EDNRB gene variants and melanoma risk in two southern European populations.

BACKGROUND: EDNRB gene variants were reported to be associated with melanoma risk in French patients, with the S305N variant showing the highest frequency. AIM: To verify the S305N association with melanoma risk in an independent larger French population (378 patients, 389 controls); to investigate the role of EDNRB variants in melanoma risk in an Italian population (133 patients, 118 controls); and to explore the association of CDKN2A or CDK4 mutations with the S305N EDNRB variant in a subgroup of patients (59 French, 12 Italian) with a suspected hereditary predisposition to melanoma (familial melanoma, sporadic multiple primary melanoma or melanoma associated with pancreatic cancer). METHODS: The S305N variant was genotyped in the French population, while the EDNRB gene in the Italian population was entirely sequenced. RESULTS: Overall, there was no significant difference in the frequency of the S305N variant between patients with sporadic melanoma and controls in either the French or the Italian population. However, a significantly higher S305N allele frequency was detected in French patients with a suspected hereditary predisposition to melanoma compared with controls (P = 0.04). In addition, in this subgroup of patients, the S305N allele was also significantly associated with the presence of CDKN2A mutations (P = 0.04). CONCLUSIONS: Our results showed no evidence of association of the S305N EDNRB polymorphism with sporadic melanoma risk in either the French or Italian populations, but there was an indication that EDNRB might be a melanoma-predisposing gene in French patients with a suspected hereditary predisposition to melanoma.

An insertion/deletion polymorphism in the angiotensin I-converting enzyme gene accounting for half the variance of serum enzyme levels.

A polymorphism consisting of the presence or absence of a 250-bp DNA fragment was detected within the angiotensin I-converting enzyme gene (ACE) using the endothelial ACE cDNA probe. This polymorphism was used as a marker genotype in a study involving 80 healthy subjects, whose serum ACE levels were concomitantly measured. Allele frequencies were 0.6 for the shorter allele and 0.4 for the longer allele. A marked difference in serum ACE levels was observed between subjects in each of the three ACE genotype classes. Serum immunoreactive ACE concentrations were, respectively, 299.3 +/- 49, 392.6 +/- 66.8, and 494.1 +/- 88.3 micrograms/liter, for homozygotes with the longer allele (n = 14), and heterozygotes (n = 37) and homozygotes (n = 29) with the shorter allele. The insertion/deletion polymorphism accounted for 47% of the total phenotypic variance of serum ACE, showing that the ACE gene locus is the major locus that determines serum ACE concentration. Concomitant determination of the ACE genotype will improve discrimination between normal and abnormal serum ACE values by allowing comparison with a more appropriate reference interval.

Haemostatic factors and the risk of cardiovascular death in patients with coronary artery disease: the AtheroGene study.

OBJECTIVE: To get a better insight into the role of hemostasis in coronary artery disease (CAD), we assessed the impact of von Willebrand factor (vWF), fibrinogen, thrombin-antithrombin (TAT) complexes, D-dimers, and plasmin-antiplasmin (PAP) complexes on the risk of cardiovascular event in a prospective cohort of CAD patients. METHODS AND RESULTS: The prospective Atherogene cohort includes 1057 individuals with an angiographically proven coronary artery disease at baseline. After a median follow-up of 6.6 years, 135 individuals died from a cardiovascular cause and 97 had a nonfatal cardiovascular event. Higher levels of all 5 hemostatic markers at baseline were associated with an increased risk of cardiovascular death, but not of nonfatal event. Except for vWF, these associations remained significant after adjustment for conventional cardiovascular risk factors and C-reactive protein (CRP) levels (P for trend according to increasing tertiles=0.20, 0.011, 0.026, 0.019, and 0.01 for vWF, fibrinogen, TAT, D-Dimer, and PAP, respectively). When including the 5 hemostatic markers in a stepwise Cox regression analysis where conventional risk factors and CRP were forced into the model, fibrinogen and D-dimers remained independently associated with the risk of cardiovascular death. Adjusted hazard ratios (95% CI) associated with one SD increase of fibrinogen and D-dimers were 1.27 (1.04 to 1.55) and 1.29 (1.09 to 1.53), respectively. CONCLUSIONS: In patients with coronary artery disease, fibrinogen and D-dimer levels are independent predictors of subsequent cardiovascular death. Our data support a role of impaired coagulation/fibrinolysis process in the complications of coronary artery disease.

