SUPPLEMENTARY INFORMATION
RESEARCH DESIGN AND METHODS
Participants
The study design, methods and measurements of the three phases of this cohort study have
5
been described in detail eklsewhere (1-4).
In brief, all participants attended a clinical examination that included standard
anthropometric measurements, medical questionnaires and a 75-g OGTT having fasted since
22.00 o’clock the previous night. Height and weight were measured with participants dressed in
light-weight clothing without shoes, and BMI was calculated [weight (kg) / height2 (m)2]. Fat
10
mass was assessed using a standard impedance technique (Bodystat, Isle of Man) and
expressed as a percentage of total weight [(fat mass/ total weight) x 100)]. Plasma samples
were obtained at fasting, 30-min, 60-min and 120-min post-glucose load. Glucose
concentrations were measured immediately in the routine laboratory using the hexokinase
method (5). Plasma insulin was measured by two-site immunometric assays. Cross-reactivity
15
was <0.2% with intact proinsulin at concentrations of 400 pmol/l and <1% with 32,33 split
proinsulin at 400 pmol/l. Interassay coefficients of variation (CV) were 6.6% at 28.6 pmol/l (n =
99), 4.8% at 153.1 pmol/l (n = 102), and 6.0% at 436.7 pmol/l (n = 99). Plasma intact proinsulin
and 32,33 split proinsulin concentrations were measured using immunoradiometric assays (6).
The intact proinsulin assay shows <1% cross-reactivity with insulin and 32,33 split proinsulin at
20
concentrations of 2500 pmol/l and 400 pmol/l, respectively. Between-batch CVs are 10.5% at
4.5pmol/l, 8.5% at 20 pmol/l and 8.1% at 92.9 pmol/l (n = 50). The 32,33 split proinsulin assay
shows 87% cross-reactivity with intact proinsulin. To obtain the specific measure of 32,33 split
proinsulin, it is necessary to take account of the intact proinsulin concentration. Cross-reactivity
with insulin is <1% at 2500 pmol/l. Between-batch CVs are 8.6% at 6.6 pmol/l, 6.1% at 41 pmol/l
25
and 5.3% at 101.2 pmol/l (n = 50).
Page 1 of 10
We calculated the insulin:glucose ratio (IGR) at 30min and at 60min as a measure of insulin
secretion [(insulin at 30 or 60min – insulin at 0 min)/(glucose at 30 or 60 min – glucose at 0
min)]. HOMA-S and HOMA-B, as measures of insulin sensitivity and -cell function respectively,
were estimated by the homeostasis model using Levy’s computer model (7).
5
Descriptive characteristics of the population are given in Supplementary Table 1.
Ethical permission was granted by the Cambridgeshire Research Ethics Committee,
and study participants provided written informed consent.
Genotyping
10
Genotyping was performed using Custom TaqMan® SNP Genotyping Assays (Applied
Biosystems, Warrington, UK). The genotyping assays were carried out on 10ng of genomic
DNA in a 5µl 384-well TaqMan® assay using a PTC-225 Thermal Cycler (MJ Research,
Watertown, MA), cycling 95°C for 10 mins, and then 40 cycles of 15 secs at 92°C and 1 min at
60°C. The ABI PRISM® 7900HT Sequence Detection System (Applied Biosystems, Warrington,
15
UK) was used for end-point detection and allele calling.
In silico analyses
In silico analyses were performed to identify TCF binding sites in the proprotein convertase 1
and 2 (PC1, PC2) and carboxypeptidase E (CPE) gene sequences. For each gene, genomic
20
sequence and associated annotation were obtained for a number of species; for PC1 from
human, cow, mouse, rat, dog, for PC2 from human, dog, mouse, and opossum, and for CPE
from human, mouse, chicken and opossum. Sequence and annotation were downloaded to
include the entire gene in question and 5 kb upstream of the start codon. A global alignment for
each gene was then performed. The consensus sequence for the TCF binding site
25
(WWCAAWG) was then used to search for any conserved sites within the alignment. The
positions for all candidate sites were added to the human annotation file and the whole
Page 2 of 10
annotated alignment viewed. All genomic sequences were exported in FASTA format from
Ensembl (www.ensembl.org (8)) and associated annotation exported in the form of a General
Feature Format (GFF) file also from Ensembl. The global alignment tool LAGAN
(http://lagan.stanford.edu/lagan_web/index.shtml (9)) was used to align the genomic sequences
5
using the following guide trees for PC1 (((human dog) cow) (mouse rat)), PC2 (((human dog)
mouse)
opossum),
and
CPE
((((human)
mouse)
opossum)
chicken).
