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Steinberg KK, Cogswell ME, Chang JC, et al. Prevalence of C282Y and H63D Mutations in the Hemochromatosis (HFE) Gene in the United States. JAMA. 2001;285(17):2216–2222. doi:10.1001/jama.285.17.2216
Author Affiliations: Division of Laboratory Sciences (Drs Steinberg, Chang, Caudill, Sampson, and Gallagher), Office of Genetics and Disease Prevention (Dr Khoury), National Center for Environmental Health, and Divisions of Nutrition and Physical Activity (Drs Cogswell and Grummer-Strawn) and Diabetes Translation (Dr Bowman), National Center for Chronic Disease Prevention and Health Promotion, Centers for Disease Control and Prevention, Chamblee, Ga; and Division of Health Examination Statistics, National Center for Health Statistics, Centers for Disease Control and Prevention, Rockville, Md (Dr McQuillan).
Context Population-based estimates of the prevalence of disease-associated mutations,
such as hemochromatosis (HFE) gene mutations, are
needed to determine the usefulness of genetic screening.
Objective To estimate the prevalence of the HFE mutations
C282Y and H63D in the US population.
Design Cross-sectional population-based study of samples in the DNA bank from
phase 2 of the Third National Health and Nutrition Examination Survey conducted
from 1992 to 1994.
Setting and Participants Genotyped samples of cells from a total of 5171 participants, cross-classified
by sex, age, and race/ethnicity in the analysis.
Main Outcome Measures Estimates of the prevalence of C282Y and H63D mutations.
Results The prevalence of C282Y homozygosity is estimated to be 0.26% (95% confidence
interval [CI], 0.12%-0.49%); 1.89% (95% CI, 1.48%-2.43%) for H63D homozygosity;
and 1.97% (95% CI, 1.54%-2.49%) for compound heterozygosity. The prevalence
estimates for C282Y heterozygosity (C282Y/wild type) are 9.54% among non-Hispanic
whites, 2.33% among non-Hispanic blacks, and 2.75% among Mexican-Americans.
The prevalence estimates of the C282Y mutation in the US population are 5.4%
(95% CI, 4.7%-6.2%) and 13.5% (95% CI, 12.5%-14.8%) for the H63D mutation.
Conclusions Estimates of prevalence of HFE mutations are
within the expected range for non-Hispanic whites and blacks but the estimated
prevalence of the C282Y mutation among Mexican-Americans is less than expected.
Mutation data now need to be linked to clinically relevant indices, such as
transferrin saturation level.
Now that the human genome has been sequenced, and as scientists elucidate
the location and function of all human genes, public health policymakers face
the daunting task of making complex decisions about the appropriateness and
usefulness of genetic screening. To make such decisions, they will first need
population-based estimates of the prevalence and penetrance of these mutations.
Hereditary hemochromatosis (HH), a major form of iron overload disease, can
serve as a model for making such decisions.
Hereditary hemochromatosis is a disorder of iron metabolism in which
excess iron absorption leads to deposition of iron in multiple organs, resulting
in cirrhosis, diabetes, cardiomyopathy, or hypogonadism, and can lead to early
death.1 Hemochromatosis is one of the most
common autosomal recessive disorders among whites in the United States. The
estimated prevalence is between 1 in 200 and 1 in 500 individuals in screening
studies conducted among patients in primary care settings, employed individuals,
blood donors, and other groups in which case definitions are based on elevated
levels of iron.2 A presumptive diagnosis of
hemochromatosis is based on elevated fasting transferrin saturation levels.3 When predisposition to iron overload is identified
early in the course of disease, organ failure can be prevented by periodic
The hemochromatosis gene (HFE) has been localized
to the short arm of chromosome 6 and has been identified as a major histocompatibility
complex class I-like gene.5 Recent evidence
shows that the protein coded for by HFE binds to
the transferrin receptor and reduces its affinity for iron-bound transferrin.6-8 Two missense mutations
in HFE, denoted C282Y and H63D, account for most
cases of HH among individuals of European descent.5,9-13
Homozygosity for the C282Y mutation accounts for an estimated 50% to 100%
of HH cases.5,9-13
Although the true prevalence remains unknown, 10% of the US population is
estimated to be heterozygous for the C282Y mutation, which can be associated
with increased levels of transferrin saturation, but which is only rarely
associated with liver damage.14-16
