A search for cyclophilin-A gene (PPIA) variation and its contribution to the risk of atherosclerosis and myocardial infarction.

Cyclophilin A is secreted by vascular smooth muscle cells in response to inflammatory stimuli, and could thus contribute to atherosclerosis. We hypothesized that the genetic variation at the cyclophilin A gene (PPIA) could affect the risk for developing atherosclerosis and myocardial infarction. This study included 250 myocardial infarction patients (all male and < 60 years; 95% are smokers). All these cases had at least one atherosclerotic diseased coronary vessel. DNA was obtained from patients and from 250 healthy controls. The variation at the PPIA gene was determined in the patients through single-strand conformation analysis and direct sequencing of seven polymerase chain reaction fragments. Allele and genotype frequencies were compared between patients and controls. The effect of a promoter polymorphism (-11 G/C) on gene expression was in vitro analysed with luciferase-reporter assays. We found two common polymorphisms in the PPIA promoter (-11 G/C) and the 5' non-translated (+36 G/A) regions. Cells transfected with luciferase-plasmids containing the -11 G had significantly higher luciferase activity. Genotype frequencies for these polymorphisms did not differ between patients and controls. In conclusion, we reported a functional variant in the PPIA promoter. However, the PPIA variation did not significantly contribute to the risk of suffering from myocardial infarction among patients with atherosclerotic diseased vessels.

Screening of the endothelin1 gene (EDN1) in a cohort of patients with essential left ventricular hypertrophy.

Our objective was to analyse the role of endothelin1 gene (EDN1) variation in essential left ventricular hypertrophy (LVH). We searched for EDN1 variants in 145 Spanish patients with an essential form of LVH (not secondary to hypertension, aortic stenosis, or any other disease that could explain the hypertrophy). The five EDN1 coding exons and 1.5 kilobases of the promoter region were analysed through single strand conformation analysis and direct sequencing. We found four nucleotide changes: -1224 C/A (promoter), -131 ins/del A (exon 1, 5'-non-translated sequence), A/G in codon 106 (exon 3, silent), and G/T in codon 198 (exon 5, lys198asn). To determine the association between these polymorphisms and cardiac hypertrophy, we compared the genotype frequencies from these 145 patients with 250 healthy controls. We found a higher frequency of patients homozygous for 198 lys (198 KK) (65% vs. 52%; p = 0.01; OR = 1.76) and for -1224 AA (73% vs. 66%; p = 0.19). Homozygotes for -1224 A + 198 K (AA+KK) were significantly more frequent in patients (62% vs. 45%; p = 0.0007; OR = 2.10; 95% CI = 1.35-3.25). The expression of the -1224 C/A and exon 5 K198N variants was analysed with cells in culture. These in vitro studies showed that these variations did not differ in their expression levels. In conclusion, our work has shown that EDN1 variation, and in particular homozygosity for the -1224A/198K haplotype, is associated with the risk of developing cardiac hypertrophy. However, these EDN1 variants do not affect in vitro gene expression.

A functional Sp1/Egr1-tandem repeat polymorphism in the 5-lipoxygenase gene is not associated with myocardial infarction.

Arachidonate 5-lipoxygenase is an enzyme encoded by the ALOX5 gene, and plays an important role in the synthesis of leukotrienes. These are inflammatory mediators, and have been involved in atherosclerosis and other pathological processes that require proinflammatory activities. Human and animal studies have suggested a role for the ALOX5 gene in atherosclerosis, including a significant association between a promoter polymorphism and a carotid intimal-medial thickness in response to dietary fat. This polymorphism was three- to six-tandem repeats of a Sp1/Egr1 binding motif (GGGCGG)(n), and the number of repeats has been linked with the amount of gene expression. We hypothesized that this ALOX5 polymorphism could influence the risk for myocardial infarction (MI). First, we analysed the effect of the four alleles on gene expression by transfecting the HEK-293 cell line with luciferase reporter-constructs. We found that luciferase activities are dependent on the number of the Sp1/Egr1 repeats, with the three and six repeats having the lowest and highest values. We genotyped 312 male MI survivors, aged < 55 years, and 376 healthy controls matched with patients for sex, age, and ethnicity. Ninety-six per cent of the patients were smokers, compared to only 42% among the controls (P < 0.001; OR = 31.84). The 55 + 56 repeat genotypes were less frequent in patients (55 = 56%, 56 = 0.6%) compared to controls (55 = 60%, 56 = 3%). However, these were non-significantly different frequencies. In addition, no difference in MI-onset age and biochemical values was found between the allele and genotypes. In conclusion, we confirmed the effect of the ALOX5-promoter polymorphism on gene expression, but our data did not support a significant effect of this functional variation on MI risk.