Association of eNOS Glu298Asp gene polymorphism with circadian blood pressure rhythm.

Hypertensive patients with altered circadian blood pressure (BP) profile experience greater repercussion of hypertension on target organs and a higher risk of cardiovascular events, compared with those with physiological variations in BP. It has been demonstrated in animal models, that circadian variations in BP depend on several regulatory systems, in particular the nitric oxide-cGMP pathway. eNOS298 Glu/Asp polymorphism is a functional variant and may alter the amount of NO generated or eNOS activity. The objective of the present study was to find out whether eNOS298 gene polymorphism affects circadian BP regulation in 110 healthy subjects and 155 never-treated hypertensive patients recruited at Hypertension Units in Grenoble, Toulouse and Lille (France).

SNPs of PSMA6 gene--investigation of possible association with myocardial infarction and type 2 diabetes mellitus.

In our preceding studies we have identified microsatellite polymorphisms inside the PSMA6 gene and in its 5' upstream region. Following the observed associations of microsatellite polymorphisms with non-insulin dependent diabetes mellitus and Graves' disease we extended the evaluation of PSMA6 genetic variations to cardiovascular disorders and non-insulin dependent diabetes mellitus. New polymorphisms in the promoter region and exon 6 of the gene were identified by direct sequencing of the promoter region and all seven exons of the gene in 30 individuals of European descent. Two SNPs at positions -110 and -8 from the translation start, in the promoter region and 5'UTR respectively, were analyzed. Neither polymorphism was associated with the risk of myocardial infarction. No significant association of the polymorphisms with plasma lipid levels or BMI was observed. A borderline association of both polymorphisms with diastolic blood pressure was observed in the control group. Genotype -8CG was significantly more frequent in type 2 diabetes patients, and haplotype C-110/G-8, compared to C-110/C-8 was associated with a higher risk of NIDDM.

Cholesteryl ester transfer protein TaqIB variant, high-density lipoprotein cholesterol levels, cardiovascular risk, and efficacy of pravastatin treatment: individual patient meta-analysis of 13,677 subjects.

BACKGROUND: Several studies have reported that the cholesteryl ester transfer protein (CETP) TaqIB gene polymorphism is associated with HDL cholesterol (HDL-C) levels and the risk of coronary artery disease (CAD), but the results are inconsistent. In addition, an interaction has been implicated between this genetic variant and pravastatin treatment, but this has not been confirmed. METHODS AND RESULTS: A meta-analysis was performed on individual patient data from 7 large, population-based studies (each >500 individuals) and 3 randomized, placebo-controlled, pravastatin trials. Linear and logistic regression models were used to assess the relation between TaqIB genotype and HDL-C levels and CAD risk. After adjustment for study, age, sex, smoking, body mass index (BMI), diabetes, LDL-C, use of alcohol, and prevalence of CAD, TaqIB genotype exhibited a highly significant association with HDL-C levels, such that B2B2 individuals had 0.11 mmol/L (0.10 to 0.12, P<0.0001) higher HDL-C levels than did B1B1 individuals. Second, after adjustment for study, sex, age, smoking, BMI, diabetes, systolic blood pressure, LDL-C, and use of alcohol, TaqIB genotype was significantly associated with the risk of CAD (odds ratio=0.78 [0.66 to 0.93]) in B2B2 individuals compared with B1B1 individuals (P for linearity=0.008). Additional adjustment for HDL-C levels rendered a loss of statistical significance (P=0.4). Last, no pharmacogenetic interaction between TaqIB genotype and pravastatin treatment could be demonstrated. CONCLUSIONS: The CETP TaqIB variant is firmly associated with HDL-C plasma levels and as a result, with the risk of CAD. Importantly, this CETP variant does not influence the response to pravastatin therapy.

Functional polymorphism in the regulatory region of gelatinase B gene in relation to severity of coronary atherosclerosis.

