1
HAMP, Transferrin (exon 7) and HFE genotyping suggests that haplotype block
2
analysis is the best strategy for predicting iron deficiency phenotype in women
3
Beatriz Sarriá 1a Ana M López-Parrab, Santiago Navas-Carreteroa, Ana M Pérez-
4
Granadosa, Carlos Baezab, Eduardo Arroyo-Pardob and M Pilar Vaqueroa
5
a
Department of Metabolism and Nutrition, Instituto del Frío (CSIC) 28040 Madrid, Spain.
6
b
Department of Toxicology and Health Legislation, Medicine Faculty (UCM) 28040 Madrid,
7
Spain.
8
9
Abstract
10
Strategies to draw associations between disease and genes and nutrients are the
11
examination of mutations and/or haplotypes. We studied, the relationship between certain
12
mutations in HAMP (hepcidin, exon 1, 2 and 3), Tf (transferrin, exon 7) and HFE
13
(hemochromatosis, H63D and C282Y) genes, as well as their associations in haplotypes, and
14
iron deficiency with or without anemia in young women. One-hundred-and-sixty-two young,
15
menstruating women were recruited. Hemoglobin (Hb g/dl), ferritin (Ft μg/l), total iron
16
binding capacity, transferrin saturation, hematocrit and mean corpuscular volume were
17
determined and subjects were divided in 3 iron status: sufficient (Hb ≥12, Ft ≥20; n=63),
18
deficient (Hb≥12, Ft<20 or Hb<12, Ft≥20; n=77) and deficient anemic (Hb <12, Ft<20;
19
n=22). Mutations in HAMP gene were detected using single strand conformational
20
polymorphism analysis, in exon 7 of the transferrin gene using DNA sequencing and C282Y
21
and H63D mutations in HFE gene using restriction enzymes. Among the iron deficient
22
women, 8 were G277S/L247L heterozygous (except 1 L247L homozygote) and 1 was I7V
23
heterozygous for the mutation in HAMP gene. Among the iron deficient anemic, 3 were
24
G277S/L247L/H63D heterozygous (except 1 H63D homozygote) and 1 I7V heterozygote.
25
Among the iron sufficient, 2 were G277S/L247L/H63D heterozygous, 1
26
G277S/L247L/H268H heterozygote and 2 G277S/L247L heterozygotes. We conclude that: 1)
27
the iron deficient anemic group presented the highest proportion of subjects carrying
28
mutations 2) there are 3 possible haplotypes G277S /L247L, G277S/L247L/H63D and
29
G277S/L247L/H268H, non restricted to any iron status 3) the analysis of haplotypes or
30
haplotype blocks is the best strategy to associate genes and iron deficiency.
1
Corresponding author: Beatriz Sarriá Telephone: +34 91 549 0038; Fax: +34 91 549 36 27; Email: beasarria@if.csic.es
1
1
Key words: Hepcidin, HAMP, transferrin, HFE, haplotype, iron deficiency, iron
2
deficiency anemia, women, humans
3
4
5
1. Introduction
If dietary recommendations are based on genotype, the influence of diet on health and
6
on the prevention of disease will be undertaken much more efficiently. A mass number of
7
genes form each individual’s genotype. Most genes have small sequence differences or
8
polymorphisms and often these polymorphisms may affect how well a protein works and how
9
it interacts with other proteins or substrates. Single nucleotide polymorphisms (SNPs) are the
10
most common form of DNA sequence variation and are useful polymorphic markers for
11
investigating genes. Haplotypes are sets of SNP alleles along a region of a chromosome. A
12
recent strategy to draw associations between disease and genes and nutrients is the
13
examination of haplotypes or haplotype blocks [1].