A small percentage of cases of HH are attributable to the C282Y/H63D compound
heterozygous state, which is estimated to have a prevalence rate of 1.4% to
2.4% in populations of European descent.16-18
Homozygosity for the H63D mutation has been associated with a significantly
increased risk for phenotypic expression of hemochromatosis (odds ratio, 9.0;
95% confidence interval [CI], 1.7-47).9,16
No study has provided a true estimate of the prevalence of the 2 mutations
within the US population. Previous prevalence estimates were derived from
small, nonrepresentative US samples, such as employed individuals (n = 1653),
or individuals recruited as control subjects in studies comparing them with
patients with hemochromatosis (sample sizes ranged from 142-384).4,8,16,19 In
addition, none of these studies included a large sample of individuals from
To obtain an estimate of the prevalence of the C282Y and H63D mutations
in the US population, we genotyped 5171 specimens from a nationally representative
sample derived from the Third National Health and Nutrition Examination Survey
(NHANES III) DNA bank.20 We then examined the
prevalence of the 2 mutations by race/ethnicity, sex, and age. Ours is the
first study using this nationally representative sample to estimate the prevalence
of disease-associated mutations.
NHANES is a series of national surveys that the National Center for
Health Statistics began conducting in 1966. Among its several objectives,
NHANES data are used to estimate the national prevalence of common diseases
and risk factors for those diseases in the US population. As part of NHANES
III, which was conducted in 2 phases from 1988 through 1994,21,22
certain populations, including non-Hispanic blacks and Mexican Americans,
were oversampled. Both phase 1 and phase 2 were nationally representative.
Prevalence estimates were weighted to account for oversampling and nonresponse
to the household interview and the examination.
Cell lines from 8205 participants in phase 2 of NHANES III (1992-1994)
were immortalized with Epstein-Barr virus. Although the Centers for Disease
Control and Prevention planned to collect DNA for storage, the decision to
establish cell lines occurred after phase 1 had already begun. Overall, 15 946
individuals aged 12 years or older (who did not list "other" as race/ethnicity)
were selected as part of phase 2. A total of 13 012 (81.6%) were interviewed,
11 960 (75.2%) were examined, and cell lines were available for 8205
(69%) of examined individuals. All estimates are weighted to represent the
total US population and to account for oversampling and nonresponse to the
household interview and physical examination. The sample for genotyping included
phase 2 participants who were 12 years or older, who were not pregnant, and
who did not have "other" listed for race/ethnicity. Because this sample was
also used to study associations between total iron-binding capacity (transferrin
saturation) level and HFE genotype (a study still
in progress), we excluded participants for whom transferrin values were missing
(3.7%). Serum iron and serum ferritin levels were also obtained (in addition
to transferrin saturation levels, these data are not reported herein). To
ensure that previously masked specimens, which had been given new identification
numbers, remained anonymous (ie, no one could link an HFE genotype to a personal identifier in the full NHANES data set), it
was necessary to guarantee that no fewer than 5 individuals had the same set
of background characteristics (age, sex, race/ethnicity). These common characteristics
constituted a sampling cell. Of the individuals within a sampling cell who
did have a common set of background characteristics, we randomly eliminated
20% or 2 of the subjects, whichever number was larger. Two additional specimens
could not be amplified for genotyping, making the final sample size 5171.
An institutional review board at the National Center for Health Statistics
approved the survey as well as the specific analysis done for this report.
All participants gave written informed consent for the survey. Principles
involved in consideration of research on the samples are represented in a
previous publication.23 The specimens were
masked, irrevocably destroying the ability to link HFE
genotype to individual participants, because informed consent did not expressly
mention genetic studies (at the time the survey was planned, there were no
specific tests planned that could be described to participants and the institutional
review board felt that a discussion of DNA would not be helpful to participants).
