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Articles |
1
Biochemistry Section, Pathcentre, QE II Medical Centre, Nedlands, Western Australia 6009, Australia.
2
Department of Public Health and
3
University Department of Medicine, University of Western
Australia, Fremantle, Western Australia 6160, Australia.
4
Department of Gastroenterology, Fremantle Hospital,
Fremantle, Western Australia 6160, Australia.
5
Busselton Population Medical Research Foundation, Perth,
Western Australia 6907, Australia.
6
Queensland Institute of Medical Research, Brisbane,
Queensland 4029, Australia.
a Author for correspondence. Fax 61-8-9346-3882; e-mail
ric.rossi{at}health.wa.gov.au.
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Abstract |
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Methods: We performed a cross-sectional analysis of 1488 females and 1522 males 20–79 years of age drawn from the Busselton (Australia) population study to assess the effects of HFE genotype, age, gender, and lifestyle on serum iron and hematology indices.
Results: Male C282Y heterozygotes had increased transferrin saturation compared with the wild-type genotype. Neither male nor female heterozygotes had significantly increased ferritin values compared with the wild-type genotype. Younger (20–29 years) wild-type males, but not heterozygous males, had significantly lower ferritin values than wild-type males in the older age groups. Compound heterozygous subjects had increased means for serum iron, transferrin saturation, corpuscular volume, and corpuscular hemoglobin compared with the wild-type genotype, and the males also had increased ferritin values (medians 323 vs 177 µg/L; P = 0.003). In both male and female wild-type subjects, an increased body mass index was associated with decreased serum iron and transferrin saturation and increased ferritin values. There was a significant increase in ferritin concentrations in both genders with increasing frequency of red meat consumption above a baseline of 1–2 times per week and alcohol intakes >10 g/day.
Conclusions: Male C282Y heterozygotes had significantly increased transferrin saturation values. Compound heterozygous (C282Y/H63D) subjects formed a separate category of C282Y heterozygotes in whom both iron and red cell indices were significantly increased compared with the wild-type genotype.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Two recent prospective population-based studies have reported an association between heterozygosity for the C282Y mutation of the HFE gene for hereditary hemochromatosis and vascular events (9)(10). These findings have led to speculation that C282Y/wt subjects had either increased serum ferritin compared with wild-type subjects or had reached the same ferritin concentrations at an earlier age (11). We therefore studied the effect of HFE genotype, age, gender, and lifestyle factors (obesity and consumption of alcohol and red meat) on iron indices.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5000 Caucasian subjects.
All blood tests were performed in the fasting state. From this group,
we randomly selected 1488 female and 1522 male nonrelated subjects
20–79 years of age. Permission was granted for this study by the Busselton Population Medical Research Foundation and The Committee for Human Rights at The University of Western Australia.
measurement of serum indices
Serum iron concentrations were measured using a standard
colorimetric method, and the transferrin concentration was determined
by rate immunoturbidimetry on a Hitachi 917 analyzer. Serum
transferrin saturation was calculated from these results as follows:
transferrin saturation (%) = serum iron (µmol/L)/[2 x
transferrin (µmol/L)] x 100. Serum ferritin concentrations were
measured by chemiluminescence immunoassay on a Chiron ACS-180 analyzer.
measurement of red cell indices
Hemoglobin, mean corpuscular volume (MCV), and mean corpuscular
hemoglobin (MCH) measurements were performed on a Coulter STKS
automated hematology analyzer.
determination of the c282Y AND H63D
MUTATIONS
Analysis was performed on DNA extracted from whole blood spotted
onto neonatal screening cards as described by Walsh et al.
(13). PCR amplification of the regions containing the
missense mutations was performed using the published primer sequences
of Feder et al. (1) (GenBank Accession No. U60319) and
cycling conditions described by Cullen et al. (14).
Mutations were identified using restriction enzyme digestion followed
by analysis on a 2% agarose gel. The C282Y missense mutation leads to
the formation of a unique SnaBI restriction site, whereas
the H63D mutation leads to the loss of a DpnII site. The
status of all C282Y homozygous subjects was confirmed by separate
testing with the primer sequence described by Jeffrey et al.