14
Iron deficiency anemia is only part of the overall syndrome of iron deficiency which
15
reaches even higher prevalence. Little is known about the genetic basis of non-diet related
16
iron deficiency. The contribution of genetic differences between women to variation in iron
17
stores outweighs the comparatively small effects of interindividual variation in iron loss
18
through menstruation and number of pregnancies [2]. A polymorphism in exon 7 (G277S) of
19
the transferrin gene (Tf), which corresponds to the transferrin electrophoretic C3 variant, has
20
been associated with a significant reduction in total iron binding capacity and higher mean
21
transferrin saturation predisposing menstruating women to iron deficiency anemia [3]. This
22
variant is found at the highest frequency in the white population (frequency of 0.0634 (111 of
23
1752 alleles; [3]). However, Aisen [4] constructed the mutation in a human transferrin
24
expression vector and obtained that mutant and native proteins were equally efficient at
25
donating iron. Accordingly, no relationship was found between transferrin concentration, iron
26
status and the G277S variant in pregnant women [5]. The G277S transferrin mutation did not
2
1
affect iron absorption in iron deficient women versus the wild-type [6]. Considering this
2
disagreement, the role of G277S polymorphisms in iron deficiency anemia also needs to be
3
clarified. The G277S mutation may be associated with other transferrin mutations, and/or
4
other polymorphisms located in other genes.
5
It is widely known that HFE (hemochromatosis) and HAMP (hepcidin) genes are
6
involved in iron metabolism. HFE mutations C282Y and H63D are responsible for iron
7
overload [7, 8]. However, the role of C282Y and H63D in iron deficiency remains
8
controversial. According to Beutler et al [9] in a large cohort of patients with HFE mutations,
9
the prevalence of non-anemic iron deficiency was significantly lower among female carriers
10
of the C282Y mutation compared with HFE wild types. However, prevalence of frank iron
11
deficiency anemia did not differ significantly among genotypes. Datz et al [10] pointed to the
12
C282Y mutation having a protective role against iron deficiency in young women. C282Y
13
heterozygote women showed significantly higher values for hemoglobin, serum iron, and
14
transferrin saturation than women homozygous for the wild-type allele, but there was no
15
significant difference in the prevalence of either iron deficiency anemia or iron depletion
16
between these groups. Similarly, Rossi et al [11] found a significantly higher mean
17
hemoglobin levels among C282Y/H63D compound heterozygotes compared with wild-type
18
homozygotes without significant difference in the prevalence of subjects with iron depletion
19
(defined as ferritin<20 µg or ferritin <12 µg/l and a transferrin saturation <15%) between
20
genotypes. The H63D mutation is relevant in certain geographical areas, such as Spain [12,
21
13, 14] but its frequency and clinical penetrance has been studied to less extent than C282Y.
22
None of these studies have looked into the association of the C282Y and H63D with other
23
mutations in iron metabolism proteins.
24
25
HAMP gene codes for hepcidin, which plays an essential role in iron homeostasis
[15]. This peptide is a novel, hepatic, antimicrobial hormone that could provide the link
3
1
between iron stores and intestinal absorption [16]. At molecular level, it seems to be the key
2
systemic regulator of iron absorption [17]. In mice, anemia seems to be the predominant
3
issue in regulating the HAMP gene [18]. Given that it plays such an important role in iron
4
homeostasis, it might be predicted that mutations in HAMP gene may result in a distinctive
5
iron-related phenotype [19, 20].
6
The study of Tf/HFE/HAMP haplotypes may yield stronger associations between iron
7
deficiency phenotype and genotype. The aim of this work is to determine HAMP, Tf (exon 7)
8
and HFE mutations and their possible linkage for predicting iron deficiency phenotype in
9
women.
10
11
2. Methods and materials
12
13
14
2.1. Subjects
Volunteer recruitment was carried out by setting up advertisements in the
15
Complutense University campus and by giving short talks about the study between lectures.
16
One hundred and sixty-two, 18-45 year-old, non-pregnant, non-smoking, white, menstruating
17
women were recruited.
18
Volunteers had not been treated with iron for the last twelve months. All subjects
19
underwent a pre-study screening which included a blood test and a health questionnaire.
20
This study was approved by the Ethics Committee of Hospital Clinica Puerta de Hierro and
21
Consejo Superior de Investigaciones Cientificas, in Madrid (Spain).
