Rectifying METTL4-Mediated N6-Methyladenine Excess in Mitochondrial DNA Alleviates Heart Failure.

BACKGROUND: Myocardial mitochondrial dysfunction underpins the pathogenesis of heart failure (HF), yet therapeutic options to restore myocardial mitochondrial function are scarce. Epigenetic modifications of mitochondrial DNA (mtDNA), such as methylation, play a pivotal role in modulating mitochondrial homeostasis. However, their involvement in HF remains unclear. METHODS: Experimental HF models were established through continuous angiotensin II and phenylephrine (AngII/PE) infusion or prolonged myocardial ischemia/reperfusion injury. The landscape of N6-methyladenine (6mA) methylation within failing cardiomyocyte mtDNA was characterized using high-resolution mass spectrometry and methylated DNA immunoprecipitation sequencing. A tamoxifen-inducible cardiomyocyte-specific Mettl4 knockout mouse model and adeno-associated virus vectors designed for cardiomyocyte-targeted manipulation of METTL4 (methyltransferase-like protein 4) expression were used to ascertain the role of mtDNA 6mA and its methyltransferase METTL4 in HF. RESULTS: METTL4 was predominantly localized within adult cardiomyocyte mitochondria. 6mA modifications were significantly more abundant in mtDNA than in nuclear DNA. Postnatal cardiomyocyte maturation presented with a reduction in 6mA levels within mtDNA, coinciding with a decrease in METTL4 expression. However, an increase in both mtDNA 6mA level and METTL4 expression was observed in failing adult cardiomyocytes, suggesting a shift toward a neonatal-like state. METTL4 preferentially targeted mtDNA promoter regions, which resulted in interference with transcription initiation complex assembly, mtDNA transcriptional stalling, and ultimately mitochondrial dysfunction. Amplifying cardiomyocyte mtDNA 6mA through METTL4 overexpression led to spontaneous mitochondrial dysfunction and HF phenotypes. The transcription factor p53 was identified as a direct regulator of METTL4 transcription in response to HF-provoking stress, thereby revealing a stress-responsive mechanism that controls METTL4 expression and mtDNA 6mA. Cardiomyocyte-specific deletion of the Mettl4 gene eliminated mtDNA 6mA excess, preserved mitochondrial function, and mitigated the development of HF upon continuous infusion of AngII/PE. In addition, specific silencing of METTL4 in cardiomyocytes restored mitochondrial function and offered therapeutic relief in mice with preexisting HF, irrespective of whether the condition was induced by AngII/PE infusion or myocardial ischemia/reperfusion injury. CONCLUSIONS: Our findings identify a pivotal role of cardiomyocyte mtDNA 6mA and the corresponding methyltransferase, METTL4, in the pathogenesis of mitochondrial dysfunction and HF. Targeted suppression of METTL4 to rectify mtDNA 6mA excess emerges as a promising strategy for developing mitochondria-focused HF interventions.

Subtype-Specific Association of Mitochondrial DNA Copy Number With Poststroke/TIA Outcomes in 10†241 Patients in China.

BACKGROUND: Mitochondrial DNA copy number (mtDNA-CN) is associated with the severity and mortality in patients with stroke, but the associations in different stroke subtypes remain unexplored. METHODS: We conducted an observational prospective cohort analysis on patients with ischemic stroke or transient ischemic attack enrolled in the Third China National Stroke Registry. We applied logistic models to assess the association of mtDNA-CN with functional outcome (modified Rankin Scale score, 3-6 versus 0-2) and Cox proportional hazard models to assess the association with stroke recurrence (treating mortality as a competing risk) and mortality during a 12-month follow-up, adjusting for sex, age, physical activity, National Institutes of Health Stroke Scale at admission, history of stroke and peripheral artery disease, small artery occlusion, and interleukin-6. Subgroup analyses stratified by age and stroke subtypes were conducted. RESULTS: The Third China National Stroke Registry enrolled 15 166 patients, of which 10 241 with whole-genome sequencing data were retained (mean age, 62.2 [SD, 11.2] years; 68.8% men). The associations between mtDNA-CN and poststroke/transient ischemic attack outcomes were specific to patients aged ≤65 years, with lower mtDNA-CN significantly associated with stroke recurrence in 12 months (subdistribution hazard ratio, 1.15 per SD lower mtDNA-CN [95% CI, 1.04-1.27]; P=5.2×10-3) and higher all-cause mortality in 3 months (hazard ratio, 2.19 [95% CI, 1.41-3.39]; P=5.0×10-4). Across subtypes, the associations of mtDNA-CN with stroke recurrence were specific to stroke of undetermined cause (subdistribution hazard ratio, 1.28 [95% CI, 1.11-1.48]; P=6.6×10-4). In particular, lower mtDNA-CN was associated with poorer functional outcomes in stroke of undetermined cause patients diagnosed with embolic stroke of undetermined source (odds ratio, 1.53 [95% CI, 1.20-1.94]; P=5.4×10-4), which remained significant after excluding patients with recurrent stroke (odds ratio, 1.49 [95% CI, 1.14-1.94]; P=3.0×10-3). CONCLUSIONS: Lower mtDNA-CN is associated with higher stroke recurrence rate and all-cause mortality, as well as poorer functional outcome at follow-up, among stroke of undetermined cause, embolic stroke of undetermined source, and younger patients.

