Ian N. M. Day

Hardy-Weinberg Equilibrium Testing of Biological Ascertainment for Mendelian Randomization Studies

Mendelian randomization (MR) permits causal inference between exposures and a disease. It can be compared with randomized controlled trials. Whereas in a randomized controlled trial the randomization occurs at entry into the trial, in MR the randomization occurs during gamete formation and conception. Several factors, including time since conception and sampling variation, are relevant to the interpretation of an MR test. Particularly important is consideration of the “missingness” of genotypes that can be originated by chance, genotyping errors, or clinical ascertainment. Testing for Hardy-Weinberg equilibrium (HWE) is a genetic approach that permits evaluation of missingness. In this paper, the authors demonstrate evidence of nonconformity with HWE in real data. They also perform simulations to characterize the sensitivity of HWE tests to missingness. Unresolved missingness could lead to a false rejection of causality in an MR investigation of trait-disease association. These results indicate that large-scale studies, very high quality genotyping data, and detailed knowledge of the life-course genetics of the alleles/genotypes studied will largely mitigate this risk. The authors also present a Web program (http://www.oege.org/software/hwe-mr-calc.shtml) for estimating possible missingness and an approach to evaluating missingness under different genetic models.

Apolipoprotein E4 and coronary heart disease in middle-aged men who smoke: a prospective study

BACKGROUND The common isoforms of apolipoprotein E (apoE), E2, E3, and E4, are important determinants of plasma lipid concentrations, and the epsilon4 allele is associated with raised risk of coronary heart disease. We investigated whether the effect of smoking on coronary heart disease risk is affected by APOE genotype. METHODS We enrolled 3052 middle-aged men who were free of coronary heart disease for prospective cardiovascular surveillance in the second Northwick Park Heart Study (NPHSII). Smoking habit was ascertained at baseline and yearly by questionnaire. APOE genotype was identified by PCR and restriction enzyme digestion. Endpoints were fatal coronary heart disease, non-fatal myocardial infarction, and coronary artery surgery and silent myocardial infarction at follow-up. FINDINGS During 18836 person years of surveillance, 96 men had an acute myocardial infarction, 26 needed coronary artery surgery, and 14 had silent myocardial infarctions. Compared with never-smokers, risk of coronary heart disease in ex-smokers was 1.34 (95% CI 0.86-2.08) and in smokers it was 1.94 (1.25-3.01). This risk was independent of other classic risk factors. In never-smokers, risk was closely similar in men with different genotypes. Risk in men homozygous for the epsilon3 allele was 1.74 (1.10-2.77) in ex-smokers and 1.68 (1.01-2.83) in smokers, whereas in men carrying the epsilon4 allele risk was 0.84 (0.40-1.75) and 3.17 (1.82-5.50), respectively, with no significant differences in risk in the epsilon2 carriers. For the epsilon3 group, the genotype effect on risk was no longer significant after adjustment for classic risk factors (including plasma lipids). However, even after adjustment, smokers who were carriers of the epsilon4 allele, showed significantly raised risk of coronary heart disease compared with the non-smoking group (2.79, 1.59-4.91, epsilon4-smoking interaction p=0.007). INTERPRETATION Smoking increases the risk of coronary heart disease in men of all genotypes but particularly in men carrying the epsilon4 allele.

Association of C-Reactive Protein With Blood Pressure and Hypertension Life Course Confounding and Mendelian Randomization Tests of Causality

Background—C-reactive protein (CRP) has repeatedly been associated with blood pressure and prevalent and incident hypertension, but whether a causal link exists is uncertain. Methods and Results—We assessed the cross-sectional relations of CRP to systolic blood pressure, pulse pressure, and prevalent hypertension in a representative sample of >3500 British women aged 60 to 79 years. For both outcomes, substantial associations were observed. However, these associations were greatly attenuated by adjustment for a wide range of confounding factors acting over the life course. We further investigated causality using a Mendelian randomization approach by examining the association of the 1059G/C polymorphism in the human CRP gene with CRP and with blood pressure, pulse pressure, and hypertension. The polymorphism was associated with a robust difference in CRP, and the expectation would be for higher blood pressure and pulse pressure and greater prevalence of hypertension among those carrying the genetic variant associated with higher CRP levels. This was not observed, and the predicted causal effects of CRP on blood pressure, pulse pressure, and hypertension using instrumental variables methods were close to 0, although with wide CIs. Conclusions—CRP levels are associated with blood pressure, pulse pressure, and hypertension, but adjustment for life course confounding and a Mendelian randomization approach suggest the elevated CRP levels do not lead to elevated blood pressure.

