Molecular Characterization of Two Hypertension Pedigrees Carrying Mitochondrial tRNAGln 4386T>C Mutation

Various mitochondrial diseases, including mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), are associated with heteroplasmic mutations in mitochondrial DNA (mtDNA). Herein, we refined a previously generated G13513A mtDNA-targeted platinum transcription activator-like effector nuclease (G13513A-mpTALEN) to more efficiently manipulate mtDNA heteroplasmy in MELAS-induced pluripotent stem cells (iPSCs). Introduction of a nonconventional TALE array at position 6 in the mpTALEN monomer, which recognizes the sequence around the m.13513G>A position, improved the mpTALEN effect on the heteroplasmic shift. Furthermore, the reduced expression of the new Lv-mpTALEN(PKLB)/R-mpTALEN(PKR6C) pair by modifying codons in their expression vectors could suppress the reduction in the mtDNA copy number, which contributed to the rapid recovery of mtDNA in mpTALEN-applied iPSCs during subsequent culturing. Moreover, MELAS-iPSCs with a high proportion of G13513A mutant mtDNA showed unusual properties of spontaneous, embryoid body-mediated differentiation in vitro, which was relieved by decreasing the heteroplasmy level with G13513A-mpTALEN. Additionally, drug-inducible, myogenic differentiation 1 (MYOD)-transfected MELAS-iPSCs (MyoD-iPSCs) efficiently differentiated into myosin heavy chain-positive myocytes, with or without mutant mtDNA. Hence, heteroplasmic MyoD-iPSCs controlled by fine-tuned mpTALENs may contribute to a detailed analysis of the relationship between mutation load and cellular phenotypes in disease modeling.

Objectives: Mutations in mitochondrial tRNA (mt-tRNA) are the important causes for maternally inherited hypertension; however, the pathophysiology of mt-tRNA mutations in clinical expression of hypertension remains poorly understood. Material and Methods: In this study, we report the molecular features of a Han Chinese pedigree with maternally transmitted essential hypertension. The entire mitochondrial genomes are PCR amplified and sequenced. Moreover, phylogenetic analysis, haplogroup analysis, as well as pathogenicity scoring system are used to assess the potential roles for mtDNA mutations. Results: Strikingly, among 10 matrilineal relatives, 3 suffer from variable degree of hypertension at different age at onset. Sequence analysis of the complete mitochondrial genomes suggests the presence of three possible pathogenic mtDNA mutations: tRNA^Asp T7561C, tRNA^His C12153T, and A12172G, together with a set of variants belonging to East Asian mitochondrial haplogroup M7a. Interestingly, the T7561C mutation occurs at position 44 in the variable region of tRNA^Asp, while the C12153T and A12172G mutations are localized at extremely conserved nucleotides in the D-arm and anticodon stem of tRNA^His gene, respectively, which are critical for tRNA steady-state level and function. Conclusions: Mitochondrial T7561C, C12153T, and A12172G mutations may lead to the failure in tRNAs metabolism and cause mitochondrial dysfunction that is responsible for hypertension. However, the homoplasmy form of mt-tRNA mutations, incomplete penetrance of hypertension suggests that T7561C, C12153T, and A12172G mutations are insufficient to produce the clinical phenotype; hence, other risk factors such as environmental factors, nuclear genes, and epigenetic modifications may contribute to the phenotypic manifestation of maternally inherited hypertension in this Chinese pedigree.