TFBSsearch
(http://hscl.cimr.cam.ac.uk/TFBSsearch.html (10)) was used to search the alignment for the TCF
consensus sequence and SYNPLOT (http://hscl.cimr.cam.ac.uk/syn_plot.html (10)) was used to
visualize the annotated alignment in order to establish the location of candidate binding sites for
10
TCF. All positions are as per the current build (NCBI 36 assembly) of the human genome
(August 2006).
REFERENCES
15
1. Williams DR, Wareham NJ, Brown DC, Byrne CD, Clark PM, Cox BD, Cox LJ, Day NE,
Hales CN, Palmer CR, Shackleton JR, Wang TWM: Undiagnosed glucose intolerance in
the community: the Isle of Ely Diabetes Project. Diabet Med 12:30-35, 1995
2. Wareham NJ, Byrne CD, Williams R, Day NE, Hales CN: Fasting proinsulin concentrations
predict the development of type 2 diabetes. Diabetes Care 22:262-270, 1999
20
3. Mesa JL, Loos RJF, Franks PW, Ong KKL, Luan J, O'Rahilly S, Wareham NJ, Barroso I:
Lamin A/C polymorphisms, type 2 diabetes and the metabolic syndrome: case-control and
quantitative trait studies. Diabetes in press, 2007
4. Forouhi NG, Luan J, Hennings S, Wareham NJ: Incidence of type 2 diabetes in England
and its association with baseline impaired fasting glucose: The Ely study 1990-2000.
25
Diabet Medin press, 2007
5. Kunst A, Draeger B, Ziegenhorn J: UV-methods with hexokinase and glucose-6-phosphate
dehydrogenase. In Methods of enzymatic analysis. IV ed. Berg-Meyer HU, Ed. Deerfield,
Weinhein Verlag Chemie, 1983, p. 163-172
Page 3 of 10
6. Sobey WJ, Beer SF, Carrington CA, Clark PM, Frank BH, Gray IP, Luzio SD, Owens DR,
Schneider AE, Siddle K, Temple RC, Hales CN: Sensitive and specific two-site
immunoradiometric assays for human insulin, proinsulin, 65-66 split and 32-33 split
proinsulins. Biochem J 260:535-541, 1989
5
7. Levy JC, Matthews DR, Hermans MP: Correct homeostasis model assessment (HOMA)
evaluation uses the computer program. Diabetes Care 21:2191-2192, 1998
8. Hubbard TJP, Aken BL, Beal K, Ballester B, Caccamo M, Chen Y, Clarke L, Coates G,
Cunningham F, Cutts T, Down T, Dyer SC, Fitzgerald S, Fernandez-Banet J, Graf S,
Haider S, Hammond M, Herrero J, Holland R, Howe K, Howe K, Johnson N, Kahari A,
10
Keefe D, Kokocinski F, Kulesha E, Lawson D, Longden I, Melsopp C, Megy K, Meidl P,
Ouverdin B, Parker A, Prlic A, Rice S, Rios D, Schuster M, Sealy I, Severin J, Slater G,
Smedley D, Spudich G, Trevanion S, Vilella A, Vogel J, White S, Wood M, Cox T, Curwen
V, Durbin R, Fernandez-Suarez XM, Flicek P, Kasprzyk A, Proctor G, Searle S, Smith J,
Ureta-Vidal A, Birney E: Ensembl 2007. Nucl Acids Res 35:D610-D617, 2007
15
9. Brudno M, Do CB, Cooper GM, Kim MF, Davydov E, Program NCS, Green ED, Sidow A,
Batzoglou S: LAGAN and Multi-LAGAN: Efficient Tools for Large-Scale Multiple Alignment
of Genomic DNA. Genome Res 13:721-731, 2003
10. Chapman MA, Donaldson IJ, Gilbert J, Grafham D, Rogers J, Green AR, Gottgens B:
Analysis of Multiple Genomic Sequence Alignments: A Web Resource, Online Tools, and
20
Lessons Learned From Analysis of Mammalian SCL Loci. Genome Res 14:313-318, 2004
Page 4 of 10
Supplementary table 1: Descriptive characteristics of the MRC Ely Study
Men
Women
780
917
Mean (SD)*
Mean (SD)*
Age (y)
61.5 (9.1)
60.8 (9.1)
BMI (kg/m2)
27.0 (3.8)
26.7 (4.9)
Fat percentage (%)
26.1 (5.2)
39.2 (6.7)
Waist circumference (%)
99.3 (10.4)
86.3 (12.1)
Fasting insulin (pmol/l)
52.0 (30.1)
46.5 (28.0)
629.1 (347)
582.6 (307)
5.7 (0.8)
5.3 (0.7)
Glucose AUC (pmol/l)
15.8 (3.7)
14.6 (3.5)
Insulin/glucose ratio at 30 min
82.7 (64)
92.5 (72)
Insulin/glucose ratio at 60 min
117.2 (103)
134.5 (120)
Fasting intact proinsulin (pmol/l)
4.3 (2.7)
3.3 (1.9)
Fasting 32,33 split proinsulin (pmol/l)
4.5 (3.7)
3.5 (2.8)
Fasting HbA1c (%)
5.4 (0.57)
5.3 (0.47)
HOMA-B (%)
80.0 (32.7)
83.6 (31.9)
HOMA-S (%)
84.9 (49.1)
97.1 (58.8)
N
Insulin AUC (pmol/l)
Fasting Glucose (pmol/l)
* Geometrics means and SD.