A case of exacerbated encephalopathy with stroke-like episodes and lactic acidosis triggered by metformin in a patient with MELAS.

Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) is a maternally inherited multisystemic disorder caused by mutations in mitochondrial DNA that result in cellular energy deficiency. MELAS affects the most metabolically active organs, including the brain, skeletal muscles, cochlea, retina, heart, kidneys, and pancreas. As a result, about 85% of carriers of m.3243A > G, the most common mutation in MELAS, develop diabetes by the age of 70. Although metformin is the most widely prescribed drug for diabetes, its usefulness in mitochondrial dysfunction remains controversial. Here, we present the case of a 32-year-old Korean patient diagnosed with MELAS who presented with exacerbated stroke-like episodes and lactic acidosis triggered by metformin.

Mitochondrial DNA mutations in Medulloblastoma.

To date, several studies on genomic events underlying medulloblastoma (MB) biology have expanded our understanding of this tumour entity and led to its division into four groups-WNT, SHH, group 3 (G3) and group 4 (G4). However, there is little information about the relevance of pathogenic mitochondrial DNA (mtDNA) mutations and their consequences across these. In this report, we describe the case of a female patient with MB and a mitochondriopathy, followed by a study of mtDNA variants in MB groups. After being diagnosed with G4 MB, the index patient was treated in line with the HIT 2000 protocol with no indications of relapse after five years. Long-term side effects of treatment were complemented by additional neurological symptoms and elevated lactate levels ten years later, resulting in suspected mitochondrial disease. This was confirmed by identifying a mutation in the MT-TS1 gene which appeared homoplasmic in patient tissue and heteroplasmic in the patient's mother. Motivated by this case, we explored mtDNA mutations across 444 patients from ICGC and HIT cohorts. While there was no statistically significant enrichment of mutations in one MB group, both cohorts encompassed a small group of patients harbouring potentially deleterious mtDNA variants. The case presented here highlights the possible similarities between sequelae caused by MB treatment and neurological symptoms of mitochondrial dysfunction, which may apply to patients across all MB groups. In the context of the current advances in characterising and interpreting mtDNA aberrations, recognising affected patients could enhance our future knowledge regarding the mutations' impact on carcinogenesis and cancer treatment.

Mitochondrial dysfunction in a rat model and the related risk of metabolic disorders.

OBJECTIVE: To explore whether kidney deficiency (KYD) is prone to metabolic disorders may be linked to impaired mitochondrial function in thermogenesis and metabolic tissues. METHODS: A rat model of KYD was used, which was established using Sprague Dawley rat dams with warm preference subjected to herbal treatment that can improve kidney . The human relevance was confirmed by reduced serum corticosterone levels, and increased preference for warm location. RESULTS: KYD Rats were underdeveloped. Adenosine-triphosphate (ATP) production was reduced in the brown fat, but increased in the muscle. However, oxidative phosphorylated complexes to generate ATP and mitochondrial biogenesis marker were reduced in both tissues. When the second insult of high-fat diet (HFD) was introduced, KYD rats gained less weight yet developed more severe lipid and glucose metabolic disorders. This may be driven by disregulated liver gluconeogenesis marker forkhead box protein O1 and lipid metabolic regulator cholesterol 7 alpha-hydroxylase. CONCLUSION: KYD rats exhibited reduced mito-chondrial function in the brown fat, but were partially compensated by skeletal muscle, associated with the phenotype of warm preference and metabolic disorder, which was further exacerbated by additional HFD consumption. Future studies can focus on treatment targetting mitochondria function to reverse this phenotype.

