Supplementary information

RESEARCH DESIGN AND METHODS

Participants

The study design, methods and measurements of the three phases of this cohort study have 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 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 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 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 CVsare 8.6% at 6.6 pmol/l, 6.1% at 41 pmol/l and 5.3% at 101.2 pmol/l (n = 50).

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).

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

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, 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 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 5kb upstream of the start codon. A global alignment for each gene was then performed. The consensus sequence for the TCF binding site (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 annotated alignment viewed.All genomic sequences were exported in FASTA format from Ensembl ( and associated annotation exported in the form of a General Feature Format (GFF) file also from Ensembl. The global alignment tool LAGAN ( was used to align the genomic sequences using the following guide trees for PC1 (((human dog) cow) (mouse rat)), PC2 (((human dog) mouse) opossum), and CPE ((((human) mouse)opossum) chicken). TFBSsearch ( was used to search the alignment for the TCF consensus sequence and SYNPLOT ( was used to visualize the annotated alignment in order to establish the location of candidate binding sites for TCF.All positions are as per the current build (NCBI 36 assembly) of the human genome (August 2006).

REFERENCES

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

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. Diabetesin 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. 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

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

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, 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

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Supplementary table 1:Descriptive characteristics of the MRC Ely Study

* Geometrics means and SD.

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Supplementary table 2: Genotype and allele frequencies, Hardy-Weinberg equilibrium (p-value) and call rates for four TCF7L2 gene variants in Europid individuals.

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.


* Geometrics means and SEM.

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Supplementary table 3: continued


* Geometrics means and SEM.

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Supplementary figure 1. Pairwise LD, calculated as r2, among SNPs in TCF7L2.

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.

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