The Pro279Leu variant in the transcription factor MEF2A is associated with myocardial infarction.

BACKGROUND: A myocyte enhancer factor 2A (MEF2A) mutation that segregated with coronary artery disease/myocardial infarction (CAD/MI) in a large family has recently been described. Missense mutations in sporadic coronary artery disease patients were also reported. These data suggest that mutations in exons 7 and 11 of MEF2A cause CAD/MI, though the association was refuted by another study. OBJECTIVE: To analyse the genetic variation of exons 7 and 11 in a large cohort of Spanish CAD/MI patients and controls. METHODS AND RESULTS: A rare polymorphism, P279L, was detected both in patients and controls. Carriers of the 279Leu allele had a threefold risk of suffering CAD/MI compared with controls (p = 0.009; odds ratio = 3.06 (95% confidence interval, 1.17 to 8.06)). In the controls the allele was found only in those under 50 years of age. Exon 11 showed a high degree of heterogeneity caused by a polyglutamine (CAG)n polymorphism, but no significant differences in genotype or allelic frequencies were found. CONCLUSIONS: The 279Leu allele appears to be a genetic risk factor for CAD/MI in the population studied. This effect could be the result of a reduced transcriptional activity on MEF2A with 279Leu.

Lack of association between polymorphisms of the coagulation factor VII and myocardial infarction in middle-aged Spanish men.

In order to determine the role of two polymorphisms in the factor VII gene (R353Q and intron 7 hypervariable region) in the susceptibility to develop early myocardial infarction, a total of 175 patients with acute myocardial infarction aged 50 years or less (mean age 41+/-7 years) and 200 controls (average age 42+/-6) without cardiovascular disease were genotyped for these polymorphisms. Gene and genotype frequencies did not differ between patients and controls. Although the 353-QQ genotype was at a higher frequency among controls (4%) compared to patients (1%), the difference did not reach statistical significance. Carriers of the H7-allele (intron 7 polymorphism) were at a slightly higher frequency among patients (51 vs. 43%; P=0.19; OR=1.36; 95% CI=1.09-1.70). Our data suggest a lack of association between both polymorphisms in the factor VII gene and early myocardial infarction in our population.

Association between the NOS3 (-786 T/C) and the ACE (I/D) DNA genotypes and early coronary artery disease.

DNA polymorphisms at the endothelium constitutive nitric oxide synthase gene (NOS3) have been linked to the risk of developing coronary artery disease (CAD). In vitro, a polymorphism in the 5' region of the NOS3 gene (-786 T/C) influences promoter activity. This polymorphism has been associated with coronary spasms among Japanese. The genetic variation at the angiotensin-converting enzyme (ACE) is associated with plasma ACE activities and has also been linked with susceptibility to cardiovascular disease. Our objective was to determine if DNA polymorphisms in the NOS3 and ACE genes were associated with early CAD. We analyzed the -786 T/C polymorphism in the 5' flanking region and the 27-bp repeat polymorphism in NOS3 intron 4, as well as the ACE-I/D polymorphism. A total of 170 male smokers (CAD patients) younger than 50 years and 300 male smokers (healthy controls) were genotyped. Frequencies were compared by the chi(2) test, and odds ratios (ORs) and their 95% confidence intervals (CI) were also calculated. Only the -786 T/C polymorphism in the 5' flanking region of the NOS3 gene was significantly associated with early CAD in our population. The frequency of the CC genotype was significantly increased (P = 0.039) in patients compared to controls (OR = 1.67; 95% CI = 1.01, 2.72). We found a synergistic effect between the NOS3-CC and the ACE-DD genotypes in the risk of developing early CAD. The frequency of CC + DD was significantly increased among patients (P = 0.002). Thus, those with a NOS3-CC and an ACE-DD genotype would have a significantly increased risk of suffering an early episode of coronary artery disease (OR = 2.82; 95% CI = 1.40, 5.70). Although based on a limited number of patients, our work suggests that individuals who are NOS3-CC + ACE-DD are at a higher risk for early CAD, probably as a consequence of increased endothelial dysfunction.

Genetic variation at the chemokine receptors CCR5/CCR2 in myocardial infarction.