Relationship between Sestrin2 and mtDNA-NLRP3 inflammasome pathway during endotoxin-induced myocardial injury in mice / 中华麻醉学杂志

Objective:To evaluate the relationship between Sestrin2 and mitochondrial DNA (mtDNA)-NOD-like receptor associated protein 3 (NLRP3) inflammasome pathway during endotoxin-induced myocardial injury in mice.Methods:One hundred and eighty-four clean-grade healthy male ICR mice, aged 8-12 weeks, weighing 20-25 g, were used in this study. One hundred and sixty-eight mice were divided into 7 groups ( n=24 each) using the random number table method: normal control group (N group), lipopolysaccaride(LPS) group (L group), mtDNA group, LPS+ mtDNA group (M group), normal control+ negative control adeno-associated virus (AAV-NC)group (NC group), LPS+ mtDNA+ AAV-NC group (MC group), and LPS+ mtDNA+ Sestrin2 overexpression adeno-associated virus (AAV-Sestrin2) group (MSgroup). Another 10 mice were used to detect the transfection effect of AAV-Sestrin2, and the left 6 mice were used for mtDNA extraction. The model of endotoxemia was developed by intraperitoneal injection of LPS 10 mg/kg. mtDNA 5 mg/kg was intraperitoneally injected in mtDNA group, and mtDNA 5 mg/kg was intraperitoneally injected at 30 min after LPS injection in M group.AAV-Sestrin2 150 μl was injected via the tail vein in MS group, and the equal volume of AAV-NC was injected via the tail vein in MC and NC groups. Four weeks after virus injection, LPS 10 mg/kg was intraperitoneally injected and 30 min later mtDNA 5 mg/kg was intraperitoneally injected in MS and MC groups. Blood samples were collected at 24 h after LPS injection for determination of serum creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) activities (by biochemical assay), concentrations of serum cardiac troponin I (cTnI), interleukin-18 (IL-18) and interleukin-1beta (IL-1β)(by enzyme-linked immunesorbent assay), and expression of mtDNA (by quantitative real-time polymerase chain reaction). The animals were sacrificed after the end of blood sampling and myocardial tissues were obtained for determination of the contents of reactive oxygen species (ROS), total antioxidant capacity (T-AOC), and adenosine triphosphate (ATP) and expression of NOD-like receptor associated protein 3 (NLRP3), active subunit p20 of caspase-1 (caspase-1p20) and apoptosis-associated microprotein (ASC) in myocardial tissues (by Western blot) and for microscopic examination of the pathological changes after HE staining (with a light microscope). Results:Compared with N group, the levels of CK-MB, LDH, cTnI, IL-1β and IL-18 in serum were significantly increased, the expression of mtDNA was up-regulated, the ROS content in myocardial tissues was increased, the T-AOC and ATP contents in myocardial tissues were decreased, the expression of NLRP3, caspase-1p20 and ASC in the myocardial tissues was up-regulated( P<0.05), and the pathological changes of myocardial tissues were aggravated in L group and mtDNA group.Compared with L group and mtDNA group, the levels of CK-MB, LDH, cTnI, IL-1β and IL-18 in serum were significantly increased, the expression of mtDNA was up-regulated, the ROS content in myocardial tissues was increased, the T-AOC and ATP contents in myocardial tissues were decreased, the expression of NLRP3, caspase-1p20 and ASC in the myocardial tissues was up-regulated( P<0.05), and the pathological changes of myocardial tissues were aggravated in M group. Compared with M group, the levels of CK-MB, LDH, cTnI, IL-1β and IL-18 in serum were significantly decreased, the expression of mtDNA was down-regulated, the ROS content in myocardial tissues was decreased, the T-AOC and ATP contents in myocardial tissues were increased, the expression of NLRP3, caspase-1p20 and ASC in the myocardial tissues was down-regulated( P<0.05), and the pathological changes of myocardial tissues were significantly attenuated in MS group. Conclusions:Sestrin2 can reduce endotoxin-induced myocardial injury in mice by alleviating mitochondrial damage, inhibiting oxidative stress, protecting mtDNA from oxidative damage, and then inhibiting mtDNA-NLRP3 inflammasome pathway.