Page 5 of 10
Supplementary table 2: Genotype and allele frequencies, Hardy-Weinberg equilibrium (p-value) and call rates for four TCF7L2 gene
variants in Europid individuals.
Major allele
Heterozygotes
homozygotes
42.7%
Minor allele
homozygotes
%
46.5%
N
779
716
181
%
49.5%
41.7%
8.8%
N
830
699
147
%
51.3%
40.2%
8.5%
N
862
676
143
%
50.3%
40.8%
8.9%
N
844
684
149
Hardy-Weinberg
Equilibrium
(p - value)
10.8%
Minor allele
frequency
(%)
67.8%
32.2%
2274
1078
70.4%
29.6%
2359
993
71.4%
28.6%
2400
962
70.7%
29.3%
2372
982
0.39
rs4506565
98.2%
0.52
rs12243326
0.54
Page 6 of 10
Call rate (%)
98.2%
0.99
rs7903146
rs12255372
Major allele
frequency
(%)
98.5%
98.3%
Supplementary table 3: Mean ( SEM) values for measures of body composition, fasting and OGTT insulin and glucose
concentrations and measures of insulin secretion, insulin sensitivity and b-cell function by genotype in 1697 Europids for four
TCF7L2 gene variants.
rs7903146
rs4506565
A/A
Mean ± SEM
A/T
Mean ± SEM
T/T
Mean ± SEM
p-value
C/C
Mean ± SEM
C/T
Mean ± SEM
T/T
Mean ± SEM
p-value
BMI (kg/m2)
26.9 ± 0.2
26.9 ± 0.2
26.8 ± 0.3
0.68
27.0 ± 0.2
26.8 ± 0.2
26.6 ± 0.4
0.24
Fat percentage (%)
31.9 ± 0.2
32.0 ± 0.2
32.2
0.4
0.61
32.0 ± 0.2
31.8 ± 0.2
31.9 ± 0.5
0.61
Waist circumference (cm)
92.7 ± 0.4
92.7 ± 0.4
91.7 ± 0.9
0.44
92.9 ± 0.4
92.5 ± 0.4
91.3 ± 0.9
0.21
Fasting insulin (pmol/l)
49.6 ± 0.9
49.1 ± 1.0
47.8 ± 1.8
0.42
49.5 ± 0.9
48.9 ± 1.0
48.6 ± 2.1
0.60
Insulin AUC (pmol/l)*
612 ± 11
598 ± 12
609 ± 23
0.65
616 ± 11
594 ± 12
621 ± 26
0.54
Fasting Glucose (pmol/l)
5.48 ± 0.02
5.54 ± 0.03
5.62 ± 0.05
0.01
5.49 ± 0.02
5.54 ± 0.03
5.61 ± 0.06
0.042
Glucose AUC (pmol/l)*
15.0 ± 0.1
15.3 ± 0.1
15.9 ± 0.3
0.003
15.0 ± 0.1
15.3 ± 0.1
15.8 ± 0.3
0.013
Insulin/glucose ratio at 30 min
88.5 ± 2.6
87.3 ± 2.7
84.1 ± 5.1
0.46
88.6 ± 2.5
86.1 ± 2.7
88.4 ± 5.9
0.70
Insulin/glucose ratio at 60 min
134.1 ± 4.6
121.0 ± 4.5
108.2 ± 7.8
0.003
134.0 ± 4.5
120.2 ± 4.5
108.5 ± 8.6
0.003
3.55 ± 0.07
3.88 ± 0.08
4.29 ± 0.17
0.0000024
3.56 ± 0.07
3.89 ± 0.08
4.46 ± 0.20
0.00000047
Fasting 32,33 split proinsulin (pmol/l) 3.82 ± 0.10
4.07 ± 0.11
4.48 ± 0.24
0.005
3.85 ± 0.10
4.06 ± 0.11
4.69 ± 0.28
0.003
Fasting HbA1c (%)
5.33 ± 0.02
5.39 ± 0.02
5.47 ± 0.04
0.0006
5.35 ± 0.02
5.38 ± 0.02
5.46 ± 0.04
0.012
HOMA-B
83.6 ± 1.1
81.2 ± 1.1
76.8 ± 2.2
0.007
83.3 ± 1.1
81.0 ± 1.2
77.7 ± 2.4
0.028
HOMA-S
89.9 ± 1.7
90.8 ± 1.8
94.1 ± 3.7
0.33
90.1 ± 1.6
91.3 ± 1.8
92.9 ± 4.0
0.45
Fasting intact proinsulin (pmol/l)
* Geometrics means and SEM.
