APPENDIX
SUBJECTS, MATERIALS AND METHODS
Subjects.
APPENDIX TABLE 1. Clinical characteristics of 15,734 examined Danish subjects
Inter99 / ADDITION / StenoDiabetesCenterTreatment-naïve subjects / Glucose tolerant
subjects / Type 2 diabetes
patients / All / Type 2 diabetes patients / Type 2 diabetes patients
n / 5,970 / 4,522 / 352 / 8,662 / 1,626 / 1,002
men/women / 2,972/2,998 / 2,102/2,420 / 215/137 / 4,729/3,933 / 924/702 / 605/397
Age (years) / 46.1 ± 7.9 / 45.2 ± 7.8 / 51.0 ± 7.1 / 60.0 ± 6.8 / 60.2 ± 6.8 / 59.0 ± 10.5
BMI (kg/m2) / 26.2 ± 4.5 / 25.5 ± 4.1 / 30.4 ± 5.6 / 28.6 ± 4.9 / 31.1 ± 5.4 / 29.6 ± 5.1
HbA1c (%) / 5.8 ± 0.5 / 5.8 ± 0.4 / 7.0 ± 1.6 / 5.9 ± 0.7 / 6.6 ± 1.3 / 8.1 ± 1.6
Data are means ± standard deviation.
Studies of quantitative metabolic traits were performed in the Inter99 and the ADDITION cohorts. The Inter99 is a population-based, randomized, non-pharmacological intervention study of middle-aged subjects for the prevention of ischemic heart disease, conducted at the Research Centre for Prevention and Health in Glostrup, Copenhagen (ClinicalTrials.gov; identifier: NCT00289237) (1). Of the 6,070 participants with available genomic DNA, 4,522 (75%) had normal glucose tolerance, 503 (8.3%) had impaired fasting glycaemia, 693 (11%) had impaired glucose tolerance, 252 (4.2%) had screen-detected and treatment-naïve type 2 diabetes and 100 (1.6%) had previously diagnosed type 2 diabetes. Studies of quantitative traits were done in all treatment-naïve participants (n=5,970) not including patients treated for type 2 diabetes (n=100). Furthermore, subjects treated with lipid-lowering drugs (n=89) were excluded in all analyses of fasting serum lipids.
The Danish ADDITION Study (Anglo–Danish–Dutch Study of Intensive Treatment in People with Screen-Detected Diabetes in Primary Care) is a high-risk screening and intervention study for type 2 diabetes in general practice sampled by Department of General Practice at University of Aarhus, Denmark(ClinicalTrials.gov ID-no: NCT00237548) (2). The 8,662 participantsfrom the initial screening cohort with available DNA included 1,626 subjects with screen-detected and untreated type 2 diabetes and 7,036 non-diabetic subjects.
For use in a case-control study patients with type 2 diabetes were recruited from the out-patient clinic at Steno Diabetes Center (n=2,111), from the population-based Inter99 cohort established at Research Centre for Prevention and Health (1) (n=352, of these 252 were screen-detected and untreated), and from the Danish ADDITION study (2) (n=1,626, all screen-detected and untreated). Glucose-tolerant subjects were recruited from the population-based Inter99 cohort (n=4,522).
At recruitment, diabetic patients with one or more of the following characteristics were excluded: (1) diabetes due to known chronic pancreatitis, haemochromatosis, severe insulin resistance, MODY, or maternally inherited diabetes and deafness; (2) a family history of first-degree relatives with type 1 diabetes; (3) a requirement for insulin within the first year after the diagnosis of diabetes; (4) a fasting serum C-peptide level ≤150 pmol/l; or patients tested GAD65 antibody positive. States of impaired glucose regulation (impaired fasting glycaemia, impaired glucose tolerance, and type 2 diabetes) were diagnosed in accordance with WHO 1999 criteria (5).
Biochemical and anthropometric measures. In all study groups height and weight were measured in light indoor clothing and without shoes, and BMI was calculated as weight (kg)/height2 (m2). Waist circumference was measured in the upright position midway between the iliac crest and the lower costal margin, and hip circumference was measured at its maximum. Blood samples were drawn after a 12-h overnight fast.
