mtDNA Deletions Research Articles

Fibroblast growth factor 21 and grow differentiation factor 15 are sensitive biomarkers of mitochondrial diseases due to mitochondrial transfer-RNA mutations and mitochondrial DNA deletions.

Diagnosis of mitochondrial diseases (MDs) is challenging, since they are multisystemic disorders, characterized by a heterogeneous symptomatology. Recently, an increase in serum levels of fibroblast growth factor 21 (FGF21) and growth differentiation factor 15 (GDF15) has been found in the majority of patients with MDs compared with healthy controls. On the other hand, the finding of low FGF21 and GDF15 levels in some patients with MDs suggests that different types of respiratory chain defects may lead to different profiles of these two proteins. In this study, we aimed to validate the diagnostic reliability of FGF21 and GDF15 assays in MDs and to evaluate a possible correlation between serum levels of the two biomarkers with genotype of MD patients. Serum FGF21 and GDF15 levels were measured by a quantitative ELISA. Our results showed increased serum FGF21 and GDF15 levels in MD patients; however, GDF15 measurement seems to be more sensitive and specific for screening tests for MD than FGF21. Moreover, we showed a positive correlation with both FGF21 and GDF15 levels and the number of COX-negative fibers. Finally, we also demonstrated that the increase of FGF21 and GDF15 was related to MDs caused by mitochondrial translation defects, and multiple and single mtDNA deletions, but not to MDs due to mutations in the respiratory chain subunits.

The Essential History of a Patient with Pearson Marrow, a Case Report

Pearson syndrome is a rare mitochondrial disorder confirmed by mt-DNA deletion which typically occurs in the first two years of life. That is to say children are at high fatal risk, most infants are marked with some common features especially anemia and pancreatitis, which results in death in early childhood. A 6-month-old Iranian female infant was presented with macrocytic anemia, required packed red blood cell transfusions. She also was affected by exocrine pancreatic dysfunction, in which she underwent Creon treatment.By first year of age she had experienced some severe metabolic crises intermittently. After hospitalized for some months she was expired unfortunately. In conclusion, Pearson syndrome, as a rare disease affects many organs, such as liver, kidney, pancreas, bone marrow, which led to anemia, failure to thrive, and multi organ failure. In such cases, a physician must consider and evaluate all possible damages, especially anemia and pancreatitis. We present a case of Pearson syndrome with anemiaKeywords: Mitochondrial disorders; Pearson syndrome; Pancreatitis; Acidosis; Infants.

OR03-04 The Study of Cell Senescence in Cortisol-Producing Adrenocortical Adenomas

Introduction Aging is associated with the pathogenesis of many endocrine disorders such as cardiovascular diseases and diabetes. Cell senescence has been reported as one of their mechanisms. In addition, stress responsiveness has been reported to be associated with cell senescence. In addition, some genetic abnormalities such as mitochondrial DNA (mtDNA) damages or telomere shortening, have been detected in some endocrine disorders. Cortisol is a well-known stress-induced hormone and closely associated with aging. We previously reported that cortisol-producing adenoma (CPA) more abundantly expressed cell senescent markers such as p16 and p21 than other hormone-producing adrenocortical adenomas. However, the detailed pathophysiology of cell senescence and its association with histological features in CPAs have remained virtually unknown. Therefore, we analyzed cell senescent markers (telomere length, mtDNA copy number, mtDNA deletion and p16 and p21 immunoreactivity) and analyzed their correlation with clinicopathological factors in CPA patients. Methods & Materials Forty CPA cases was immunohistochemically evaluated. Twenty CPA, ten adjacent ZF and six non-functional adenoma (NFA) were examined for mtDNA abnormalities. mtDNA deletion was evaluated by nested-PCR and mtDNA copy number and telomere length were measured using real-time PCR. Results p21 immunoreactivity was significantly higher in CPA than that of adjacent ZF (P=0.0001) and significantly inversely correlated with tumor size (P=0.0004). Telomere length was much longer in CPA than that in adjacent ZF(P=0.0038), and NFA (P=0.0018). mtDNA copy number of NFA was significantly higher than that of CPA and adjacent ZF (P=0.0038). mtDNA copy number of compact cells was significantly higher than that of clear cells (P=0.0432). mtDNA copy number of compact cells was positively correlated with urinary free cortisol (UFC) (P=0.0428) and plasma cortisol (F) (P=0.0609). mtDNA copy number of clear cells were inversely correlated with F (0.0497). 4977 bp mtDNA deletion was more frequently detected in CPA (54%) and in adjacent ZF (50%) than in NFA (17%). Discussion Results of our present study did reveal that CPA harbored more senescent phenotype as demonstrated by abundant p16 and p21, marked telomere shortening, frequent mtDNA 4977bp deletion and relatively low mtDNA copy number, possibly caused by long-term exposure of excessive cortisol in situ compared to NFA. In addition, clear tumor cells could represent more senescent histological phenotype because of their lower mtDNA copy numbers. This is the first study to demonstrate that compact tumor cells were biologically more active than clear tumor cells and could reflect clinical cortisol biosynthesis, resulting in marked functional intratumoral heterogeneity in CPAs.