Also, the penetrance and clinical significance of HFE
mutations have yet to be established. Finally, study participants were made
aware of abnormalities in results of testing (all participants in NHANES III
were notified regarding serum ferritin levels, although it is possible that
they could have had HFE mutations without altered
Specimens were genotyped in the Molecular Biology Branch of the Division
of Laboratory Sciences, National Center for Environmental Health, Centers
for Disease Control and Prevention, using genomic DNA extracted from Epstein-Barr
virus–transformed cell lines. The wild type (WT) and C282Y and H63D
mutations were genotyped using TaqMan technology24-26
in which amplification and genotyping are simultaneously performed using the
ABI PRISM 7700 (Applied Biosystems, Foster City, Calif).
Separate polymerase chain reactions were performed for C282Y and H63D
sites using primers and probes that were designed using Primer Express (Applied
Biosystems) based on published sequences. The H63D site sequences used were
as follows: forward primer, 5′TCTTTCCTTGTTTGAAGCTTTGG; reverse primer,
5′TCCCACCCTTTCAGACTCTGA; WT probe, 5′ FAM CACGGCGACTCTCATGATCATAGAACAC;
mutant probe, 5′ VICCACGGCGACTCTCATCATCATAGAACAC. The C282Y site sequences
were: forward primer, 5′GGCTGGATAACCTTGGCTGTAC; reverse primer, 5′TCACATACCCCAGATCACAATGA;
WT probe, 5′ FAM TGCTCCACCTGGCACGTATATCTCTG; mutant probe, 5′
Discrimination between alleles was accomplished by running allelic discrimination
using ABI PRISM 7700, following the manufacturer's protocol. In addition,
all allele calls were confirmed by visual inspection of the results of the
multicomponent analysis of the real-time polymerase chain reaction. We randomly
selected approximately 5% of the specimens for genotype confirmation using
the restriction fragment-length polymorphism method described by Lynas.27 In addition, we confirmed all samples with the C282Y/C282Y
genotype. Genotype calls, as determined by the restriction fragment-length
polymorphism method, were 100% concordant with genotype calls obtained by
TaqMan polymerase chain reaction analysis.
NHANES III was designed so that both phase 1 and phase 2 would be national
probability samples. Prevalence estimates are weighted to give an estimate
representing the US population.22 Weighting
accounts for oversampling of non-Hispanic black and Mexican American populations,
probability of selection, noncoverage, and nonresponse. For each sample cell,
we determined average weights and assigned them to each individual in that
cell. The average weights were used to calculate the weighted prevalence estimates
using SAS software (SAS Institute Inc, Cary, NC).28
We computed weighted prevalence estimates of HFE
mutations in the population by determining the appropriate functions that
relate allele frequency to genotype frequencies and then applying the average
weights, as before, using SAS. For example, to compute the weighted prevalence
estimate for the C282Y mutation, we determined the weighted estimate of the
variable C282Y using the following definitions: C282Y = (2 × C282_HO
+ C282_HE + C282_H6)/2 in which C282_HO is the NHANES III variable corresponding
to the homozygous C282Y/C282Y genotype, C282_HE is the variable corresponding
to the heterozygous C282Y/WT genotype, and C282_H6 is the variable corresponding
to the compound heterozygous C282Y/H63D genotype.
We were unable to calculate SEs accounting for the complex sample design
because anonymity requirements prevented access to cluster variables. Therefore,
to account for the complex sampling design, we used the binomial distribution
to construct approximate CIs for the weighted estimates using sample sizes
determined by dividing actual sample sizes by an assumed design effect of
1.5. In reality, the design effect for this analysis may be lower or higher
than 1.5. Random selection within strata, for example, tends to lower the
Using an average design effect of 1.5, we found that genotypes from
2454 participants would be required to ensure 80% power to estimate a mean
(SE) prevalence of 0.4% (0.2%), which was the expected prevalence of homozygosity.
At least 354 participants would be needed to estimate a 10% (2.5%) prevalence,
which was the expected prevalence of C282Y/WT heterozygosity.
We randomly selected 5171 specimens for study from participant cells
cross-classified by sex, age, race/ethnicity, and transferrin saturation level.