22
23
2.2. Study design
24
4
1
This is an observational study. Subjects were divided into three groups attending to
2
their iron phenotype, established by hemoglobin (g/dl) and ferritin (μg/l) values: iron
3
sufficient (Hb ≥12, Ft ≥20), deficient (Hb≥12 and Ft<20 or Hb<12 and Ft≥20) and anemic
4
(Hb<12 and Ft<20).
5
Exon 7 of Tf gene, C282Y and H63D mutations in the HFE gene, and the 3 exons of
6
the HAMP gene were genotyped in the former three groups. Genetic results were used to
7
establish haplotypes in order to study associations between haplotypes and iron status.
8
9
2.3. Blood sampling
10
11
Blood samples were collected by venipuncture into EDTA tubes. DNA was extracted
12
from whole blood using standard phenol-chloroform methodology with proteinase K (adapted
13
from Sambrook et al [21].
14
15
2.4. Biochemical and hematological measurements
16
17
Measurements of hemoglobin concentration (Hb), serum ferritin (Ft), hematocrit
18
(Hct), mean corpuscular volume (MCV), serum transferrin (Tf) and serum iron (Fe) were
19
carried out following standard laboratory techniques and using the Symex NE 9100
20
automated hematology (Symex, Kobe, Japan) and the Modular Analytics Serum Work Area
21
(Roche, Basel, Switzerland) analyzers. Total iron binding capacity (TIBC (µmol/l) = 25.1 x
22
Tf (g/l)) and transferrin saturation (TfS (%) = [Fe (g/dl)/TIBC(g/dl)] x 100) were
23
calculated.
24
25
2.5. Genetic analysis
5
1
2
Amplification of exon 7 was performed using polymerase chain reaction (PCR) in a 25
3
μl reaction volume containing 1X Standard Buffer (Tris-HCl (pH 9,0) 75mM, KCl 50 mM,
4
(NH4)2SO4 20 mM), 2.5 mM MgCl2, 200 μM dNTPs mix, 1 U Taq polymerase (Biotools) and
5
0.200 μM of each primer. PCR programming was carried out in a eppendorf mastercycler
6
using the following conditions: for initial denaturizing 1 cycle at 95 ºC – 3 min; followed 32
7
cycles of amplification at 93 ºC – 30 s, 62 ºC – 30 s, 72 ºC – 30 s and 1 cycle of final
8
extension at 72 ºC - 7 min. Exon 7 of transferrin gene was amplified in the 162 samples using
9
primers described by Beutler et al., [22]. The PCR product was purified with a QIAquick gel
10
extraction kit (QIAGEN) and sequenced on an Applied Biosystems automatic sequencer
11
model 3730. Results were compared to a consensus sequence for transferrin gene found in
12
Ensembl database v34, Gen ID ENST00000264998. Hemochromatosis gene mutations,
13
C282Y and H63D, were analyzed using restriction enzymes according to Alvarez et al [14] in
14
the all the samples. The 3 exons of the HAMP gene were amplified in all 162 subjects
15
according to Zaahl et al [23]. Amplicons were then analyzed by SSCP in an ALF Sequencer
16
(Pharmacia) [24], according to the manufacturer’s protocol with slight modifications
17
(ALFexpress, Pharmacia Biotech). Mutants yielded an abnormal pattern of double peaks
18
which was clearly detected (Fig. 1). PCR products of mutated individuals were purified with a
19
QIAquick gel extraction kit (QIAGEN) and sequenced on an Applied Biosystems automatic
20
sequencer model 3730. The sequences obtained were compared to a consensus for HAMP
21
gene found in Ensembl database v34, Gen ID ENSG00000105697.