Decision letter: The multi-tissue landscape of somatic mtDNA mutations indicates tissue-specific accumulation and removal in aging

Author response: The multi-tissue landscape of somatic mtDNA mutations indicates tissue-specific accumulation and removal in aging

Editor's evaluation: The multi-tissue landscape of somatic mtDNA mutations indicates tissue-specific accumulation and removal in aging

A Novel Next-Generation Sequencing–Based Approach for Concurrent Detection of Mitochondrial DNA Copy Number and Mutation

Mitochondrial tRNALeu(UUR) C3275T, tRNAGln T4363C and tRNALys A8343G mutations may be associated with PCOS and metabolic syndrome

Mitochondrial DNA (mtDNA) mutations are associated with essential hypertension (EH), but the molecular mechanism remains largely unknown. The aim of this study is to explore the association between mtDNA mutations and EH. Two maternally inherited families with EH are underwent clinical, genetic and biochemical assessments. mtDNA mutations are screened by PCR-Sanger sequencing and phylogenetic, and bioinformatics analyses are performed to evaluate the pathogenicity of mtDNA mutations. We also generate cytoplasmic hybrid (cybrid) cell lines to analysis mitochondrial functions. Matrilineal relatives exhibit variable degree of clinical phenotypes. Molecular analysis reveals the presence of m.A14693G and m.A14696G mutations in two pedigrees. Notably, the m.A14693G mutation occurs at position 54 in the TψC loop of tRNAGlu, a position which is critical for post-transcriptionally modification of tRNAGlu. While the m.A14696G mutation creates a novel base-pairing (51C-64G). Bioinformatic analysis shows that these mutations alter tRNAGlu secondary structure. Additionally, patients with tRNAGlu mutations exhibit markedly decreased in mtDNA copy number, mitochondrial membrane potential (MMP) and ATP, whereas the levels of reactive oxygen species (ROS) increase significantly. The m.A14696G and m.A14693G mutations lead to failure in tRNAGlu metabolism and cause mitochondrial dysfunction that is responsible for EH.

Introduction: Systemic lupus erythematosus (SLE) is a common autoimmune disease with unknown etiology. Recently, a growing number of evidence suggested that mitochondrial dysfunctions played active roles in the pathogenesis of SLE, but its detailed mechanism remains largely undetermined. The aim of this study was to analyze the frequencies of mitochondrial tRNA (mt-tRNA) variants in Chinese individuals with SLE. Methods: We carried out a mutational screening of mt-tRNA variants in a cohort of 200 patients with SLE and 200 control subjects by PCR-Sanger sequencing. The potential pathogenicity of mt-tRNA variants was evaluated by phylogenetic conservation and haplogroup analyses. In addition, trans-mitochondrial cybrid cell lines were established, and mitochondrial functions including ATP, reactive oxygen species (ROS), mitochondrial DNA (mtDNA) copy number, mitochondrial membrane potential (MMP), superoxide dismutase (SOD), and mt-RNA transcription were analyzed in cybrids with and without these putative pathogenic mt-tRNA variants. Results: We identified five possible pathogenic variants: tRNAVal G1606A, tRNALeu(UUR) A3243G, tRNAIle A4295G, tRNAGly T9997C, and tRNAThr A15924G that only found in SLE patients but were absent in controls. Interestingly, these variants were located at extremely conserved nucleotides of the corresponding tRNAs and may alter tRNAs’ structure and function. Furthermore, cells carrying these tRNA variants had much lower levels of ATP, mtDNA copy number, MMP, and SOD than controls; by contrast, the levels of ROS increased significantly (p < 0.05 for all). Furthermore, a significant reduction in mt-ND1, ND2, ND3, ND5, and A6 mRNA expression was observed in cells with these mt-tRNA variants, while compared with controls. Thus, failures in tRNA metabolism caused by these variants would impair mitochondrial translation and subsequently lead to mitochondrial dysfunction that was involved in the progression and pathogenesis of SLE. Conclusions: Our study suggested that mt-tRNA variants were important causes for SLE, and screening for mt-tRNA pathogenic variants was recommended for early detection and prevention for this disorder. Introduction: Systemic lupus erythematosus (SLE) is a common autoimmune disease with unknown etiology. Recently, a growing number of evidence suggested that mitochondrial dysfunctions played active roles in the pathogenesis of SLE, but its detailed mechanism remains largely undetermined. The aim of this study was to analyze the frequencies of mitochondrial tRNA (mt-tRNA) variants in Chinese individuals with SLE. Methods: We carried out a mutational screening of mt-tRNA variants in a cohort of 200 patients with SLE and 200 control subjects by PCR-Sanger sequencing. The potential pathogenicity of mt-tRNA variants was evaluated by phylogenetic conservation and haplogroup analyses. In addition, trans-mitochondrial cybrid cell lines were established, and mitochondrial functions including ATP, reactive oxygen species (ROS), mitochondrial DNA (mtDNA) copy number, mitochondrial membrane potential (MMP), superoxide dismutase (SOD), and mt-RNA transcription were analyzed in cybrids with and without these putative pathogenic mt-tRNA variants. Results: We identified five possible pathogenic variants: tRNAVal G1606A, tRNALeu(UUR) A3243G, tRNAIle A4295G, tRNAGly T9997C, and tRNAThr A15924G that only found in SLE patients but were absent in controls. Interestingly, these variants were located at extremely conserved nucleotides of the corresponding tRNAs and may alter tRNAs’ structure and function. Furthermore, cells carrying these tRNA variants had much lower levels of ATP, mtDNA copy number, MMP, and SOD than controls; by contrast, the levels of ROS increased significantly (p < 0.05 for all). Furthermore, a significant reduction in mt-ND1, ND2, ND3, ND5, and A6 mRNA expression was observed in cells with these mt-tRNA variants, while compared with controls. Thus, failures in tRNA metabolism caused by these variants would impair mitochondrial translation and subsequently lead to mitochondrial dysfunction that was involved in the progression and pathogenesis of SLE. Conclusions: Our study suggested that mt-tRNA variants were important causes for SLE, and screening for mt-tRNA pathogenic variants was recommended for early detection and prevention for this disorder.