Double Trouble from POLG1 and CLCN1 Variants with Intrafamilial Phenotypic Heterogeneity.

I read with interest the article by Pauly and colleagues1 about a family in which the father carried the variants c.844T>G in POLG1 and c.937G>A in CLCN1. The POLG1 variant manifested with ptosis, ophthalmoparesis, parkinsonism, and depression, and the CLCN1 variant with myotonia (myoclonic twitches and muscle cramping triggered by rapid movements or sneezing) and muscle hypertrophy.1 All of his 3 children carried his POLG1 variant and 1 the CLCN1 variant, but only 2 manifested clinically.1 The study has a number of shortcomings. Missing are the results of cerebral magnetic resonance imaging and the electroencephalogram. “Twitching” and “cramping” may not only be attributable to the CLCN1 variant but also to the POLG1 variant.2 POLG1 variants may manifest with myoclonic epilepsy, which needs to be excluded as it requires a completely different therapy. This is of relevance as the deceased daughter had Alpers-Huttenlocher disease, thus intractable epilepsy. Because “twitching” was triggerable,1 we should know if the clinical phenomenon correlated with the occurrence of myotonic discharges on needle electromyography. Because the father and his son both had myopathy of the extraocular eye muscles and POLG1 variants can also manifest with myopathy of the skeletal muscles,3 we should know if the father or his son had undergone muscle biopsy to see if features for a mitochondrial disorder (MID) were present or not. Missing are results of mitochondrial DNA (mtDNA) investigations. Because POLG1 variants may secondarily cause mtDNA depletion or multiple mtDNA deletions,4 we should know if the amount of mtDNA was reduced or if there were multiple mtDNA deletions. There is no explanation provided why the second girl did not manifest clinically from the POLG1 variant. Because MIDs frequently manifest subclinically, we should know if the girl was investigated for subclinical involvement of the brain, eyes, ears, endocrine organs, heart, intestines, or kidneys. Early detection of clinical manifestations is crucial as early treatment may improve the outcome in these patients. Assuming that the girl did not even manifest subclinically, the missing penetrance of the variant should be explained. It is conceivable that secondary damage of mtDNA was not present in this girl but only in her sister and brother. It is also not discussed why the mother of the 3 children who carried the POLG1 variant c.1399GA>A did not manifest clinically. Cardiac complications may not only derive from the CLCN1 variant but also from the POLG1 variant.5 Because cardiac involvement in MIDs strongly determines the outcome of these patients, it is crucial that they undergo regular follow-up investigations and long-term electrocardiogram recordings. If ventricular arrhythmias are recorded, implantation of an implantable cardioverter-defibrillator may strongly improve the survival and outcome of these patients. Cardiac surveillance is crucial in the light of the side effects of flecainide. It should be mentioned that, among the syndromic MIDs, POLG1 variants can also cause Leigh syndrome; mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes; myoclonic epilepsy with ragged-red fibers; myoclonic epilepsy myopathy sensory ataxia; sensory ataxic neuropathy, dysarthria, ophthalmoparesis; and chronic progressive external ophthalmoplegia syndrome. Overall, this study has a number of shortcomings that should be addressed and considered for modifying the conclusions. Genetic findings need to be related to the clinical presentation, and factors determining phenotypic variability need to be discussed. We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this work is consistent with those guidelines. Informed patient consent and the approval of an institutional review board was not necessary for this work. There are no conflicts of interest and no funding was received for this work. There are no disclosures to report.