Age-related change in mitochondrial DNA copy number and its correlation with intrinsic capacity and body composition / 中华老年医学杂志

Objective:To investigate the correlation of peripheral blood relative mitochondrial DNA copy number(mtDNAcn)with intrinsic capacity and body composition, and to identify potential biomarkers for healthy aging.Methods:Clinical data of 416 patients admitted to our hospital from September 2019 to June 2021 were consecutively collected.MtDNA was extracted from peripheral blood of these subjects, and mtDNAcn was determined by a real-time fluoresence quantitative reverse transcription-polymerase chain reaction(qRT-PCR). Intrinsic capacity assessment included 5 aspects that were exercise[Morse Fall Scale(MFS), Physiological Frailty Phenotype(PFP), Sarcopenia Questionnaire(SARC-CALF), Short Physical Performance Battery(SPPB), Time Up and Go Test(TUG)]; vitality[Mini Nutritional Assessment(MNA), Multidimensional Prognostic Index(MPI)]; cognition[Mini-Mental State Examination(MMSE)scale]; psychology[Geriatric Depression Scale(GDS), Self-rating Anxiety Scale(SAS)]; sensory capacities[Cumulative Illness Rating Scale-the Comorbidity Index(CIRS-CI)]. To assess body composition, dual-energy X-ray absorptiometry was used to measure body fat, including trunk fat, total body fat, fat in the abdominal region, fat in the buttock region, and then to calculate fat index(FMI)and limb skeletal muscle mass index(ASMI).Results:Spearman correlation analysis showed that mtDNAcn had a negatively correlation with age( r=-0.176, P<0.05). After adjustment for gender and body mass index, partial correlation analysis showed mtDNAcn were still negatively correlated with age( r=-0.144, P<0.05). Furthermore, mtDNAcn was significantly correlated with 4 m gait speed, the scores of SARC-CalF, MFS, MNA, MMSE, MPI and its sub-scale's Activities of Daily Living(ADL)and Short Portable Mental Status Questionnaire(SPMSQ)( r=0.171, -0.207, -0.163, 0.221, 0.184, -0.210, 0.241, -0.269, all P<0.05). After adjustment for age, gender and body mass index, partial correlation analysis showed mtDNAcn still had a significant correlation with gait speed, the scores of MFS, MNA, MPI and SPMSQ( r=0.170, -0.170, 0.148, -0.242, -0.188, all P<0.05). In addition, the Spearman correlation analysis showed that mtDNAcn was positively correlated with FMI, trunk fat, total body fat, abdominal fat and fat in the buttock region( r=0.168, 0.143, 0.175, 0.116, 0.199, all P<0.05). However, after adjustment for age and gender, mtDNAcn was only correlated with FMI, total body fat, fat in the buttock region( r=0.126, 0.131, 0.127, all P<0.05). On the other hand, multiple linear regression analysis showed that mtDNAcn was significantly correlated with age, gait speed, FMI, total body fat, fat in the buttock region, the scores of MFS, PFP, MNA and MPI( β=-0.191, 0.156, 0.126, 0.131, 0.125, -0.119, -0.145, 0.151, -0.171, all P<0.05). Conclusions:MtDNAcn is correlated with physical function, frailty, nutrition, falling, cognition and body composition, and may be considered as a biomarker for the evaluation of the locomotion and vitality of human intrinsic capacity.

Heteroplasmy of Wild-Type Mitochondrial DNA Variants in Mice Causes Metabolic Heart Disease With Pulmonary Hypertension and Frailty.