In the Inter99 study plasma glucose was analyzed by a glucose oxidase method (Granutest; Merck, Darmstadt, Germany) and serum insulin (excluding des-31,32 and intact proinsulin) was measured using the AutoDELFIA insulin kit (Perkin-Elmer/Wallac, Turku, Finland). Serum triglycerides and serum total and HDL cholesterol were analyzed using enzymatic colorimetric methods (GPO-PAP and CHOD-PAP; Roche Molecular Biochemicals, Mannheim, Germany). HbA1C was measured by ion-exchange HPLC (normal range 4.1–6.4%).
In the ADDITION study fasting blood glucose was measured on capillary whole blood using a HemoCue B-glucose analyser (HemoCueAB, Ängelholm, Sweden). The Hitachi 971 system (Roche Diagnostics GmbH, Mannheim, Germany) was used to measure total serum cholesterol, serum HDL and serum triglyceride by enzymatic tests.HbA1cwas analysed using liquid chromatography.
Insulinogenic indexinsulin was calculated as (s-insulin at 30 minutes [pmol/l] - fasting s-insulin [pmol/l]) / p-glucose at 30 minutes (mmol/l). Homeostasis model assessment of insulin resistance (HOMA-IR) (mmol/l×pmol/l) was calculated as fasting plasma-glucose (mmol/l) multiplied by fasting serum-insulin (pmol/l) and divided by 22.5.BIGTT-insulin sensitivity index (BIGTT-SI) uses information on sex and BMI combined with analysis of plasma-glucose and serum-insulin levels at the time points 0, 30, and 120 min to provide an index for Si that is highly correlated with indexes obtained during an intravenous glucose tolerance test and was calculated as described elsewhere (4).
Genotyping.
APPENDIX TABLE 2.Genotype distributions, minor allele frequencies and Hardy-Weinberg equilibrium of variants genotyped in SREBF1 in study subgroups
Variant / Success/mismatch rate (%) / Inter99 / ADDITION / Type 2 diabetes patients / Glucose-tolerant control subjects
Treatment-naïve subjects / All / All
rs4925118 / 97.6/0.0
Genotypes / 4640/984/48 / 4957/1055/49 / 6870/1507/84 / 2292/525/32 / 3537/734/38
MAF (95%CI) / 9.5 (9.0-10.1) / 9.5 (9.0-10.0) / 9.9 (9.4-10.3) / 10.3(9.6-11.2) / 9.4(8.8-10.0)
HWE / 0.6 / 0.4 / 0.9 / 0.8 / 1
rs1889018 / 97.5/0.2
Genotypes / 2517/2529/626 / 2682/2714/661 / 3745/3723/982 / 1217/1321/320 / 1936/1914/468
MAF (95%CI) / 33.3 (32.5-34.2) / 33.3 (32.5-34.2) / 33.7 (32.9-34.4) / 34.3(33.1-35.6) / 33.0(32.0-34.0)
HWE / 0.8 / 0.5 / 0.2 / 0.2 / 0.9
rs2297508 / 97.1/0.1
Genotypes / 2510/2462/644 / 2672/2643/681 / 3672/3789/986 / 1192/1328/322 / 1921/1874/481
MAF (95%CI) / 33.4 (32.5-34.3) / 33.4 (32.6-34.2) / 34.1 (33.4-34.8) / 34.7(33.5-35.9) / 33.2(32.2-34.2)
HWE / 0.3 / 0.5 / 0.9 / 0.1 / 0.5
rs11868035 / 97.7/0.1
Genotypes / 3013/2238/439 / 3212/2404/483 / 4460/3361/651 / 1473/1153/221 / 2304/1702/326
MAF (95%CI) / 27.6 (26.7-28.4) / 27.6 (26.8-28.4) / 27.5 (26.8-28.2) / 28.0(26.8-29.2) / 27.2(26.2-28.1)
HWE / 0.4 / 0.7 / 0.6 / 0.8 / 0.6
Mismatch rate is based on 968 samples genotyped in duplicates.