Our objective was to examine the association between myocardial infarction (MI) and two DNA-polymorphisms at the proinflammatory chemokine receptors CCR2 (I64V) and CCR5 (32 bp deletion, (Delta)ccr5), defining if these polymorphisms influence the age for the onset of MI. A total of 214 patients with an age at the first MI episode <55 years, 96 patients that suffered the first MI episode when older than 60 years, and 360 population controls were polymerase chain reaction genotyped for the CCR2-V64I and CCR5-Delta32/wt polymorphisms. Patients and controls were male from the same Caucasian population (Asturias, northern Spain). The frequency of the Deltaccr5 allele was significantly higher in controls compared to patients <55 years (P = 0.004), or in patients >60 years compared to patients <55 years (P = 0.002). Taking the patients >60 years as the reference group, non-carriers of the (Delta)ccr5-allele would have a three-fold higher risk of suffering an episode of MI at <55 years of age (OR = 3.06; 95% CI = 1.46-6.42). Gene and genotype frequencies for the CCR2 polymorphism did not differ between patients <55 years and controls or patients >60 years. Our data suggest that the variation at the CCR5 gene could modulate the age at the onset of MI. Patients carrying the (Delta)ccr5-allele would be protected against an early episode of MI. CCR5 and the CCR5-ligands are expressed by cells in the arteriosclerotic plaque. Thus, the protective role of (Delta)ccr5 could be a consequence of an attenuated inflammatory response, that would determine a slower progression of the arteriosclerotic lesion among (Delta)ccr5-carriers. Our work suggests that the pharmacological blockade of CCR5 could be a valuable therapy in the treatment of MI.

Synergistic effect between apolipoprotein E and angiotensinogen gene polymorphisms in the risk for early myocardial infarction.

BACKGROUND: Several studies based on different populations worldwide have described an association between cardiovascular diseases and genetic variations in the apolipoprotein E (A:POE), angiotensinogen (A:GT), angiotensin receptor type 1 (A:T1R), and angiotensin-converting enzyme (A:CE) genes. In addition, there is growing evidence of an interaction between hypercholesterolemia and the renin-angiotensin system in the risk for hypertension and atherosclerosis. METHODS: To determine whether the DNA polymorphisms in A:POE (epsilon2, epsilon3, and epsilon4 alleles), A:GT (M235T), A:T1R (1166 A:/C:), and ACE (I:/D:) are associated with early onset of myocardial infarction (MI), we genotyped 220 patients and 200 controls <55 years of age. Patients and controls were males from the same homogeneous Caucasian population. Data concerning hypertension, diabetes, and tobacco consumption were recorded. The lipid profiles of patients and controls were also determined. RESULTS: APOE, ACE, AGT, and AT1R allele and genotype frequencies did not differ between patients and controls. None of these polymorphisms was related to the biochemical values in patients or controls. The frequency of individuals who were both APOE epsilon4 allele carriers and AGT-TT homozygotes was significantly higher in patients than in controls (11% vs 3.5%; P: = 0.0037). In patients, the frequency of epsilon4 carriers was significantly higher (P: <0.00001) in those who were AGT-TT (46%) than those who were AGT-MT/MM (14%). Mean cholesterol was significantly higher in AGT-TT + APOE epsilon34/44 patients than in the TM/MM + epsilon34/44 or TT + epsilon23/33 genotypes (P: = 0. 029). CONCLUSIONS: Our data suggest a synergistic effect between the APOE and AGT polymorphisms and early MI. The increased risk could be mediated in part through higher cholesterol concentrations among individuals who are AGT-TT + APOE epsilon4 allele carriers.

Genetic variation in the renin-angiotensin system and athletic performance.

The D allele at the angiotensin-I-converting enzyme (ACE)-insertion/deletion polymorphism has been associated with an increased risk of developing several pathological processes, such as coronary heart disease and ventricular hypertrophy. Individuals with the DD genotype show a significantly increased left-ventricular mass in response to physical training, compared to the II genotype (which would be associated with the lowest plasma ACE levels) and the ID genotype. The II genotype has been linked to a greater anabolic response. In accordance with a role for ACE in the response to rigorous physical training, a higher frequency of the I allele has been reported to exist among elite rowers and high-altitude mountaineers. Sixty elite (professional) athletes (25 cyclists, 20 long-distance runners, and 15 handball players), and 400 healthy controls were genotyped for the DNA polymorphisms of the ACE, angiotensinogen (Ang) and angiotensin receptor type 1 (AT1) genes. Plasma ACE levels showed a strong correlation with the I/D genotype in our population. The I-allele occurred at a significantly higher frequency in athletes compared to controls (P = 0.0009). Gene and genotype frequencies for the Ang and AT1 polymorphisms did not differ between athletes and controls. Since the frequency of the ACE I allele was significantly increased among our elite athletes, we conclude that the ACE polymorphism represents a genetic factor that contributes to the development of an elite athlete.