BACKGROUND: In most eukaryotic cells, the mitochondrial DNA (mtDNA) is transmitted uniparentally and present in multiple copies derived from the clonal expansion of maternally inherited mtDNA. All copies are therefore near-identical, or homoplasmic. The presence of >1 mtDNA variant in the same cytoplasm can arise naturally or result from new medical technologies aimed at preventing mitochondrial genetic diseases and improving fertility. The latter is called divergent nonpathologic mtDNA heteroplasmy (DNPH). We hypothesized that DNPH is maladaptive and usually prevented by the cell. METHODS: We engineered and characterized DNPH mice throughout their lifespan using transcriptomic, metabolomic, biochemical, physiologic, and phenotyping techniques. We focused on in vivo imaging techniques for noninvasive assessment of cardiac and pulmonary energy metabolism. RESULTS: We show that DNPH impairs mitochondrial function, with profound consequences in critical tissues that cannot resolve heteroplasmy, particularly cardiac and skeletal muscle. Progressive metabolic stress in these tissues leads to severe pathology in adulthood, including pulmonary hypertension and heart failure, skeletal muscle wasting, frailty, and premature death. Symptom severity is strongly modulated by the nuclear context. CONCLUSIONS: Medical interventions that may generate DNPH should address potential incompatibilities between donor and recipient mtDNA.

Gene therapy for Leber hereditary optic neuropathy / 中华眼底病杂志

Leber hereditary optic neuropathy (LHON) is a matrilineal hereditary optic neuropathy in which mitochondrial DNA mutations lead to retinal ganglion cell degeneration. At present, the treatment for LHON is limited. Early symptomatic treatment and medical treatment may improve the vision of patients. In recent years, rapid progress has been made in gene therapy. Many clinical studies have confirmed its safety and efficacy. Monocular gene therapy is helpful to improve the visual function of LHON patients, and it can also improve the visual acuity of uninjected eyes. Patients do not have serious eye or systemic adverse events during the treatment period, showing good safety and tolerance. Studies with larger sample size and longer follow-up time are needed to further verify the efficacy and safety of gene therapy in the future. Gene therapy is expected to become a safe and effective treatment, bringing hope to LHON patients.

Increased Mitochondrial DNA Copy Number and Oxidative Damage in Patients with Hashimoto's Thyroiditis.

BACKGROUND: The purpose of present study was to investigate mitochondrial DNA copy number (mtDNAcn) and mtDNA damage in peripheral blood of patients with Hashimoto's thyroiditis (HT) and healthy controls (HC). METHODS: The relative mtDNAcn and oxidative DNA damage in this case-control study were measured in peripheral blood of 50 patients with Hashimoto's thyroiditis and 50 healthy controls using quantitative real-time PCR. The study was conducted in Tehran University of Medical Sciences hospital, Tehran, Iran in 2018. RESULTS: HT patients had significantly higher mitochondrial DNA copy number and mitochondrial oxidative damage than the comparison group. CONCLUSION: These data suggest the possible involvement of mitochondria and oxidative stress in the pathophysiology of HT.

Neuromitochondrial Disorders : Genomic Basis and an Algorithmic Approach to Imaging Diagnostics.

Mitochondrial disorders have been an enigma for a long time due to the varied clinical presentations. Although a genetic confirmation will be mandatory most of the time, half the number of Leigh syndrome would be negative for genetic mutations. There are a growing number of mutations in clinical practice, which escape detection on routine clinical exome sequencing. Imaging would render help in pointing towards a mitochondrial disorder. There are a few case reports which brief about specific mitochondrial mutations and their specific imaging appearance. This article tries to provide a comprehensive review on the imaging-genomic correlation of mitochondrial disorders with an objective of performing a specific genetic testing to arrive at an accurate diagnosis.

Extracellular Tuning of Mitochondrial Respiration Leads to Aortic Aneurysm.