HWE, Hardy-Weinberg equilibrium; MAF, minor allele frequency
RESULTS
APPENDIX TABLE 3.Quantitative metabolic traits in the population-based Inter99 cohort including 5,970 middle-aged subjects with normal glucose tolerance, impaired fasting glycemia, impaired glucose tolerance or screen-detected and treatment-naïve type 2 diabetes stratified according to genotype of variants in SREBF1
rs4925118Trait / GG / GA / AA / P
n (men/women) / 4640 (2321/2319) / 984 (486/498) / 48 (29/19)
Age (years) / 46.2±7.9 / 45.8±7.9 / 46.8±8.2
BMI (kg/m2) / 26.17±4.46 / 26.29±4.66 / 26.29±5.91 / 0.4
Waist (cm) / 86.43±13.14 / 86.67±13.3 / 89.12±12.74 / 0.3
s-Triglyceride (mmol/l) / 1.10 (0.80-1.50) / 1.1 (0.80-1.60) / 1.15 (0.85-1.50) / 0.8
s-Cholesterol (mmol/l) / 5.53±1.06 / 5.52±1.08 / 5.58±1.04 / 0.9
s-HDL cholesterol (mmol/l) / 1.44±0.4 / 1.43±0.4 / 1.37±0.33 / 0.8
Fasting s-insulin (pmol/l) / 35 (24-51) / 34 (24-53) / 41 (32-58) / 0.1
s-insulin at 30 min (pmol/l) / 245 (175-354) / 251 (173-355) / 291 (184-373) / 0.8
s-insulin at 120 min (pmol/l) / 155 (96-256) / 161 (99-256) / 171 (124-236) / 0.1
Fasting p-glucose (mmol/l) / 5.53±0.79 / 5.56±0.86 / 5.64±0.53 / 0.2
p-glucose at 30 min (mmol/l) / 8.69±1.87 / 8.73±1.95 / 9.1±1.88 / 0.2
p-glucose at 120 min (mmol/l) / 6.17±2.06 / 6.33±2.38 / 5.92±1.37 / 0.2
HbA1c (%) / 5.83±0.5 / 5.84±0.54 / 5.8±0.4 / 1
Insulinogenic indexinsulin / 24.4 (16.9-36.7) / 25.3 (16.3-36.6) / 23.4 (15.8-34.2) / 0.6
HOMA-IR (mmol/l×pmol/l) / 8.32 (5.66-12.88) / 8.24 (5.66-13.40) / 10.16 (7.97-14.11) / 0.07
BIGTT-Si / 9.24±4.05 / 9.15±4.15 / 8.56±4.18 / 0.3
rs1889018
Trait / TT / TC / CC / P
n (men/women) / 2517 (1266/1251) / 2529 (1266/1263) / 626 (315/311)
Age (years) / 46.2±7.9 / 46.1±8.0 / 45.7±8.0
BMI (kg/m2) / 26.16±4.46 / 26.21±4.53 / 26.12±4.56 / 0.9
Waist (cm) / 86.4±13.16 / 86.62±13.21 / 86.2±13.18 / 0.8
s-Triglyceride (mmol/l) / 1.10 (0.80-1.60) / 1.10 (0.80-1.50) / 1.10 (0.80-1.60) / 0.4
s-Cholesterol (mmol/l) / 5.56±1.09 / 5.52±1.06 / 5.52±1.08 / 0.3
s-HDL cholesterol (mmol/l) / 1.44±0.39 / 1.43±0.41 / 1.42±0.4 / 0.2
Fasting s-insulin (pmol/l) / 34 (23-50) / 34 (24-51) / 35 (24-54) / 0.2
s-insulin at 30 min (pmol/l) / 243 (175-355) / 246 (174-350) / 256 (182-365) / 0.4
s-insulin at 120 min (pmol/l) / 152 (91-247) / 156 (98-261) / 165 (101-260) / 0.001
Fasting p-glucose (mmol/l) / 5.5±0.68 / 5.57±0.88 / 5.55±0.92 / 0.005
p-glucose at 30 min (mmol/l) / 8.64±1.84 / 8.73±1.92 / 8.84±1.92 / 0.002
