Hemochromatosis gene (HFE) mutations in patients with type 2 diabetes and their control group in an Iranian population.

Abstract

To assess the frequency of 2 different forms of hemochromatosis HFE gene mutations (C282Y and H63D mutations) in a normal population in comparison with type 2 diabetic patients.
This case control study was undertaken in Zanjan Diabetic Care Center, Zanjan, western Tehran, in 2005. Two hundred and two individuals were included in this study: 101 type 2 diabetes mellitus (T2DM) patients, and 101 age, and gender-matched controls. The patients were examined for mutations in the HFE gene. Nucleotide 845 (C282Y) and 187 (H63D) alleles were amplified by polymerase chain reaction PCR with lymphocyte deoxy-ribonucleic acid. The PCR products were analyzed by restriction enzyme digestion. Chi-square, student's t test, and Fisher's exact tests were used for comparison, and odds' ratio was calculated.
Two hundred and two individuals were studied. The frequency of wild/C282Y alleles was 98/2% in T2DM patients, and 99/1% in controls p=0.6. The frequency of wild/H63D alleles was 68.3/31.7% in diabetics p=0.08, and 73.4/26.3% in control subjects p=0.08. The distribution of genotypes was not statistically different.
Based on our data, HFE mutations were not found in excess in patients with T2DM, and there was no evidence that a population-based search for an excess of these alleles in type 2 diabetes was indicated.

Full text

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Hemochromatosis gene (HFE) mutations in patients with
type 2 diabetes and their control group in an Iranian
population
Faranak Sharifi, MD, Abdulreza Esmaeilzadeh, PhD, Mohammadreza Zali, MD.
808
ABSTRACT
ينلجا ةرفط نم يننثا ينلكش راركت مييقت لجأ نم :فادهلأا
ناكسلا ىدل H63DوC282Y ةرفطو ،HEF يومدلا يغابصلا
عونلا نم يركسلا ءادب ينباصلما ىضرلما عم ةنراقلماب ينميلسلا
.T2DM يناثلا
،ركسلا ضرلم نانجاز زكرم يف ةساردلا هذه تيرجأ :ةقيرطلا
202 ةساردلا تلمش .م2005 ماع يف ،ناريإ - نانجاز ميلقإب
يناثلا عونلا نم يركسلا ءادب ينباصلما ىضرلما نم 101 :ًادرف
سنلجا عونل ةقباطم مكتح ةعومجمك ًادرف 101و ،T2DM
يومدلا يغابصلا ينلجا ةرفط لجأ نم مهصحف تم .رمعلاو
H63D 187و C282Y 845 ديتولكوين داضم ريبكت تم .HFE
جتنم ليلتح تم .PCR ةلقانلا رئاملخا ةلسلس لعافت ةطساوب
.مضهلا يمزنأ دييقت ةطساوب PCR ةلقانلا رئاملخا ةلسلس لعافت
تمو ،ةنراقملل رشيف رابتخاو ريوكس-يشت رابتخا انمدختسإ
.ةلصالحا تانايبلا )Odd's( لدعم باسح
داضلم عساولا راركتلا غلب .ًادرف 202 ةسارد تتم :جئاتنلا
عونلا نم يركسلا ءادب ينباصلما ىضرلما ىدل 98/2%-C282Y
.)p=0.6( مكحتلا ةعومجم يف 99/1% غلبو ،T2DM يناثلا
ىضرلما يف 68.3/31.7%-H63D داضلم عساولا راركتلا غلب
ةعومجم يف 73.4/26.3%و )p=0.08( يركسلاب ينباصلما
ينلجا عون عيزوت نكي مل .يلاوتلا ىلع ،)p=0.08( مكحتلا
.ظوحلم لكشب ًافلتخم
مل ،HEF يومدلا يغابصلا ينلجا ةرفطل انتانايب ىلع ًءانب :ةتماخ
يركسلا ءادب ينباصلما ىضرلما ىدل طرفم لكشب هدوجو ينبتي
هذه طرفب ريشي ليلد كلانه سيلو ،T2DMيناثلا عونلا نم
ىلع ءانب T2DM يركسلا ءاد نم يناثلا عونلا يف تاداضلما
.ناكسلا ىلع ينبلما ثحبلا
Objective: To assess the frequency of 2 different
forms of hemochromatosis (HFE) gene mutations
(C282Y and H63D mutations) in a normal
population in comparison with type 2 diabetic
patients.