(15) to avoid possible false-positive results attributable
to the G5569A polymorphism. The H63D mutation was determined only in
subjects who were heterozygous for the C282Y mutation to ascertain the
prevalence of C282Y wild-type heterozygous (C282Y/wt) and compound
heterozygous (C282Y/H63D) genotypes. Wild-type refers to absence of the
C282Y mutation.
statistical analysis
The Fisher exact test, the 
2 test, and
generalized linear models adjusted for multiple means testing using the
least significant difference method were used. The normality test was
carried out on all variables. Ferritin was highly skewed, and
log transformation was used for all subsequent analyses. Statistical
analyses were performed with SAS software (16).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The prevalences of the population with one mutated C282Y
allele (C282Y/wt and C282Y/H63D genotypes combined) were 14.1% in
females and 14.5% in males. These prevalences were consistent with
those predicted with the Hardy-Weinberg equation, based on an allelic
frequency of 7.6% for the C282Y mutation. There were no significant
between-gender differences in genotype prevalence
(2 test, P = 0.95).
Homozygosity for the C282Y mutation occurred in 0.53% (1 in 188) of
the subjects, nine females and seven males. Phenotypic presentation and
clinical data for these subjects with hereditary hemochromatosis have
been reported separately (7).
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iron and hematology studies
The data for serum iron, red cell indices, and body mass index
(BMI) according to genotype in 1479 females are shown in Table 2
. A threshold of 45% for transferrin saturation has been
proposed for population screening for hereditary hemochromatosis
(17), and the proportions exceeding this value are shown.
Iron depletion was defined by a ferritin concentration <20 µg/L
(18) and iron deficiency by a ferritin concentration <12
µg/L and a transferrin saturation <15% (19). The
significance values are shown for comparison of either C282Y/wt or
C282Y/H63D genotypes to the wild type.
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The iron indices in C282Y/wt females were not significantly different from those in the wild-type genotype; however, the mean MCV and MCH values were significantly increased. Compound heterozygous females had increased means for serum iron, transferrin saturation (21.9% exceeded a saturation of 45%), MCV, and MCH compared with the wild-type genotype. There were no significant differences between genotypes for the prevalence of iron depletion or deficiency.
Comparable data for serum iron, red cell indices, and BMI according to
genotype in 1515 males are shown in Table 3
. Compared with the wild-type genotype, C282Y/wt males had
significantly increased means for serum iron, transferrin saturation,
MCV, and MCH, and the C282Y/H63D males also had increased means for
ferritin. The prevalences of both C282Y/wt and C282Y/H63D males with
transferrin saturation exceeding a threshold of 45% were significantly
higher than the prevalence for wild-type males. There were no
significant differences between genotypes for the prevalence of iron
depletion or deficiency.
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iron indices and age
Box and whisker plots for ferritin according to deciles of age in
wild-type and C282Y/wt genotype males are shown in Fig. 1
. Initial univariate regression analyses showed that there was
no age-related increase in ferritin. A significant overall decrease of
ferritin with age occurred in men with the wild-type genotype
(coefficient ± SE, -0.0029 ± 0.0014; P =
0.05) but not the C282Y/wt genotype. Subsequent multiple comparison
tests of age deciles showed that ferritin values in wild-type males
significantly increased between the 20–29 years and 30–39 years
deciles (geometric means, 126 and 181 µg/L, respectively;
P < 0.0001), and no subsequent changes occurred.
Similar analysis by age deciles for C282Y/wt men confirmed the
univariate regression analysis results and showed that there were no
significant differences in ferritin values throughout the age range we
examined (20–79 years). The results in Table 3
for the entire age
range show that there were no significant differences between ferritin
values in wild-type males and their C282Y/wt counterparts.