BACKGROUND: Gelatinase B, a matrix metalloproteinase that has proteolytic activity against connective tissue proteins, has been suggested to be important in the connective tissue remodeling processes associated with atherogenesis and plaque rupture. This study tested the hypothesis that sequence variation in the promoter region of the gelatinase B gene influences its expression, predisposing individuals carrying certain genetic variants to more severe atherosclerosis. METHODS AND RESULTS: Single-strand conformation polymorphism analysis was carried out to search the promoter region of the gene encoding gelatinase B for naturally occurring genetic variation. As a result, an unreported common polymorphism was detected, which arose from a cytosine (C) to thymidine (T) transition at position -1562 relative to the start of transcription. Transient transfection experiments and DNA-protein interaction assays indicated that the T allele had a higher promoter activity than the C allele, which appeared to be due to preferential binding of a putative transcription repressor protein to the C allelic promoter. A sample of 584 male patients with myocardial infarction and 645 age-matched male healthy control subjects were genotyped. The allele frequencies were not significantly different between the cases and control subjects. However, in 374 patients with available angiographic data, 26% of those carrying 1 or 2 copies of the T allele had >50% stenosis in 3 coronary arteries, whereas only 15% of C/C homozygotes had triple-vessel disease. CONCLUSIONS: These data suggest that this functional genetic variation influences gelatinase B gene promoter activity in an allele-specific manner and has an effect on atherosclerotic phenotype.

Polymorphism screening of four genes encoding advanced glycation end-product putative receptors. Association study with nephropathy in type 1 diabetic patients.

Advanced glycation end-products (AGEs) may play an important role in the pathogenesis and progression of cardiovascular and renal complications of diabetes. Four putative AGE receptors (RAGEs), AGE-R1, AGE-R2, and AGE-R3 have been described. In this study, we scanned the sequence of the genes encoding these AGE receptors in 48 patients with type 1 diabetes and investigated the identified polymorphisms (n = 19) in 199 type 1 diabetic patients with nephropathy and 193 type 1 diabetic patients without nephropathy. Overall, none of the polymorphisms was strongly associated with nephropathy. The minor allele of a polymorphism located in the promoter region of the RAGE gene (C-1152A) conferred a weak protective effect (P < 0.05) and was associated with a longer duration of nephropathy-free diabetes (P = 0.08).

Angiotensin I-converting enzyme gene polymorphism modulates the consequences of in utero growth retardation on plasma insulin in young adults.

In utero growth retardation has been linked to a reduced rate of cell division in the fetal organs that undergo rapid growth and to permanent changes and adaptations (programming) that may affect the physiology in adult life. In particular, in utero growth retardation as reflected by a low birth weight for gestational age has been shown to be associated with a relative insulin resistance in adults. How programming may influence glucose metabolism is not completely understood, and the possible role of genetic factors has not been explored. The angiotensin I-converting enzyme gene insertion/deletion (ACE I/D) polymorphism may predispose to insulin resistance and modulate the expression of several common cardiovascular and renal disorders, especially in people with diabetes. The possible impact of this polymorphism on plasma glucose and insulin levels was investigated in a group of young adults born at term whose length or weight at birth were in the lowest 3% of the sex and gestational age-adjusted distribution (SGA, n = 172) and a group of control individuals born with an appropriate birth weight for gestational age (AGA, n = 207). In this study, we have previously demonstrated an association between SGA and relative insulin resistance, especially in those with shorter gestational age. In the SGA group, fasting plasma glucose and insulin levels were significantly correlated (R = 0.196, P < 0.015), with this association being significant only in ACE II individuals (R = 0.539, P < 0.0009). In the AGA group, fasting plasma glucose and insulin levels were not significantly correlated. Consistent with this observation, the relationship between the ACE polymorphism and the insulin response to a glucose load was significantly heterogeneous between the AGA and SGA groups (P < 0.05); this was due to a tendency for ACE II individuals in the SGA group to exhibit increased 30-min plasma insulin levels (P < 0.05). In the SGA group, there was a significant interaction between gestational age and genotype on the insulin area (P < 0.0004); this index was inversely associated with gestational age in ACE II (P < 0.0005) and ACE ID (P < 0.005) subjects, but not in DD homozygotes (P > 0.05). The ACE D allele may thus attenuate the additive consequences of SGA and relatively short duration of gestation on insulin resistance in young adults.

Relationships between angiotensin I converting enzyme gene polymorphism, plasma levels, and diabetic retinal and renal complications.