Methods: This case control study was undertaken
in Zanjan Diabetic Care Center, Zanjan, western
Tehran, in 2005. Two hundred and two individuals
were included in this study: 101 type 2 diabetes
mellitus (T2DM) patients, and 101 age, and gender-
matched controls. The patients were examined
for mutations in the HFE gene. Nucleotide
845 (C282Y) and 187 (H63D) alleles were amplified
by polymerase chain reaction (PCR) with lymphocyte
deoxy-ribonucleic acid. The PCR products were
analyzed by restriction enzyme digestion. Chi-square,
student’s t test, and Fisher’s exact tests were used for
comparison, and odds’ ratio was calculated.
Results: Two hundred and two individuals were
studied. The frequency of wild/C282Y alleles was
98/2% in T2DM patients, and 99/1% in controls
(p=0.6). The frequency of wild/H63D alleles was
68.3/31.7% in diabetics (p=0.08), and 73.4/26.3%
in control subjects (p=0.08). The distribution of
genotypes was not statistically different.
Conclusion: Based on our data, HFE mutations were
not found in excess in patients with T2DM, and
there was no evidence that a population-based search
for an excess of these alleles in type 2 diabetes was
indicated.
Saudi Med J 2008; Vol. 29 (6): 808-812
From the Department of Endocrinology (Sharifi), Zanjan Metabolic
Diseases Research Center, and the Department of Molecular
Immunology (Esmaeilzadeh), Zanjan University of Medical Sciences,
Zanjan, Iran, and the Department of Gastroenterology (Zali), Research
Center for Gastroenterology and Liver Disease, Tehran, Iran.
Received 5th January 2008. Accepted 30th April 2008.
Address correspondence and reprint request to: Dr. Faranak Sharifi,
Associate Professor, Department of Endocrinology, Vali-e-asr
Hospital, Zanjan Metabolic Diseases Research Center, Zanjan,
Iran. Tel. +98 (241) 7270815. Fax. +98 (241) 4262486.
E-mail: faranaksharifi@hotmail.com

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www.smj.org.sa Saudi Med J 2008; Vol. 29 (6)
HFE mutation in diabetes mellitus ... Sharifi et al
Disclosure: This study was supported by the Zanjan
University of Medical Sciences and Research Center for
Gastroenterology and Liver Disease in Tehran.
H
is an autosomal recessive condition, in which excess
iron is absorbed by the intestine, and accumulates as
excess tissue iron over many years.4 Although effective
treatments for HH can cause normal life expectancy,
iron overload is dangerous for the body.5 Although
HH can be detected via biochemical blood analyses
and liver biopsies,6 these methods may be unreliable in
some cases.7,8 The discovery of the hemochromatosis
HFE (HLA-H) gene has provided a sensitive test for the
chromosome 6-linked component of hemochromatosis.9
Two disease-linked alleles were described. The allele that
is of major importance in symptomatic HH, is a G-to-A
transition of nucleotide 845 of the c-deoxy-ribonucleic
acid (DNA), which causes a cysteine 282 to tyrosine
amino acid change in the predicted protein. A second,
less-penetrant, allele is a C-to-G change of nucleotide
187 that causes a histidine 63 to aspartic acid change.
This mutation is in complete linkage disequilibrium with
the 845A mutation (in other words, no chromosome 6
with the 845A mutation carries the 187G mutation).
However, the 187G mutation is found in excess only
in HH patients when present with the 845A mutation
as a compound heterozygote (heterozygosity for both
845A and 187G). Most patients with HH (87-100%)
are homozygous for the C282Y allele, or are compound
heterozygotes for these mutations.2,3,9 Hereditary
hemochromatosis was recognized as a cause of diabetes
mellitus,10 and there are some reports of the prevalence
of HH with insensitive markers in subjects with
T2DM.11-13 The potential role of serum ferritin in the
pathogenesis of type 2 diabetes has become interesting
in recent years.14,15 Most studies have revealed elevated
ferritin concentration in the diabetic group of patients,
and pre diabetes situations in comparison with normal
subjects.16-18 This evidence suggest that excessive ferritin
concentration can be a marker of iron overload, and
sub clinical hemochromatosis in diabetic patients. Is
the high serum ferritin in type 2 diabetic patients the
consequence of mutations in HFE gene? It was not
revealed. Iron overload was revealed to decrease insulin
sensitivity,15,19 and can cause earlier complications
in diabetes mellitus.20 As we have no sufficient data
regarding the frequency of mutant HH gene in Iran,
this study was designed to search for an association
of HFE mutations with T2DM in the population of
Zanjan, north-western Iran.
