Full text of DOI:10.1038/sj.ejhg.5200723

European Journal of Human Genetics (2001) 9, 843 ± 848
ã 2001 Nature Publishing Group All rights reserved 1018-4813/01 $15.00
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ARTICLE
Comparative study of the two more frequent HFE
mutations (C282Y and H63D): significant different
allelic frequencies between the North and South of
Portugal
Carla S Cardoso1,2, Pedro Oliveira3, GracËa Porto1,2,4, Christian Oberkanins5,
2
Monica Mascarenhas , Pedro Rodrigues1,2, Fritz Kury5 and Maria de Sousa*,1,2
Â
2
1Molecular Immunology and Pathology, ICBAS, Porto, Portugal; Molecular Immunology, IBMC, Porto, Portugal;
4
3Department of Production and Systems Engineering, University of Minho, Braga, Portugal; Clinical Hematology,
5
Â
Santo Antonio General Hospital, Porto, Portugal; ViennaLab, Vienna, Austria
An earlier study of reference values of iron parameters in Portugal showed significant differences between
populations from northern and southern villages. This study addresses the question of the geographical
distribution in Portugal of the two main mutations (C282Y and H63D) of the hereditary hemochromatosis
gene, HFE. For that purpose, a stratified sample of 640 anonymous dried blood spot samples was randomly
selected from the major regions of Portugal: North, Center, Lisbon and the Tagus Valley, Alentejo and
Algarve. Differences in the geographical distribution of these two mutations were observed thus confirming
the presumed differences between the age of the two mutations which is compatible with the postulated
Celtic/Nordic origin of the C282Y mutation. The finding of a significantly higher allelic frequency of the
C282Y mutation in the North (0.058) than in the South (0.009) could also point to an effect of differential
selective forces acting in the different geographical areas of the country. Data on archaeological,
ethnographic and linguistic records and on the North/South distribution of Portuguese cattle breeds of
European or African origin have also been reported. In addition to their interest for population genetics, the
results represent a reminder of the need to take into account regional differences in the design of strategies
for population screening of hereditary hemochromatosis. European Journal of Human Genetics (2001) 9, 843 ±
848.
Keywords: HFE; hemochromatosis; C282Y; H63D; Iberian peninsula; population genetics
Introduction
consequences of iron accumulation include cirrhosis of the
liver, hepatocellular carcinoma, diabetes, heart failure,
arthritis and hypogonadism.
Hereditary hemochromatosis (HHC) is one of the most
common hereditary metabolic diseases in Caucasians. It is
an autosomic recessive disorder of iron metabolism char-
acterised by increased iron absorption that leads to iron
overload of parenchymal cells in several organs.1 ± 3 Clinical
Feder et al4 reported a candidate gene for HHC on
chromosome 6 (6p22.1) as a non classical MHC-class I gene
denominated HFE, in which two mutations were identified:
C282Y and H63D. The C282Y mutation predicts substitution
of the cysteine residue at codon 282 by a tyrosine in the
alpha3 domain of the molecule4 and prevents the interaction
with beta2-microglobulin and consequently disrupts the HFE
structure and function.5,6 The C282Y mutation in homo-
zygosity is responsible for the majority of HHC cases (for
*Correspondence: Maria de Sousa, MD, PhD, Molecular Immunology
Laboratory, Institute for Molecular and Cell Biology (IBMC), Rua do
Campo Alegre, 823, 4150-180 Porto, Portugal. Tel: +351 22 6074956;
Fax: +351 22 6098480; E-mail: mdesousa@ibmc.up.pt
Received 9 April 2001; revised 12 July 2001; accepted 19 August 2001

HFE mutations: North/South differences
CS Cardoso et al
844
review see Porto and De Sousa7). The H63D mutation results
in a substitution in the aminoacid 63 in the alpha1 domain
changing a histidine to an aspartate.4 The role of the H63D
mutation in the pathophysiology of the disease is still
unclear.
previous estimated allele frequency of the C282Y mutation in
a control population from Northern Portugal.12 The sample
size, taking into consideration the cost and time required for
the analysis, was calculated in order to assure a global error
below 1% for a 95% confidence interval.