Insulin-dependent diabetes mellitus (IDDM), cardiovascular morbidity, and vital prognosis are linked to diabetic nephropathy, which is probably determined by renal hemodynamic abnormalities and by a genetic predisposition. Angiotensin I converting enzyme (ACE) regulates systemic and renal circulations through angiotensin II formation and kinins metabolism. Plasma and cellular ACE levels are genetically determined; an insertion/deletion polymorphism of the ACE gene is strongly associated with ACE levels, subjects homozygote for insertion (genotype II) having the lowest plasma values. We studied the relationship between the ACE gene polymorphism or plasma levels and microcirculatory disorders of IDDM through two independent studies: one involved 57 subjects with or without diabetic retinopathy, and the other compared 62 IDDM subjects with diabetic nephropathy to 62 diabetic control subjects with the same characteristics (including retinopathy severity) but with normal kidney function. The ACE genotype distribution was not different in diabetic subjects with or without retinopathy and in a healthy population. Conversely, an imbalance of ACE genotype distribution, with a low proportion of II subjects, was observed in IDDM subjects with diabetic nephropathy compared with their control subjects (P = 0.006). Plasma ACE levels were mildly elevated in all diabetic groups, independently of retinopathy, but they were higher in subjects with nephropathy than in those without nephropathy (P = 0.0022). The II genotype of ACE gene is a marker for reduced risk for diabetic nephropathy.

Lack of relationship between an insertion/deletion polymorphism in the angiotensin I-converting enzyme gene and diabetic nephropathy and proliferative retinopathy in IDDM patients.

Genotypic abnormalities of the renin-angiotensin system have been suggested as a risk factor for the development of diabetic nephropathy and proliferative retinopathy. We studied the relationship between an insertion(I)/deletion (D) polymorphism in the angiotensin-converting enzyme (ACE) gene in insulin-dependent diabetes mellitus (IDDM) patients with diabetic nephropathy (121 men and 77 women, age 40.9 +/- 10 years, diabetes duration 27 +/- 8 years) and in IDDM patients with normoalbuminuria (118 men and 74 women, age 42.7 +/- 10 years, diabetes duration 26 +/- 8 years). A total of 155 patients (40%) had proliferative retinopathy, and 67 patients (17%) had no diabetic retinopathy. There was no difference in genotype distribution between IDDM patients with diabetic nephropathy and those with normalbuminuria: 63 (32%)/95 (48%)/40 (20%) vs. 67 (35%)/77 (41%)/46 (24%) had DD/ID/II genotypes, respectively. Patients with nephropathy had higher plasma ACE levels (609 [151-1,504] micrograms/l) compared with patients with normoalbuminuria (428 [55-1,630] micrograms/l) (P < 0.001). Multiple linear regression analysis revealed that the plasma ACE level in patients with nephropathy is partially determined by ACE/ID polymorphism, mean arterial blood pressure, and glomerular filtration rate (r2 = 0.30, P < 0.001). There was no difference in genotype distribution between IDDM patients with proliferative retinopathy and those without diabetic retinopathy: 52 (34%)/74 (48%)/29 (19%) vs. 26 (39%)/25 (37%)/16 (24%) had DD/ID/II genotypes, respectively. There was also no difference in plasma ACE concentration detected among patients with no, simplex, or proliferative retinopathy.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensinogen gene polymorphisms in IDDM patients with diabetic nephropathy.

Genotypic abnormalities of the renin-ANG system have been suggested as a risk factor for the development of diabetic nephropathy. Cleavage of angiotensinogen is the rate-limiting step in the activation of the renin-ANG system. The TT genotype of a polymorphism encoding threonine instead of methionine (M235T) has been associated not only with increased plasma angiotensinogen concentration but also with essential hypertension. In addition, a polymorphism in the angiotensinogen gene substituting methionine for threonine (T174M) has been associated with hypertension in nondiabetic populations. We studied the relationship between these polymorphisms in the angiotensinogen gene in IDDM patients with diabetic nephropathy (121 men, 74 women, age 40.9 +/- 10 years, diabetes duration 27 +/- 8 years). There was no difference in M235T genotype distribution between IDDM patients with diabetic nephropathy and those with normoalbuminuria: 73/97/25 (37/50/13%) vs. 67/95/23 (36/52/12%) had MM/MT/TT genotypes, respectively. No difference in distribution of T174M genotypes between nephropathic and normoalbuminuric IDDM patients was observed either: 148/44/1 (77/23/0.5%) vs. 141/42/2 (76/23/1%) had TT/TM/MM genotypes, respectively. In patients with nephropathy, systolic blood pressure was higher (161 +/- 22 mmHg [mean +/- SD]) in patients carrying TT genotype of the M235T angiotensinogen polymorphism as compared with patients with MM or MT genotypes (150 +/- 23 mmHg; P = 0.03). We conclude that neither the M235T nor the T174M polymorphism in the angiotensinogen gene contributes to genetic susceptibility to diabetic nephropathy in white IDDM patients, whereas the TT genotype of the M235T is associated with elevated blood pressure in patients with diabetic nephropathy.

