Genetic polymorphisms in folate and homocysteine metabolism as risk factors for DNA damage.

Epidemiological studies indicated a role for polymorphisms in genes of folate and homocysteine (Hcy) metabolism in the etiology of neurodegenerative disease, congenital defects and coronary artery disease (CAD). This study investigated the effect of several polymorphisms [C677 T, A1298C of methylenetetrahydrofolate reductase (MTHFR) and A66G of methionine synthase reductase (MTRR) genes] on Hcy levels and DNA damage in 68 patients who underwent coronary angiography. Plasma Hcy concentrations were higher in patients with multivessel disease with respect to monovessel disease and no-CAD patients (19.4+/-2.6 vs 11.6+/-1.2 and 13.7+/-1.4 micromol/l, respectively; P=0.03). 677TT patients had higher Hcy levels than those with 677CC or 677CT genotypes (26.2+/-4.3 vs 13.1+/-1.4 and 13.0+/-1.4 micromol/l, respectively; P=0.0006). No significant associations were found between A1298C and A66G polymorphisms and plasma Hcy levels. Among patients with 677CC genotype, 66GG individuals tended to have higher levels of Hcy than 66AA homozygotes (14.5+/-1.9 vs 8.9+/-0.7 micromol/l, P=0.06). Multivessel disease patients showed an increased frequency of DNA damage, measured by the micronucleus (MN) frequency, as compared to monovessel disease and no-CAD subjects (12.5+/-1.1 vs 8.5+/-0.8 and 8.2+/-0.9, respectively; P=0.006). The MN were positively correlated with Hcy levels (r=0.33, P=0.006) and were significantly higher in subjects with the 677TT genotype compared with the 677CC or 677CT genotypes (14.4+/-2.0 vs 8.8+/-1.2 and 9.5+/-0.7, respectively; P=0.006). A1298C and A66G polymorphisms had no effect on MN frequency. However, among 677TT patients, 66GG subjects tended to have higher levels of MN than those 66AG and 66AA (18.2+/-3.6 vs 13.8+/-4.0 and 10.3+/-1.7, respectively; P=NS). Our results indicate that genetic instability may be associated with increased risk for multiple Hcy-related diseases.

Interactive effect of the glutathione S-transferase genes and cigarette smoking on occurrence and severity of coronary artery risk.

Cardiovascular diseases and cancer are the main causes of death in developed countries. Mortality trends for these diseases suggest that they share common pathogenetic mechanisms. Glutathione S-transferase (GST) is a family of enzymes that detoxify reactive electrophiles, particularly present in tobacco smoke. Glutathione S-transferase null M1 and T1 (GSTM1 and GSTT1) genotypes have often been associated with increased risk of developing cancer. Our hypothesis was that the polymorphic GSTM1 and GSTT1 genes modulate the risk of smoking-coronary artery disease (CAD). We evaluated the distribution of GST genotypes in 430 angiographically defined patients (308 CAD and 122 non-CAD). The frequencies of GST null genotypes did not differ significantly between patients with CAD and without CAD. However, smokers with GSTM1 and GSTT1 null genotypes had a significantly higher risk of CAD than never-smokers with these genotypes present (OR 2.2 and 3.4 for smokers with null GSTM1 and GSTT1 genes, respectively). There was also evidence of multiple interaction between GSTM1 and GSTT1 deleted genotypes and smoking. In nonsmokers carrying both null genotypes the risk of CAD was 0.66. In smokers with both present genotypes the OR was 1.5 and was significantly increased in smokers with concurrent lack for GSTM1 and GSTT1 genes (OR=4.0). Moreover, smokers lacking GST genes had both more stenosed vessels and a higher Duke score than smokers expressing the genes. We also examined the levels of DNA damage in 66 men patients using the micronucleus test, a sensitive assay for evaluating chromosome damage. Micronucleus levels were higher in smokers with null genes than in smokers with present genes. These observations suggest that GST-null genotypes strengthen the effect of smoking on CAD risk by modulating the detoxification of genotoxic atherogens.

Endothelial nitric oxide synthase gene polymorphisms and risk of coronary artery disease.