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Box and whisker plots for ferritin according to deciles of age in
wild-type and C282Y/wt females are shown in Fig. 2
. For females 20–49 years of age, both univariate analysis and
multiple comparison tests of age deciles showed no significant
differences in mean ferritin values for either the wild-type or the
C282Y/wt genotype. There was a significant increase in ferritin values
for both wild-type and C282Y/wt genotypes occurring between the fourth
and fifth decades, consistent with the average age of menopause for our
population (51 years). There were no differences in the mean ferritin
values between wild-type and C282Y/wt genotypes either before
(geometric means for 20–49 years, 42 and 36 µg/L, respectively) or
after menopause (geometric means for 50–79 years, 84 and 87 µg/L,
respectively).
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Serum iron and transferrin saturation values were not correlated with age in either gender.
iron indices and obesity
A BMI between 20 and 25 kg/m2 is usually
considered normal, and obesity is defined as a value >30. Obesity as
assessed by BMI had highly significant effects on iron indices. Table 4
shows coefficients ± SE for univariate regression
analyses of the relationship between age-adjusted BMI (dependent
variable) and serum iron, transferrin saturation, and log ferritin
(independent variables) according to HFE genotype. In
wild-type subjects of both genders, the age-adjusted BMI was negatively
correlated with serum iron and transferrin saturation and positively
correlated with ferritin. Obesity tends to increase ferritin, whereas
it decreases serum iron and transferrin saturation. In male C282Y/wt
subjects, the age-adjusted BMI was not correlated with serum iron,
transferrin saturation, or ferritin, but in female C282Y/wt subjects,
it was correlated with serum iron. No correlations were detected
between BMI and serum iron, transferrin saturation, or log ferritin in
C282Y/H63D subjects.
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alcohol consumption and ferritin
Males admitted to a higher alcohol consumption than females, and
63% of males compared with 28% of females estimated their intake to
be >10 g/day. Fig. 3
shows box and whisker plots for ferritin according to alcohol
consumption and gives ferritin concentrations and levels of
significance compared with a baseline intake of 1–10 g/day. Ferritin
concentrations (median, interquartile range) for both males and females
showed a significant increase with increasing alcohol consumption.
Ferritin values for males consuming either 11–50 g of alcohol/day or
>50 g/day were both significantly higher than for those who consumed
1–10 g/day (P = 0.0007 and <0.0001, respectively).
Similarly, females consuming either 11–50 g of alcohol/day or >50
g/day had significantly higher ferritin values than those who consumed
1–10 g/day (P = 0.0002 and 0.006, respectively).
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meat consumption and ferritin
The frequency of red meat (beef) consumption was high, with 89%
of men and 81% of women reporting eating red meat three or more times
per week. Fig. 4
shows box and whisker plots for ferritin according to the
frequency of red meat consumption and gives ferritin concentrations and
levels of significance compared with a baseline intake of one to two
times per week. Ferritin concentrations (median, interquartile range)
for both males and females were significantly increased with increasing
frequency of consumption. Ferritin values for males consuming red meat
either three to six times per week or every day were significantly
higher than for those who consumed one to two times per week
(P = 0.0002 and 0.0001, respectively). Similarly,
ferritin values for females consuming red meat either three to six
times per week or every day were significantly higher than for those
who consumed one to two times per week (P = 0.007 and
0.005, respectively). We compared the log-ferritin values for C282Y/wt
and wild-type subjects who consumed meat every day by two-sample
t-test: although male C282Y/wt subjects had higher median
ferritin values than wild-type subjects, the difference did not achieve
significance (medians, 221 vs 190 µg/L; P = 0.098),
and there was no difference in the females (medians, 75 vs 70 µg/L;
P = 0.691).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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); 14.1% of the females and 14.5% of the males therefore had
one mutated C282Y allele. These relatively high prevalence rates for
the C282Y mutation make this population ideal for studying the effect
of HFE genotype.
