Stefano Turchi

Development of a new multiplex quantitative real‐time PCR assay for the detection of the mtDNA4977 deletion in coronary artery disease patients: A link with telomere shortening

Mitochondrial DNA (mtDNA) and telomere shortening have been proposed as important contributors to vascular disease and atherogenesis. The role of mitochondrial and telomere alterations has been examined frequently, but usually separately. Recently, an integrated model in which DNA damage and metabolic pathways intersect in age‐associated cardiovascular disease has been proposed. In this study we developed a fast and reliable real‐time PCR‐based procedure to investigate relative quantification of the 4,977 bp mitochondrial DNA deletion (also indicated as “mtDNA4977 deletion”), employing TaqMan probes with a multiplex approach. As a validation of the assay, a nested PCR coamplification was performed. Telomere shortening was evaluated by a real‐time monochrome multiplex PCR technique employing a SybrGreen‐based analysis. The study of mtDNA4977 deletion and telomere shortening was carried out in atrial biopsies from 11 patients undergoing coronary artery (n = 5) and valve surgery (n = 6). The relative quantifications showed that the amount of mtDNA4977 deletion was greater in tissue of patients with coronary artery disease (CAD) (P = 0.01) and that telomere length (expressed as telomere length relative to a single copy reference gene) was significantly shorter in tissue of CAD patients, compared to patients without CAD (P = 0.03). Moreover, most conventional risk factors were significantly more frequent in CAD patients, smoking and dyslipidemia having the strongest association with the degree of mtDNA4977deletion and a significant correlation with telomere attrition (P = 0.02 and P = 0.006, respectively). In conclusion, the present study suggests that mtDNA4977 deletion and telomere shortening may represent additional and synergic major risk factors for the pathogenesis of CAD and its complications. Environ. Mol. Mutagen. 54:299–307, 2013.

Increased circulating cell-free DNA levels and mtDNA fragments in interventional cardiologists occupationally exposed to low levels of ionizing radiation

Circulating cell‐free DNA (ccf‐DNA) and mtDNA (ccf‐mtDNA) have often been used as indicators of cell death and tissue damage in acute and chronic disorders, but little is known about changes in ccf‐DNA and ccf‐mtDNA concentrations following radiation exposure. The aim of the study was to investigate the impact of chronic low‐dose radiation exposure on serum ccf‐DNA levels and ccf‐mtDNA fragments (mtDNA‐79 and mtDNA‐230) of interventional cardiologists working in high‐volume cardiac catheterization laboratory to assess their possible role as useful radiation biomarkers. We enrolled 50 interventional cardiologists (26 males; age = 48.4 ± 10 years) and 50 age‐ and gender‐matched unexposed controls (27 males; age = 47.6 ± 8.3 years). Quant‐iT™ dsDNA High‐Sensitivity assay was used to measure circulating ccf‐DNA isolated from serum samples. Quantitative analysis of mtDNA fragments was performed by real‐time PCR. No significant relationships were found between ccf‐DNA and ccf‐mtDNA, and age, gender, smoking, or other clinical parameters. Ccf‐DNA levels (44.2 ± 31.1 vs. 30.6 ± 19.2 ng/ml, P = 0.013), ccf‐mtDNA‐79 (2.6 ± 2.1 vs. 1.1 ± 0.8, P < 0.01), and ccf‐mtDNA‐230 copies (2.0 ± 1.8 vs. 1.04 ± 0.9, P = 0.02) were significantly higher in interventional cardiologists compared with the non‐exposed group. In a subset (n = 15) of interventional cardiologists with a reliable reconstruction of cumulative professional exposure (59.7 ± 48.4 mSv; range: 1.4–182 mS), ccf‐DNA (53.2 ± 41.3 vs. 36.4 ± 22.9 and 32.2 ± 20.5, P = 0.08), mtDNA‐79 (2.4 ± 2.1 vs. 2.03 ± 1.7 and 1.09 ± 0.82, P = 0.05), and mtDNA‐230 (2.0 ± 2.2 vs. 1.5 ± 1.4 and 1.04 ± 0.9, P = 0.09) tended to be significantly increased in high‐exposure subjects compared with both low‐exposure interventional cardiologists and controls. Our results provide evidence for a possible role of circulating DNA as a relevant biomarker of cellular damage induced by exposure to chronic low‐dose radiation. Environ. Mol. Mutagen. 56:293–300, 2015.

Stromal cell-derived factor-1-3'A polymorphism is associated with decreased risk of myocardial infarction and early endothelial disturbance.