BACKGROUND: Endothelial nitric oxide synthase (eNOS) could be a candidate gene for coronary artery disease (CAD). This study investigated the relationship of the eNOS Glu(298)-->Asp and T(786)-->C polymorphisms with the presence and severity of CAD in the Italian population. METHODS: We enrolled 415 unrelated individuals who underwent coronary angiography. The severity of CAD was expressed by means of the Duke score. The eNOS Glu(298)-->Asp and T(786)-->C variants were analyzed by PCR. RESULTS: There was significant linkage disequilibrium between the two eNOS polymorphisms (P <0.0001). Both variants were significantly associated with the occurrence and severity of CAD (P = 0.01 and 0.004 for Glu(298)-->Asp and T(786)-->C, respectively). The risk of CAD was increased among individuals homozygous for the C allele of the T(786)-->C polymorphism compared with individuals homozygous for the T allele (odds ratio = 2.5; P <0.01) and was independent of the other common risk factors (P = 0.04). Moreover, individuals with both the Asp/Asp genotype of the Glu(298)-->Asp polymorphism and at least one C allele of the T(786)-->C variant in the promoter region of the eNOS gene had an increased risk of CAD (odds ratio = 4.0; P <0.001) and a significantly higher mean Duke score (26.2 +/- 2.9 vs 45.2 +/- 3.7; P = 0.002) compared with individuals with the TT genotype and the Glu allele. CONCLUSIONS: The present study provides evidence that the Glu(298)-->Asp and T(786)-->C polymorphisms of the eNOS gene are associated with the presence and severity of angiographically defined CAD in the Italian population and that those individuals carrying both eNOS variants simultaneously might have a higher risk of developing CAD.

Methylenetetrahydrofolate reductase gene C677T polymorphism, homocysteine, vitamin B12, and DNA damage in coronary artery disease.

Elevated levels of plasma homocysteine (Hcy), a risk factor for coronary artery disease (CAD), can result from genetic errors, e.g., the methylenetetrahydrofolate reductase (MTHFR) polymorphism, or nutritional deficiencies, e.g., in vitamin B12 and folate. The mechanism by which Hcy induces atherosclerosis is not fully understood. Recently, Hcy has also been observed to induce DNA damage. In this study, we have investigated whether DNA damage is related to the C677T variant in the MTHFR gene and to plasma levels of Hcy, B12, and folate in patients with CAD. Patients ( n=46) with angiographically proven CAD were studied by using the micronucleus (MN) test, an accepted method for evaluating genetic instability. TT patients had plasma Hcy levels higher than those with the CT or CC genotypes (27.8+/-5.2 vs 13.7+/-2.2 and 12.9+/-1.9 micro mol/l, respectively; P=0.02). Patients with multi-vessel disease had higher plasma Hcy levels (11.6+/-1.2, 22.0+/-4.7, 19.3+/-3.9 micromol/l for one-, two- and three-vessel disease, respectively; P=0.05). The MN index increased with the number of affected vessels (8.4+/-0.7, 11.1+/-2.0, 14.2+/-1.7 for one-, two-, and three-vessels disease, respectively; P=0.02) and was significantly higher in subjects with the TT genotype compared with the CC or CT genotypes (15.7+/-2.4 vs 8.9+/-1.7 and 9.9+/-0.8; P=0.02). The MN index was also correlated negatively with plasma B12 concentration ( r=-0.343; P=0.019) and positively with plasma Hcy ( r=0.429, P=0.005). These data indicate that the MN index is associated with the severity of CAD and is related to the MTHFR polymorphism, suggesting an interesting link between coronary atherosclerosis and genetic instability in humans.

Elevated levels of oxidative DNA damage in patients with coronary artery disease.

BACKGROUND: Somatic DNA damage has been suggested to contribute to the pathogenesis of atherosclerosis. However, little is known about the role of oxidative DNA damage in patients with coronary artery disease (CAD). METHODS: In this study, we used the comet assay to measure oxidative DNA damage (DNA strand breaks and enzyme-sensitive sites) in peripheral blood lymphocytes from 13 patients with angiographically documented CAD and 11 age- and sex-matched control participants. RESULTS: Mean values of DNA strand breaks, oxidized pyrimidines and altered purines were significantly higher in CAD patients than in the control group (11.9 +/- 1.4, 18.0 +/- 2.7 and 18.1 +/- 3.1 compared with 3.3 +/- 0.2, 2.7 +/- 0.5 and 4.5 +/- 1.1; P < 0.0001, P < 0.0001 and P = 0.0009, respectively). Moreover, oxidized purines (for example, 8-oxo-guanine) increased with the number of affected vessels and positively correlated with the extent of CAD measured by means of the number of the coronary lesions (P = 0.76, P = 0.003) and the Duke scoring system (P = 0.66, P = 0.01). Diabetic patients showed higher levels of oxidized pyrimidines (31.3 +/- 5.5 compared with 14.1 +/- 2.7; P = 0.013), while patients with dyslipidemia had elevated altered purines compared with normal patients (20.4 +/- 2.6 compared with 4.9 +/- 3.1; P = 0.03). CONCLUSIONS: These data indicate an overall elevation of oxidative DNA damage in CAD patients correlated with the severity of the disease and some atherogenic risk factors, suggesting a possible role of oxidative genetic damage in the pathogenesis of atherosclerosis.