Significantly increased serum iron and transferrin saturation values
were observed in females with the C282Y/H63D but not the C282Y/wt
genotype (Table 2
). Thus, 21.9% of female C282Y/H63D subjects exceeded
a transferrin saturation value of 45%, which has been proposed as a
threshold for the investigation of subjects for hereditary
hemochromatosis (17). Previous studies have reported
increased transferrin saturation values in female heterozygotes
(20)(21), but these studies were conducted
before the availability of genotyping for HFE mutations and
putative heterozygotes were identified on the basis of HLA typing in
family studies of hereditary hemochromatosis probands. Subjects with
the C282Y/H63D genotype would have been included as heterozygotes, and
we have shown that this group has significantly increased transferrin
saturation values. No significant between-genotype differences were
observed in females for ferritin or hemoglobin, although both C282Y/wt
and C282Y/H63D genotypes had increased MCV and MCH indices.
Both C282Y/wt and C282Y/H63D males had significantly increased serum
iron, transferrin saturation, MCV, and MCH values compared with
wild-type subjects (Table 3
). These results agree with previous studies
of heterozygous males (20)(21).
The mean ferritin values for both female and male C282Y/wt subjects
were not significantly different from wild-type subjects (Tables 2
and 3
, respectively). The assertion that heterozygotes have significantly
increased serum ferritin compared with wild-type subjects is based on
studies conducted before the availability of genotyping for
HFE mutations of heterozygotes identified in family studies
of hereditary hemochromatosis patients (20)(21).
Both studies identified putative heterozygotes on the basis of HLA
typing in family studies of hereditary hemochromatosis probands. A US
study reported significantly higher mean ferritin concentrations for
209 male and 260 female heterozygotes 31–60 years of age
compared with healthy controls in the same age range
(20). Another study of 255 heterozygotes in Canada
(21) found that the mean ± SE for serum ferritin for
heterozygotes was significantly higher than in control subjects
(140 ± 10.2 vs 87 ± 8.5 µg/L; P <0.05). There
may have been a selection bias resulting from studying hereditary
hemochromatosis families rather than a community population. In the
case of males, the inadvertent inclusion of the C282Y/H63D genotype as
heterozygotes would have increased the mean ferritin values obtained in
both studies (Table 3
).
Our finding that ferritin did not differ significantly between C282Y/wt and wild-type subjects has been corroborated in a large community sample of 1233 complete pairs of Australian twins (22) in whom both HFE mutations were assessed. The latter study concluded that the effects of the HFE gene on serum ferritin are minor compared with the effects on serum iron and transferrin saturation. We found that the C282Y/wt genotype caused a significant increase in transferrin saturation in males, whereas no such effect was observed for ferritin in either gender. The mechanism for the effects of the C282Y/wt genotype on iron absorption and iron stores have not been fully studied. However, the main effect of C282Y heterozygosity seems to be to change the interactions between transferrin, its receptor, and the HFE protein (23), which may account for the increased transferrin saturation and implies that the effects on iron stores and hence ferritin may be secondary consequences.
Two recent prospective population-based studies have reported an association between heterozygosity for the C282Y mutation of the HFE gene for hereditary hemochromatosis and vascular events. A study of 12 239 Dutch women showed that C282Y/wt subjects were at significantly increased risk of death from either myocardial infarction or cerebrovascular disease compared with wild-type subjects (9). Similar findings were reported in a prospective study of 1150 Finnish men where C282Y/wt subjects were at a 2.3-fold increased risk of acute myocardial infarction (10). In an editorial accompanying these studies, it was stated that heterozygous subjects had higher mean serum ferritin concentrations than wild-type subjects and that it was likely that even heterozygotes with statistically normal ferritin concentrations achieved them at an earlier age (11). We have demonstrated that C282Y/wt subjects do not have higher ferritin concentrations than their wild-type equivalents. However, we present evidence that C282Y/wt males have achieved steady-state ferritin concentrations by their 20–29 years age decile, whereas wild-type males do not do so until the 30–39 years decile. Thus, there is a longer period of steady-state ferritin concentrations in C282Y/wt males than in wild-type subjects. This effect was not observed in females, and there were no significant increases of ferritin when the population was segregated into premenopausal (ages 20–49 years) and postmenopausal (ages 50–79 years) age groups.