The frequency of the HFE mutations in control subjects has
been reported in several European populations and in
populations of European descent. The highest allelic
frequencies for the C282Y mutation were found in the
Northern European countries, namely in Ireland (an average
of 10%),8,9 with a lower frequency (1%) observed in countries
from southern Europe, namely Italy.10 The H63D mutation
was found in highest frequencies in the Iberian Peninsula
(above 20%).11,12 In non-Caucasian populations HFE muta-
tions were either absent or found in lower frequencies.13,14
The present work was motivated by a previous study of
reference values of iron parameters in Portugal done before
the discovery of the HFE gene.15 That study reported regional
differences in iron parameters between Northern and South-
ern villages in Portugal. These differences were observed both
in serum ferritin levels in females and in serum iron levels in
males from the two regions, with the highest values reported
in subjects from the North. These differences were also
reflected on the prevalence of iron overload and iron
deficiency in the two regions. Iron deficiency was found
significantly more frequently in the South, in contrast with
iron overload that was found more commonly in the
North.15
The distribution of the 640 genotyped dried blood spot
samples according to the five Portuguese regions was as
follows: 129 from the North, 130 from the Center, 133 from
Lisbon and the Tagus Valley, 132 from the Alentejo and 116
from the Algarve.
DNA purification
The DNA purification of the dried blood samples was done
using the InstaGeneTM Dry Blood Kit (Bio-Rad Laboratories),
according to the manufacturer's recommendations. This
procedure consists in the selective extraction of amplifica-
tion reaction inhibitors present in the blood without
removing any of the filter-bound DNA. The extracted dry
blood spot is added directly to the amplification reaction
mixture prior to thermocycling.
HFE genotyping
The HFE genotyping was done using two commercial kits
(Haemochromatosis Gene Mutation Assay I and II, Vienna-
Lab, Vienna, Austria). Briefly, exon 4 (for C282Y) or exon 2
(for H63D) sequences of the HFEgene were amplified in vitro
and terminally labelled with fluorescein as a reporter
molecule. The amplification products were alkali-denatured,
and 25 ml aliquots were selectively hybridised to allele-
specific (wild type or mutant) oligonucleotide probes
immobilised in two separate cavities of a microwell plate.
After hybridisation and stringent washes at 378C, bound
sequences were detected using a horseradish peroxidase-
labelled anti-fluorescein antibody and colour reaction with
tetramethylbenzidine. The methodology, as well as its
validation on samples of known genotype (RFLP-typed) and
application for typing were presented elsewhere.16 ± 18
The aim of the present work was to estimate the frequency
of the C282Y and H63D HFE mutations in Portugal,
comparing its distribution through different regions.
Material and methods
The National Institute of Statistics (INE) divides Portugal in
five major regions: North, Center, Lisbon and the Tagus
Valley, Alentejo and Algarve (see Figure 1). Lisbon and the
Tagus Valley, being an immigration region from other regions
in the country reflects, to a large extent, the whole
population in the country.
Statistical analysis
The differences among the allelic frequencies in the different
regions were tested by contingency table analysis (Chi-square
Test). Given that the proportions were close to zero, the
confidence limits for the proportions were calculated using a
relationship between the F distribution and the binomial
distribution. The multiple comparisons for the proportions
were based on a procedure analogous to the Tukey test, based
on the angular transformation of each proportion.19
Sample selection
In order to compare the prevalence of the C282Y and H63D
HFE mutations among those five regions, an anonymous
random stratified sample was used. The Portuguese Neonatal
Screening Program covers 95% of all newborns in Portugal. A
single laboratory is responsible for the screening at the
Medical Genetics Institute. The information of each card is
kept in a database and the only accessed information
regarding the subjects is the record number and the
geographical location, constituting the sampling base.
From this base, a stratified random sample of 640
anonymous dried blood spot samples was selected, through
a random number generator, from the five regions of
Portugal. The selection of the sample size was based on the
Results
The results obtained in terms of HFE genotypes and the
corresponding allelic frequencies of the two mutations by
region are summarised in Table 1 and illustrated in Figure 1.
The lower and upper 95% confidence limits for the allelic
frequencies in the five regions are also presented in Table 1.
European Journal of Human Genetics

HFE mutations: North/South differences
CS Cardoso et al
845
Figure 1 Maps representing the geographical distribution of the allelic frequencies of the C282Y (A) and H63D (B) HFE mutations in the
five Portuguese regions.
