S.E. Humphries

Association of Angiotensin-Converting Enzyme Gene I/D Polymorphism With Change in Left Ventricular Mass in Response to Physical Training

BACKGROUND The absence (deletion allele [D]) of a 287-base pair marker in the ACE gene is associated with higher ACE levels than its presence (insertion allele [I]). If renin-angiotensin systems regulate left ventricular (LV) growth, then individuals of DD genotype might show a greater hypertrophic response than those of II genotype. We tested this hypothesis by studying exercise-induced LV hypertrophy. METHODS AND RESULTS Echocardiographically determined LV dimensions and mass (n=140), electrocardiographically determined LV mass and frequency of LV hypertrophy (LVH) (n=121), and plasma brain natriuretic peptide (BNP) levels (n=49) were compared at the start and end of a 10-week physical training period in male Caucasian military recruits. Septal and posterior wall thicknesses increased with training, and LV mass increased by 18% (all P<.0001). Response magnitude was strongly associated with ACE genotype: mean LV mass altered by +2.0, +38.5, and +42.3 g in II, ID and DD, respectively (P<.0001). The prevalence of electrocardiographically defined LVH rose significantly only among those of DD genotype (from 6 of 24 before training to 11 of 24 after training, P<.01). Plasma brain natriuretic peptide levels rose by 56.0 and 11.5 pg/mL for DD and II, respectively (P<.001). CONCLUSIONS Exercise-induced LV growth in young males is strongly associated with the ACE I/D polymorphism.

Variation in the PPARα gene is associated with altered function in vitro and plasma lipid concentrations in Type II diabetic subjects

Aims/hypothesis. Peroxisome proliferator activated receptor alpha (PPARα) regulates genes involved in lipid metabolism, haemostasis and inflammation, in response to fatty acids and fibrates, making it a candidate gene for risk of dyslipidaemia, atherosclerosis and coronary artery disease. Plasma non-esterified fatty acids are increased in subjects with Type II (non-insulin-dependent) diabetes mellitus, suggesting that PPARα could link Type II diabetes and dyslipidaemia, and affect response to fibrates. This has been investigated in association studies in healthy and diabetic subjects and in vitro studies. Methods. The human PPARα gene was isolated and screened for variation by single strand conformation polymorphism analysis. Genotypes were determined for 129 Type II diabetic subjects and 2508 healthy men. The association with plasma lipid concentrations was examined. The function of the V162 variant was examined in co-transfection assays. Results. We identified two polymorphisms, one in intron 3 and a missense mutation, leucine 162 to valine, in the DNA binding domain. In Type II diabetic patients, V162 allele carriers had higher total cholesterol, HDL cholesterol and apoAI whereas intron 3 rare allele carriers had higher apoAI concentrations. By contrast, no effect was observed in healthy rare allele carriers. In vitro, the V162 variant showed greater transactivation of a reporter gene construct. Conclusion/interpretation. Naturally occurring variation alters PPARα function, influencing plasma lipid concentrations in Type II diabetic patients but not healthy people. This demonstrates that PPARα is a link between diabetes and dyslipidaemia, and so could influence the risk of coronary artery disease, the greatest cause of morbidity and mortality in Type II diabetes. [Diabetologia (2000) 43: 673–680]

Plasma Thrombin-Activatable Fibrinolysis Inhibitor Antigen Concentration and Genotype in Relation to Myocardial Infarction in the North and South of Europe

