Barry R. Palmer

The dam and dcm strains of Escherichia coli--a review

The construction of a variety of strains deficient in the methylation of adenine and cytosine residues in DNA by the methyltransferases (MTases) Dam and Dcm has allowed the study of the role of these enzymes in the biology of Escherichia coli. Dam methylation has been shown to play a role in coordinating DNA replication initiation, DNA mismatch repair and the regulation of expression of some genes. The regulation of expression of dam has been found to be complex and influenced by five promoters. A role for Dcm methylation in the cell remains elusive and dcm- cells have no obvious phenotype. dam- and dcm- strains have a range of uses in molecular biology and bacterial genetics, including preparation of DNA for restriction by some restriction endonucleases, for transformation into other bacterial species, nucleotide sequencing and site-directed mutagenesis. A variety of assays are available for rapid detection of both the Dam and Dcm phenotypes. A number of restriction systems in E. coli have been described which recognise foreign DNA methylation, but ignore Dam and Dcm methylation. Here, we describe the most commonly used mutant alleles of dam and dcm and the characteristics of a variety of the strains that carry these genes. A description of several plasmids that carry dam gene constructs is also included.

Angiotensin-converting enzyme gene polymorphism interacts with left ventricular ejection fraction and brain natriuretic peptide levels to predict mortality after myocardial infarction.

OBJECTIVES The goal of this study was the exploration of the associations between the angiotensin-converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism and post-myocardial infarction (MI) outcomes, especially any interaction with the accepted clinical prognostic markers brain natriuretic peptide (BNP) and left ventricular ejection fraction (LVEF). BACKGROUND The ACE gene I/D polymorphism has been implicated in the development of MI, hypertension, and left ventricular hypertrophy. We examined the association of ACE I/D and prognosis after acute MI. METHODS Patients incurring acute MI were genotyped for the ACE I/D polymorphism. Clinical data included assays of neurohormones, radionuclide ventriculography, and mortality over a mean 2.6 years of follow-up. RESULTS Patients (n = 978) had a mean age of 62.1 years, and 78% were male. Overall genotype frequencies were II 23.2%, ID 49.5%, and DD 27.3%. Chi-square analysis revealed an association between the ACE D allele and death after MI (88 of 103 who died were DD or ID; p < 0.05), with an odds ratio for mortality of 8.03 (95% confidence interval, 2.16 to 29.88). Patients with the DD genotype had higher (p < 0.05) plasma BNP, N-terminal BNP (N-BNP), and endothelin-1 levels within 96 h after MI than grouped ID/II patients. Multivariate analysis indicated ACE genotype, age, and previous MI were independent predictors of death (p < 0.05). Patients with an ACE D allele in combination with either a lower than median LVEF or greater than median BNP had a higher mortality (p < 0.001 and p < 0.025, respectively) than the risk associated with the D allele itself. CONCLUSIONS Angiotensin-converting enzyme genotyping may provide additional prognostic information in patients after MI in combination with the proven utility of LVEF, plasma BNP, and N-BNP measurements.

Inflammatory biomarkers for predicting cardiovascular disease.

The pathology of cardiovascular disease (CVD) is complex; multiple biological pathways have been implicated, including, but not limited to, inflammation and oxidative stress. Biomarkers of inflammation and oxidative stress may serve to help identify patients at risk for CVD, to monitor the efficacy of treatments, and to develop new pharmacological tools. However, due to the complexities of CVD pathogenesis there is no single biomarker available to estimate absolute risk of future cardiovascular events. Furthermore, not all biomarkers are equal; the functions of many biomarkers overlap, some offer better prognostic information than others, and some are better suited to identify/predict the pathogenesis of particular cardiovascular events. The identification of the most appropriate set of biomarkers can provide a detailed picture of the specific nature of the cardiovascular event. The following review provides an overview of existing and emerging inflammatory biomarkers, pro-inflammatory cytokines, anti-inflammatory cytokines, chemokines, oxidative stress biomarkers, and antioxidant biomarkers. The functions of each biomarker are discussed, and prognostic data are provided where available.