When weighted properly, these specimens should be representative of the US
Based on our results, homozygosity for the C282Y mutation was estimated
to occur in 0.26% (95% CI, 0.12%-0.49%) of the total US population, and compound
heterozygosity (C282Y/H63D) in approximately 2% (Table 1). Among WT heterozygous genotypes (C282Y/WT and H63D/WT),
the genotype H63D/WT was about 2.5-fold more common than the genotype C282Y/WT.
Homozygosity for the H63D mutation was estimated to occur in 1.89% (95% CI,
1.48%-2.43%) of the total US population.
When we estimated prevalence of genotypes by ethnic group, we found
that the estimate for the C282Y/C282Y genotype was 5- to 10-fold higher in
non-Hispanic whites than in Mexican Americans or non-Hispanic blacks (Table 1). The CIs for prevalence estimates
for C282Y/C282Y overlapped among the 3 groups, but the estimates were made
on the basis of only 1 person each in the Mexican American and non-Hispanic
black groups. The estimated prevalence for the H63D/H63D genotype was highest
among non-Hispanic whites and lowest among non-Hispanic blacks, with estimates
for Mexican Americans falling between the 2 previous groups. However, differences
among the groups were statistically significant only between non-Hispanic
blacks and non-Hispanic whites. Prevalence estimates for compound heterozygotes
(C282Y/H63D) were also significantly higher for non-Hispanic whites than for
the other groups. Although the C282Y/WT genotype was estimated to be significantly
more common in non-Hispanic whites than in other groups, non-Hispanic whites
and Mexican Americans had similar prevalence estimates for the H63D/WT genotype,
and both of these groups had significantly higher prevalence estimates for
H63D/WT than did non-Hispanic blacks.
Calculations using these genotype frequency data indicated that the
C282Y mutation is estimated to be present in 5.4% of the total US population
and the H63D mutation in 13.5% (Table 2). These percentages are slightly higher when data from the non-Hispanic
white population are used in separate calculations.
We found no significant differences in prevalence estimates for genotypes
between men and women when ethnic groups were combined (Table 3). For all genotypes, CIs around the estimates for men and
women overlapped. Although there were generally no differences in prevalence
of genotype by age, these estimates are based on a small number of individuals,
as only 3 individuals younger than 60 years had the C282Y/C282Y genotype (Table 4).
We evaluated the prevalence of the C282Y and H63D mutations in the HFE gene in a representative sample of the US population.
These estimates are derived from a nationally representative sample of sufficient
size to provide power for a more precise estimate of mutations associated
with HH. Data from NHANES III indicate that the estimated prevalence of the
C282Y/C282Y genotype, which is associated with 50% to 100% of HH in the US
population of European descent,5,9-13
is 0.26% or approximately 1 in 385 individuals. This frequency level falls
within published estimates of between 1 in 200 and 1 in 500 individuals and
translates into approximately 718 000 individuals who are homozygous
for the C282Y mutation in the United States.2
Prevalence estimates for all other genotypes among non-Hispanic whites were
similar to those reported in other studies that genotyped large samples from
populations of European descent (Table 5). It is reasonable to assume that the group of NHANES III participants
who identified themselves as non-Hispanic white is likely to represent many
individuals of European descent.
Although NHANES was not designed to estimate the prevalence of rare
genotypes, this population did offer a sample of sufficient size to provide
power to estimate prevalence for both the C282Y and H63D mutations. However,
the power to estimate prevalence of homozygosity for the C282Y mutation with
precision was sufficient only for the total population of 5171 participants.
We compared demographic variables of race/ethnicity, sex, and age between
the original sample of 8502 and our final sample of 5171 and found no significant
differences, thus indicating that the sample of 5171 was not biased.
The largest and most recent estimates of genotype and allele frequencies
by ethnic group32 were reported from a study
of 10 198 adult members of a California health maintenance organization.