p-glucose at 120 min (mmol/l) / 6.1±1.99 / 6.28±2.23 / 6.29±2.18 / 0.001
HbA1c (%) / 5.81±0.46 / 5.84±0.54 / 5.86±0.6 / 0.006
Insulinogenic indexinsulin / 24.2 (16.9-37.1) / 24.6 (16.6-35.9) / 25.7 (16.9-37.2) / 0.8
HOMA-IR (mmol/l×pmol/l) / 8.25 (5.55-12.63) / 8.32 (5.66-12.98) / 8.56 (5.76-13.65) / 0.07
BIGTT-Si / 9.36±4.09 / 9.15±4.04 / 9.09±4.1 / 0.03
rs11868035
Trait / CC / CT / TT / P
n (men/women) / 3013 (1515/1498) / 2238 (1113/1125) / 439 (216/223)
Age (years) / 46.2±7.9 / 46.0±7.9 / 45.7±8.0
BMI (kg/m2) / 26.27±4.58 / 26.08±4.51 / 26.18±4.24 / 0.3
Waist (cm) / 86.63±13.23 / 86.27±13.15 / 86.19±12.46 / 0.5
s-Triglyceride (mmol/l) / 1.10 (0.80-1.50) / 1.00 (0.80-1.50) / 1.20 (0.80-1.60) / 0.02
s-Cholesterol (mmol/l) / 5.55±1.08 / 5.51±1.06 / 5.54±1.03 / 0.8
s-HDL cholesterol (mmol/l) / 1.44±0.4 / 1.43±0.41 / 1.4±0.4 / 0.05
Fasting s-insulin (pmol/l) / 34 (24-51) / 34 (24-51) / 35 (23-52) / 0.6
s-insulin at 30 min (pmol/l) / 243 (175-352) / 248 (174-354) / 249 (181-356) / 0.2
s-insulin at 120 min (pmol/l) / 153 (92-248) / 159 (99-261) / 170 (101-262) / 0.0008
Fasting p-glucose (mmol/l) / 5.52±0.67 / 5.55±0.86 / 5.55±0.99 / 0.06
p-glucose at 30 min (mmol/l) / 8.64±1.83 / 8.73±1.89 / 8.78±1.9 / 0.006
p-glucose at 120 min (mmol/l) / 6.11±1.99 / 6.26±2.17 / 6.29±2.15 / 0.002
HbA1c (%) / 5.81±0.45 / 5.84±0.53 / 5.88±0.62 / 0.0005
Insulinogenic indexinsulin / 24.2 (16.8-36.4) / 24.7 (16.7-36.6) / 25.7 (17.4-36.2) / 0.6
HOMA-IR (mmol/l×pmol/l) / 8.28 (5.66-12.72) / 8.32 (5.65-12.96) / 8.36 (5.61-13.01) / 0.4
BIGTT-Si / 9.29±4.08 / 9.20±4.05 / 9.10±3.95 / 0.2
Data are mean ± standard deviation or median (interquantile range) (values of s-insulin, s-triglyceride, HOMA-IR and insulinogenic index). Values of p-glucose, s-insulin, insulinogenic index insulin and triglyceride were logarithmically transformed before statistical analysis. Calculated P-values were adjusted for age, sex, and BMI, where appropriate assuming an additive model. Insulinogenic indexinsulin was calculated as (s-insulin at 30 minutes [pmol/l] - fasting s-insulin [pmol/l]) / p-glucose at 30 minutes (mmol/l).BIGTT-insulin sensitivity index (BIGTT-Si) uses information on sex and BMI combined with analysis of p-glucose and s-insulin levels at the time points 0, 30, and 120 min to provide an index for Si which highly correlates with indexes obtained during an IVGTT, and were calculated as described elsewhere (4). P, plasma; s, serum.