Angiotensinogen M235T and T174M Gene Polymorphisms in Combination Doubles the Risk of Mortality in Heart Failure

Angiotensinogen M235T and T174M polymorphisms have individually been associated with elevated levels of plasma angiotensinogen, hypertension, and left ventricular hypertrophy. In this study, heart failure patients (n=451) were genotyped for the angiotensinogen M235T and T174M polymorphisms to investigate association with survival (recorded over 4 years of follow-up) and prognostic hormone markers. Patients carrying the 235TT genotype (n=86) were 3 years younger at admission (P=0.011), and, in those with hypertension, diagnosis was made ≈10 years earlier than other patients. Patients carrying ≥1 174M allele (n=94) were more likely to have a previous history of heart failure (P=0.044) and increased mortality during follow-up (risk ratio: 1.69, 95% CI: 1.03 to 2.79; P=0.038) compared with 174TT homozygotes (n=355), despite having a higher left ventricular ejection fraction (P=0.009). “High-risk” genotype combinations (defined a priori as 235TT and/or ≥1 174M allele; n=144; 32%) were independently predictive of mortality, conferring a 2-fold greater risk of dying during the follow-up period (odds ratio: 2.0; 95% CI: 1.3 to 3.0; P=0.001). This study suggested that angiotensinogen gene variants M235T and T174M may provide prognostic information for long-term survival in heart failure patients.

A Common Variant at Chromosome 9P21.3 Is Associated With Age of Onset of Coronary Disease but Not Subsequent Mortality

Background—Chromosome 9p21.3 (chr9p21.3) recently was identified by several genome-wide association studies as the genomic region most strongly associated with the risk of coronary artery disease. Within the chr9p21.3 locus, the single-nucleotide polymorphism rs1333049 has been demonstrated to be most strongly associated with susceptibility to developing coronary artery disease. However, the effect of rs1333049 on clinical outcomes in patients with established coronary disease has yet to be determined. Methods and Results—Coronary Disease Cohort Study (CDCS) (n=1054) and Post-Myocardial Infarction (PMI) (n=816) study participants were genotyped for rs1333049. Clinical history, circulating lipids, neurohormones, cardiac function, and discharge medications were documented. All-cause mortality and cardiovascular hospital readmissions were recorded over a median follow-up period of 4.0 years for the CDCS cohort and 9.1 years for the PMI cohort. The CDCS patients homozygous for the high-risk C allele had an age of onset 2 to 5 years earlier for coronary disease (P=.005), angina (P=.025), myocardial infarction (P=.022), and percutaneous transluminal coronary angioplasty (P=.009). Patients with the CC genotype also had higher levels of total cholesterol (P=.033) and triglycerides (P=.003). The PMI participants with the CC genotype were 3 years younger on admission (P=.009). Cox proportional hazards analysis adjusting for established predictors of increased risk showed no significant association between rs1333049 genotype and mortality in either the CDCS (P=.214) or the PMI (P=.696) cohorts. Conclusions—The chr9p21.3 polymorphism rs1333049 was associated with an earlier age of disease onset in 2 coronary disease cohorts but not with poorer clinical outcome in either cohort.

Genetic polymorphism rs6922269 in the MTHFD1L gene is associated with survival and baseline active vitamin B12 levels in post-acute coronary syndromes patients.

Background and Aims The methylene-tetrahydrofolate dehydrogenase (NADP+ dependent) 1-like (MTHFD1L) gene is involved in mitochondrial tetrahydrofolate metabolism. Polymorphisms in MTHFD1L, including rs6922269, have been implicated in risk for coronary artery disease (CAD). We investigated the association between rs6922269 and known metabolic risk factors and survival in two independent cohorts of coronary heart disease patients. Methods and Results DNA and plasma from 1940 patients with acute coronary syndromes were collected a median of 32 days after index hospital admission (Coronary Disease Cohort Study, CDCS). Samples from a validation cohort of 842 patients post-myocardial infarction (PMI) were taken 24–96 hours after hospitalization. DNA samples were genotyped for rs6922269, using a TaqMan assay. Homocysteine and active vitamin B12 were measured by immunoassay in baseline CDCS plasma samples, but not PMI plasma. All cause mortality was documented over follow-up of 4.1 (CDCS) and 8.8 (PMI) years, respectively. rs6922269 genotype frequencies were AA n = 135, 7.0%; GA n = 785, 40.5% and GG n = 1020, 52.5% in the CDCS and similar in the PMI cohort. CDCS patients with AA genotype for rs6922269 had lower levels of co-variate adjusted baseline plasma active vitamin B12 (p = 0.017) and poorer survival than patients with GG or GA genotype (mortality: AA 19.6%, GA 12.0%, GG 11.6%; p = 0.007). In multivariate analysis, rs6922269 genotype predicted survival, independent of established covariate predictors (p = 0.03). However the association between genotype and survival was not validated in the PMI cohort. Conclusion MTHFD1L rs6922269 genotype is associated with active vitamin B12 levels at baseline and may be a marker of prognostic risk in patients with established coronary heart disease.

Single nucleotide polymorphisms in an intron of the ovine calpastatin gene.