ereditary hemochromatosis (HH) is one of the
most common genetic disease of Caucasians,1-3and
Methods. One hundred and one patients with
type 2 diabetes, including 70 female and 31 males
were recruited serially from the Zanjan Diabetes
Centre, Zanjan, Iran, in 2005. Patients included were
more than 20 years of age at the time of diagnosis,
and were not taking oral hypoglycemics for at least
6 months. Insulin treatment was not a reason for
exclusion, as long as the patients were without insulin,
6 months after diagnosis. Patients with type 2 diabetes
resulting from chronic pancreatitis, or steroid use were
excluded. In families with more than one diabetic
patient, one of them was randomly included in the
study, and others were excluded. One hundred and
one control subjects were age- and gender-matched
healthy people, who came for check up without
known diabetes. Peripheral venous blood sampling
was undertaken for lymphocyte DNA and iron studies.
Each patient was classified as one of 6 HFE genotypes,
as follows: 1) 845G/845G homozygote (normal), 2)
845A/845A mutant homozygote (potentially affected),
3) 845G/845A heterozygote 4 with 187C/187C
(normal), 5) 187G/187G homozygote (potentially
affected); (6) 187C/187G heterozygote. Approval from
the ethics committee of Zanjan University of Medical
Sciences was obtained. All participants were informed
regarding the aims of the study, and informed consent
was obtained from all of them.
DNA amplification.
extracted by salting out method,26 and amplified by
polymerase chain reaction (PCR). A PCR reaction
was performed in 25 ml volume with 12 pmol of HFE
exon 2 primers (5’-ACATGGTTAAGGCCTGTTGC-
3’ and 5’-GCCACATCTGGCTTGAAATT-3’), and
another PCR with 12 pmol of HFE exon 4 primers
(5’-CCTTCCTCCAACCTATAGAA-3’
CAGCCCATCCCCTAACAAAG-3’).27 For both, we
used 50-100 ng of genomic DNA, 1 unit Taq DNA
polymerase (Super Taq, HT Bioscience, UK), 0.2 mM of
each dATP, dGTP, dCTP, dTTP (HT Bioscience, UK), 2
mM magnesium chloride, and 1 x PCR buffer (50 mM
potassium chloride, 20 mM Tris-hydrochloric acid, pH
8.4). After an initial temperature of 94°C for one minute,
35 amplification cycles were performed on a personal
thermocycler (Eppendorf, Gmbh, Hamburg, Germany).
Each cycle consisted of denaturation at 94°C for 20
seconds, annealing at 55°C (C282Y), and 58°C (H63D)
for 20 seconds, and extension at 72°C for 40 seconds. A
final extension step (72°C for 10 minutes) terminated
the process. To verify proper amplification conditions,
5 µl of each PCR product from the amplification of
the HFE exons were then analyzed electrophoretically
on an ethidium bromide-stained 1% agarose gel at 100
volts (V) for 40 minutes. The amplification of exon 2
Genomic DNA was
and 5’-

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(for H63D mutation)were detected by the presence of
bands at 208 base pair (bp), and exon 4 (for C282Y
mutation) were detected by the presence of bands at
486 bp. To determine the polymorphism of exon 2,
5 µl of amplified DNA was digested with one µl of
BclI (10u/µl) restriction endonucleases (Fermentas,
Germany) treated with one µl of buffer G+ (Fermentas,
Germany) at 55°C for 3 hours, and to determine the
polymorphism of exon 4, 5 µl of amplified DNA
was digested with 0.5 µl of RsaI (10u/µl) restriction
endonucleases (Fermentas, Germany) treated with one
µl of buffer Y+ (Fermentas, Germany), at 37°C for 3
hours. Ten percent polyacrylamide gel electrophoresis at
150 V for 120 minutes was used to separate restriction
fragments. For HFE gene codon 63, bands at 138,
and 70 bp after endonuclease digestion revealed a
wild type allele, characterized by the presence of BclI
restriction site. The presence of a single band at 208
bp indicated homozygous mutant alleles, characterized
by the absence of BclI restriction site. The heterozygous
alleles were characterized by the presence of bands at
208, 138, and 70 bp. Polymorphism in codon 282
was investigated after RsaI digestion by the presence or
absence of bands at 193 and 164 bp. For this codon,
mutant allele contains 2 RsaI sites (GTAC) to split PCR
product into fragments of 293, 164, and 29 bp, whereas
wild type allele was characterized by the absence of one
site, and was revealed by presence of bands at 293 and
193 bp. Homozygosity for tyrosine at codon 282 was
determined by the presence of bands at 293 and 164 bp
(the 29 bp band is often not visualized), whereas 3 bands
at 293, 193, and 164 bp indicated heterozygosity.
