Definition of cohorts
The Medical Research Council (MRC) Ely Study is a population-based cohort study of the aetiology and pathogenesis of type 2 diabetes and related metabolic disorders in the UK . This analysis included 1,721 white Europid men and women aged 35 to 79 years without diagnosed diabetes. Participants underwent standard anthropometric measurements and a 75-g OGTT. Plasma glucose was measured using the hexokinase method.
Plasma-specific insulin was determined by two-site immunometric assays with either 125I or alkaline phosphatase labels. Cross-reactivity was <0.2% with intact proinsulin at 400 pmol/l and <1% with 32–33 split proinsulin at 400 pmol/l. Interassay CVs 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). Informed consent was obtained from all participants and ethical approval for the study was granted by the Cambridge Local Research Ethics Committee.
The Hertfordshire Cohort Study was recruited from the cohort of people born in Hertfordshire between 1931 and 1939. The cohort definition has been described previously . Intact insulin was measured by in-house immunofluorimetric two-site assays (DELFIA system) based on published methods . The CV for interassay imprecision ranged from 6 to 10% for low-, medium- and high-quality control samples for intact insulin and from 7 to 15% for insulin precursors. Glucose was assayed on an Advia 1650 autoanalyser (Bayer Diagnostics, Leverkusen, Germany). In this study we attempted to genotype 2,341 samples from North and East Hertfordshire.
DNA was available for 1,706 Ely cohort samples and 2,341 Hertfordshire cohort samples. The Leu262Val SNP (rs3732581) was genotyped in Ely using a multiplex kit according to the manufacturer’s instructions (ABI PRISM SNaPshot; Applied Biosystems, Foster City, CA, USA). Analysis was carried out using GeneMapper software (version 3.0; Applied Biosystems). The analysis included 120 water blanks, and 86 DNA samples (2.2%) were in duplicate on the plates. The genotyping success rate was 95%, with no discordance between replicate samples. Hertfordshire cohort samples were genotyped using a custom SNP assay (TaqMan; Applied Biosystems) on 10 ng of degenerate oligonucleotide primed DNA. Allele calling was performed on a sequence detection system (ABI PRISM 7900HT; Applied Biosystems). The analysis included 155 water blanks and 91 duplicates. The genotyping success rate was 96%. There was no discordance between replicate samples.
All analyses were carried out in Stata version 8 (StataCorp, College Station, TX, USA). Deviation of Leu262Val genotype from Hardy–Weinberg equilibrium was assessed using a χ
2 test. Only participants with genotype, fasting insulin and BMI data were included in the analysis, i.e. 1,608 Ely samples and 2,058 Hertfordshire samples (total=3,666). Linear regression analysis was used to assess the association between genotype and fasting plasma insulin or BMI in Ely and Hertfordshire cohorts separately. Based on the previous reported association, our primary analysis was for a dominant genetic model and we tested genotype–age interactions using log-likelihood ratio tests. In secondary exploratory analyses, we used regression analysis to assess the association between Leu262Val genotype and three further quantitative traits: fasting plasma glucose, 2-h glucose, and 30-min insulin increment (the difference between 30 min and fasting insulin concentrations divided by the 30-min glucose concentration) in an OGTT test. We also investigated whether the association between genotype and fasting insulin was modified by BMI. To increase power we undertook all analyses on both cohorts together including an indicator term for study.