22
23
2.6. Statistical methods
24
6
1
An explanatory analysis of the data was carried out considering the number of subjects
2
studied and the fact that we obtained, within each iron status, different number of subjects,
3
carrying different number of mutations. With this purpose three variables were generated: a)
4
iron status or degree of iron deficiency, b) number of mutations carried and c) proportion of
5
subjects carrying mutations in different genes within each iron status. These variables were
6
globally transformed and normalized by “optimal scaling” [25]. CATPCA-Principal
7
components analysis for categorical data by Data Theory Scaling System Group (DTSS)
8
(Leiden, The Netherlands) was used.
9
10
11
3. Results
12
13
Attending to the hemoglobin (g/dl) and ferritin (μg/l) values obtained, among the 162
14
subjects, 63 were iron sufficient (Hb ≥12, Ft ≥20), 77 iron-deficient (Hb≥12 and Ft<20 or
15
Hb<12 and Ft≥20) and 22 iron deficient anemic (Hb<12 and Ft<20). The hematological and
16
biochemical parameters data in the three iron status groups are shown in table 1. The values
17
for the other iron status indicators examined: total iron binding capacity, transferrin
18
saturation, hematocrit and mean corpuscular volume were within the range corresponding to
19
iron sufficiency, deficiency and deficiency anemia, respectively. Only the transferrin
20
saturation data (%) was slightly high in the iron deficient group attending to the cutoff point
21
for abnormal values proposed by Dallman et al [26] of <16%, as well as the mean corpuscular
22
volume in the iron deficient anemic group according to Gibson [27] of 80 fl.
23
Table 2 shows all the different genotypes obtained attending to HFE (C282Y and
24
H63D), transferrin (exon 7) and HAMP (3 exons) gene mutations as well as genotype
25
frequencies in the iron sufficient, iron deficient and iron deficient anemic groups. Three
7
1
mutations were observed in exon 7 of the transferrin gene: L247L, G277S and H268H. As far
2
as the HFE gene, H63D heterozygotes and 1 homozygote were obtained. Concerning the
3
hepcidin gene, one SNP, I7V, was obtained in exon 1. Among the group of 63 iron sufficient
4
women, 5 subjects carried mutations: 2 subjects were heterozygous for H63D, G277S and
5
L247L mutations, one was heterozygous for G277S, L247L and H268H and two were
6
heterozygous for G277S and L247L. Among the 77 iron deficient women, 8 were
7
heterozygous for G277S and L247L (except 1 L247L homozygote) and 1 subject was
8
heterozygous for the I7V mutation in the HAMP gene. Among the subgroup of 22 iron
9
deficient anemic, 3 G277S/L247L heterozygous individuals who also presented the H63D
10
mutation (1 was homozygote) were obtained and 1 subject was heterozygous for the I7V
11
mutation in the HAMP gene.
12
Attending to these results, there seems to be an association between these mutations
13
and there are three possible haplotypes, G277S/L247L which was obtained in the iron
14
sufficient and deficient groups, G277S/L247L/ H63D in the iron sufficient and iron deficient
15
anemic groups and G277S/L247L/H268H/ corresponding to an iron sufficient subject.
16
The prevalence of iron deficiency in women with genotype G277S/G277G was 18.7%
17
and 13.0% in G277G/G277G. In the 3 iron status groups, subjects carrying the G277S
18
mutation showed a slight reduction in TIBC [G277G/G277S 62.9, 74.4 and 73.0 versus
19
G277G/G277G 70.4, 79.2 and 83.6 for iron sufficient, iron deficient and iron deficient anemic
20
subjects, respectively].
21
In order to further interpret our results, an explanatory analysis was carried out and
22
three variables were generated: a) iron status or degree of iron deficiency (3 levels: 1 iron
23
sufficient, 2 iron deficient and 3 iron deficient anemic) b) number of mutations (from 1 to 5)
24
c) proportion of subjects carrying mutations in different genes within each iron status
25
(intervals). The distribution of the number subjects carrying mutations, the number of
8
1
mutations and the proportion of subjects carrying mutations in different genes within the three
2
iron status levels is reported in table 3.