We observed a striking correlation of age-adjusted BMI with iron
indices for wild-type but not heterozygous subjects (Table 4
). Obesity
tended to increase ferritin, whereas it decreased serum iron and
transferrin saturation in wild-type subjects of either gender. These
results agree with findings in the study of Australian twins
(22). The physiological mechanism responsible for these
effects is unknown at this time.
We confirm a previous report showing that the frequency of meat intake and quantity of alcohol consumed are important lifestyle factors affecting serum ferritin concentrations for both genders (24). The heme content of red meat provides a dietary iron content of very high bioavailablity. Intestinal absorption of heme iron is relatively unaffected by the status of body iron stores, whereas absorption of non-heme iron is regulated according to the demands of iron stores (25). We observed significant increases in median ferritin concentrations with increasing frequency of red meat consumption above a baseline of one to two times per week, consistent with lack of regulation of iron uptake from dietary heme.
Median ferritin concentrations for both males and females showed a significant increase with increasing alcohol consumption above a baseline of 1–10 g/day. The mechanism for the effect of alcohol consumption on serum ferritin is poorly understood. Alcohol appears to have many modes of action that can affect serum ferritin concentrations. These include induction of an inflammatory response in the liver with resulting de novo ferritin synthesis, causing ferritin release from liver cells and changing gut permeability, thereby altering iron absorption (26).
In conclusion, we report iron and red cell indices on a large community population in whom the prevalence of heterozygosity for the C282Y mutation is relatively high. Previous studies found increased transferrin saturation and ferritin concentrations in putative heterozygotes; however, we confirmed significantly increased transferrin saturation only in male C282Y/wt subjects. Ferritin values for our C282Y/wt subjects were not significantly different from the wild-type genotype, although male C282Y/wt subjects achieved maximal ferritin concentrations in their second rather than their third decade. Compound heterozygous (C282Y/H63D) subjects formed a separate category of C282Y heterozygotes in whom both iron and red cell indices were significantly increased compared with the wild-type genotype.
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Acknowledgments |
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Footnotes |
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References |
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The following articles in journals at HighWire Press have cited this article:
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 D J Lehmann, M Worwood, R Ellis, V L J Wimhurst, A T Merryweather-Clarke, D R Warden, A D Smith, and K J H Robson Iron genes, iron load and risk of Alzheimer's disease. J. Med. Genet., October 1, 2006; 43(10): e52 - e52. [Abstract] [Full Text] [PDF]
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 D. W. Swinkels, M. C.H. Janssen, J. Bergmans, and J. J.M. Marx Hereditary Hemochromatosis: Genetic Complexity and New Diagnostic Approaches Clin. Chem., June 1, 2006; 52(6): 950 - 968. [Abstract] [Full Text] [PDF]
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 M. A Roe, A.-L. M Heath, S. L Oyston, C. Macrow, J. A Hoogewerff, R. Foxall, J. R Dainty, G. Majsak-Newman, G. Willis, and S. J Fairweather-Tait Iron absorption in male C282Y heterozygotes Am. J. Clinical Nutrition, April 1, 2005; 81(4): 814 - 821. [Abstract] [Full Text] [PDF]
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J. R Hunt and H. Zeng Iron absorption by heterozygous carriers of the HFE C282Y mutation associated with hemochromatosis Am. J. Clinical Nutrition, October 1, 2004; 80(4): 924 - 931. [Abstract] [Full Text] [PDF]
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 O. T. Njajou, M. Hollander, P. J. Koudstaal, A. Hofman, J. C.M. Witteman, M. M.B. Breteler, and C. M. van Duijn Mutations in the Hemochromatosis Gene (HFE) and Stroke Stroke, October 1, 2002; 33(10): 2363 - 2366. [Abstract] [Full Text] [PDF]
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, wt/wt; 
, C282Y/wt.