Table 1 Number of subjects genotyped for the C282Y and H63D HFE mutations, the correspondent allelic frequencies and
95% confidence limits for the allelic frequencies in the five regions of Portugal. (n=total number of subjects genotyped)
North
Center
Lisbon and Tagus Valley
Alentejo
Algarve
HFE genotype
(n=129)
(n=130)
(n=133)
(n=132)
(n=116)
C282Y/C282Y
C282Y/wt
C282Y/H63D
H63D/wt
H63D/H63D
wt/wt
1
10
3
31
8
1
5
5
35
6
78
0
7
1
27
6
92
0
5
1
37
5
84
0
0
2
23
7
84
76
C282Y allelic frequency
95% confidence interval
H63D allelic frequency
95% confidence interval
0.058
[0.033; 0.095]
0.194
0.046
[0.024; 0.079]
0.200
0.030
[0.013; 0.058]
0.150
0.023
[0.008; 0.049]
0.182
0.009
[0.001; 0.031]]
0.168
[0.151; 0.247]
[0.152; 0.250]
[0.112; 0.200]
[0.140; 0.233]
[0.126; 0.221]
P50.05). In spite of a decreasing allele frequency of the
C282Y from the North to the South, no other statistically
significant differences were seen with the present sample size
(Table 2).
C282Y HFE mutation
The overall distribution of the C282Y mutation includes two
homozygous and 39 heterozygous samples (12 of the 39 are
compound heterozygous for both mutations) (Table 1).
The allelic frequency of the C282Y mutation decreases
from the North to the South. In the North, an allelic
frequency of 0.058 ([0.033, 0.095] 95% CI) was observed
contrasting with the allelic frequency of 0.009 ([0.001, 0.031]
95% CI) observed in the Algarve (Figure 1a, Table 1). The
frequency of the C282Y mutation is not independent of the
regions tested (w2=11.57, P50.05). When the frequency of
the C282Yallele was compared between the different regions,
applying the Tukey test (Table 2), a significant difference was
observed between the North and the Algarve (q=4.711,
P50.05) and between the Center and the Algarve (q=3.873,
H63D HFE mutation
In terms of global genotype frequencies, 32 homozygous and
165 heterozygous samples (12 of the 165 are compound
heterozygous for both mutations) for the H63D mutation
were observed. The allelic frequency of the H63D is
independent of the region tested (w2=2.59, P40.05), ranging
from 0.19 ([0.151, 0.247] 95% CI) in the North to 0.17
([0.126, 0.221] 95% CI) in the Algarve (Figure 1b, Table 1).
In summary, the regional differences seen in the allelic
frequency of the C282Y mutation between the North and the
South contrasted remarkably with the homogeneous dis-
European Journal of Human Genetics

HFE mutations: North/South differences
CS Cardoso et al
846
Table
2
Multiple comparisons of the C282Y allelic
of many population settlements, coming from different parts
of Europe and Africa.27 These settlements, however, did not
have the same influence in the whole country. A geographi-
cal and cultural boundary could be defined between the
north and south, documented by archeological, ethnogra-
phical and linguistic records.28 This is further supported by
more recent studies of the European or African origin of
Portuguese cattle breeds.29
frequencies between the five different Portuguese regions
(Tukey test). The significant differences are highlighted in
bold in the table. No other significant differences between
regions were found
Region 1 Region 2 Allelic Diff. Transf. Diff. SE
q
North
North
North
North
Center
Center
Centro
Lisbon
Lisbon
Center
Lisbon
0.011986 71.54692 1.77839 0.86984
0.028065 73.96590 1.76839 2.24266
Simon et al30 postulated that the geographical distribution
of hemochromatosis in the world was similar to the
migration pattern of Celts, a postulate discussed more
recently by Lucotte.20 The occupation of the Celts in Portugal
occurred in the 6th century BC and it is believed that the
Alentejo 0.035413 75.28201 1.77168 2.98136
Algarve 0.049519 78.62528 1.83095 4.71083
Lisbon
0.016079 72.41897 1.76494 1.37057
Alentejo 0.023427 73.73509 1.76824 2.11233
Algarve 0.037533 77.07836 1.82762 3.87300
Alentejo 0.007348 71.31612 1.75818 0.74857
Algarve
0.021454 74.65939 1.81789 2.56308
0.014106 73.34327 1.82109 1.83586
influence of the Celts was predominant in the North.28
A
Alentejo Algarve
strong founder effect could thus be an explanation for the
marked differences observed. However, it goes against the
putative age estimated to be on average 60 generations.24,25
Considering that one generation corresponds to about 20
years, the C282Y mutation should have occurred 1200 years
ago, which is more recent than the early Celtic occupation of
Portugal. Another possible explanation for the distribution of
the C282Y mutation could be the Nordic/Suevian occupation
and settlement that occurred between the 5th ± 6th century
only in the North of the country.28 A possible later Viking
origin has been discussed by others.21,31
Allelic Diff: Difference of the allelic frequency in the two regions;
Transf. Diff: Transformed difference of the allelic frequency, based
on the angular transformation of each proportion, in the two
regions; SE: standard error of the difference; q: Tukey statistics q.
tribution of the H63D mutation. Statistically significant
differences were observed in the C282Y allelic frequencies
between the global North (comprising the North and the
Center regions) and the global South (comprising the
Alentejo and the Algarve) (w2=3.841, P50.01 Fisher's exact
test).