The thrombin-activatable fibrinolysis inhibitor (TAFI) is a recently described inhibitor of fibrinolysis that decreases plasminogen binding to the fibrin surface. The plasma TAFI concentration is almost entirely genetically determined. We investigated whether plasma TAFI levels and polymorphisms located in the TAFI gene could constitute risk markers of myocardial infarction (MI). Plasma TAFI antigen (Ag) levels were assayed by ELISA and 2 TAFI gene polymorphisms (Ala147Thr and C+1542G in the 3′ untranslated region) were determined in a large European case-control study. This study compared 598 men recruited 3 to 6 months after MI with 653 age-matched controls from North Europe (Stockholm, Sweden, and London, England) and South Europe (Marseilles, France, and San Giovanni Rotondo, Italy). A TAFI Ag value above the 90th percentile was associated with a significantly lower risk of MI (odds ratio 0.55, P <0.02), indicating that elevated TAFI may be protective against MI. As previously shown, the 2 TAFI gene polymorphisms were in strong linkage disequilibrium and were associated with the TAFI Ag concentration, with carriers of the Thr147 and 1542C alleles having higher levels (P <0.0005). These effects were similar in controls and cases and in each center. There was a difference in allele frequency between cases and controls for the Ala147Thr polymorphism, with Thr147 allele carriers being more frequent in controls than in cases in 2 centers, Stockholm (P =0.03) and San Giovanni Rotondo (P =0.03); the odds ratio for the entire cohort was 0.78 (P <0.05). In conclusion, patients with a recent MI presented lower values of TAFI Ag and higher frequencies of the “TAFI-decreasing” alleles. The geographical differences observed do not contribute to explaining the North-South gradient in MI risk in Europe.

DNA polymorphisms of the apolipoprotein genes — their use in the investigation of the genetic component of hyperlipidaemia and atherosclerosis

DNA probes for all eight of the major apolipoprotein genes are now available. The chromosomal location, the basic structure and in many cases the nucleotide sequences of the normal genes are known. Common DNA polymorphisms of all of the genes have been detected. These have been been used in a number of ways to investigate rare inherited defects of the apolipoprotein genes, to study the potential involvement of different variants of the genes in the development of hyperlipidaemia in patients, and to investigate the contribution of common variation in these genes in the determination of serum lipid levels in the normal population.

DNA polymorphisms of the gene for apolipoprotein B in patients with peripheral arterial disease

We have determined the frequency of DNA polymorphisms of the gene for human apolipoprotein B, detected with XbaI and EcoRI, in 205 patients with documented peripheral arterial disease. Of the patients, 78 have no evidence of disease in the coronary and carotid arteries, 64 have coexisting coronary artery disease but no evidence of carotid artery disease, 26 patients have coexisting carotid artery disease but no evidence of coronary artery disease, and 37 have coexisting coronary and carotid artery disease. Levels of triglycerides, cholesterol and apolipoprotein B were measured for each patient, and RFLP frequency was determined in all the patients. Lipid, lipoprotein and apolipoprotein levels were not significantly different between the different patient groups. Compared with a sample from the clinically well London population, the frequency of the R2 allele of the polymorphism detected with EcoRI, and the frequency of the X1 allele of the XbaI polymorphism was significantly higher in the patient group. The frequency of these alleles was not significantly different in the different patient groups. In patients with only peripheral arterial disease, individuals with the XbaI genotype X1X1 have the lowest and those with the genotype X2X2 have the highest mean levels of serum cholesterol. However, in all other patient groups this trend was reversed (X1X1 highest and X2X2 lowest). Our observations suggest that variation at the apo B locus is one of the factors involved in predisposing an individual to develop arterial disease but does not determine where in the arterial system the disease develops.

Angiotensin II Type 1 Receptor 1166C Polymorphism Is Associated With Abdominal Aortic Aneurysm in Three Independent Cohorts

Objectives—Although polymorphic variations in genes of the RAS system have previously been associated with susceptibility to AAA, such studies have been significantly limited by small sample sizes. This study was undertaken, using the largest case series yet reported, to determine whether common genetic variants of the RAS are associated with either susceptibility or severity of AAA. Methods and Results—The frequencies of 4 common genetic variants of genes related to the renin-angiotensin system were investigated in 3 geographically distinct, but ethnically similar, case-control cohorts, resulting in comparison of 1226 AAA cases with 1723 controls. In all 3 the AGTR1 1166C allele was significantly more common in AAA patients than controls (overall adjusted OR 1.60, 95% CI 1.32 to 1.93, P=1.1×10−6). Overall, the ACE ID genotype was associated with AAA (OR 1.33, 95% CI 1.06 to 1.67, P<0.02). The AGT 268T allele appeared to have an epistatic effect on large aneurysm size. Conclusion—This study has identified a strong and repeated association between the AGTR1 1166C allele and susceptibility to AAA, and a weaker effect associated with the ACE deletion allele, in 3 geographically distinct, but ethnically similar, case-control cohorts. This study highlights the key role of the RAS in AAA and emphasizes the need for replication and validation of results in suitable independent cohorts.