BACKGROUND: Marfan syndrome (MFS) is an autosomal dominant disorder of the connective tissue caused by mutations in the FBN1 (fibrillin-1) gene encoding a large glycoprotein in the extracellular matrix called fibrillin-1. The major complication of this connective disorder is the risk to develop thoracic aortic aneurysm. To date, no effective pharmacologic therapies have been identified for the management of thoracic aortic disease and the only options capable of preventing aneurysm rupture are endovascular repair or open surgery. Here, we have studied the role of mitochondrial dysfunction in the progression of thoracic aortic aneurysm and mitochondrial boosting strategies as a potential treatment to managing aortic aneurysms. METHODS: Combining transcriptomics and metabolic analysis of aortas from an MFS mouse model (Fbn1c1039g/+) and MFS patients, we have identified mitochondrial dysfunction alongside with mtDNA depletion as a new hallmark of aortic aneurysm disease in MFS. To demonstrate the importance of mitochondrial decline in the development of aneurysms, we generated a conditional mouse model with mitochondrial dysfunction specifically in vascular smooth muscle cells (VSMC) by conditional depleting Tfam (mitochondrial transcription factor A; Myh11-CreERT2Tfamflox/flox mice). We used a mouse model of MFS to test for drugs that can revert aortic disease by enhancing Tfam levels and mitochondrial respiration. RESULTS: The main canonical pathways highlighted in the transcriptomic analysis in aortas from Fbn1c1039g/+ mice were those related to metabolic function, such as mitochondrial dysfunction. Mitochondrial complexes, whose transcription depends on Tfam and mitochondrial DNA content, were reduced in aortas from young Fbn1c1039g/+ mice. In vitro experiments in Fbn1-silenced VSMCs presented increased lactate production and decreased oxygen consumption. Similar results were found in MFS patients. VSMCs seeded in matrices produced by Fbn1-deficient VSMCs undergo mitochondrial dysfunction. Conditional Tfam-deficient VSMC mice lose their contractile capacity, showed aortic aneurysms, and died prematurely. Restoring mitochondrial metabolism with the NAD precursor nicotinamide riboside rapidly reverses aortic aneurysm in Fbn1c1039g/+ mice. CONCLUSIONS: Mitochondrial function of VSMCs is controlled by the extracellular matrix and drives the development of aortic aneurysm in Marfan syndrome. Targeting vascular metabolism is a new available therapeutic strategy for managing aortic aneurysms associated with genetic disorders.

Mitochondrial DNA Heteroplasmy of Hair Shaft Using HID Ion GeneStudioTM S5 Sequencing System.

ABSTRACT: Objective To study the heteroplasmy of the whole mitochondrial genome genotyping result of hair shaft samples using HID Ion GeneStudioTM S5 Sequencing System. Methods The buccal swabs and blood of 8 unrelated individuals, and hair shaft samples from different parts of the same individual were collected. Amplification of whole mitochondrial genome was performed using Precision ID mtDNA Whole Genome Panel. Analysis and detection of whole mitochondrial genome were carried out using the HID Ion GeneStudioTM S5 Sequencing System. Results The mitochondrial DNA sequences in temporal hair shaft samples from 2 individuals showed heteroplasmy, while whole mitochondrial genome genotyping results of buccal swabs, blood, and hair samples from the other 6 unrelated individuals were consistent. A total of 119 base variations were observed from the 8 unrelated individuals. The numbers of variable sites of the individuals were 29, 40, 38, 35, 13, 36, 40 and 35, respectively. Conclusion Sequence polymorphism can be fully understood using HID Ion GeneStudioTM S5 Sequencing system.

Atlantic Forest Malaria: A Review of More than 20 Years of Epidemiological Investigation.

In the south and southeast regions of Brazil, cases of malaria occur outside the endemic Amazon region near the Atlantic Forest in some coastal states, where Plasmodium vivax is the recognized parasite. Characteristics of cases and vectors, especially Anopheles (Kerteszia) cruzii, raise the hypothesis of a zoonosis with simians as reservoirs. The present review aims to report on investigations of the disease over a 23-year period. Two main sources have provided epidemiological data: the behavior of Anopheles vectors and the genetic and immunological aspects of Plasmodium spp. obtained from humans, Alouatta simians, and Anopheles spp. mosquitoes. Anopheles (K.) cruzii is the most captured species in the forest canopy and is the recognized vector. The similarity between P. vivax and Plasmodium simium and that between Plasmodium malariae and Plasmodium brasilianum shared between simian and human hosts and the involvement of the same vector in the transmission to both hosts suggest interspecies transfer of the parasites. Finally, recent evidence points to the presence of Plasmodium falciparum in a silent cycle, detected only by molecular methods in asymptomatic individuals and An. (K.) cruzii. In the context of malaria elimination, it is paramount to assemble data about transmission in such non-endemic low-incidence areas.