Aim Genome-wide association studies have identified single-nucleotide polymorphisms at the 10q11 locus as risk factors for myocardial infarction (MI). This locus lies upstream (∼80 kb) of the stromal cell-derived factor-1 (SDF1) gene that codify for a chemokine with protective atherogenetic effects and with a major role in the mobilization, homing, and differentiation of endothelial progenitor cells (EPCs). The purpose of this study was to investigate the possible association of SDF1–3′A polymorphism, that upregulates SDF1 protein expression, with MI and early endothelial dysfunction and atherosclerosis in young healthy subjects. Methods 200 patients (181 men age 57.3 ± 7.7 years) and 230 healthy controls (96 men, age 52 ± 11.9 years) were recruited to investigate the association between MI and SDF1–3′A polymorphism. The relationship between SDF1–3′A polymorphism and brachial artery flow-mediated dilation and the number of circulating EPCs was examined in 50 healthy young adults. Results A significant difference in SDF1–3′A genotype distribution was observed between patients and controls (P = 0.006). Patients carrying the A allele had a significantly reduced MI risk compared with subjects with GG genotype (odds ratio = 0.5, 95% CI = 0.3–0.9, P = 0.001). SDF1–3′A polymorphism presented a significant interaction with other cardiovascular risk factors (Pinteraction < 0. 0001). Controls carrying the A allele showed significantly higher flow-mediated dilation (13.9 ± 4.9 vs 10.8 ± 4.3, P = 0.03) and significantly higher values of EPCs (0.029 ± 0.009 vs 0.022 ± 0.008, P = 0.02) compared with GG homozygotes. Conclusion SDF1–3′A polymorphism is associated with a decreased risk of MI and early endothelial dysfunction, strongly confirming the important atherogenic role of SDF1 gene at clinical level.

Effects of Highly Polluted Environment on Sperm Telomere Length: A Pilot Study

High environmental pressure may impair male fertility by affecting sperm quality, but the real effect remains controversial. Herein, we assessed the influence of environmental exposure on telomere length (TL) in both leukocytes (LTL) and sperm cells (STL). A pilot biomonitoring study was conducted in 112 clinically healthy, normospermic men living in various areas of Campania region (South of Italy) with high (n = 57, High Group) or low (n = 55, Low Group) environmental pressure. TL analysis was assessed by quantitative real time-PCR. STL was not significantly correlated with either age (p = 0.6) or LTL (p = 0.7), but was significantly longer in the High Group compared with the Low Group (p = 0.04). No significant difference was observed between leukocyte TL in the High or Low Group. Our results showed that male residents in areas with high environment exposure had a significant increase in STL. This finding supports the view that the human semen is a sentinel biomarker of environmental exposure.

Prognostic value of mitochondrial DNA4977 deletion and mitochondrial DNA copy number in patients with stable coronary artery disease

BACKGROUND AND AIMS Mitochondrial DNA copy number (mtDNA-CN) depletion has been recently associated with an increased cardiovascular risk. However, the integrity of mtDNA is another key aspect of the energy metabolism and mitochondrial function. We investigated the prognostic role of peripheral blood common mitochondrial deletion (mtDNA4977) and mtDNA-CN on long-term major adverse cardiac events (MACEs) and all-cause mortality in a cohort of patients with coronary artery disease (CAD). METHODS Within the Italian GENOCOR (Genetic Mapping for Assessment of Cardiovascular Risk) cohort, we studied 515 patients (450 males, 65 ± 8 years) with known or suspected stable CAD. mtDNA4977 deletion and mtDNA-CN were assessed in peripheral blood using qRT-PCR. RESULTS During a mean follow-up of 4.5 ± 1.1 years, 78 (15%) patients had MACEs (15 cardiac deaths, 17 nonfatal myocardial infarction and 46 coronary revascularizations) and 28 patients died for non-cardiac causes. Patients with high levels of mtDNA4977 deletion (>75th) had increased risk of MACEs (log rank = 7.2, p=0.007) and all-cause mortality (log rank = 5.7, p=0.01) compared with patients with low mtDNA4977 deletion (≤75th). Multivariate Cox regression analysis showed that log mtDNA4977 was a significant predictor of MACEs (HR = 2.17; 95% CI, 1.31-3.59; p=0.003) and all-cause mortality (HR = 2.03; 95% CI: 1.13-3.65, p=0.02). Log mtDNA-CN was not significantly associated with MACEs or all-cause mortality. However, patients with high mtDNA4977 deletion (>75th) and low mtDNA-CN (<25th) had significantly increased risk for MACEs (HR: 3.73; 95% CI: 1.79-7.79; p=0.0005). CONCLUSIONS Mitochondria DNA damage was associated with an increased risk of MACEs and all-cause mortality in patients with stable CAD, confirming the critical role of mitochondrial dysfunction in atherosclerosis.