Inter- and intra-laboratory study to determine the reproducibility of toxicogenomics datasets.

The application of toxicogenomics as a predictive tool for chemical risk assessment has been under evaluation by the toxicology community for more than a decade. However, it predominately remains a tool for investigative research rather than for regulatory risk assessment. In this study, we assessed whether the current generation of microarray technology in combination with an in vitro experimental design was capable of generating robust, reproducible data of sufficient quality to show promise as a tool for regulatory risk assessment. To this end, we designed a prospective collaborative study to determine the level of inter- and intra-laboratory reproducibility between three independent laboratories. All test centres (TCs) adopted the same protocols for all aspects of the toxicogenomic experiment including cell culture, chemical exposure, RNA extraction, microarray data generation and analysis. As a case study, the genotoxic carcinogen benzo[a]pyrene (B[a]P) and the human hepatoma cell line HepG2 were used to generate three comparable toxicogenomic data sets. High levels of technical reproducibility were demonstrated using a widely employed gene expression microarray platform. While differences at the global transcriptome level were observed between the TCs, a common subset of B[a]P responsive genes (n=400 gene probes) was identified at all TCs which included many genes previously reported in the literature as B[a]P responsive. These data show promise that the current generation of microarray technology, in combination with a standard in vitro experimental design, can produce robust data that can be generated reproducibly in independent laboratories. Future work will need to determine whether such reproducible in vitro model(s) can be predictive for a range of toxic chemicals with different mechanisms of action and thus be considered as part of future testing regimes for regulatory risk assessment.

Emerging homogeneous DNA-based technologies in the clinical laboratory.

BACKGROUND: Advances in molecular diagnostic technologies have enabled genetic testing in single closed-tube reactions. The purpose of this review is to highlight some of the platforms and technologies currently available for the homogeneous detection of targets and the application of the technologies in the clinical setting. Validation issues surrounding the technologies, which may need to be addressed before they can become widely accepted, will also be discussed. APPROACH: This review discusses the principles of several of the major technologies available for performing homogeneous genetic analyses. Publications arising from the application of the technologies in a wide range of clinical areas are used to highlight and compare the potential advantages and shortcomings of the various technologies. CONTENT: This review is descriptive and focuses on three areas: the technologies available for performing homogeneous analysis, the clinical applications where the technologies are being used, and validation issues surrounding the acceptance of the technologies in the general clinical setting. SUMMARY: This review intends to give the reader a greater understanding of the various technologies available for performing homogeneous genetic testing in the clinical laboratory. Through insight into the principles and performance characteristics underlying these technologies, the end user can evaluate their value and limitations in the clinical diagnostic setting.

Angiotensin converting enzyme and angiotensin II type 1-receptor gene polymorphisms and risk of ischaemic heart disease.

OBJECTIVE: Polymorphisms in several genes of the renin-angiotensin system have been implicated as risk factors for myocardial infarction and ischaemic heart disease. In particular, it has been suggested that the angiotensin converting enzyme insertion/deletion (I/D) polymorphism and the angiotensin II type 1 receptor A1166C polymorphisms might act synergistically to increase the risk of myocardial infarction. The aim of this study was to investigate associations between the angiotensin converting enzyme I/D polymorphism and angiotensin II type 1 receptor polymorphisms and ischaemic heart disease. METHODS: We screened 331 white European patients who were recruited for routine angiographic investigation of chest pain, and 287 healthy white European controls for the angiotensin converting enzyme I/D and angiotensin II type 1 receptor A1166C polymorphisms, and related the genotype frequencies to angiotensin converting enzyme levels and the clinical phenotypes of atheroma and history of myocardial infarction. RESULTS: Angiotensin converting enzyme levels were related to I/D polymorphism but not to angiotensin II type 1 receptor polymorphism genotypes. I/D polymorphism and angiotensin II type 1 receptor genotypes did not relate individually to risk of myocardial infarction or atheroma in univariate or multivariate analysis. However, evidence of a synergistic relationship between the AC/II and CC/DD genotypes and coronary stenosis in the major arteries was found. No evidence of any relationship between these polymorphisms and history of myocardial infarction by World Health organisation (WHO) criteria was detected. CONCLUSION: These findings suggest that there is a weak relationship between the angiotensin converting enzyme I/D and angiotensin II type 1 receptor A1166C polymorphisms and coronary atheroma, but no evidence of a relationship with history of myocardial infarction.

Angiotensin-1-converting enzyme (ACE) gene polymorphism, plasma ACE levels, and their association with the metabolic syndrome and electrocardiographic coronary artery disease in Pima Indians.