Mitochondrial DNA Heteroplasmy of Hair Shaft Using HID Ion GeneStudioTM S5 Sequencing System / 法医学杂志

Objective To study the heteroplasmy of the whole mitochondrial genome genotyping result of hair shaft samples using HID Ion GeneStudioTM S5 Sequencing System. Methods The buccal swabs and blood of 8 unrelated individuals, and hair shaft samples from different parts of the same individual were collected. Amplification of whole mitochondrial genome was performed using Precision ID mtDNA Whole Genome Panel. Analysis and detection of whole mitochondrial genome were carried out using the HID Ion GeneStudioTM S5 Sequencing System. Results The mitochondrial DNA sequences in temporal hair shaft samples from 2 individuals showed heteroplasmy, while whole mitochondrial genome genotyping results of buccal swabs, blood, and hair samples from the other 6 unrelated individuals were consistent. A total of 119 base variations were observed from the 8 unrelated individuals. The numbers of variable sites of the individuals were 29, 40, 38, 35, 13, 36, 40 and 35, respectively. Conclusion Sequence polymorphism can be fully understood using HID Ion GeneStudioTM S5 Sequencing system.

Research progress in cell and animal models of Leber hereditary optic neuropathy / 中华眼底病杂志

Leber hereditary optic neuropathy (LHON) is a blinding disease caused by mutations in mitochondrial DNA. It is a classic disease model for studying mitochondrial abnormalities. Its main mutation sites are m11778G.A, m.3460G.A and m.14484T.C. LHON cell models are mainly produced by lymphoblasts, fibroblasts, cell hybrids and induced pluripotent stem cells, while LHON animal models are mainly mice, which are produced by rotenone and ND4 mutants. Although the research on the LHON model has achieved good results, there are still many difficulties in constructing an ideal experimental model, which severely limit the exploring to the pathogenesis and therapeutic drugs of LHON. A detailed understanding of the application and characteristics of existing models in LHON will help improve experimental design and construct new models.

Changes in peripheral mitochondrial DNA copy number in metformin-treated women with polycystic ovary syndrome: a longitudinal study.

BACKGROUND: Patients with polycystic ovarian syndrome (PCOS) are associated with known alterations in mitochondria DNA copy number (mtDNA-CN). The aim of this study is to study the change in mtDNA-CN in patients with PCOS who were treated with metformin. METHODS: This is a prospective cohort of patients with PCOS, who received metformin for one year. From 2009 to 2015, 88 women diagnosed with PCOS, based on the Rotterdam criteria, were enrolled. Serial measurements of mtDNA-CN, 8-hydroxydeoxyguanosine (8-OHdG), anthropometric, metabolic, endocrine, and inflammatory markers were obtained before and after 3, 6, and 12 months of treatment. RESULTS: A significant decrease in mtDNA-CN was seen over the course of one year. Other markers, including 8-OHdG, testosterone, free androgen index, blood pressure and liver enzymes, also decreased in the same interval. On regression analysis, there was a significant association between the change in mtDNA-CN and serum total testosterone, and no association between mtDNA-CN and metabolic factors. CONCLUSIONS: Treatment with metformin is associated with a time-dependent decrease in mtDNA-CN in patients with PCOS who are treated over the course of one year. This may signify a reduction in mitochondria dysfunction. The change in mtDNA-CN corresponds to a similar change in serum total testosterone, and suggests a possible relationship between mtDNA-CN and testosterone. TRIAL REGISTRATION: ClinicalTrials.gov , NCT00172523 . Registered September 15, 2005.

The heart in m.3243A>G carriers.

OBJECTIVES: Little is known about cardiac involvement in m.3243A>G variant carriers. Thus, this study aimed to assess type and frequency of cardiac disease in symptomatic and asymptomatic m.3243A>G carriers. METHODS: Systematic literature review. RESULTS: The m.3243A>G variant may manifest phenotypically as mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), maternally inherited diabetes and deafness (MIDD), myoclonic epilepsy with ragged red fiber (MERRF), Leigh syndrome, or MELAS/KSS (Kearns-Sayre syndrome) overlap. Only few systematic studies which prospectively investigated m.3243A>G carriers for cardiac involvement were found. Cardiac abnormalities reported in m.3243A>G carriers include myocardial abnormalities, arrhythmias, or conduction defects. Myocardial abnormalities include myocardial thickening, hypertrophic cardiomyopathy, dilated cardiomyopathy, noncompaction, myocardial fibrosis, systolic dysfunction, heart failure, or arterial hypertension. Arrhythmias reported in m.3243A>G carriers include paroxysmal supraventricular or ventricular arrhythmias, including sinus tachycardia, atrial fibrillation and nonsustained ventricular tachycardia, and sudden cardiac death. Conduction defects in this group of patients include Wolff-Parkinson-White syndrome and left/right bundle branch block. Asymptomatic m.3243A>G carriers usually do not develop clinical or subclinical cardiac disease. CONCLUSIONS: Cardiac involvement in m.3243A>G carriers has been only rarely systematically studied, which is perhaps why the incidence of cardiac diseases in MELAS is lower than would be expected. Myocardial abnormalities are much more frequent than arrhythmias or conduction defects. All symptomatic and asymptomatic m.3243A>G carriers should be systematically investigated for cardiac disease.