Nicotine induced changes in gene expression by human coronary artery endothelial cells

The primary role of cigarette smoking in the development of coronary heart disease is to cause damage to the vascular endothelium, leading to endothelial cell dysfunction and initiating the pathogenesis of coronary atherosclerosis. We studied the response of human coronary artery endothelial cells to nicotine exposure by examining the expression of a panel of genes encoding molecules that have been shown to be involved in atherogenesis. Treatment of primary human coronary artery endothelial cells with nicotine for 24 h at concentrations (10(-5) and 10(-7) M) similar to those in the blood of smokers resulted in increased mRNA levels of endothelial nitric oxide synthase, angiotensin-I converting enzyme, tissue-type plasminogen activator, plasminogen activator inhibitor-1, von Willebrand factor, and vascular cell adhesion molecule-1. No change was detected in the expression levels of the genes encoding basic fibroblast growth factor, endothelin-1, endothelial leukocyte adhesion molecule-1 and matrix metalloproteinase-2 under these conditions. These data indicate that nicotine alters the expression of a number of endothelial genes whose products play major roles in regulating the vascular tone and thrombogenicity, making a contribution to the understanding of the effects of cigarette smoking on the development of coronary atherosclerosis.

Influences of matrix metalloproteinase-3 gene variation on extent of coronary atherosclerosis and risk of myocardial infarction

OBJECTIVES The aim of this study was to assess matrix metalloproteinase-3 (MMP3) gene variation in relation to the degree of coronary atherosclerosis and risk of myocardial infarction (MI) in patients with coronary artery disease. METHODS In this study, we systematically screened the promoter and coding regions for sequence variants. All polymorphisms identified were analyzed in 1,240 individuals undergoing coronary angiography. Functional analyses of the polymorphisms were carried out with the use of report assays and electrophoretic mobility shift assays. RESULTS Six novel polymorphisms were identified. The 6A/6A genotype was associated with greater number of coronary arteries with significant stenosis (odds ratio [OR] 1.52, p = 0.008), whereas the 5A/5A and 5A/6A genotypes were associated with increased risk of MI (OR 2.02 and 1.78, p = 0.016 and 0.032, respectively). A stepwise logistic regression analysis with all polymorphisms taken into account showed that the effect of MI susceptibility was largely attributed to the 5A/6A polymorphism. In a stepwise logistic regression analysis with all haplotypes as independent variables, the most common haplotype (T-5A-A-A-G-A), and two rare haplotypes, all containing the 5A allele, were associated with MI susceptibility. Functional studies showed that the T-5A-A-A-G-A haplotype had a higher promoter activity in macrophages. CONCLUSIONS These data indicate that the effect of MMP3 gene variation is attributable to the 5A/6A polymorphism and that individuals carrying the 6A/6A genotype may be predisposed to developing atherosclerotic plaques with significant stenosis, whereas those carrying the 5A allele may be predisposed to developing unstable plaques.

Determinants of the rate of nicotine metabolism and effects on smoking behavior

Studies on cytochrome P450 (CYP) 2A6 suggest that genotype affects the rate of nicotine metabolism and, consequently, cigarette consumption. However, known alleles of CYP2A6 associated with fast or slow metabolism are relatively uncommon, and there remains considerable variation in metabolic activity among those with presumed wild‐type CYP2A6 alleles, suggesting that other genetic or environmental factors also influence the rate of nicotine metabolism.

Apal polymorphism in insulin-like growth factor II (IGF2) gene and weight in middle-aged males.

In 1474 healthy Caucasoid men aged 45–65 y, insulin-like growth factor II (IGF2) Apal AA homozygotes showed a mean body weight 4 kg lower than Apal GG homozygotes (77.6±10.9 kg vs 81.6±11.5 kg, P=0.003) with heterozygotes (GA) intermediate (80.1±11.9 kg). The mean serum IGF-II concentration in 44 Apal AA individuals was significantly higher than in 48 Apal GG individuals (683.3±146.9 ng/ml vs 614.0±124.0 ng/ml, P=0.01). An INS Pstl polymorphism showed no association with weight and it was also found to be in minimal linkage disequilibrium with the IGF2 Apal site (coefficient 0.016). The IGF2 Apal AA genotype is therefore associated with lower mean body weight but higher serum IGF-II concentrations than the GG genotype. Apal GG homozygotes incur a 1.67-fold risk of pathological Body Mass index (BMI) (>30 kg/m2) compared with AA homozygotes.