3
Once these variables were globally transformed and normalized by optimal scaling,
4
using the new scale it was possible to calculate correlations, such as Pearson’s correlations,
5
and express the results in a subspace where both variables and objects (subjects with a certain
6
iron status, number of mutations, and proportion of subjects carrying mutations in different
7
genes) were projected. Figure 2 allows our results to be visually interpreted. Virtual axis X
8
represents the variables proportion of subjects carrying mutations and iron status, which are
9
highly correlated, and virtual axis Y shows the number of mutations, which is independent
10
from the degree of iron deficiency.
11
The results of this study indicate that the distribution of the number of subjects
12
carrying mutation(s), the number of mutations and the proportion of subjects carrying
13
mutations within the iron sufficient, iron deficient and iron deficient anemic groups was not
14
homogenous, but the iron deficient anemic group presented the highest proportion of subjects
15
carrying mutations followed by the iron deficient and the least the iron sufficient (table 3, Fig.
16
2).
17
18
4. Discussion
19
20
The percentages of iron-deficient menstruating white women within the G277G
21
transferrin genotype are comparable to those described by Lee et al [3] who worked with a
22
larger group of white women (n=1511) and obtained a higher prevalence of iron deficiency in
23
menstruating white women with genotype G277S/G277S and G277S/G277G than in the wild-
24
type subjects. Accordingly, we obtained a higher prevalence of iron deficiency in women with
25
genotype G277S/G277G (18.7%) compared to G277G/G277G (13.0%) although we do not
9
1
disregard that we worked with a smaller number of subjects. Furthermore, in accordance with
2
the above indicated authors G277S carriers also showed a slight reduction in TIBC.
3
Nevertheless, the outcome of the present work does not point to a clear association between
4
the G277S mutation and iron deficiency.
5
The L247L mutation in exon 7 was detected in all 16 subjects who carried the G277S
6
mutation, this indicates that they are closely enough linked to be inherited as a unit. The
7
G277S/L247L haplotype has not been described previously in the literature. L247L, on its
8
own, has recently been identified in a patient with atransferrinemia [28] who presented severe
9
hypochromic, microcytic anemia. Another silent mutation, H268H, was also identified in one
10
G277S carrier. Haplotypes G277S/L247L and G277S/L247L/H268 need to be further
11
investigated as well as their association to iron deficiency.
12
As far as the HFE gene, we obtained a higher frequency for the H63D mutation than
13
for C282Y which is in agreement with population studies that indicate that the H63D
14
mutation is more common, particularly in Spain compared to other countries in Europe [12,
15
13, 14]. Mutations in the HFE gene have been extensively studied and shown different
16
degrees of clinical penetrance. The relationship between HFE mutations and iron deficiency
17
anemia is controversial and has been mostly evaluated in individuals carrying the C282Y
18
mutation rather than H63D. The presence of the G277S allele in the heterozygous state in
19
HFE patients homozygous for the C282Y mutation has been suggested to reduce mean TIBC
20
levels to values similar to those of Caucasian individuals with a normal HFE gene [3]. In the
21
present work, there were 2 heterozygotes and 1 mutant homozygote for the H63D mutation,
22
all being G277S carriers, who were iron deficient and showed TIBC (µmol/L) values of 69
23
and 71.8, the heterozygotes, and 73.8 the homozygote, which are close to the cutoff point of
24
73 indicating low iron status [29]. In contrast, 2 subjects who were H63D/G277S carriers
25
were iron sufficient (TIBC (µmol/L) values of 67 and 50). Given this small number, it was not
10
1
possible to draw any conclusion as to how the H63D/G277S genotype affects TIBC levels and
2
consequently iron status. This outcome points to the existence of another possible haplotype
3
G277S/L247/H63D which role in iron status needs to be studied in more detail.
4
Mutations in the hepcidin peptide can originate abnormal peptides which are involved
5
in dysfunctions of iron metabolism [20]. The I7V hepcidine gene mutation affects leader
6
peptide sequence and substitutes a hydrophobic amino acid (Isoleucine) with another
7
hydrophobic one (Valine). It is interesting that the indicated mutation was found in an iron-
8
deficient and iron-deficient anemic subject. However, from two volunteers, no reliable
9
conclusion concerning the relationship between I7V and iron status may be drawn. This
10
11
mutation was not associated to any other in the genes studies.