Mitochondria are the primary source of cellular energy. Mitochondrial DNA (mtDNA) copy number in peripheral blood leukocytes (PBLs) has high genetic heritability and inter-individual variation of mtDNA copy number in PBLs has been associated with the risk of several human cancers. In this study, we investigated into the link between mtDNA and clear-cell renal cell carcinoma (ccRCC) in a large population-based case control study. We measured relative mtDNA copy number in PBLs from 616 Caucasian ccRCC cases and 603 controls frequency-matched on age, gender, and ethnicity. In the analyses of controls only, there was a significant inverse correlation between age and mtDNA copy number (P=0.001); men had significantly lower mtDNA copy number than women (P&amp;lt;0.001); and both smoking and hypertension had a negative effect on mtDNA copy number (P=0.03 and 0.04, respectively). We found a significantly lower mtDNA copy number in cases compared to controls (mean±SD: 1.16±0.37 versus 1.21±0.39, P=0.04). Dichotomized at the median level of mtDNA copy number in controls, individuals with lower mtDNA copy number were at an increased risk of ccRCC (OR=1.20, 95% CI: 0.96-1.51, P=0.11) adjusting for age, gender, smoking status, BMI, and history of hypertension in a multivariate logistic regression analysis. In stratified analyses, the association between lower mtDNA copy number and increased ccRCC risks reached statistical significance in younger aged (age&amp;lt;60) individuals (OR=1.56, 95% CI: 1.12-2.15, P=0.01), women (OR=1.57, 95% CI: 1.06-2.35, P=0.03), and individuals without history of hypertension (OR=1.48, 95% CI: 1.08-2.04, P=0.02). Interestingly, there was a significant interaction between age and mtDNA copy number, and between a history of hypertension and mtDNA copy number in elevating ccRCC risk (P for interaction=0.02 and 0.03, respectively). In summary, our data showed that several established risk factors of ccRCC, including advanced age, male sex, smoking, and history of hypertension, negatively impacted mtDNA copy number in PBLs. Lower mtDNA copy number in PBLs was associated with significantly increased risk of ccRCC in younger aged individuals, women, never smokers, and those without a history of hypertension. There was a significant interaction between age and mtDNA, and a history of hypertension and mtDNA in elevating the risk of ccRCC. These findings indicate that the association between mtDNA copy number in PBLs and ccRCC risk may be modified by various host characteristics (age, gender, smoking, and history of hypertension), providing novel insight into the role of mtDNA in modulating RCC tumorigenesis. Citation Format: Xin Wang, Stephanie Melkonian, Jian Gu, David Chang, Nizar Tannir, Christopher Wood, Xifeng Wu. Mitochondrial DNA copy number in peripheral blood and the risk of clear-cell renal cell carcinoma: Effect of age, gender, smoking, and history of hypertension. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4320. doi:10.1158/1538-7445.AM2014-4320