Deoxyribonucleic acid damage in human lymphocytes after percutaneous transluminal coronary angioplasty.

UNLABELLED: OBJECTIVES; We investigated the presence of oxidative deoxyribonucleic acid (DNA) damage in the peripheral lymphocytes of patients undergoing percutaneous transluminal coronary angioplasty (PTCA) by using the micronucleus test and comet assay, which are sensitive biomarkers of DNA damage. BACKGROUND; Although it has recognized that ischemia-reperfusion can induce oxidative DNA damage, its occurrence in patients undergoing PTCA has not yet been demonstrated. METHODS: Three groups of patients were enrolled: 30 patients with documented coronary heart disease who underwent elective PTCA (group I); 25 patients who underwent elective coronary angiography for diagnostic purpose (group II); and 27 healthy, age- and gender-matched subjects (group III). For each subject, the frequency of micronucleated binucleated (MNBN) cells, DNA single-strand breaks (SSBs), endonuclease III-sensitive sites, and sites sensitive to formamidopyrimidine glycosylase (FPG) were analyzed before and after diagnostic procedures. RESULTS: The mean basal values of MNBN cells (p = 0.04), DNA-SSBs (p = 0.001), endonuclease III-sensitive sites (p = 0.002), and FPG sites (p < 0.0001) were significantly higher in groups I and II than in group III. A high significant increase of MNBN cell frequency was observed in group I after the PTCA procedure (11.0 +/- 1.3 vs. 19.8 +/- 1.6, p < 0.0001), whereas no significant difference was observed in group II (10.2 +/- 1.3 vs. 12.9 +/- 1.4, p = 0.18). A significant positive correlation was observed between the increase in the MNBN cell rate and total inflation time during PTCA (R = 0.549, p = 0.0017). The levels of DNA-SSBs (11.7 +/- 1.4 vs. 26.5 +/- 3.0, p = 0.0003) and FPG sites (13.8 +/- 1.8 vs. 22.5 +/- 2.4, p = 0.01) were also higher after PTCA. CONCLUSIONS: Our results provide evidence for oxidative DNA damage after PTCA, likely related to ischemia-reperfusion injury.

P53 codon 72 polymorphism in coronary artery disease: no evidence for association with increased risk or micronucleus frequency.

A common polymorphism at codon 72 (Arg72Pro) of the p53 gene, a gene which codes for a tumor-suppressor protein with both antiproliferative and pro-apoptotic actions, has recently been reported to be a risk factor for coronary luminal narrowing after angioplasty. However, the association of the polymorphism with coronary artery disease (CAD) risk has not been studied. We evaluated the distribution of the Arg72Pro genotype in 250 patients, 180 with angiographically documented CAD and 70 with normal coronary angiography, by using polymerase chain reaction amplification of patient DNA followed by restriction enzyme digestion. We also examined the association between the Arg72Pro genotype and chromosome damage in 82 male patients (60 CAD and 22 no-CAD) by the micronucleus (MN) test in human lymphocytes, a sensitive assay for chromosome breakage and aneuploidy. The frequencies of Pro/Pro, Pro/Arg, and Arg/Arg genotypes in CAD patients were not significantly different from those who were CAD-free (chi(2) = 0.20, P = 0.90) and not significantly associated with the extent and severity of CAD. A significant increase in MN frequency was observed in relation to smoking status (8.4 +/- 0.6, 11.9 +/- 1 and 12.0 +/- 1.6, for non smokers, ex-smokers and smokers, respectively; P = 0.02). Moreover, diabetic patients showed higher levels of MN than normal patients (13.5 +/- 1.4 vs. 9.6 +/- 0.5, P = 0.0025). Also, MN frequency was significantly higher in CAD patients than in no-CAD patients (11.2 +/- 0.7 vs. 8.0 +/- 0.9, P = 0.02) and increased with the number of affected vessels (9.3 +/- 0.1, 12.2 +/- 1.5 and 12.5 +/- 1.3 for one-, two-, and three-vessel disease, respectively; P = 0.02). However, there were no associations between MN frequency and the Arg72Pro polymorphism. Although there appears to be an association between CAD and MN frequency, our results indicate that the Arg72Pro polymorphism does not have a significant impact on CAD or MN frequencies.