The results of the present study indicate that although SNPs may hold true for some
12
conditions, iron status pathologies are complex from a genetic point of view and thus the
13
phenotype predominantly depends on the combined variation of several genes apart from
14
environmental and behavioral factors. The probable existence of linkage disequilibria among
15
mutations can be very helpful for a better prediction of iron deficiency. In agreement with
16
Trujillo et al [2], we point to haplotype block analysis as the best strategy in understanding
17
the distribution of risk alleles in human populations which could lead to tailoring prevention
18
strategies to those at increased risk.
19
We conclude that the iron deficient anemic group presented the highest proportion of subjects
20
carrying mutations of genes associated with iron deficiency as well as overload, although the
21
sample size of this group was small. We found that there are 3 possible haplotypes G277S
22
/L247L, G277S/L247L/H63D and G277S/L247L/H268H, non restricted to any iron status.
23
Further studies should be focused on the analysis of haplotypes or haplotype blocks to
24
associate genes and iron deficiency.
11
1
This study opens a new pathway to the understanding of the genetic factors involved in iron
2
deficiency, which leads to considering the use of diets based upon individual genotype, iron
3
requirements and status.
4
5
Acknowledgments
6
This study was funded by the EU (MERG-CT-2003-506368) and the Spanish
7
government (AGL 2002-04411-C02-02 ALI). Santiago Navas is granted with a Comunidad
8
de Madrid fellowship and Social European Funds. The authors thank the volunteers who took
9
part in this study and Laura Barrios for the statistical analysis.
10
11
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[27] Gibson R. Principals of Nutritional Assessment: A Laboratory Manual. New York:
Oxford University Press, 1993.
[28] Knisely AS, Gelbart T, Beutler E. Molecular characterization of a third case of human
atransferrinemia. Blood 2004; 104(8): 2607.
[29] Cook JD, Finch CA. Assessing iron status of a population. Am J Clin Nutr 1979; 32(10):
2115-9.
12
13
14
Abreviations list
15
Hemoglobin: Hb
16
Serum ferritin: Ft
17
Single nucleotide polymorphisms: SNPs
18
Hematocrit: Hct
19
Mean corpuscular volume: MCV
20
Serum transferrin: Tf
21
Serum iron: Fe
15
1
Total iron binding capacity: TIBC
2
Transferrin saturation: TfS
3
Polymerase chain reaction: PCR
4
Iron sufficient: IS
5
Iron deficient: ID
6
Iron deficient anemic: IDA
7
8
16
1
Figure legends
2
3
Figure 1. SSCP electropherograms (left) and sequence (right) of HAMP genes (exon 1) of a
4
homozygous individual with the A/A genotype (A) and a heterozygous individual with the
5
I7V mutation (A->G) (B).
6
7
Figure 2. Bidimensional dispersion diagram. Virtual axis X represents the variables
8
proportion of subjects carrying mutations and iron status and virtual axis Y the number of
9
mutations which are independent from the degree of iron deficiency. The objects represent
10
the subjects with a certain iron status, number of mutations, and proportion of subjects
11
carrying mutations in different genes.
12
17
1
18
1
2
3
Table 1. Hematological and biochemical characterization of the volunteers who participated in the study.
Iron status
Iron sufficient
(n=63)
Iron deficient
(n=77)
Iron deficient
anemic
(n=22)
Hemoglobin (g/dl)
13.5±0.8
13.3±0.7
11.3±0.5
Serum ferritin (µg/l)
37.0 (36-50)
11.5 (11-16)
6.2 (5.5-10)
70±10
79±12
82±13
25.1±11.6
22.1±10.4
13.9±11.2
Hematocrit (%)
40±2
40±2
35±1
Mean corpuscular
volume (fl)
89±4
88±4
83±7
Total iron binding
capacity (µmol/l)
Transferrin
saturation (%)
4
5
6
7
Values are arithmetic mean ± standard deviation
Ferritin values are expressed as geometric mean (confidence interval).