Lack of association between polymorphisms of eight candidate genes and idiopathic dilated cardiomyopathy: the CARDIGENE study.

OBJECTIVES: The study investigated the potential role of eight candidate genes in the susceptibility to idiopathic dilated cardiomyopathy (IDC). BACKGROUND: Idiopathic dilated cardiomyopathy has a familial origin in 20% to 25% of cases, and several genetic loci have been identified in rare monogenic forms of the disease. These findings led to the hypothesis that genetic factors might also be involved in sporadic forms of the disease. In complex diseases that do not exhibit a clear pattern of familial aggregation, the candidate gene approach is a strategy widely used to identify susceptibility genes. All genes coding for proteins involved in biochemical or physiological abnormalities of cardiac function are potential candidates for IDC. METHODS: We studied 433 patients with IDC and 401 gender- and age-matched controls. Polymorphisms investigated were the I/D polymorphism of the angiotensin I-converting enzyme (ACE) gene, the T174M and M235T polymorphisms of the angiotensinogen (AGT) gene, the A-153G and A+39C polymorphisms of the angiotensin-II type 1 receptor (AGTR1) gene, the T-344C polymorphism of the aldosterone synthase (CYP11B2) gene, the G-308A polymorphism of the tumor necrosis factor-alpha (TNF) gene, the R25P polymorphism of the transforming growth factor beta1 (TGFB1) gene, the G+11/in23T polymorphism of the endothelial nitric oxide synthase (NOS3) gene and the C-1563T polymorphism of the brain natriuretic peptide (BNP) gene. RESULTS: None of the polymorphisms were significantly associated with the risk or the severity of the disease. CONCLUSIONS: We did not find evidence for an involvement of any of the 10 investigated polymorphisms in the susceptibility to IDC.

TAFI gene haplotypes, TAFI plasma levels and future risk of coronary heart disease: the PRIME Study.

OBJECTIVES: To evaluate the association of thrombin-activatable fibrinolysis inhibitor (TAFI) gene polymorphisms with the risk of coronary heart disease (CHD) and with TAFI levels measured by a newly developed enzyme-linked immunosorbent assay (ELISA) (TAFI-1B1), shown to be a reliable method for detecting quantitative variations in circulating TAFI. PATIENTS/METHODS: Six polymorphisms (C-2599G, G-438A, Ala147Thr, Thr325Ile, C + 1542G and T + 1583A) were genotyped and baseline plasma concentrations of TAFI were measured in a nested case-control design as part of the Prospective Epidemiological Study of Myocardial Infarction (PRIME) Study. Participants from France and Northern Ireland who had developed a CHD event during a 5-year follow-up (n = 321) were compared with 645 population- and age-matched control subjects. RESULTS: In France, the Thr147 allele was more frequent in cases than in controls (0.41 vs. 0.32; P = 0.02), whereas the reverse was observed in Northern Ireland (0.33 vs. 0.38; P = 0.19) (P = 0.01 for interaction). No other polymorphism was associated with CHD risk. Consistent with the results derived from the single-locus analysis, haplotype analysis revealed that the haplotype carrying the Thr147 allele was associated with increased risk of CHD in France while the reverse tended to hold in the Northern Ireland population. Single-locus and haplotype analyses revealed that two polymorphisms, C-2599G and Ala147Thr (or T + 1583A that is in nearly complete association with it), had additive effects on TAFI levels and explained >18% of TAFI variability. This effect was homogeneous in France and Northern Ireland, and in cases and controls who exhibited similar TAFI levels. CONCLUSIONS: Thrombin-activatable fibrinolysis inhibitor gene polymorphisms are strongly associated to plasma TAFI levels, but the relation to CHD risk is less clear.

Association between P-selectin gene polymorphisms and soluble P-selectin levels and their relation to coronary artery disease.