Essential respiratory chain subunits encoded by mitochondrial DNA control plasma cell development

Abstract The contribution of mitochondrial metabolism to plasma cell development is largely unknown. To define the function of the mitochondrial respiratory chain during B cell activation and differentiation, we used a mouse model in which CD23-Cre drives the expression of a dominant-negative mutant of the mitochondrial helicase Twinkle (DNTwinkle) and the reporter GFP in mature B cells. DNTwinkle expression induces mitochondrial DNA (mtDNA) deletions in dividing cells and results in impaired respiratory chain activity. Flow cytometry in non-immunized DNTwinkle mice did not reveal a difference in either total cell numbers or frequencies of mature B cell subsets. However, only about 8% of the plasma cells in the bone marrow were positive for DNTwinkle/GFP, indicating that these cells have a significant disadvantage in the presence of wildtype plasma cells. T-dependent immunization showed a decreased primary immune response, whereas the secondary immune response was not affected. Strikingly, in immunized mice, all antigen-specific plasma cells in the bone marrow were GFP-negative, again highlighting the advantage of escaper cells. Comparing to in vitro stimulated wild-type B cells, DNTwinkle-positive splenic B cells showed comparable survival, but proliferation, Ig class switch, and plasmablast differentiation were impaired. Also, OxPhos was reduced, but glycolysis and lactate secretion were increased. Finally, AMPK activation was enhanced along with reduced mTOR activity, which could explain the observed impaired Blimp-1 expression and plasmablast differentiation of DNTwinkle B cells. Our results place mitochondrial DNA replication and respiratory chain activity upstream of mTOR and Blimp-1 via AMPK.

Joint effects of mitochondrial DNA4977 deletion and serum folate deficiency on coronary artery disease in type 2 diabetes mellitus.

The 4977-bp mitochondrial deletion (mtDNA4977 deletion), as a hallmark of mitochondrial oxidative damage, may play an important role in coronary artery disease (CAD), but its interaction with folate deficiency among diabetic patients is largely unknown. We aimed to explore the joint association of leukocyte mtDNA4977 deletion and serum folate status with obstructive CAD in Chinese adults with type 2 diabetes. We cross-sectionally analyzed the angiographic data of 2017 diabetic patients without B-vitamin supplementation. Of the 2017 participants, 756 who received percutaneous coronary intervention (PCI) completed prospective follow-up of one year. Invitro, we explored the mediation effects of mitochondrial reactive oxygen species (mtROS) in folic acid (FA)-deficient human aortic smooth muscle cells (HASMCs) under hyperglycemic conditions. Cross-sectionally, the multivariate odds ratios (ORs) for obstructive CAD were 1.41 (95% CI: 1.29-1.55) for greater mtDNA4977 deletion, and 1.15 (95% CI: 1.05-1.25) for lower folate levels. Particularly, the combination of high mtDNA4977 deletion (top tertile) and folate deficiency (serum folate<6ng/mL) was associated with more than 2-fold increased odds of having obstructive CAD and higher degrees of coronary stenosis. Prospectively, the hazard ratio for all-cause death at 1-year after PCI was up to 2.37 (95% CI: 1.21-4.63) for folate-deficient participants in the top tertile of mtDNA4977 deletion. In HASMCs, the adverse effects of FA deficiency were aggravated by induction of mtROS, and attenuated by scavenging of mtROS. The risk of obstructive CAD may be greatly increased by the interaction between greater mtDNA4977 deletion and folate deficiency among diabetic patients.