In Caucasian subjects, an insertion/deletion (I/D) polymorphism of the angiotensin-converting enzyme (ACE) gene is associated with coronary artery disease (CAD) and fatal myocardial infarction. The underlying mechanism(s) of this association is not fully understood. Pima Indians have a low incidence of nonfatal and fatal CAD despite a high prevalence of diabetes. In Pima Indians, circulating ACE levels are related to ACE genotype, but the frequency of the D allele is significantly lower than in Caucasians. A lower frequency of the D allele may underlie a low risk of CAD in this population. We examined the relationship of the ACE genotype and plasma ACE level with electrocardiographic evidence of CAD (Tecumseh criteria), hypertension, and metabolic variables associated with insulin resistance in 305 (146 men and 159 women aged 47+/-9.0 years) Pima Indians characterized for the ACE I/D genotype. The distribution of ACE genotypes was unrelated to diabetes and obesity. Fasting plasma insulin, plasminogen activator inhibitor-1 (PAI-1) activity, plasma triglyceride concentrations, and systolic (SBP) and diastolic (DBP) blood pressure were not significantly different between the three ACE genotypes among nondiabetic and diabetic subjects. There was no significant association of ACE genotype with electrocardiographic evidence of CAD or with hypertension. Plasma ACE concentrations were not significantly different between nondiabetic and diabetic subjects (median, 77 [range, 21 to 1691 v 83 [7 to 238] IU/mL, P=NS). In all subjects, plasma ACE levels were associated weakly with plasma triglyceride (partial r=.20, P < .01) and total cholesterol (partial r=.13, P <.03) concentrations, but not with fasting plasma insulin or PAI-1 activity. In diabetic subjects, ACE levels were related to fasting plasma glucose concentrations (partial r=.15, P=.07). These findings would suggest that ACE gene I/D polymorphism is unlikely to be a major determinant of susceptibility to CAD in Pima Indians. Plasma ACE levels, but not ACE genotype, correlated with lipids, plasma glucose, and blood pressure, suggesting that elevated plasma ACE levels may contribute to the link between insulin resistance and CAD disease or may be a consequence of it.

Angiotensin-converting enzyme (ACE) gene polymorphisms in patients characterised by coronary angiography.

The angiotensin converting enzyme (ACE) gene is implicated as a risk factor for coronary artery disease and myocardial infarction (MI). An insertion/deletion (I/D) polymorphism is believed to be in linkage disequilibrium with a functional site elsewhere. Ten polymorphisms have recently been identified in the ACE gene. We screened patients undergoing coronary angiography (n = 258) for six of these polymorphisms (T-5491C, T-93C, A-240T, T1237C, D/I and 4656(CT)2/3), and identified a further two rare polymorphisms. ACE levels were associated with genotype for all polymorphisms analysed individually by one way ANOVA (P < 0.0005). The polymorphisms occurring in the 5' region were in negative linkage disequilibrium with the exonic and 3' region polymorphisms. The A-240T polymorphism had the greatest association with ACE levels (R2 = 14%); none of the others were significantly associated with levels when adjustment was made for A-240T. None of the polymorphisms were associated with the extent of coronary atheroma. Two of the promoter polymorphisms (A-240T and T-93C) were weakly related to the occurrence of MI (P = 0.03 and P = 0.05, respectively, by chi 2 analysis). The TT genotype of A-240T appeared to be protective against MI with an odds ratio of 0.31 (95% confidence interval, 0.12, 0.83). These findings indicate that polymorphisms in the ACE gene promoter region may have a stronger association with disease than the I/D polymorphism.

PCR-RFLP detection of PAI-2 gene variants: prevalence in ethnic groups and disease relationship in patients undergoing coronary angiography.

PAI-2 is a fibrinolytic inhibitor produced predominantly by monocytes. Most PAI-2 is intracellular making study in clinical conditions difficult. Abnormalities in production may be associated with inflammation and fibrinolysis at sites of tissue damage such as the atherosclerotic plaque. PAI-2 gene variants have been described: variant A consists of Asn120, Asn404 and Ser413 and variant B consists of Asp120, Lys404 and Cys413. We designed a PCR-RFLP assay using primers spanning the region containing Asn/Lys404 and Ser/Cys413. Variant B contains an MwoI restriction site. We analysed 302 Pima Indians and 286 healthy Caucasian volunteers. To investigate relationships between genotype and vascular disease we analysed 333 Caucasian patients undergoing coronary angiography. Gene variant B was more common in the Pimas than in Caucasians (p < 0.0001). There was no significant difference in genotype distribution between the volunteers and patients. In the patients there was no association between genotype and either a history of MI or extent of coronary atheroma.

Genes and the development of vascular disease.

Clinical vascular disease occurs as the result of the chronic development of atherosclerosis, often with acute occlusive thrombus formation as the final event. Both atherosclerotic and thrombotic disorders are complex processes resulting from genetic and environmental interactions. We review genes currently implicated as risk factors and the approaches for identifying novel genetic risk factors.