Data analysis. Statistical analysis was conducted
using SPSS software (version 11.5). Proportions were
compared using the Chi-square test. Group means
were compared by using student’s t test. Differences in
allele frequencies between groups were assessed with
the Fisher’s exact test. Odds ratio was calculated for the
prevalence of HFE mutations in diabetic patients and
for both genders. Significance at p<0.05 was taken for
2-sided tests.
Results. Two hundred and two people, including
141 females and 61 males were recruited in this study.
Genotyping failed in 6 of the blood samples, and
finally we reported the genotype of 196 subjects. Table
1 presents general characteristics and biochemical data
of the diabetic and the control group. The mean serum
iron in subjects with mutant H63D alleles was 90±29
µg/l, and was not different from participants without
this mutation (90±37 µg/l, p=0.5). The serum iron
concentration in subjects with C282Y mutant alleles
was not different from those without this mutation
either (105±48 µg/l versus 88±36 µg/l, p=0.3). None of
Table 1 - General characteristics and biochemical markers of diabetic
and control group.
Parameters Diabetic group
(n=101)
55.4±11.4
31/70
6.5±5.9
160±40
7.8±2.1
91.3±38.9
Control group
(n=101)
55.2±11.3
30/71
-
80±14
-
84.7±34
p-value
Age, years
Male/female
Duration of diabetes, years
FPG, mg/dl
HbA1c, %
Serum iron, mg/dl
0.9
0.5
-
0.01
-
0.2
Hb, g/dl12.9±0.5 13.5±1.20.1
FPG - fasting plasma glucose, HbA1c - glycosated hemoglobin,
Hb - hemoglobin
Table 2 - Frequency of different types of HFE mutations in males and
females (n: males=60, females=136).
Frequencies of mutationGenderTotal
Female Male
Frequency of H63 mutation
Non, n (%) 100 (73.5) 39 (65)
139
Heterozygous n (%)
Homozygous n (%)
Frequency of C282Y mutation
Non, n (%)
Heterozygous, n (%)
Homozygous, n (%)
33 (24.3) 19 (31.7)
3 (2.2) 2 (3.4)
52
5
134 (98.5) 59 (98.3)
2 (1.5) 1 (1.7)
-
193
3
-
-
Table 3 - Clinical and biochemical data in homozygotes or heterozygotes
for mutant alleles of HFE gene in comparison with normal
genotype subjects (N=196).
GenotypeAge, yearsFrequency of
diabetes (%)
Serum iron
concentration
(mg/dl)
105±48
90.6±29
84±35.8
88±36
845G/845A
187G/187G
187C/187G
No mutation
p-value is non-significant, continous variables expressed as mean±SD
48.3±6.6
52.6±13.8
56.6±11.3
55±11.4
66.7
100
51.9
49.6
the 101 patients, and none of the 101 control subjects
were 845A/845A mutant homozygote (C282Y) (p=1.0).
Three of the 202 participants (1.5%, 95% (confidence
interval (CI): 0-3%) including 2 of the diabetic patients
(2%, 95% CI: 0-4%), and one of the control group
(1.1%) were 845G/845A mutant heterozygote (p=0.5).
Five of the 202 people participating in this study (2.6%,
95% CI: 0.4-4%) were homozygous for the H63D
mutation (187G/187G homozygote). All the subjects

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HFE mutation in diabetes mellitus ... Sharifi et al
with this type of mutation were diabetic (5%, 95% CI:
0.8-9.2%), and none of the control group were mutant
in this type (p=0.08).
Fifty-two of the 202 participants (25.7%,
95% CI: 20.4-32.6%) were heterozygote for mutation
H63D (187C/187G heterozygote) including 27 diabetic
(26.7%, 95% CI: 16.6-36.8%), and 25 normal (24.7%)
subjects (p=0.8). Odd’s ratio for mutation H63D in
diabetes mellitus was estimated to be 1.3 (95% CI:
0.6-2.4, p=0.4) and for mutation C282Y was 1.9 (95%
CI: 0.16-21, p=0.6). Table 2 presents the frequency of
the mutations in diabetic and control people in terms
of their gender. Table 3 illustrates the clinical and
biochemical data in homozygotes or heterozygotes for
mutant alleles of HFE gene in comparison with normal
genotype subjects.