Abstract Calpastatin is the specific inhibitor of the ubiquitous calcium‐dependent proteases μ‐calpain and m‐calpain. Enzyme assay data from sheep and cattle inversely correlates post‐mortem muscle calpastatin levels with ultimate meat tenderness. Genetic markers of meat quality may therefore be found linked to the calpastatin gene (CAST). A three‐allele system detected by polymerase chain reaction ‐ single strand conformational polymorphism (PCR‐SSCP) has been observed in the ovine CAST. The three allele amplimers have been fully nucleotide sequenced and their differences in terms of single nucleotide polymorphism (SNPs) in the intron region of the amplimer are reported and compared to a consensus sequence of the orthologous region of the cattle CAST. A PCR‐RFLP for more rapid CAST genotyping of all three ovine alleles was also developed.

Ile164 variant of β2‐adrenoceptor does not influence outcome in heart failure but may interact with β blocker treatment

The Ile164 variant of the β2‐adrenoceptor has been shown to alter cardiovascular phenotypes and adversely affect survival in heart failure patients.

The common G-866A polymorphism of the UCP2 gene and survival in diabetic patients following myocardial infarction

BackgroundA variant in the promoter of the human uncoupling protein 2 (UCP2) gene, the G-866A polymorphism, has been associated with future risk of coronary heart disease events, in those devoid of traditional risk factors and in those suffering from diabetes. We thus examined the impact of the G-866A polymorphism on 5-year survival in a cohort of 901 post-myocardial infarction patients, and the impact of type-2 diabetes on this relationship. The association of UCP2 with baseline biochemical and hormonal measurements, including levels of the inflammatory marker myeloperoxidase, was also examined.MethodsUCP2 G-866A genotypes were determined using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) protocol. Myeloperoxidase levels were measured in plasma samples taken from 419 cohort patients 24–96 hours after admission.ResultsGenotypes were obtained for 901 patients with genotype frequencies AA 15.5%, GA 45.5%, and GG 39.0%. Genotype was not associated with survival in the overall cohort (mortality: AA 15.6%, GA 16.8%, GG 19.4%, p = 0.541). However, amongst diabetics, AA and GA genotype groups had significantly worse survival than GG diabetic patients (p < 0.05) with an attributable risk of 23.3% and 18.7% for those of AA and GA genotype respectively. Multivariate analysis using a Cox proportional hazards model confirmed that the interaction of diabetes with genotype was significantly predictive of survival (p = 0.031). In the cohorts diabetic subgroup AA/GA patients had higher myeloperoxidase levels than their GG counterparts (GA/AA, n = 51, 63.9 ± 5.23; GG, n = 34, 49.1 ± 3.72 ng/ml, p = 0.041). Further analysis showed that this phenomenon was confined to male patients (GA/AA, n = 36, 64.3 ± 6.23; GG, n = 29, 44.9 ± 3.72 ng/ml, p = 0.015).ConclusionDiabetic patients in this post-myocardial infarction cohort with UCP2 -866 AA/GA genotype have poorer survival and higher myeloperoxidase levels than their GG counterparts.

Genetic variation in the renin-angiotensin-aldosterone system is associated with cardiovascular risk factors and early mortality in established coronary heart disease.

This study examined renin–angiotensin–aldosterone (RAAS) system gene variants for associations with cardiovascular risk factors and outcomes in coronary heart disease. Coronary disease patients (n=1186) were genotyped for 21 single-nucleotide polymorphisms (SNPs) within angiotensinogen (AGT), angiotensin-converting enzyme (ACE), angiotensin-II type-1 receptor (AGTR1) and aldosterone synthase (CYP11B2). Associations with all-cause mortality and cardiovascular readmissions were assessed over a median of 3.0 years. The AGT M235T ‘T’ allele was associated with a younger age of clinical coronary disease onset (P=0.006), and the AGT rs2478545 minor allele was associated with lower circulating natriuretic peptides (P=0.0001–P=0.001) and E/E1 (P=0.018). Minor alleles of AGT SNPs rs1926723 and rs11122576 were associated with more frequent history of renal disease (P⩽0.04) and type-2 diabetes (P⩽0.02), higher body mass index (P⩽0.02) and greater mortality (P⩽0.007). AGT rs11568054 minor allele carriers had more frequent history of renal disease (P=0.04) and higher plasma creatinine (P=0.033). AGT rs6687360 minor allele carriers exhibited worse survival (P=0.02). ACE rs4267385 was associated with older clinical coronary disease onset (P=0.008) and hypertension (P=0.013) onset, increased plasma creatinine (P=0.01), yet greater mortality (P=0.044). Less history of hypertension was observed with the AGTR1 rs12685977 minor allele (P=0.039). Genetic variation within the RAAS was associated with cardiovascular risk factors and accordingly poorer survival.