MIDAS: software for analysis and visualisation of interallelic disequilibrium between multiallelic markers

BackgroundVarious software tools are available for the display of pairwise linkage disequilibrium across multiple single nucleotide polymorphisms. The HapMap project also presents these graphics within their website. However, these approaches are limited in their use of data from multiallelic markers and provide limited information in a graphical form.ResultsWe have developed a software package (MIDAS – Multiallelic Interallelic Disequilibrium Analysis Software) for the estimation and graphical display of interallelic linkage disequilibrium. Linkage disequilibrium is analysed for each allelic combination (of one allele from each of two loci), between all pairwise combinations of any type of multiallelic loci in a contig (or any set) of many loci (including single nucleotide polymorphisms, microsatellites, minisatellites and haplotypes). Data are presented graphically in a novel and informative way, and can also be exported in tabular form for other analyses. This approach facilitates visualisation of patterns of linkage disequilibrium across genomic regions, analysis of the relationships between different alleles of multiallelic markers and inferences about patterns of evolution and selection.ConclusionMIDAS is a linkage disequilibrium analysis program with a comprehensive graphical user interface providing novel views of patterns of linkage disequilibrium between all types of multiallelic and biallelic markers.AvailabilityAvailable from http://www.genes.org.uk/software/midas and http://www.sgel.humgen.soton.ac.uk/midas

Candidate-Gene Studies of the Atherogenic Lipoprotein Phenotype: A Sib-Pair Linkage Analysis of DZ Women Twins

There is a growing body of evidence supporting the roles of small, dense LDL and plasma triglyceride (TG), both features of the atherogenic lipoprotein phenotype, as risk factors for coronary heart disease. Although family studies and twin studies have demonstrated genetic influences on these risk factors, the specific genes involved remain to be determined definitively. The purpose of this study was to investigate genetic linkage between LDL size, TG, and related atherogenic lipoproteins and candidate genes known to be involved in lipid metabolism. The linkage analysis was based on a sample of 126 DZ women twin pairs, which avoids the potentially confounding effects of both age and gender, by use of a quantitative sib-pair linkage-analysis approach. Eight candidate genes were examined, including those for microsomal TG-transfer protein (MTP), hepatic lipase, hormone-sensitive lipase, apolipoprotein (apo) B, apo CIII, apo E, insulin receptor, and LDL receptor. The analysis suggested genetic linkage between markers for the apo B gene and LDL size, plasma levels of TG, of HDL cholesterol, and of apo B, all features of the atherogenic lipoprotein phenotype. Furthermore, evidence for linkage was maintained when the analysis was limited to women with a major LDL-subclass diameter >255 A, indicating that the apo B gene may influence LDL heterogeneity in the intermediate-to-large size range. In addition, linkage was found between the MTP gene and TG, among all the women. These findings add to the growing evidence for genetic influences on the atherogenic lipoprotein phenotype and its role in genetic susceptibility to atherosclerosis.

A mitochondrial DNA sequence variant associated with schizophrenia and oxidative stress

We have previously reported a changed mitochondrial (mt) gene expression in brain from patients with schizophrenia [Schizophr. Res. 14 (1995) 203]; now, we describe the distribution in the mtDNA from lymphocytes of a heteroplasmic sequence variation that was originally found in the mtDNA from the postmortem brain of a patient with schizophrenia. The variant is m.12027T>C and results in the change from isoleucine to threonine at position 423 of the ND4 subunit of NADH-ubiquinone reductase. Using a PCR-RFLP method, we have determined the heteroplasmy as the ratio of variant to total (variant ratio) at m.12027 in 184 controls and 181 patients with schizophrenia as well as 24 postmortem brain samples. The distribution of variants is bimodal having peaks at variant ratios of 0.262 and 0.732. The variant-rich fraction is very significantly associated with schizophrenia in males (47%), while there is only 18% in control males. There are significantly more variant-rich control females (36%) than control males (18%), suggesting that the female population is less sensitive to the presence of a variant in terms of liability to schizophrenia. In variant-rich samples from postmortem brain originating from both sexes, there is an increased superoxide production, suggesting that the variation contributes to oxidative stress. Antioxidant glycosides, such as quercetin rutoside, quench the superoxide production without (in contrast to neuroleptic drugs) interfering with the electron transfer activity of the reductase.