Catabolic rate of low density lipoprotein is influenced by variation in the apolipoprotein B gene.

This study examines the potential influence of genetic variation on the metabolism of LDL. Restriction fragment length polymorphisms (RFLP) of the gene coding for apo B were identified using the endonucleases Xba I, Eco RI, and Msp I in a group of 19 subjects with moderate hyperlipidemia. There was a significant association between the Xba I polymorphism and the total fractional clearance rate (FCR) of LDL. The individuals with the X1X1 genotype had, on average, a 22% higher FCR (P less than 0.025) than those with the genotype X2X2 (X2 allele = presence of Xba I cutting site). This difference was attributable to increased clearance by the receptor-mediated pathway of LDL catabolism. In this group of subjects, there was no association of LDL kinetic parameters and RFLPs of the LDL receptor gene or the AI- CIII- AIV gene cluster. The data suggest that variation in apo B itself, presumably acting through variable binding to the LDL receptor, makes a significant contribution to the rate of catabolism of LDL.

Lack of association between the insertion/deletion polymorphism of the angiotensin-converting enzyme gene and idiopathic dilated cardiomyopathy

OBJECTIVES We sought to investigate the role of polymorphisms of the gene for angiotensin-converting enzyme in the development and progression of idiopathic dilated cardiomyopathy. BACKGROUND Cardiovascular renin-angiotensin systems may be involved in cardiac remodeling and fibrosis. The absence (deletion [D]) of a 287-base pair marker in the angiotensin-converting enzyme gene (introm 16) is associated with increased serum angiotensin-converting enzyme levels. The DD genotype may be a risk factor for the development of end-stage heart failure due to cardiomyopathy. We therefore examined the relation of the angiotensin-converting enzyme genotype to idiopathic dilated cardiomyopathy and to markers of disease severity. METHODS We studied 364 control subjects and 99 consecutive patients with idiopathic dilated cardiomyopathy. When the incidence of the DD genotype in our control group was assumed to be similar to that previously reported (27%), this study had a power of 0.9 to detect a different incidence in the patient group, if the true incidence in patients was 42%. Deoxyribonucleic acid (DNA) was isolated from blood samples, and angiotensin-converting enzyme genotype was determined by specific polymerase chain reaction and separation of amplified fragments by agarose gel electrophoresis. We also compared genotype distribution with that in previously reported European control subjects. Functional status, clinical course over a mean +/- SD of 28 +/- 33 months and outcome were documented. Cardiac morphology and function and evidence of rhythm disturbance were noninvasively determined. RESULTS Angiotensin-converting enzyme genotype distribution and allele frequencies were similar in patients and control subjects to within 10% (with 95% confidence) and were also similar between patients and European control subjects. No markers of disease severity or progression other than duration of symptoms before diagnosis and the number of ventricular ectopic beats/h were significantly associated with the presence of the DD alleles. CONCLUSIONS We find no evidence to support an association between angiotensin-converting enzyme genotype and either the diagnosis of idiopathic dilated cardiomyopathy itself or progression of the disease.