Discussion. The prevalence of the HFE mutation
in the Iranian population was not previously reported.
We did not detect an excess of HFE mutations in
patients with T2DM as compared with matched
control subjects without known diabetes in this study.
Most of the previous studies using biochemical or tissue
markers in patients with diabetes have failed to detect
an excess of HH,23,24 although there are exceptions.25,26
Some of the methods used in these studies have poor
diagnostic accuracy,11,27 and glycemic control may have
an influence on some of these measurements.27 The
present study, using a more specific genetic marker for
mutations in the HFE gene, has failed to demonstrate
any significant excess in the mutations in patients with
T2DM. The prevalence of 845A homozygosity of 0/101
in our population with T2DM was identical to the age-
and gender-matched control subjects. Despite the small
sample size in our study, our results for the frequency
of 845A homozygosity is very close to the frequency of
1/212 observed in a population of diabetic patients in
the United Kingdom.28
No excess of HFE 845A homozygosity in T2DM has
been reported in different populations recently.29-35 When
available data from these studies14,29-38 are combined
with the present data, the overall prevalence of HFE
845A homozygosity becomes 0.46% in T2DM, as
compared with 0.365% in those without diabetes
(p=0.76). Although the majority of these studies (and
the present study), have not shown an excess of mutant
alleles in the type 2 diabetes populations, there are
some exceptions. Kwan et al33 studied 105 patients
with T2DM, and 103 patients with type 1 diabetes as
a control group. Although there was no excess of 845
(C282Y) homozygotes in the type 2 population, 22
heterozygotes were present. A borderline significant
increase in the frequency of this allele in the type 2
patients (odds ratio 2.1; 95% CI: 1-4.5; p=0.048) was
detected. Some researchers pointed out, that these data
in a small sample should be interpreted cautiously. In
another report from Fernandez-Real et al,34 in 170
Spanish patients when compared with 108 control
subjects without diabetes, a borderline significant
increase in the H63D allele frequency was detected
(odds ratio 1.17; 95% CI: 1.01-1.36; p=0.04). The
frequency of the H63D mutation as reported in this
study was lower than the frequency of 26.3%, reported
for a different Spanish population.2 This finding may in
part be explained by the small control group. We have
found that neither homozygosity nor heterozygosity for
the H63D mutation was elevated in an Iranian diabetic
population, as compared with results in control subjects.
One previous observation in England revealed, that the
penetrance of the HFE gene with respect to disease
manifestations may be low in the local population as
most of the HFE 845A homozygotes are not diagnosed
with the manifestations of hemochromatosis.3 In
another study, patients carrying at least one C282Y
allele had a younger age of onset of diabetes as well as
a longer duration of the disease (p=0.007).39 We could
not detect an earlier age of onset in type 2 diabetic
patients carrying HFE mutations in our study. We
did not search for diabetes complications in this
study, however, an increased prevalence of retinopathy
(p=0.014), and of nephropathy (p=0.04) were detected
in individuals carrying at least one C282Y allele in some
other studies.39-41
In our study, the 2 845A heterozygous patients
identified in the group with diabetes did not have elevated
iron levels, suggesting the probable low penetrance of the
HFE gene with respect to biochemical manifestations
in this population. In one previous study,19 T2DM
patients, females in particular, with mono-allelic C282Y
mutation, had slightly increased iron parameters. In
that study, no difference in insulin sensitivity or B cell
function was observed in the presence of mono-allelic
HFE mutations. The treatment of hemochromatosis
may retard the progression of T2DM42 it is still
necessary to consider it for individual T2DM patients
who may warrant screening, such as those with a family
history of hemochromatosis, those with an associated
cardiomyopathy or gonad failure, and perhaps those
with abnormal liver function tests.
In conclusion, the present data from Iranian
population, and other similar reports from other
countries do not support population-based genetic
screening of patients with T2DM, to detect the alleles
C282Y and H63D.
Acknowledgment. We are grateful to Dr. Kalantari from
the laboratory of Booali for his contribution in this study, and Dr.
Mousavinasab for the statistical analysis.
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