The frequency of the H63D mutation did not vary between
regions supporting an older origin of this mutation. Similarly
to the present result, a reported study of 101 Portuguese
unrelated males (53 from the north and 48 from the south)
studied for 18 nuclear polymorphic markers, showed that 16
out of the 18 markers, had no differences in allelic
frequencies between North and South suggesting a genetic
homogeneity of the country.32
Discussion
The present study demonstrates the existence of significant
regional differences in the frequency of the C282Y mutation
and a homogeneous frequency of the H63D mutation in
Portugal. The results are in accordance with previously
reported differences between biochemical parameters of the
iron status in the normal population.15 The highest allelic
frequency of the C282Y mutation was observed in the North
(0.058), approaching allelic frequencies reported in Northern
European countries (reviewed in Lucotte,20 Merryweather-
Clarke,21 Porto and De Sousa,7 Fairbanks22), in contrast with
the frequency in the Algarve (0.009), which is similar to that
reported in Italy.10,23 The allelic frequency of the H63D
mutation observed in this study (0.15 ± 0.20) is among the
highest reported in Europe. Similar frequencies were seen in
Spain (0.22)11 with the highest frequency reported in Basques
(0.30).13
Thus, the high C282Y allelic frequency in the North
supports the notion of acting selective forces.25,33 ± 35 The
C282Y heterozygous subjects could be protected from iron
deficiency when dietary iron intake is limited. In the case
of the females, they could be protected from blood loss
due to menstruation and multiple pregnancies. Interest-
ingly, in the population from the North of Portugal a very
high average number of pregnancies (410 children) was
reported in females older than 40 years.36 In addition one
could speculate that
a mutation that favoured iron
overload could be negatively selected in areas of high
incidence of malaria such as European regions round the
Mediterranean.37 In Portugal, the highest mortality rate
due to malaria was observed in the South.38 Similar North/
South discrepancies have been reported in France39 and
Italy.23, 40
The regional differences in the C282Yand the constancy of
the H63D allelic frequencies raise two questions: (1) The
putative age of the two HFE mutations; (2) The possible
evolutionary significance of the differences observed in the
C282Y allele frequency.
In conclusion, in addition to their interest for European
population genetics, the present and similar results in other
countries are a reminder of the need to take into account
regional differences in the design of strategies for population
screening of hereditary hemochromatosis in Europe and/or
in countries with populations of European descent.
The findings are compatible with the notion of the recent
origin of the C282Y mutation24,25 and of an older origin for
the H63D mutation as suggested by Risch26
.
The strategic localisation of Portugal between the Atlantic
and the Mediterranean places the country at the convergence
European Journal of Human Genetics

HFE mutations: North/South differences
CS Cardoso et al
847
Acknowledgements
15 Porto G, Vicente C, Fraga J, Martins da Silva B, De Sousa M:
Importance to establishing appropriate local reference values
for the screening of hemochromatosis: A study of three different
control populations and 136 hemochromatosis family mem-
bers. J Lab Clin Med 1992; 119: 295 ± 305.
16 Oberkanins C, Kazemi-Shirazi L, Kury F et al: Genotyping of the
hereditary haemochromatosis mutation (HLA-H:C282Y) in
microtiter plates. Am J Hum Genet 1997; 61 (Suppl.): A225.
17 Oberkanins C, Kazemi-Shirazi L, Kury F et al: Genotyping of
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Carla Cardoso is recipient of a PhD fellowship PRAXIS XXI (BD/
13383/97). We thank Vienna Lab (Austria) for providing the study
with kits for genotyping of HFE mutations. We gratefully acknowledge
Â
Â
Drs R Vaz Osorio, C Sa Miranda and L Vilarinho from the Institute of
Medical Genetics (Porto, Portugal) for their interest and support of the
present study. We also thank Drs J Vieira, C Vieira and AT
Merryweather-Clarke for the helpful discussion of the manuscript.
The work was funded in part by a joint grant of the C Gulbenkian and
the National Science and Technology Foundations and the financial
support of the American Portuguese Biomedical Research Fund
(APBRF, USA).
18 Oberkanins C, Kazemi-Shirazi L, Kury F et al: Genotyping of
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19 Zar JH: Biostatistical Analysis, 4th edn. Prentice Hall, New Jersey,
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