Effect Of The 5178C>A Mutation Heteroplasmy On The ND2 Gene Of Mitochondria Dna In Essential Hypertension Generation And Development In Chinese General Population

Part I Correlation of heteroplasmy level of the 5178C>A mutation in the ND2 gene of mitochondria DNA and essential hypertension. Objective: To date, accumulated evidences supported the contribution of the 5178C>A mutation of mitochondria DNA(mt5178C>A) to essential hypertension(EH).The genetic predisposition of the mt5178C>A mutation to EH may be influenced by heteroplasmy level. However, it is still uncertain whether the heteroplasmy level for mt5178C>A mutation could attribute to the susceptibility of EH. This study aimed to investigate the relationship between mt5178C>A mutation heteroplasmy level and EH in Chinese general population. Methods: EH cases and normotensive controls were recruited consecutively from general population. Demographic, clinical characteristics and blood leukocytes were collected. The mt5178C>A mutation heteroplasmy level was quantified by the rapid and sensitive real-time polymerase chain reaction(PCR) method for each participant. Results: A total of 108 EH cases and 109 controls were recruited. The mt5178C>A heteroplasmy level were(42±11%) from EH patients and(54±13%)from control subjects, was significantly different between the two groups(P<0.001).Using a two-step cluster analysis, the mt5178C>A heteroplasmy level exceeding 44% was associated with decrease risk of EH(OR=0.18, 95%CI: 0.10-0.31, P<0.001).Correlational analyses showed mt5178C>A heteroplasmy level was significantly negative correlated both with systolic blood pressure(r=-0.38, P<0.001)and diastolic blood pressure(r=-0.49, P<0.001) in 109 controls. Logistic regression analysis singly in univariate models demonstrated that mt5178C>A heteroplasmy level(OR: 0.82, 95%CI: 0.77-0.87, P<0.001) was protective factor, however, BMI(OR:1.30, 95%CI:1.12-1.45, P<0.001), total cholesterol(OR: 2.13,95%CI: 1.39-3.28, P= 0.001), triglyceride(OR: 7.62,95%CI:3.45-16.84,P<0.001)and blood urea nitrogen(OR: 1.35,95%CI:1.04-1.77, P=0.03) were risk factors for EH. And analysis simultaneously in a multivariate model showed that mt5178C>A heteroplasmy level(OR: 0.83, 95%CI: 0.78-0.89, P<0.001) was independent protective factor, however, only total cholesterol(OR: 2.17, 95%CI: 1.58-2.98, P=0.02) and low density lipoprotein cholesterol(OR: 0.06, 95% CI: 0.01-0.83, P = 0.04) were independent risk factors for EH, and the level of P value at 0.05 critical. Conclusion: Mitochondrial ND2 gene 5178C>A mutation heteroplasmy level exert protective role against EH in Chinese population.Part II Effect of the 5178C>A mutation heteroplasmy on the ND2 gene of mitochondria DNA on mitochondrial function. Objective: This study is aimed to investigate the mechanism of the mt5178C>A mutation heteroplasmy level in the generation and development of EH in Chinese general population. Methods: The mt5178C>A heteroplasmy level was classified as “low heteroplasmy”(mt5178C>A low-carriers) from EH cases and “high heteroplasmy”(mt5178C>A high- carriers) from controls, and between the two groups of gender, age, body mass index and laboratory indexes were matched. Lymphoblastoid cell lines were immortalized by transformation with Epstein-Barr virus from the peripheral whole blood of each participant. Effect of the mt5178C>A mutation heteroplasmy level on ND2 gene expression and Mitochondrial function analyzed by real-time PCR, flow cytometry, Seahorse XF-96 analyzer and bioluminescence technique for each lymphoblastoid cell line. The mechanism of the mt5178C>A mutation heteroplasmy level in the generation and development of EH in Chinese general population wasexplained. Results: A total of 6 mt5178C>A low-carriers from EH and 5 high-carriers from controls were selected. The mt5178C>A heteroplasmy level was significantly different between the two groups(34±2 vs. 70±3 %, P<0.001). The expression of the ND2 gene was no significantly different between the two groups(0.39±0.08 vs. 0.40±0.03, P=0.93). Compared with mt5178C>A low-carriers group, the population DT(73.2±9.9 vs. 37.5±2.1h, P<0.001), ROS production(89.12±9.22 vs. 33.53±9.10, P<0.001), cytosolic Ca2+ concentration(64.32±5.92 vs. 37.85±2.19, P<0.001) and rate of apoptosis cells(20.33±3.72 vs. 10.80±2.59 %, P=0.002) were decreased markedly, while mitochondrial membrane potential(ΔΨm)(1.62±0.52 vs. 5.79±0.93, P<0.001), basal respiration OCR(23.74±1.20 vs. 37.23±2.23, P<0.001), ATP production OCR(13.91±1.17 vs. 24.56±3.18, P<0.001), maximal respiration OCR(32.00±4.18 vs. 65.58±11.23, P<0.001), spare capacity OCR(8.26±1.84 vs. 28.35±9.76, P=0.009) and ATP concentration(1456186.0±79046.9 vs. 738920.1±32589.59, P<0.001) significantly elevated in mt5178C>A high-carriers group. In short, cell proliferation were promoted, OCR were increased, ATP were generated, ROS were decreased, ΔΨm were stabilized and rate of apoptosis cells were decreased in mt5178C>A high-carriers group. Conclusion: The mt5178C>A mutation heteroplasmy level may play an important role in improving the mitochondrial function to protect mechanism of generation and development of EH.