8
9
10
11
12
1
1
2
Table 2. Genotypes obtained attending to mutations in HFE, transferrin and HAMP genes. Genotype frequencies in the iron status groups
Genes
Genotypes
3
4
5
HFE
Genotype frequencies (%)
HAMP
1
Position
282
-/-
Position
63
-/-
Position
247
-/-
Transferrin
Position
268
-/-
2
-/-
-/-
-/-
-/-
-/-
3
-/-
-/-
+/-
-/-
4
-/-
-/-
+/-
5
-/-
-/-
6
-/-
7
Iron
sufficient
Iron
deficient
Iron deficient
anemic
18.52
19.75
3.70
+/-
0
0.62
0.62
-/-
-/-
4.94
1.85
0.62
+/-
-/-
-/-
0
0.62
0
+/-
+/-
+/-
-/-
0.62
0
0
-/-
+/-
-/-
+/-
-/-
1.23
3.70
0
-/-
-/-
+/
-/-
-/-
-/-
0
0
0.62
8
-/-
-/-
+/
-/-
+/-
-/-
0
0.62
0.62
9
-/-
+/-
-/-
-/-
-/-
-/-
5.55
14.81
4.32
10
-/-
+/-
+/-
-/-
-/-
-/-
1.85
1.85
1.23
11
-/-
+/-
+/-
-/-
+/-
-/-
1.23
0.62
0.62
12
-/-
+/
-/-
-/-
-/-
-/-
1.85
1.85
0
13
-/-
+/
+/-
-/-
+/-
-/-
0
0
0.62
14
+/-
-/-
-/-
-/-
-/-
-/-
2.47
1.23
0
15
+/-
-/-
+/-
-/-
-/-
-/-
0
0
0.62
16
+/-
-/-
-/-
+/-
-/-
-/-
0.62
0
0
Position
277
-/-
Position
7
-/-
Black font represent: homocygote wild-types, red font: heterocygote mutants; blue font: homocygote mutants.
6
2
1
2
3
4
Table 3. Distribution of the number of subjects carrying mutations, the number of mutations and the proportion of subjects carrying mutations in different
genes within the three iron status levels *
Iron status (degree
of iron deficiency)
Number of subjects
carrying one or
more mutations
Number of
mutations
Proportion of
subjects carrying
mutations **
5
6
7
8
9
1. Iron sufficient
(n=63)
2. Iron deficient
(n=77)
3. Iron deficient anemic
(n=22)
2
1
2
7
1
1
2
1
1
3
3
2
2
2
1
3
3
1
0,03
0,02
0,03
0,09
0,01
0,01
0,09
0,05
0,05
* Each column corresponds to subjects carrying identical mutations within each iron status group.
** Calculated as Number of subject / n of the corresponding iron status group
10
11
12
13
3
1
4
1
2
3
4
5
6
7
Figure 1.Wild-type and Mutant patterns in the HAMP gene determined by Single Strand
Conformational Polymorphism
Pattern A: Peak for Wild-type genotype in HAMP gene.
Pattern B: Abnormal double peak for mutant genotype in HAMP gene
8
9
10
11
12
13
14
1
1
2
Figure 2. Distribution of subjects attending to the number of mutations present, the proportion
of subjects carrying mutations and iron status.
2
Number of mutations
ID
IS
IS
Proportion of subjects carrying mutations
IDA
IDA
IS
ID
0
2
Dimension 2
1
Iron status
-1
IDA
ID
-2
-1,5
-1,0
-0,5
0,0
0,5
1,0
1,5
2,0
Dimension 1
3
4
5
6
7
8
9
10
11
12
13
14
IS= iron sufficient group (n= 63)
ID=iron deficient group (n= 77)
IDA=iron deficient anemic (n= 22)
Normalization and distribution of subjects performed by Pearson’s correlation test.
15
2