The angiotensin-I converting enzyme (ACE) gene I/D polymorphism and ACE levels in Pima Indians.

An insertion/deletion (I/D) polymorphism of the angiotensin converting enzyme (ACE) gene is associated with plasma ACE levels in white populations. The occurrence of the I/D polymorphism and relationship to ACE levels was examined in a Pima Indian group (n = 305). The frequency of the D allele was lower in Pimas than whites (0.29 v 0.52 respectively). ACE levels were significantly associated with genotype in both groups (p = 0.0001), which accounted for 6.5% of the variation in ACE levels in Pimas and 18% in whites. The association of the I/D polymorphism with ACE levels confirms the relationship across ethnic groups. The low frequency of the D allele in Pima Indians shows that ethnic differences should be accounted for when studying the ACE gene.

Commingling analysis of the distribution of a phenotype conditioned on two marker genotypes: application to plasma angiotensin-converting enzyme levels.

Commingling analysis is a statistical method for distinguishing between one (usually normal) distribution and a mixture of two or more distributions. It is used in genetic studies, since one mechanism giving rise to a mixture of distributions is the effect of a major gene. Plasma levels of angiotensin-converting enzyme (ACE) have been shown to be related to an insertion/deletion (I/D) polymorphism in the ACE gene. Recently, Cambien et al. [(1994) Circulation 90:669-676] used commingling analysis in a study of ACE, extending the method to condition on information about the I/D polymorphism in the gene. A further common polymorphism has been discovered recently by our group [Foy et al. (1995) Blood Coagulation Fibrinolysis 6:590] in the promoter region of the gene. In this paper we extend the method of commingling analysis to condition on two marker loci. The method is illustrated by application to plasma ACE levels in subjects for whom genetic information at both the I/D and promoter loci has been recorded. The results confirm strong evidence for a mixture of three normal distributions in preference to one or two, and also show that neither polymorphism is identical to a putative functional polymorphism. To assess the performance of the method, a simulation study was carried out. Parameters may be estimated more efficiently conditioning on two marker loci than on one or none, but this is not always the case if the underlying distributions are well separated. Conditioning on marker loci can increase the power to distinguish between alternative hypotheses of interest.

Detection of common viruses using the polymerase chain reaction to assess levels of viral presence in type 1 (insulin-dependent) diabetic patients.

The polymerase chain reaction was used to detect a range of common viruses in the peripheral blood of Type 1 diabetic and non-diabetic control patients in order to identify any abnormal viral presence, with possible roles in the pathogenesis of Type 1 diabetes. Peripheral blood from 17 newly diagnosed Type 1 diabetic patients, 38 Type 1 diabetic patients with disease of longer duration, and 43 age and sex matched non-diabetic controls was obtained. Samples were screened for cytomegalovirus, Epstein-Barr virus, enterovirus (including coxsackie), and mumps virus. Cytomegalovirus was detected in control patients only (5%), Epstein-Barr virus was detected equally in newly diagnosed and control patients (12%), and enterovirus was detected slightly more frequently in diabetic than non-diabetic patients (41% and 31%, respectively). Mumps virus was not detected in any of the samples. It is concluded that Type 1 diabetic individuals are neither more prone to persistence of common viruses nor to more frequent acute infections with the viruses tested for than non-diabetic individuals. If common viruses are involved in the pathogenesis of Type 1 diabetes then they act either as non-specific agents to which the host has abnormal immune responses, or, the diabetogenic viruses are eliminated from the body by the time of disease diagnosis.

A search for candidate viruses in type 1 diabetic pancreas using the polymerase chain reaction.

In order to investigate a possible viral aetiology for Type 1 diabetes the polymerase chain reaction (PCR) technique was used. Pancreatic tissue from Type 1 diabetic subjects was examined for the presence of a panel of common viruses. Primers specific for mumps, measles, cytomegalovirus, and Epstein Barr virus, as well as primers located in a highly conserved region of the enterovirus genome which are capable of detecting all of the following family members: Coxsackie B, echovirus, polio, mengovirus, and encephalomyocarditis virus were used to screen 18 Type 1 diabetic subjects of whom 3 had proven insulitis, 12 Type 2 diabetic subjects and 18 non-diabetic controls. Epstein Barr virus was detected in two Type 1 (13%), two Type 2 (22%), and three of the normal nondiabetic pancreases (20%), and the DNA sequences confirmed by direct sequencing. Cytomegalovirus was detected in one of the normal pancreases only and no evidence of any of the other viruses was found. It is concluded that the Type 1 diabetic pancreatic samples studied did not show persistence of infection with any of the above viruses. Non-persistent acute infection of the pancreas by the above viruses cannot be excluded in the aetiology of Type 1 diabetes from this study.