival.
The results suggest that there is a survival difference, but more specific
data on the relationship of the C282Y allele to mortality rates are needed
before such a conclusion can be made because the design used here is vulnerable
to migration and association to twin status. However, it seems unlikely that
migration or twin status should be associated with HFE
mutation status. Another limitation is that our study does not give an idea
of the mechanisms behind this survival difference. It is tempting to suggest
that the difference is owing to accumulation of iron, but this should be further
investigated. It is well known that heterozygotes for mutations in HFE have a higher iron content, but it has until now been assumed that
this was without significant influence on morbidity and mortality rates. Our
finding that heterozygotes for C282Y may have reduced life expectancy raises
the question of whether the elevated iron stores affect mortality and morbidity
rates, although some other effect of C282Y is possible.
It is well known that there is a low incidence of myocardial infarction
in menstruating women. The finding that heterozygosity for mutations in HFE confers a significant increase in risk for cardiovascular
events in men and women18 is compatible with
the hypothesis that iron plays an important role in ischemic injury. In that
case, iron depletion could have a large protective effect.19
However, if the accumulation of iron is in fact the mechanism behind this
apparently increased mortality rate among heterozygotes for C282Y, then our
study would probably underestimate the increased mortality rate because several
individuals without this mutation may nonetheless have higher-than-normal
stored iron levels owing to other known or unidentified iron-loading mutations.
Several studies have demonstrated a familial aggregation of premature
myocardial infarction.30 Some of this can be
explained by hypertension or hyperlipidemia; however, in a large proportion
of high-risk families, no aggregation of the known risk factors is seen. It
is possible that this familial aggregation is due to a gene that favors iron
absorption.31 A relevant question then is whether
the increased mortality can be prevented by deliberate iron depletion by regular
blood donation, by recommending a diet with a low iron content, or by warning
against taking vitamins that contain iron.
The finding of 3 homozygotes for C282Y (2 aged 93 years and 1 aged 70
years) is not surprising because earlier studies have shown that the penetrance
of hereditary hemochromatosis in homozygotes for C282Y is low—probably
less than 50%. According to the Hardy-Weinberg law, we should have found 6
participants homozygous for C282Y, with an overall allele frequency of 6%.
This study shows an age-related reduction in the carrier frequency of
C282Y in the HFE gene, suggesting that carriers have
a shorter life expectancy. Our study does not shed light on which mechanisms
are behind this shorter life expectancy or whether this increased mortality
can be prevented by, for instance, venesection. Future research needs to elucidate
this before a decision concerning population-based screening for mutations
in HFE is made.
AUTHOR INFORMATION
Accepted for publication July 24, 2001.
This study was supported by research grant NIA-PO1-AG08761 from the
National Institute on Aging; the Danish National Research Foundation, Copenhagen,
Denmark; A. J. Andersen and Wifes Foundation, Odense; the Grant Committee
of the Consultancy Council, Odense University Hospital, Odense; the Medical
Research Projects Foundation of Funen County, Odense; and the Danish Medical
Association Research Fund, Copenhagen.
The valuable technical assistance of Alice Jensen and Susanne Knudsen
is highly appreciated.
Corresponding author and reprints: Lise Bathum, MD, PhD, Department
of Clinical Biochemistry, Odense University Hospital, Sdr. Blvd 29, DK-5000
Odense C, Denmark (e-mail: Lise.Bathum{at}ouh.fyns-amt.dk).
From the Department of Clinical Biochemistry, University Hospital,
Odense, Denmark (Drs Bathum, Christiansen, and Petersen); The Danish Center
for Demographic Research and Epidemiology, Institute of Public Health, University
of Southern Denmark, Odense (Drs Nybo, Andersen Ranberg, Gaist, Jeune, Vaupel,
and Christensen); the Max Planck Institute for Demographic Research, Rostock,
Germany (Dr Vaupel); and the Terry Stanford Institute, Duke University, Durham,
NC (Drs Vaupel and Christensen).
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