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.SUPPLEMENTARY MATERIAL
Title: A candidate gene approach to identifying differential iron responses of young
overweight women to an energy-restricted haem iron-rich diet
Running head: Genetic iron responses to a haem iron-rich diet
Authors and affiliations: Hoi Lun Cheng1, Dale P. Hancock2, Kieron B. Rooney1, Katharine
S. Steinbeck3, Hayley J. Griffin1, and Helen T. O’Connor1
1
Discipline of Exercise and Sport Science, Faculty of Health Sciences, The University of
Sydney, Australia
2
School of Molecular Bioscience, The University of Sydney, Australia
3
Academic Department of Adolescent Medicine, The University of Sydney, Australia
Author for correspondence and reprint requests:
Hoi Lun Cheng
Discipline of Exercise and Sport Science, Faculty of Health Sciences, University of Sydney,
P.O. Box 170, Lidcombe, NSW 1825, Australia
Telephone +61 404343872
Fax +61 2 9351 9204 Email: hche3056@uni.sydney.edu.au
Conflict of interest: A research grant supporting this work was awarded to HTO, KSS and
KBR from Meat and Livestock Australia. The authors declare no other conflicts of interest.
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SUPPLEMENTARY METHODS
Clinical Trial Registry and Ethics
Both studies were registered with the Australian New Zealand Clinical Trials Registry
(ACTRN12613000072718; ACTRN12609000307202) and approved by the Sydney South
West Area Health Service Ethics Review Committee.
Estimation of dietary intake
Three-day food records were collected after the first, second, third, sixth and twelfth month
of intervention, totalling 15 days over the longitudinal study period. To maximise accuracy,
each participant received kitchen scales and training on recording food intake from a
dietitian. When required, recorded food portions were verified with the assistance of a visual
aid.1 Nutrient analysis was performed using FoodWorks Version 6.0.25175 (Xyris Software,
Brisbane, Australia).
Biochemical analysis
Fasting morning venous blood samples were collected at baseline, six and 12 months.
Haemoglobin was measured using the Sysmex Instrument (Roche Diagnostics Australia,
Sydney, Australia) with anaemia defined as haemoglobin <120 g/l. Serum iron, transferrin
saturation and serum ferritin were measured using the Roche Modular E170 Immunoassay
Analyser (Roche Diagnostics Australia, Sydney, Australia). Plasma sTfR and CRP were
analysed using commercial ELISA kits (R&D Systems, Minneapolis, USA). Plasma hepcidin
was measured only at baseline by on-line extraction coupled to liquid chromatographytandem mass spectrometry using the Xevo TQ MS (Waters Corporation, Milford, USA).2
Inter-assay accuracy and coefficient of variation for plasma hepcidin analysis were 95% and
8.2% respectively. Assay sensitivity was 2.00 ng/ml, with values below the detectable range
defined as 1.00 ng/ml. The reference ranges used were: 120-165 g/l for haemoglobin; 10.0-
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30.0 µmol/l for serum iron; 12.0-45.0% for transferrin saturation; and 15.0-165.0 µg/l for
serum ferritin; 0.74-2.39 mg/l for sTfR; and 1.92-32.40 ng/ml for hepcidin.3, 4
Genetic analysis – DNA extraction
DNA was extracted from 1 ml frozen packed cells using a Wizard® genomic DNA extraction
kit (Promega Corporation, Madison, USA) in accordance with the manufacturer’s
instructions.
Genetic analysis – DNA target sequence amplification
A pair of forward and reverse oligonucleotide primers intended to amplify 167 base pairs
surrounding the SNP rs855791 were generated using a primer design programme,5 and
verified using an in-silico polymerase chain reaction (PCR) tool.6 The custom primers were
obtained from Sigma-Aldrich Australia.
The target sequence was amplified by polymerase chain reaction (PCR) using DNA from five
participants. The custom primer and target sequences are shown in Supplementary Table 1,
with details of the PCR reaction set-up outlined in Supplementary Table 2.