Page 7 of 10
Supplementary table 3: continued
rs12243326
rs12255372
C/C
Mean ± SEM
C/T
Mean ± SEM
T/T
Mean ± SEM
p-value
G/G
Mean ± SEM
G/T
Mean ± SEM
T/T
Mean ± SEM
p-value
BMI (kg/m2)
26.9 ± 0.2
26.9 ± 0.2
26.6 ± 0.4
0.77
26.8 ± 0.2
27.0 ± 0.2
26.8 ± 0.4
0.69
Fat percentage (%)
31.9 ± 0.2
32.1 ± 0.2
31.6 ± 0.5
0.99
31.8 ± 0.2
32.1 ± 0.2
31.7 ± 0.4
0.72
Waist circumference (cm)
92.6 ± 0.4
92.9 ± 0.4
91.3 ± 1.0
0.53
92.5 ± 0.4
93.0 ± 0.4
91.5 ± 0.9
0.80
Fasting insulin (pmol/l)
49.5 ± 0.9
48.7 ± 1.0
48.4 ± 2.1
0.47
49.6 ± 0.9
48.6 ± 1.0
48.6 ± 2.1
0.48
Insulin AUC (pmol/l)*
615 ± 11
593 ± 12
606 ± 27
0.32
614 ± 11
592 ± 12
606 ± 26
0.37
Fasting Glucose (pmol/l)
5.49 ± 0.02
5.55 ± 0.03
5.59 ± 0.06
0.05
5.50 ± 0.02
5.53 ± 0.03
5.60 ± 0.06
0.10
Glucose AUC (pmol/l)*
15.1 ± 0.1
15.2 ± 0.1
15.9 ± 0.3
0.02
15.1 ± 0.1
15.2 ± 0.1
15.8 ± 0.3
0.04
Insulin/glucose ratio at 30 min
88.1 ± 2.4
86.1 ± 2.7
87.7 ± 6.0
0.71
87.7 ± 2.4
86.2 ± 2.7
89.1 ± 6.0
0.93
Insulin/glucose ratio at 60 min
134.0 ± 4.4
118.5 ± 4.6
105.5 ± 8.7
0.001
133.2 ± 4.4
118.9 ± 4.6
108.3 ± 8.7
0.004
Fasting intact proinsulin (pmol/l)
3.56 ± 0.06
3.91 ± 0.08
4.39 ± 0.20
0.00000059
3.55 ± 0.07
3.89 ± 0.08
4.37 ± 0.19
0.0000012
Fasting 32,33 split proinsulin (pmol/l) 3.85 ± 0.09
4.07 ± 0.11
4.65 ± 0.28
0.004
3.84 ± 0.09
4.04 ± 0.11
4.64 ± 0.27
0.004
Fasting HbA1c (%)
5.35 ± 0.02
5.40 ± 0.02
5.44 ± 0.04
0.014
5.34 ± 0.02
5.40 ± 0.02
5.43 ± 0.04
0.007
HOMA-B
83.2 ± 1.1
80.6 ± 1.2
78.2 ± 2.5
0.026
83.1 ± 1.1
80.8 ± 1.2
78.1 ± 2.4
0.039
HOMA-S
90.0 ± 1.6
91.5 ± 1.8
93.3 ± 4.1
0.38
89.9 ± 1.6
91.8 ± 1.8
92.9 ± 4.0
0.35
* Geometrics means and SEM.
Page 8 of 10
Supplementary figure 1. Pairwise LD, calculated as r2, among SNPs in TCF7L2.
Page 9 of 10
Supplementary figure 2. Geometric mean ( SEM) values for insulin [A] and glucose [B]
concentrations during OGTT by genotype in 1697 Europids for the rs7903146 TCF7L2 gene
variant.
[A]
C/C
500
C/T
T/T
Insulin (pmol/l)
400
300
200
100
0
0
[B]
30
60
Minutes (OGTT)
90
120
C/C
9.0
C/T
T/T
Glucose (pmol/l)
8.0
7.0
6.0
5.0
0
30
60
Minutes (OGTT)
Page 10 of 10
90
120