P-selectin is a cellular adhesion molecule that mediates the interaction of activated endothelial cells or platelets with leukocytes. Increased levels of soluble P-selectin have been reported in various cardiovascular disorders. We measured serum soluble P-selectin levels as well as 3 polymorphisms of the P-selectin gene (C-2123G, A-1969G, and Thr715Pro) in a large cohort of patients with documented coronary artery disease (n=869) and a healthy control group (n=334). The 3 P-selectin polymorphisms were strongly associated with P-selectin levels and altogether explained 7.3% and 18.6% of the P-selectin variability in patients and controls, respectively. Genotype distributions did not significantly differ between patients and controls. P-selectin levels were increased in patients younger than 55 years of age compared with controls (135.2 vs 114.3 ng/mL, P<0.01). On the contrary, patients older than 65 years of age had significantly lower P-selectin levels than did controls (121.5 vs 134.7 ng/mL, P<0.02). In intermediate age groups, P-selectin levels did not significantly differ between the 2 groups. In conclusion, this study revealed a strong association between P-selectin gene polymorphisms and serum P-selectin levels and a complex age-dependent relation between soluble P-selectin levels and coronary artery disease, which suggests that this molecule might have different roles in the atherothrombotic process.

Characterization of polymorphic structure of cathepsin G gene: role in cardiovascular and cerebrovascular diseases.

Cathepsin G (CTSG), a serine protease released from activated neutrophils, may cause platelet activation, leading to intravascular thrombosis, thus contributing to cardiovascular and cerebrovascular disease. Applying the candidate gene approach, we screened the 5'-flanking region and the entire coding region of the CTSG gene for genetic variation by using polymerase chain reaction/single-strand conformation polymorphism analysis from 96 patients at high risk for myocardial infarction (MI). We identified 4 polymorphisms in the 5'-flanking region (G-618C, G-315A, C-179T, and C-160T) and 1 polymorphism in the coding region (Asn125Ser) of the gene and genotyped the participants in the Etude Cas-Temoins sur l'Infarctus du Myocarde (ECTIM Study), a case-control study for MI, and in the Etude du Profil Génétique de l'Infarctus Cérébral (GENIC Study), a case-control study for brain infarction (BI), for all identified genetic variants. The potential in vitro functionality of the 4 variants in the 5'-flanking region was investigated with transient transfection analyses in U937 cells with different allelic promoter constructs by using a luciferase assay. Our in vitro analyses did not reveal any differences for the investigated allelic constructs with respect to promoter activity, and none of the polymorphisms in the 5'-flanking region was associated with the available phenotypes in either study. Allele and genotype distributions of all identified polymorphisms did not globally differ between cases and controls in the ECTIM Study. However, in patients from the ECTIM Study, the Ser125 allele was significantly associated with elevated plasma fibrinogen levels (P=0.006), but this effect was not seen in controls (case-control heterogeneity, P=0.04). There was a significant interaction between CTSG Asn125Ser and the beta-fibrinogen gene polymorphism G-455A on plasma fibrinogen levels (P=0.04). In the GENIC Study, the odds ratio for BI associated with CTSG Ser125 carrying was 1.82 (95% CI 1.16 to 2.84, P=0.008) in patients without a history of cardiovascular or cerebrovascular diseases. Our results indicate that the CTSG Ser125 allele is associated with plasma fibrinogen levels in MI patients from the ECTIM Study and with BI in the GENIC Study. Further studies should be carried out to define the underlying mechanisms.

Polymorphisms of the human matrix gla protein (MGP) gene, vascular calcification, and myocardial infarction.

The matrix Gla protein (MGP) is an important inhibitor of vessel and cartilage calcification that is strongly expressed in human calcified, atherosclerotic plaques and could modulate plaque calcification and coronary heart disease risk. Using a genetic approach, we explored this possibility by identifying polymorphisms of the MGP gene and testing their possible association with myocardial infarction (MI) and plaque calcification. Eight polymorphisms were identified in the coding and 5'-flanking sequences of the MGP gene. All polymorphisms were investigated in 607 patients with MI and 667 control subjects recruited into the ECTIM Study (Etude Cas-Témoins de l'Infarctus du Myocarde) and in 717 healthy individuals with echographically assessed arterial calcification and atherosclerosis who were participating in the AXA Study. In the ECTIM Study, alleles and genotypes were distributed similarly in patients and controls in the whole study group; in only 1 subgroup of subjects defined as being at low risk for MI were the concordant A-7 and Ala 83 alleles more frequent in patients with MI than in controls (P<0.003). In the AXA Study among subjects with femoral atherosclerosis, the same alleles were more common in the presence than the absence of plaque calcification (P<0.025). The other MGP polymorphisms were not associated with any investigated clinical phenotype. Transient transfection experiments with allelic promoter-reporter gene constructs and DNA-protein interaction assays were carried out to assess possible in vitro functionality of the promoter variants detected at positions -814, -138, and -7 relative to the start of transcription. When compared with the -138 T allele, the minor -138 C: allele consistently conferred a reduced promoter activity of -20% (P<0.0001) in rat vascular smooth muscle cells and of -50% (P<0.004) in a human fibroblast cell line, whereas the other polymorphisms, including -7, displayed no evidence of in vitro functionality. We conclude that the A-7 or Ala 83 alleles of the MGP gene may confer an increased risk of plaque calcification and MI; however, the observed relationships are weak or limited to subgroups of patients and therefore need confirmation.