Although this was not a population-based sample, estimates of genotype prevalences
were similar to those reported in our analysis for non-Hispanic whites (Table 5) and the same was true for the
C282Y and H63D mutation frequencies. Small differences between our estimates
and those from the California study for mutation frequencies among Hispanics
(C282Y: 2.7%; H63D: 12.4%)32 and blacks (C282Y:
1.1%; H63D: 5.1%)32 may have been due to the
small numbers of mutations found in those groups. However, the California
study did not report CIs, so the significance of those differences could not
Prevalence estimates for the C282Y/WT genotype were between 9% and 13%
in other studies.17,18,30-33
We found a slightly lower prevalence of 8.33% in the general population and
9.54% in the population who is presumably of European descent. Others have
estimated the prevalence of the C282Y/WT genotype to be approximately 2% in
black populations32,33; our estimate
was 2.3% in non-Hispanic blacks. Because the C282Y mutation has not been demonstrated
in African populations,34-36
haplotype analysis is likely to show that the C282Y mutation found in blacks
is the result of admixture with the white population, as is the case for C282Y
mutations found in Chinese, Pacific Islander, and Australian aboriginal populations.37
The frequency of the C282Y/WT genotype in a group of Spanish blood donors
was estimated to be 4.1%,13 similar to the
3.7% for Hispanics in California,32 but slightly
higher than the 2.7% (95% CI, 1.89%-3.99%) found in our analysis of Mexican
Americans. In a previous study, researchers found no C282Y mutations in a
study of 54 chromosomes from Mexican individuals, but found a frequency of
3.2% in a study of 78 chromosomes from Spanish individuals.34
That was not a population-based study, thus the representativeness of the
data from the 54 chromosomes regarding genetic characteristics of the general
population is unknown. It is possible that, due to population admixture, the
prevalence of C282Y is lower among individuals of Mexican origin than among
individuals of Spanish origin. Seemingly contrary to this finding, a study
based on California health maintenance organization data indicated that phenotypic
expression of hemochromatosis was as frequent among Hispanics as among non-Hispanic
whites.38 Additionally, among Mexican Americans,
the prevalence of elevated transferrin saturation levels, a phenotypic indicator
of hemochromatosis, was similar to or slightly less than that found among
non-Hispanic whites.39 Hence, it is possible
that another yet undiscovered mutation exists that may explain phenotypic
expression of hemochromatosis in Mexican American populations. However, the
association of the C282Y mutation with hemochromatosis among Mexican Americans
has not been reported.
The frequency of the H63D mutation among non-Hispanic whites and Mexican
Americans is similar to previously reported frequencies of between 6% and
30% in European populations and a mean (SE) of 6.6% (4.7%) found in Mexican
populations.32,34 The frequency
of this mutation among non-Hispanic blacks, present at a low frequency in
sub-Saharan African populations, is probably the result of population admixture.32,35,36
Because mutations accounting for a large proportion of HH have been
identified, and because expression of the disease is preventable, the question
of screening healthy populations for HH using genetic testing has arisen.
Experts recently reviewed the implications of screening for these common mutations
and concluded that population-based screening for HH is not appropriate because
the prevalence and penetrance of HFE mutations and
the optimal care of asymptomatic individuals who have the mutations are unknown.9 Information presented here represents the first national,
population-based prevalence estimate, thereby adding an important piece of
the puzzle needed for making policy decisions about screening.
These findings have implications for use of genetic tests for HH screening.
Information on prevalence of the mutations among ethnic groups is important
for targeted screening when appropriate. However, if the prevalence for the
homozygous genotype that has been associated with expression of disease is
higher than the rate of the disease, that is, if penetrance of the genetic
mutations is low, then the positive predictive value of the relevant genetic
test may be low.
Results of our study suggest that the prevalence of homozygosity for
C282Y among non-Hispanic whites and for the total US population may not be
equivalent to (because of penetrance, for example) but may parallel the prevalence
of hemochromatosis as defined by elevated serum iron levels.16,40
We need additional information about other genetic and environmental factors
affecting expression of hemochromatosis-associated mutations. We also need
to identify new hemochromatosis-associated mutations in populations not of
European descent, such as Mexican Americans and non-Hispanic blacks, to fully
understand this relatively common and treatable genetic disorder.
Finally, an important step in understanding the public health significance
of HFE mutations will be to relate these mutations
to clinically relevant information, such as transferrin saturation levels.41 An analysis of the relationship between measures
of transferrin saturation level and HFE mutations
in the NHANES III population is in progress.
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