Supplementary Table 1. Custom oligonucleotide primer and target sequences
Primers
TMPRSS6fwd
TMPRSS6rev
Target sequence
Tm (ºC)
Any SC
3’ SC
Sequence (5’ to 3’)
64.1
63.5
8.00
2.00
2.00
0.00
TCTGCAGAAAGTGGATGTGC
GCATCCTTTCTCCCTCCTCT
6.00
2.00
TCTGCAGAAAGTGGATGTGCAGTTG
ATCCCACAGGACCTGTGCAGCGAGG
[C/T]CTATCGCTACCAGGTGACGC
CACGCATGCTGTGTGCCGGCTACCG
CAAGGGCAAGAAGGATGCCTGTCAG
GTGAGTCCCCCGGGCATGGGAGGGA
GAGAGGAGGGAGAAAGGATGC
Tm, melting temperature; SC, self-complementarity
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Supplementary Table 2. PCR reaction set-up and thermal cycler program
PCR reaction set-up (total volume 50 µl)
Thermal cycler program
- 5 µl genomic DNA
95ºC × 5 min
- 1.5 µl TMPRSS6fwd primer (5 µM)*
95ºC × 1 min
repeated for
- 1.5 µl TMPRSS6rev primer (5 µM)*
60ºC × 1 min
35 cycles
- 5 µl 10× PCR buffer†
72ºC × 1 min
- 5 µl dNTPs (2 mM)
72ºC × 7 min
- 1 µl Taq polymerase (5 units/µl)
4ºC × ∞
- 31 µl sterile nuclease free water
DNA, deoxyribonucleic acid; dNTPs, deoxyribonucleotide triphosphates; PCR,
polymerase chain reaction; ∞, indefinite amount of time
*rehydrated in 10mM Tris-HCl (pH 7.4) and 1mM EDTA (1× TE buffer)
†10× PCR buffer contains 200 mM Tris-HCl (pH 8.4) and 500 mM KCl
Genetic analysis – purification of the PCR product
DNA was purified by gel electrophoresis (Supplementary Figure 1), using a 3.5 % (w/v)
agarose gel. Electrophoresis was carried out in 1 × TAE (40 mM Tris, pH 8.0; 20 mM
acetate; 1 mM EDTA) for ~40 min at 80 V. Gel-purified DNA bands were cut out and
recovered by Spin-X centrifuge filtration (Corning Incorporated, NY, USA) at 13,000 × g for
15 minutes. PCR products were precipitated from solution using 3 M sodium acetate (0.1
volume), 100% ethanol (2.5 volumes) and 2 µl of glycogen (1 mg/ml) and stored at -20ºC for
overnight precipitation. Samples were centrifuged the following day at 13,000 × g for 15
minutes (at 4 ºC) and a final wash with 500 µl of 70% ethanol and centrifugation (13,000 × g
for 15 minutes at 4 ºC) were performed before the PCR products were air-dried and
rehydrated in 10 µl of sterile nuclease free water.
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A
1
B
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16
17 18
19
20
21
22
23
167bp
Supplementary Figure 1. Agarose gels visualised under ultraviolet (UV) light. Gels show
single bands of purified PCR product generated from the five DNA samples initially selected
for sequencing (numbered according to gel loading order). Four samples (each taking three
wells) were loaded onto gel A with each sample divided by a minimum of one empty lane to
ensure clear separation of DNA. For logistical reasons, one sample was loaded onto gel B.
Genetic analysis –Sequencing of the PCR product
PCR products were sent to an accredited national research facility (Australian Genome
Research Facility Ltd, Sydney, Australia) for sequence determination using Sanger
sequencing techniques. Results showed one participant as homozygous for the C allele,
another as homozygous for the T allele, and the remaining three participants as
heterozygotes. Sequencing results of three participants (one of each zygosity) are presented in
Supplementary Figure 2.
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A
B
C
D
E
F
Supplementary Figure 2. Sequencing outcomes from three participants. Sequencing results
show C allele homozygosity as reflected by the clear cytosine signal spike (in blue) on the
(A) forward and (B) reverse complement sequences; T allele homozygosity by the clear
thymine signal spike (in red) on the (C) forward and (D) reverse complement sequences; and
CT heterozygosity by the mixed cytosine (in blue) and thymine (in red) signal spikes on the
(E) forward and (F) reverse complement sequences.
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Genetic analysis – Genotyping
Genotyping was performed using TaqMan® SNP Allelic Discrimination (Applied
Biosystems, Foster City, USA). Three of the five sequenced samples were selected to be used
as positive controls for genotyping analysis on the remaining participants (n=71). Prior to
analysis, genomic DNA samples were diluted to a concentration of 30 ng/µl with sterile
nuclease free water. Details of the genotyping reaction set-up are outlined in Supplementary
Table 3.