Alterations in mitochondrial DNA (mtDNA) copy number are observed in human gliomas. However, whether variations in mtDNA copy number in whole blood play any role in glioma carcinogenesis is still largely unknown. In current study with 395 glioma patients and 425 healthy controls, we intended to investigate the association between mtDNA copy number in whole blood and glioma risk. Overall, we found that levels of mtDNA copy number were significantly higher in glioma cases than healthy controls (mean: 1.48 vs. 1.32, P < 0.01). In both cases and controls, levels of mtDNA copy number were inversely correlated with age (P < 0.01, respectively). And in cases, newly diagnosed, glioblastoma (GBM), and high grade glioma patients had significantly lower mtDNA copy number than their counterparts (P = 0.02, P < 0.01, and P = 0.04, respectively). In the multivariate analysis, elevated mtDNA copy number levels were associated with a 1.63-fold increased risk of glioma (adjusted odds ratio (OR) = 1.63, 95% confidence interval (CI) = 1.23-2.14). In further quartile analysis, study subjects who had highest levels of mtNDA copy number had 1.75-fold increased risk of gliomas (adjOR = 1.75, 95%CI = 1.18-2.61). In brief, our findings support the role of mtDNA copy number in the glioma carcinogenesis. © 2016 Wiley Periodicals, Inc.

Mitochondrion-targeted RNA therapies as a potential treatment strategy for mitochondrial diseases

Objective Studies on etiopathogenesis of attention deficit/hyperactivity disorder (ADHD) are increasingly focussing on mitochondrial dysfunction. Children diagnosed with ADHD who had significantly higher mitochondrial DNA (mtDNA) copy numbers than healthy children in our first study were re-examined in 1-year follow-up to investigate effects of severity and treatment of ADHD on mtDNA. Methods Twenty-eight patients who participated in previous study were included in this follow-up study. Patients were equally divided into two groups according to whether they had been receiving treatment. Kiddie Schedule for Affective Disorders and Schizophrenia for School-Age Children–Present and Lifetime Version, and Conners Parent Rating Scale (CPRS) were used. Polymerase chain reaction was performed. Results Means of the first and second mtDNA copy were similar in all patients. mtDNA copy numbers did not change between two measurements in treated and non-treated groups. There was a correlation between CPRS ADHD index and inattention scores and mtDNA copy number in treated group. mtDNA copy number did not change in patients with ADHD over a period of 1 year regardless of treatment. Conclusions There may be a relationship between decreased ADHD severity with treatment and positive effects of mitochondrial functions. Mitochondrial dysfunction may play a role in pathophysiology of ADHD. KEY POINTS This was the first study to follow up ADHD patients in order to investigate mitochondrial dysfunction by measuring mtDNA copy numbers 1 year after the initial measurements. mtDNA copy number, one of the best markers of mitochondrial dysfunction, did not change in ADHD patients over a period of 1 year regardless of treatment. Mitochondrial dysfunction may play a role in the pathophysiology of ADHD, where it may be involved with or without treatment. In the treated group, there was an association between decreased ADHD severity and reduced mtDNA copy numbers. There may be a relationship between decreased ADHD severity with treatment and the positive effects of mitochondrial functions.

