Biologia molecolare della paraoxonasi

Human serum paraoxonase (PON1) is an HDL-associated enzyme involved in the protection of lipoproteins from oxidation. Endothelial dysfunction has been documented in subjects with diabetes mellitus and is probably related to increased oxidation of circulating LDL. A genetic polymorphism of the PON1 (Gln192Arg) has been suggested to influence its anti-oxidant capacity, with the Arg allele associated with lesser protection of LDL against the accumulation of lipid peroxides. The first step of the present study was to test a fluorescent 5’ exonuclease assay to detect the point mutation in the paraoxonase gene (Q192R). This assay takes advantage of 5’ exonuclease activity of Taq DNA polymerase to cause the cleavage of two allelic-specific probes, which hybridized to template DNA during the PCR and that determine an increase of reporter fluorescence in solutions. This assay was applied to 120 sample of genomic DNA, extracted from whole blood, and we had a perfect allelic discriminations between sample wild-type, mutant and heterozygote . This results show the complete concordance of Real Time fluorescent 5’ Exonuclease Assay with a standard Polymerase Chain Reaction and restriction analysis and is an assay easy to use, rapid and accurate for the study of single nucleotide polymorphisms that permits to analyse a great number of samples in a short period of time. The second step was to evaluate, in vivo, the contribution of the PON1-192 polymorphism to the protective effect of HDLs. Three hundred and forty seven subjects in the upper and lower decile of sex-specific HDL cholesterol distribution were selected from participants in a cardiovascular disease prevention study. PON1 genotypes were determined by PCR amplification and restriction analysis and, at the same time, by Real Time PCR. Blood pressure, height, weight, smoking and alcohol habits, as well as the presence of familial or personal history of CHD were recorded. Plasma lipids and blood glucose were measured by routine enzymatic methods. As a measure of antiatherogenic HDL effect, carotid atherosclerosis was assessed by ultrasonography. Allele and genotype frequencies did not differ significantly between low and high HDL groups. Similarly, no significant difference was observed among genotypes in all variables studied. Subjects with Gln/Arg or Arg/Arg had more carotid abnormalities than Gln/Gln with an adjusted odds ratio (OR) of 3.27 (95% CI, 1.61-6.64, P=0.001) for abnormal carotid score. Stepwise logistic regression analysis showed that in the whole population age and presence of low-HDL were the only independent predictors for abnormal carotid score. In low-HDL, age was the only independent predictor entered into the model (OR 1.09/year, P<0.0001); in high-HDL, age entered first (OR, 1.07/year, P=0.001), followed by the presence of Gln/Arg or Arg/Arg (OR, 2.94, 95% CI, 1.47-5.91, P=0.002). In conclusion, in subjects with elevated concentration of HDL cholesterol, the presence of carotid atherosclerosis is significantly associated with the arginine variant in position 192 of thePON1 gene. Since there is a relative dearth of data on the association between the genetic polymorphism of PON1 and endothelial function in subjects with diabetes mellitus, we have also investigate the possible association between the PON1 Gln192Arg polymorphism and the brachial artery endothelial function in subjects with diabetes mellitus. Sixty-nine patients with type2 diabetes and 41 sex- and age-matched controls have been enrolled. The clinical and biochemical characteristics and Gln/Arg polymorphism were determined and endothelial function has been evaluated as flow mediated vasodilation (FMD) of the brachial artery after forearm ischemia. In controls no difference was observed in FMD between subjects homozygous for the Gln allele and those carrying the Arg allele. In diabetic patients, FMD was lower among those carrying the Arg allele (1.90±1.65% vs. 3.69±2.40%, p=0.02). This difference became more marked after exclusion of subjects with hypertension (2.01±1.27% vs. 5.72±3.24%, p=0.01). In multiple regression analysis, hypertension and PON1 gene polymorphism were independently and significantly associated with FMD. In conclusion Gln>Arg polymorphism of PON1 influences endothelial dysfunction in diabetic subjects, especially in those with normal blood pressure.