Use of degenerate oligonucleotide primed PCR (DOP-PCR) for the genotyping of low-concentration DNA samples.

Degenerate oligonucleotide primed amplification (DOP-PCR) is an efficient method for performing whole genome amplification. We analysed the yield of DNA using this technique starting with various quantities of material. We used DOP-PCR products to genotype single nucleotide polymorphisms and insertion/deletion polymorphisms. DOP-PCR also proved usable for SSCP analysis.

The Pro12Ala polymorphism in the peroxisome proliferator-activated receptor gamma2 gene is not associated with postprandial responses to glucose or fat tolerance tests in young healthy subjects: the European Atherosclerosis Research Study II.

This study investigated whether the Pro12Ala polymorphism of the peroxisome proliferator-activated receptor gamma2 (PPARgamma2) gene is associated with glucose and lipid metabolism in young healthy subjects participating in the European Atherosclerosis Research Study II. Men aged 18-28 years (n=675) were recruited from 14 university student populations in 11 European countries. At their first visit subjects had an oral glucose tolerance test and 1 week later an oral fat tolerance test. Lipid variables and genotype were measured centrally. The Ala allele frequency exhibited a clearcut north-to-south gradient through Europe, decreasing from 0.21 in Baltic countries to 0.07 in Mediterranean countries. There was no significant effect of the Pro12Ala polymorphism on fasting lipid, glucose, or insulin levels, nor on the postprandial changes in these variables after glucose and fat tolerance tests. Neither was the Pro12Ala polymorphism associated with body mass index. This study provides no evidence for a major effect of the Pro12Ala polymorphism on glucose and lipid metabolism in young healthy subjects. Since PPARgamma has a major role in adipogenesis, the differential effect of its polymorphism on weight and related metabolic disorders may become apparent only later in life.

Interleukin-6 gene polymorphisms and susceptibility to myocardial infarction: the ECTIM study. Etude Cas-Témoin de l'Infarctus du Myocarde.

There is growing evidence that interleukin (IL) 6 plays an important role in the atherosclerotic process because of its role in mediating immune and inflammatory responses and inducing cell proliferation. The present study examined whether molecular variations at the IL-6 locus are involved in the predisposition to myocardial infarction. The entire coding region, 1,158 bp of the 5' flanking region and 237 bp of the 3' flanking region of the IL-6 gene were screened. We detected three nucleotide substitutions in the 5' region at positions -174 (G/C), -572 (G/C), and -596 (G/A) from the transcription start site, and one insertion/deletion in the 3' region at position +528 after the Stop codon. These polymorphisms were genotyped in the Etude Cas-Témoin de l'Infarctus du Myocarde study comparing male patients (n=640) and age-matched controls (n=719) from Northern Ireland and France. The IL-6/G-174C and IL-6/G-596A polymorphisms were in nearly complete association. Carriers of the IL-6/-174 C allele were more frequent in patients than in controls. The population-adjusted odds ratio for myocardial infarction associated with genotype CC+CG vs. GG was estimated as 1.34. In French patients the number of coronary arteries with greater than 50% stenosis was assessed by angiography. The IL-6/-174 C allele was more frequent in patients with two or fewer stenosed vessels than in patients with three-vessel lesions. These results suggest that genetic variation at the IL-6 locus is associated with susceptibility to myocardial infarction, especially events occurring on less extended lesions. These findings would be compatible with a lower IL-6 secretion associated with the IL-6/-174 C allele, itself or in combination with other promoter polymorphisms, leading to more unstable plaques.