Mitochondrial DNA (mtDNA) copy number in leukocytes has been regarded as a biomarker for various environmental exposures and chronic diseases. Our previous study showed that certain demographic factors (e.g. age, gender, BMI, etc.) significantly affect levels of leukocyte mtDNA copy number in Mexican Americans. However, the effect of the built environment on leukocyte mtDNA copy number has not been studied previously. In this cross-sectional study, we examined the association between multiple components of the built environment with leukocyte mtDNA copy number among 5,502 Mexican American adults enrolled in Mano-A-Mano, the Mexican American Cohort Study (MACS). Based on the median levels of mtDNA copy number, the study population was stratified into low mtDNA copy number group (< median) and high mtDNA copy number group (≥ median). Among all built environment exposure variables, household density and road/intersection ratio were found to be statistically significant between groups with low and high mtDNA copy number (P < 0.001 and 0.002, respectively). In the multivariate logistic regression analysis, individuals living in areas with elevated levels of household density had 1.24-fold increased odds of having high levels of mtDNA copy number [Odds ratio (OR) = 1.24, 95% confidence interval (CIs) 1.08, 1.36]. Similarly, those living in areas with elevated levels of road/intersection ratio had 1.12-fold increased odds of having high levels of mtDNA copy number (OR = 1.12, 95% CI 1.01, 1.27). In further analysis, when both variables were analyzed together in a multivariate logistic regression model, the significant associations remained. In summary, our results suggest that selected built environment variables (e.g. population density and road/intersection ratio) may influence levels of mtDNA copy number in leukocytes in Mexican Americans.

Background Mitochondria, essential for energy production, can inadvertently produce reactive oxygen species. Measuring mitochondrial DNA (mtDNA) copy number provides a window into mitochondrial function. Changes in mtDNA copy number can occur due to genetic mutations, environmental factors, and inflammation, influencing various health outcomes. Dysfunctional mitochondria are linked to increased oxidative stress and inflammation, contributing to diverse diseases. In BD, mitochondrial dysfunction is intricately tied to the disease's pathological manifestations, affecting energy metabolism and intracellular calcium signaling. Alterations in mtDNA copy number have been observed in manic and depressed BD patients. Additionally, childhood trauma has been identified as a major risk factor for BD and has been associated with increased susceptibility to and severity of the disorder, although the specific role of each trauma subtype (emotional, physical, or sexual abuse) remains a topic of debate. Previous research has established that both BD and childhood trauma are independently associated with mitochondrial abnormalities. However, the potential association between childhood trauma, mitochondrial dysfunction, and BD has not been thoroughly investigated. Aims &amp; Objectives Prior research has shown an increased incidence of childhood trauma in individuals diagnosed with bipolar disorder (BD). It has also been independently established that both childhood trauma and mitochondrial dysfunction have connections with BD. Consequently, the aim of this study is to explore whether there might be a link between mitochondrial dysfunction and childhood trauma in people suffering from BD. Method The study involved 60 participants diagnosed with BD and 26 individuals serving as healthy controls. To gather data, the Childhood Trauma Questionnaire (CTQ) was utilized, and blood samples were collected for the purpose of determining mitochondrial DNA copy number (MCN) from leukocytes. An examination was then conducted to identify any correlation between CTQ scores and MCN. Results Compared to the healthy controls, the BD group exhibited slightly lower MCN, which did not reach significance. However, the CTQ sum scores were significantly higher in the BD group. After controlling age, gender, and BMI, negative correlations were found between CTQ scores, particularly emotional neglect (r = -0.431, p = 0.04), emotional abuse (r = -0.425, p = 0.043), and total scores (r = - 0.513; p = 0.012), and MCN within the healthy control group. Nevertheless, no substantial correlation was observed between CTQ and MCN in the participants with BD. Discussion &amp; Conclusion Childhood traumata have different effects on BD compared to healthy individuals. Therefore, a comprehensive investigation of the complex relationship between childhood trauma, BD, and medication effects is crucial to better understand how trauma impacts individuals living with this psychiatric condition.