Mutation scanning by meltMADGE: validations using BRCA1 and LDLR, and demonstration of the potential to identify severe, moderate, silent, rare, and paucimorphic mutations in the general population.

We have developed a mutation-scanning approach suitable for whole population screening for unknown mutations. The method, meltMADGE, combines thermal ramp electrophoresis with MADGE to achieve suitable cost efficiency and throughput. The sensitivity was tested in blind trials using 54 amplicons representing the BRCA1 coding region and a panel of 94 unrelated family breast cancer risk consultands previously screened in a clinical diagnostic laboratory. All 10 common polymorphisms, 15/15 previously identified disease-causing mutations, and three previously untested single base changes were identified. Assays of LDLR exons 3 and 8 were validated in 460 familial hypercholesteremics and detected 8/9 known variants. We then applied the exon 3 assay in several DNA banks representing approximately 8000 subjects with known cholesterol values and applied both assays in one DNA bank (n = 3600). In exon 3 we identified one previously reported moderate mutation, P84S (n = 1), also associated with moderate hypercholesteremia in this subject; an unreported silent variant, N76N (n = 1); and known severe hypercholesteremia splice mutation 313+1G-->A (n = 2). Around exon 8 we identified a paucimorphism (n = 35) at the splice site 1061-8T-->C (known to be in complete linkage disequilibrium with T705I) and unreported sequence variants 1186+11G-->A (n = 1) and D335N G-->A (n = 1). The cholesterol value for D335N was on the 96.2 percentile and for T705I, 2/35 carriers were above the 99th percentile. Thus, variants with predicted severe, moderate, and no effect were identified at the population level. In contrast with case collections, CpG mutations predominated. MeltMADGE will enable definition of the full population spectrum of rare, paucimorphic, severe, moderate (forme fruste), and silent mutations and effects.

The association of the PON1 Q192R polymorphism with coronary heart disease: findings from the British Women's Heart and Health cohort study and a meta-analysis.

BACKGROUND: There have been inconsistent results from case-control studies assessing the association of the PON1 Q192R polymorphism with coronary heart disease (CHD). Most studies have included predominantly men and the association in women is unclear. Since lipid levels vary between the sexes the antioxidant effect of PON1 and any genes associated with it may also vary by sex. We have examined the association of the PON1 Q192R polymorphism with CHD in a large cohort of British women and combined the results from our cohort study with those from all other published studies. RESULTS: The distribution of genotypes was the same among women with CHD and those without disease. The odds ratio (95% confidence interval) of having CHD comparing those with either the QR or RR genotype to those with QQ genotype (dominant model of association) was 1.03 (0.89, 1.21) and the per allele odds ratio was 0.98 (0.95, 1.01). In a meta-analysis of this and 38 other published studies (10,738 cases and 17,068 controls) the pooled odds ratio for the dominant effect was 1.14 (1.08, 1.20) and for the per allele effect was 1.10 (1.06, 1.13). There was evidence of small study bias in the meta-analyses and the dominant effect among those studies with 500 or more cases was 1.05 (0.96, 1.15). Ethnicity and reporting of whether the genotyping was done blind to the participants clinical status also contributed to heterogeneity between studies, but there was no difference in effect between studies with 50% or more women compared to those with fewer women and no difference between studies of healthy populations compared to those at high risk (with diabetes, renal disease of familial hypercholesterolaemia). CONCLUSION: There is no robust evidence that the PON1 Q192R polymorphism is associated with CHD risk in Caucasian women or men.

Polymorphism of the IGF2 gene, birth weight and grip strength in adult men.

BACKGROUND: Grip strength is a simple measure of skeletal muscle function but a powerful predictor of disability, morbidity and mortality. Recent evidence has shown that prenatal and infant growth influence grip strength in later life; this may reflect genetic influences on muscle size and function, although strong candidate genes have not been identified. IGF II has proliferative effects in adult muscle and is one of the major determinants of fetal growth; polymorphism in the IGF2 gene could therefore link early growth to adult grip strength. OBJECTIVES: To determine whether polymorphism of the IGF2 gene influences adult grip strength and mediates the association between size at birth and grip strength in later life. METHODS: Polymorphism of the ApaI marker in the IGF2 gene was determined for 693 Hertfordshire men and women born between 1920 and 1930 who had taken part in a study linking early growth to ageing. Grip strength was measured using isometric dynamometry. Genotyping assay development was undertaken in Southampton Genetic Epidemiology Laboratories (http://www.sgel.humgen.soton.ac.uk). RESULTS: In univariate analyses, IGF2 genotype and birth weight were both significant predictors of adult grip strength in the men after adjustment for age and current height. Significant associations were not seen in the women. When IGF2 genotype and birth weight in men were studied simultaneously, both contributed significantly to grip strength after adjustment for age and adult height. CONCLUSIONS: These results show that polymorphism of the IGF2 gene and birth weight have independent effects on adult grip strength in men and suggest that IGF2 polymorphism does not explain the association between size at birth and grip in later life. This study provides preliminary evidence for independent genetic and early environmental programming of adult muscle strength.

Null mutation in human ciliary neurotrophic factor gene confers higher body mass index in males.

Ciliary neurotrophic factor (CNTF) administration reduces weight in leptin-resistant mice via the signalling pathway normally activated by leptin. A G>A null mutation in the CNTF gene results in complete absence of protein. We hypothesised that absence of CNTF could lead to diminished initiation of anorectic pathways, with consequent increase in body mass. In 575 Caucasian men aged 59-73 years, the A/A genotype (frequency 1.9%) was associated with a 10 kg increase in weight (P=0.03, 2 df) and 3 kg/m(2) greater BMI (P=0.02, 2 df). There was no effect in women. The CNTF G>A null mutation therefore confers a moderate effect on obesity in males of A/A genotype, who represent 1% of the general population.