Quantification of mitochondrial DNA damage and copy number in circulating blood of patients with systemic sclerosis by a qPCR-based assay

Abstract Background: Although not fully understood, oxidative stress has been implicated in the pathogenesis of different autoimmune diseases such as systemic sclerosis. Accumulating evidence indicates that oxidative stress can induce mitochondrial DNA (mtDNA) damage and variations in mtDNA copy number (mtDNAcn). Objective: The aim of this study was to explore mtDNAcn and oxidative DNA damage byproducts in peripheral blood of patients with systemic sclerosis and healthy controls. Methods: Forty six patients with systemic sclerosis and forty nine healthy subjects were studied. Quantitative real-time PCR used to measure the relative mtDNAcn and the oxidative damage (oxidized purines) of each sample. Results: The mean mtDNAcn was lower in patients with systemic sclerosis than in healthy controls whereas the degree of mtDNA damage was significantly higher in cases as compared to controls. Moreover, there was a negative correlation between mtDNAcn and oxidative DNA damage. Study limitations: The lack of simultaneous analysis and quantification of DNA oxidative damage markers in serum or urine of patients with systemic sclerosis and healthy controls. Conclusion: These data suggest that alteration in mtDNAcn and increased oxidative DNA damage may be involved in the pathogenesis of systemic sclerosis.

Relationships between blood leukocyte mitochondrial DNA copy number and inflammatory cytokines in knee osteoarthritis / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Osteoarthritis (OA) is a degenerative articular disorder manifested by cartilage destruction, subchondral sclerosis, osteophytes, and synovitis, resulting in chronic joint pain and physical disability in the elderly. The purpose of this study was to investigate mitochondrial DNA copy number (mtDNACN) and inflammatory cytokines in primary knee OA patients and healthy volunteers. A total of 204 knee OA patients and 169 age-matched healthy volunteers were recruited. Their relative blood leukocyte mtDNACN was assessed by quantitative real-time polymerase chain reaction (qRT-PCR), and ten inflammatory cytokines in their plasma were detected by multiplex immunoassay. Blood leukocyte mtDNACN in the OA group was significantly lower than that in the control group. Leukocyte mtDNACN in the control group was negatively correlated with their age (r=-0.380, P<0.0001), whereas mtDNACN in the OA group was positively correlated with their age (r=0.198, P<0.001). Plasma interleukin-4 (IL-4) and IL-6 were significantly higher in the knee OA group than in the control group. The plasma IL-6 level was positively correlated with blood leukocyte mtDNACN in the OA group (r=0.547, P=0.0014). IL-5 showed as a major factor (coefficient 0.69) in the second dimension of principle components analysis (PCA)-transformed data and was significantly higher in the OA group (P<0.001) as well as negatively correlated with mtDNACN (r=-0.577, P<0.001). These findings suggest that elevation of plasma IL-4 and IL-6 and a relative reduction in mtDNACN might be effective biomarkers for knee OA. IL-5 is a plausible factor responsible for decreasing blood leukocyte mtDNACN in knee OA patients.

Mitochondrial DNA 4977bp Deletion Mutation in Peripheral Blood Reflects Atrial Remodeling in Patients with Non-Valvular Atrial Fibrillation

PURPOSE: Recently, mitochondrial DNA 4977bp deletion (mtDNA4977-mut), a somatic mutation related to oxidative stress, has been shown to be associated with atrial fibrillation (AF). We hypothesized that patient age, as well as electroanatomical characteristics of fibrillating left atrial (LA), vary depending on the presence of mtDNA4977-mut in peripheral blood among patients with non-valvular AF. MATERIALS AND METHODS: Analyzing clinical and electroanatomical characteristics, we investigated the presence of the mtDNA4977-mut in peripheral blood of 212 patients (51.1+/-13.2 years old, 83.5% male) undergoing catheter ablation for non-valvular AF, as well as 212 age-matched control subjects. RESULTS: The overall frequency of peripheral blood mtDNA4977-mut in patients with AF and controls was not significantly different (24.5% vs. 19.3%, p=0.197). When the AF patient group was stratified according to age, mtDNA4977-mut was more common (47.4% vs. 20.0%, p=0.019) in AF patients older than 65 years than their age-matched controls. Among AF patients, those with mtDNA4977-mut were older (58.1+/-11.9 years old vs. 48.8+/-11.9 years old, p<0.001). AF patients positive for the mtDNA mutation had greater LA dimension (p=0.014), higher mitral inflow peak velocity (E)/diastolic mitral annular velocity (Em) ratio (p<0.001), as well as lower endocardial voltage (p=0.035), and slower conduction velocity (p=0.048) in the posterior LA than those without the mutation. In multivariate analysis, E/Em ratio was found to be significantly associated with the presence of mtDNA4977-mut in peripheral blood. CONCLUSION: mtDNA4977-mut, an age-related somatic mutation detected in the peripheral blood, is associated with advanced age and electro-anatomical remodeling of the atrium in non-valvular AF.