The plasminogen activator inhibitor-1 -675 4G/5G genotype influences the risk of myocardial infarction associated with elevated plasma proinsulin and insulin concentrations in men from Europe: The HIFMECH Study

Summary.  Although the potential role of plasminogen activator inhibitor‐1 (PAI‐1) in the development of coronary artery disease is strongly supported by its biological characteristics, results of clinical studies remain controversial. Objectives: To investigate whether plasma PAI‐1 concentrations and the −675 4G/5G polymorphism located in the PAI‐1 gene could constitute risk markers for myocardial infarction (MI). Patients and methods: We used a European case–control study, the HIFMECH study, comparing 598 men with MI and 653 age‐matched controls. Results: Insulin resistance explained a major part of the variation in PAI‐1 (24%) whereas inflammation had only a minor contribution (0.01%). For both cases and controls plasma PAI‐1 concentrations were significantly higher in the North than the South, and in both regions were higher in individuals with MI compared with control subjects [overall odds ratio (OR) for a 1 SD increase = 1.54, 95% confidence interval (CI) 1.34, 1.77]. This difference was observed in all the centers studied. Overall, the difference between cases and control subjects remained significant after controlling for inflammation variables (OR = 1.30, 95% CI 1.08, 1.57), but lost significance after controlling for insulin resistance variables (OR = 1.17, 95% CI 0.98, 1.40). The 4G allele was associated with significantly higher PAI‐1 levels in cases but not controls and, taken independently, did not modify the risk of MI (P = 0.9). However, a significant interaction was observed with both insulin or proinsulin and the risk of MI (P = 0.05 and 0.02, respectively), but not with triglycerides or body mass index (BMI). The insulin or proinsulin effect on risk was observed only in the carriers of the 4G/4G genotype. This interaction appeared not to be mediated by plasma PAI‐1 antigen concentrations (P = 0.01 and 0.02 after adjustment for PAI‐1 plasma levels). The interaction with proinsulin but not insulin remained statistically significant after further adjustment for other factors associated with insulin resistance (triglycerides and BMI) and C‐reactive protein (P = 0.01). Conclusion: This study suggests that PAI‐1 has a role in risk of MI in the presence of underlying insulin resistance. A significant interaction between insulin or proinsulin and the −675 4G/5G polymorphism was observed in risk for MI. The mechanisms for these interactions remain to be determined.

Association between the LPL-D9N mutation in the lipoprotein lipase gene and plasma lipid traits in myocardial infarction survivors from the ECTIM study

Using polymerase chain reaction (PCR) based techniques, we have identified individuals in the ECTIM study of myocardial infarction survivors (cases) and healthy matched controls who are carriers for a mutation of the gene for lipoprotein lipase (LPL) which alters amino acid 9 from aspartic acid to asparagine (LPL-D9N). The frequency of carriers in the cases from Belfast and France (3 separate centres) was 2.5 and 3.7%, respectively (mean 3.3%, 95% CI 1.9-4.7) and in the controls 2.0 and 2.9%, respectively (mean 2.7%, 95% CI 1.6-3.8%), but this difference was not statistically significant. In the cases, carriers of the allele for LPL-N9 had higher levels of several plasma lipid traits including total triglycerides (TG) (30%), very low density lipoprotein (VLDL) cholesterol (19%), apo E (24%), apo C-III (17%), lipoprotein particles (Lp) containing both apo E and apo B (LpE:B) (32%), and particles containing both apo C-III and apo B (LpCIII:B) (39%), and this effect was consistent in cases both from Belfast and from the French centres combined. By contrast, in the controls there were no differences in any lipid trait between carriers and non-carriers of the mutation that was consistent between the French centres and Belfast. There were no significant differences in the levels of any measured factor between cases and controls that could explain the different effect on plasma lipid traits associated with the mutation. However, compared to the non-carriers, in both cases and controls who carried the mutation, plasma TG concentrations were higher in those whose body mass index (BMI) was above the mean of the sample (26.0 kg/m2), with statistically significant interaction seen between BMI and genotype and levels of apo C-III, and lipoprotein particles containing both apo C-III and apo B (P < 0.02). The data suggest that carriers for the LPL-N9 mutation have a mild genetic predisposition to developing hyperlipidaemia and an atherogenic lipid profile, but that this requires the presence of other genetic or environmental factors for full expression, one of which appears to be increasing obesity.