Supplementary Table 3. Genotyping reaction set-up and thermal cycler program
Genotyping reaction set-up (total volume 25 µl)
- 2.5 µl genomic DNA (30 ng/µl)
- 1.25 µl 20× TaqMan® SNP Genotyping Assay
- 12.5 µl 2× TaqMan® Universal PCR Master Mix
- 8.75 µl sterile nuclease free water
Thermal cycler program
60ºC × 1 min
95ºC × 10 min
repeated for
95ºC × 15 sec
40 cycles
60ºC × 1 min
60ºC × 1 min
DNA, deoxyribonucleic acid; dNTPs indicate deoxyribonucleotide triphosphates; PCR,
polymerase chain reaction
Statistical analysis
Data were analysed using IBM SPSS Statistics 20 (IBM Corporation, Armonk, USA). A
dominant genetic model (T allele carriers combined into a single group and compared against
C homozygotes) was employed for biochemical comparisons. Univariate ANOVA
(covariates: BMI, waist, lnCRP, ethnicity, contraceptives, polycystic ovary syndrome) was
used assess genotypic differences in the baseline data. The longitudinal impact of rs855791
on iron was analysed using repeated measures ANOVA (covariates: percent weight loss, iron
intake, contraceptives), with effect sizes reported as partial eta-squared (ηp2).
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SUPPLEMENTARY RESULTS
Supplementary Table 4. Comparison of baseline biochemical markers between participants
with/without available DNA and between those who were recruited into the cross-sectional
study only and those who completed the clinical weight management trial
Participants
Participants
without DNA
with DNA
(n=38)
(n=76)
22.5 ± 1.9
22.1 ± 2.5
European
73.7
61.8
Asian
5.3
15.8
African
0.0
5.3
South American
7.9
Other
Biochemical marker
Participants with DNA
CSS only
Clinical trial
(n=49)
only (n=27)
22.0 ± 2.5
22.5 ± 2.4
52.8
81.5
18.9
7.4
5.7
3.7
2.6
1.9
3.7
13.1
14.5
20.7
3.7
BMI (kg/m )
33.8 ± 4.8
33.6 ± 4.2
0.81
33.5 ± 4.4
33.8 ± 3.8
0.77
Hb (g/l)
131 ± 9
130 ± 9
0.80
130 ± 9
131 ± 8
0.50
Serum iron (µmol/l)
14.9 ± 7.6
15.5 ± 6.5
0.67
15.3 ± 6.5
15.7 ± 6.0
0.40
Tsat (%)
22.4 ± 11.7
22.5 ± 10.1
0.96
22.0 ± 10.3
23.2 ± 9.5
0.71
Serum ferritin (µg/l)*
41.5 (44.0)
33.0 (31.0)
0.47
31.0 (34.0)
34.0 (26.0)
0.73
sTfR (mg/l)
1.52 ± 1.47
1.65 ± 0.45
0.14
1.67 ± 0.42
1.60 ± 0.51
0.67
sTfR-F*
0.89 (0.74)
1.05 (0.59)
0.20
1.08 (0.59)
1.01 (0.40)
0.86
Hepcidin (ng/ml)*
6.50 (11.0)
6.95 (8.00)
0.36
8.40 (9.25)
5.60 (4.60)
0.99
Hepcidin/ferritin ratio*
0.20 (0.21)
0.20 (0.17)
0.63
0.21 (0.19)
0.15 (0.14)
0.64
Hepcidin/Tsat ratio*
0.34 (0.56)
0.31 (0.48)
0.19
0.36 (0.55)
0.29 (0.21)
0.86
CRP*
3.59 (5.79)
3.85 (5.91)
0.83
3.52 (6.08)
3.85 (5.56)
0.77
Age (y)
p value
0.35
p value
0.82
Ethnicity (%)
2
0.23
0.20
Mean ± SD or median (interquartile range). Differences assessed using unpaired t-tests and chi-square tests.
BMI, body mass index; CRP indicates C-reactive protein; CSS, cross-sectional study; DNA,
deoxyribonucleic acid; Hb, haemoglobin; sTfR, soluble transferrin receptor; sTfR-F, soluble transferrin
receptor-ferritin index; Tsat, transferrin saturation.
*Natural log transformation performed for the serum ferritin, sTfR-F, hepcidin, hepcidin-ferritin, hepcidinTsat and CRP variables
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Weight loss
Both diets independently led to significant weight loss after 12 months (HP: 9.79 ± 13.0%,
p=0.003; LP: 4.56 ± 7.15%, p=0.027). Weight loss in the HPHI group was approximately
double than that of LP, although this was not statistically significant (p=0.16). Similar
outcomes were also observed for waist circumference (HP: -7.9 ± 1.8; LP: -2.4 ± 0.8 cm;
p=0.36), with other anthropometric outcomes published elsewhere.7
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