APPENDIX TABLE 4. Quantitative metabolic traits in the DanishADDITION study consisting of 8,662 subjects stratified according to genotype of variants in SREBF1
rs4925118Trait / GG / GA / AA / P
n / 6854 / 1503 / 84
men/women / 3760/3094 / 802/701 / 48/36
Age (years) / 60.0±6.8 / 60.0±6.8 / 58.5±7.5
BMI (kg/m2) / 28.58±4.9 / 28.48±4.75 / 28.99±4.27 / 0.7
Waist (cm) / 97.03±13.48 / 97.05±13.5 / 98.23±11.07 / 0.02
s-Cholesterol (mmol/l) / 5.81±1.07 / 5.83±1.02 / 5.81±0.96 / 0.7
s-HDL cholesterol (mmol/l) / 1.55±0.43 / 1.55±0.42 / 1.47±0.39 / 0.4
Fasting b-glucose (mmol/l) / 5.38±1.21 / 5.38±1.16 / 5.42±1.04 / 0.8
HbA1c (%) / 5.85±0.75 / 5.86±0.68 / 5.82±0.61 / 0.6
rs1889018
Trait / TT / TC / CC / P
n / 3733 / 3719 / 978
men/women / 2064/1669 / 1981/1738 / 544/434
Age (years) / 59.9±6.8 / 60.1±6.7 / 59.7±7.0
BMI (kg/m2) / 28.71±5.02 / 28.47±4.8 / 28.42±4.63 / 0.02
Waist (cm) / 97.42±13.57 / 96.71±13.41 / 96.98±13.34 / 0.5
s-Cholesterol (mmol/l) / 5.79±1.08 / 5.84±1.05 / 5.85±1.05 / 0.06
s-HDL cholesterol (mmol/l) / 1.54±0.43 / 1.56±0.43 / 1.54±0.43 / 0.9
Fasting b-glucose (mmol/l) / 5.38±1.2 / 5.39±1.2 / 5.4±1.27 / 0.3
HbA1c (%) / 5.84±0.73 / 5.86±0.74 / 5.87±0.74 / 0.03
rs11868035
Trait / CC / CT / TT / P
n / 4447 / 3356 / 649
men/women / 2428/2019 / 1805/1551 / 373/276
Age (years) / 60.0±6.8 / 60.0±6.7 / 59.8±7.0
BMI (kg/m2) / 28.65±4.98 / 28.52±4.79 / 28.31±4.55 / 0.06
Waist (cm) / 97.2±13.59 / 96.86±13.36 / 96.99±13.14 / 0.4
s-Cholesterol (mmol/l) / 5.79±1.07 / 5.84±1.04 / 5.88±1.08 / 0.006
s-HDL cholesterol (mmol/l) / 1.55±0.43 / 1.56±0.43 / 1.53±0.41 / 0.9
Fasting b-glucose (mmol/l) / 5.38±1.2 / 5.38±1.16 / 5.45±1.44 / 0.2
HbA1c (%) / 5.84±0.72 / 5.86±0.73 / 5.9±0.84 / 0.02
Data are mean ± standard deviation. Values of b-glucose were logarithmically transformed before statistical analysis. Calculated P-values were adjusted for age, sex, and BMI, where appropriate, assuming an additive model. B, blood; s: serum.
APPENDIX TABLE 5. Haplotype association analysis of SREBF1 variants in relation to type 2 diabetes in 2,980 type 2 diabetic patients and 4,522 glucose-tolerant control subjects
# / rs4925118 / rs1889018 / rs2297508 / rs11868035 / Haplotype frequency / OR (95% CI) / P-value1 / 1 / 1 / 1 / 1 / 0.65 / 1 (base)
2 / 1 / 1 / 2 / 2 / 0.018 / 1.35 (0.92-1.99) / 0.1
3 / 1 / 2 / 1 / 1 / 0.017 / 0.99 (0.66-1.49) / 1
4 / 1 / 2 / 2 / 2 / 0.22 / 1.21 (1.07-1.38) / 0.003
5 / 2 / 2 / 2 / 1 / 0.057 / 1.17 (0.93-1.46) / 0.2
6 / 2 / 2 / 2 / 2 / 0.037 / 1.35 (1.02-1.77) / 0.03
1 designate the common allele of each variant and 2 designate minor allele. All odds ratios and P-values are relative to haplotype #1 consisting of the common allele of every variant.
APPENDIX REFERENCES
1. Jørgensen T, Borch-Johnsen K, Thomsen TF, Ibsen H, Glumer C, Pisinger C: A randomized non-pharmacological intervention study for prevention of ischaemic heart disease: Baseline results Inter99 (1). Eur J Cardiovasc Prev Rehab 10:377-386, 2003
2. Lauritzen T, Griffin S, Borch-Johnsen K, WarehamNJ, Wolffenbuttel BH, Rutten G, for the ADDITION Study Group: The ADDITION study: proposed trial of the cost-effectiveness of an intensive multifactorial intervention on morbidity and mortality among people with Type 2 diabetes detected by screening. Int J Obes Relat Metab Disord 24 Suppl 3:S6-11, 2000
3. Matthews DR, Hosker JP, RudenskiAS, Naylor BA, Treacher DF, Turner RC: Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 28:412-419, 1985
4. Hansen T, Drivsholm T, Urhammer SA, Palacios RT, Vølund A, Borch-Johnsen K, Pedersen O: The BIGTT test: a novel test for simultaneous measurement of pancreatic -cell function, insulin sensitivity, and glucose tolerance. Diabetes Care 30:257-262, 2007