Characterization of mitochondrial COX-1 gene of Sarcoptes scabiei from rabbits in East Java, Indonesia.

OBJECTIVE: The purpose of this study was to characterize the mitochondrial COX-1 gene of Sarcoptes scabiei in rabbits from three districts of Malang, Nganjuk, and Kediri, East Java, Indonesia. The gene was aligned with a DNA isolated from S. scabiei of Chong'qing rabbit (accession number: EU256388.1) to construct a molecular analysis of phylogenetic in S. scabiei COX-1 gene. MATERIALS AND METHODS: This study has been verified by the Committee Ethics (Faculty of Veterinary Medicine, Universitas Airlangga). The mites were collected and identified from rabbits that have an indication of scabies infection. DNA was extracted with QIAamp DNA mini kit and polymerase chain reaction (PCR) analysis was done. The PCR products were purified with the protocol of the BigDye XTerminator™ Purification Kit (Thermo Scientific) and were double-sequenced with the forward and reverse PCR primers of ABI PRISM 310 Genetic Analyzer. The sequence product was confirmed with Clone Manager Professional 9 (Sci-Ed Software) and the Neighbor-Joining method was done with MEGA6 to build a phylogenetic tree. RESULTS: The target product of DNA amplification in this PCR was around 290-bp. The amplicon was visualized in 2% of agarose gel electrophoresis. The homology analysis of these sequences showed that it had more than 99% similarity. CONCLUSION: COX-1 gene sequences of S. scabiei from rabbits in Malang, Nganjuk, and Kediri were very similar to COX-1 gene sequences in S. scabiei acquired from several hosts according to NCBI data.

Application of Multiple Genetic Markers in Determination of Full and Half Sibling Relationship： A Case Report.

ABSTRACT: Objective To investigate the application of the comprehensive use of multiple genetic markers in full and half sibling relationship testing through the identification of a case of suspected sibling relationship. Methods Genomic DNA were extracted from bloodstain samples from 4 subjects （ZHANG-1, ZHANG-2, male; ZHANG-3, ZHANG-4, female）. Autosomal STR loci, X-STR, Y-STR loci and polymorphisms of mtDNA HV-Ⅰ and Ⅱwere genotyped by EX20 STR kit, X19 kit, Data Y24 STR kit, and Sanger sequencing, respectively. Results According to autosomal STR based IBS scoring results, full sibling relationships were indicated among ZHANG-2, ZHANG-3 and ZHANG-4, but those were not indicated between ZHANG-1 and ZHANG-2 or ZHANG-3 or ZHANG-4. According to autosomal STR based FSI and HSI, with ITO method and discriminant function method, full sibling relationships among ZHANG-2, ZHANG-3 and ZHANG-4 were indicated, and half sibling relationships between ZHANG-1 and ZHANG-2 or ZHANG-3 or ZHANG-4 were also indicated. X-STR and mtDNA sequencing results showed that all the 4 samples came from a same maternal line, and Y-STR results showed that ZHANG-1 and ZHANG-2 did not come from a same paternal line, which supported the half sibling relationship between ZHANG-1 and ZHANG-2 or ZHANG-3 or ZHANG-4, verified by parental genotype reconstruction based on autosomal STR genotyping. Conclusion For the identification of sibling relationships, it is effective to have reliable results with the mutual verification and support of multiple genetic markers （autosomal STR, sex chromosomal STR and mtDNA sequence） and calculations （IBS, ITO, discriminant function method and family reconstruction）.