Effects of Carnitine on Peripheral Blood Mitochondrial DNA Copy Number and Liver Function in Non-Alcoholic Fatty Liver Disease / 대한소화기학회지

BACKGROUND/AIMS: Functional and anatomical abnormalities of mitochondria play an important role in developing steatohepatitis. Carnitine is essential for enhanced mitochondrial beta oxidation through the transfer of long-chain fatty acids into the mitochondria. We examined the impact of carnitine complex on liver function and peripheral blood mitochondria copy number in NAFLD patients. METHODS: Forty-five NAFLD patients were enrolled. Patients were categorized into the carnitine complex-administered group and control group. Before and 3 months after drug administration, a liver function test and peripheral blood mitochondrial DNA and 8-oxo-dG quantitive analysis were conducted. RESULTS: In carnitine treatment group, ALT, AST, and total bilirubin were reduced after medication. There was no difference in AST, ALT, and total bilirubin between before and after treatment in control group. In carnitine group, peripheral mitochondrial DNA copy number was significantly increased from 158.8+/-69.5 copy to 241.6+/-180.6 copy (p=0.025). While in control group the mitochondrial copy number was slightly reduced from 205.5+/-142.3 to 150.0+/-109.7. 8-oxo-dG level was also tended to decrease in carnitine group (p=0.23) and tended to increase in control group (p=0.07). CONCLUSIONS: In NAFLD, the carnitine improved liver profile and peripheral blood mitochondrial DNA copy number. This results suggest that carnitine activate the mitochondria, thereby contributing to the improvement of NAFLD.

Higher mitochondrial DNA copy number is associated with lower prevalence of microalbuminuria

It has been suggested that mitochondrial dysfunction contributes to the initiation and development of atherosclerosis and cardiovascular disease. We examined the association between mitochondrial DNA (mtDNA) copy number and microalbuminuria in a cross-sectional community-based study. We measured peripheral blood mtDNA copy number in 694 adults without chronic kidney disease by a real-time PCR method. The overall prevalence of microalbuminuria (defined as an albumin creatinine ratio of 30 to 299 mg/g) was 4.5%. The prevalence of microalbuminuria decreased progressively from the lower to the upper quartiles of mtDNA copy number (6.9%, 5.7%, 2.9%, and 2.3% in quartiles 1, 2, 3, and 4, respectively, P = 0.017 for trend). Multiple logistic regression models showed that the quartile of mtDNA copy number was independently associated with the prevalence of microalbuminuria (P = 0.01 for trend). Compared with the lowest quartile, the highest quartile had an odds ratio of 0.22 for microalbuminuria (95% confidence interval, 0.05 to 0.87; P = 0.03). Higher mtDNA copy number was associated with the lower prevalence of microalbuminuria in a community-based population.

Potential relationship between peripheral blood mitochondrial DNA content and insulin resistance and secretion in offspring of type 2 diabetic patients

Both qualitative and quantitative changes in mitochondrial DNA [mtDNA] have been implicated in the pathogenesis of diabetes mellitus. In this study, we investigate whether peripheral blood mtDNA [pb-mtDNA] is decreased and if there is any relation between its content and the parameters of both insulin resistance and secretion in offspring of diabetic subjects. The pb-mtDNA content was measured by real time polymerase chain reaction with mitochondrial- specific fluorescent probe, normalized by a nuclear DNA, 28S rRNA gene, in 42 offspring of type 2 diabetic patients and 12 age-, sex- and body mass index [BMI]- matched normal subjects. The correlations between pb-mtDNA content and the parameters of insulin resistance and secretion were studied. Our results indicated that the level of pb-mtDNA was lower in offspring of diabetic subjects than in control subjects [1.230 +/- 0.05 vs. 1.513 +/- 0.02 in the offspring and control subjects, respectively, P <0.05]. Also, pb-mtDNA content was significantly correlated with logarithmically transformed insulin sensitivity index [r = 0.5, P<0.05], fasting C-peptide [r = -0.8, P<0.05], acute insulin response [r = -0.8, P<0.05] and late insulin response [r = -0.7, P <0.05] in offspring of diabetic subjects. In conclusion, quantitative mtDNA status might be a hereditary factor associated with type 2 diabetes and is correlated negatively with indexes of insulin resistance and insulin secretion in offspring of diabetic patients. So, pb-mtDNA content could serve as an indicator of insulin sensitivity and insulin secretion in those subjects