Mitochondrial DNA content and deletion ratio are associated with metabolic syndrome in a general population exposed to pesticide

Mitochondrial DNA (mtDNA) copy number and deletion ratio may be asymmetrically determined using physiologic or pathologic conditions. Mitochondrial dysfunction is associated with an increased risk of developing metabolic syndrome (MetS). Here, we investigated the association between mtDNA copy number and/or deletion ratio and the risk of developing MetS in a general population exposed to the pesticide. We examined 215 randomly sampled adults who were exposed to pesticide but did not present with MetS in a prospective cohort study. Both mtDNA copy number and deletion ratio were determined using quantitative real-time polymerase chain reaction. During the average 2.8-year follow-up period, 76 (35.3%) participants developed new-onset MetS. The numbers of mtDNA copies were significantly lower in participants with new-onset MetS than in those without MetS (112.15 ± 26.15 vs. 133.13 ± 44.44, p < 0.001), whereas the mtDNA deletion ratios were higher in participants with MetS than in those without MetS (10.59 ± 11.96 vs. 4.52 ± 6.74, p < 0.001). In the multivariate-adjusted models, the participants with a higher tertile of mtDNA copy number were 0.355 times more likely to develop MetS than those with the lowest tertile [odds ratio (OR) 0.355, 95% confidence interval (CI) 0.150–0.841, p = 0.004]. A higher mtDNA deletion ratio was significantly associated with the risk of developing MetS in populations exposed to pesticide (OR 5.062, 95% CI 1.164–22.004, p = 0.004). A lower mtDNA copy number and higher deletion ratio were independent predictors for new-onset MetS in a general population exposed to the pesticide.

Heart failure after pressure overload in autosomal-dominant desminopathies: Lessons from heterozygous DES-p.R349P knock-in mice

BackgroundMutations in the human desmin gene (DES) cause autosomal-dominant and -recessive cardiomyopathies, leading to heart failure, arrhythmias, and AV blocks. We analyzed the effects of vascular pressure overload in a patient-mimicking p.R349P desmin knock-in mouse model that harbors the orthologue of the frequent human DES missense mutation p.R350P.Methods and resultsTransverse aortic constriction (TAC) was performed on heterozygous (HET) DES-p.R349P mice and wild-type (WT) littermates. Echocardiography demonstrated reduced left ventricular ejection fraction in HET-TAC (WT-sham: 69.5 ± 2.9%, HET-sham: 64.5 ± 4.7%, WT-TAC: 63.5 ± 4.9%, HET-TAC: 55.7 ± 5.4%; p<0.01). Cardiac output was significantly reduced in HET-TAC (WT sham: 13088 ± 2385 μl/min, HET sham: 10391 ± 1349μl/min, WT-TAC: 8097 ± 1903μl/min, HET-TAC: 5793 ± 2517μl/min; p<0.01). Incidence and duration of AV blocks as well as the probability to induce ventricular tachycardias was highest in HET-TAC. We observed reduced mtDNA copy numbers in HET-TAC (WT-sham: 12546 ± 406, HET-sham: 13526 ± 781, WT-TAC: 11155 ± 3315, HET-TAC: 8649 ± 1582; p = 0.025), but no mtDNA deletions. The activity of respiratory chain complexes I and IV showed the greatest reductions in HET-TAC.ConclusionPressure overload in HET mice aggravated the clinical phenotype of cardiomyopathy and resulted in mitochondrial dysfunction. Preventive avoidance of pressure overload/arterial hypertension in desminopathy patients might represent a crucial therapeutic measure.

Deep sequencing of mitochondrial DNA and characterization of a novel POLG mutation in a patient with arPEO.

ObjectiveTo determine the pathogenicity of a novel POLG mutation in a man with late-onset autosomal recessive progressive external ophthalmoplegia using clinical, molecular, and biochemical analyses.MethodsA multipronged approach with detailed neurologic examinations, muscle biopsy analyses, molecular genetic studies, and in vitro biochemical characterization.ResultsThe patient had slowly progressive bilateral ptosis and severely reduced horizontal and vertical gaze. Muscle biopsy showed slight variability in muscle fiber size, scattered ragged red fibers, and partial cytochrome c oxidase deficiency. Biallelic mutations were identified in the POLG gene encoding the catalytic A subunit of POLγ. One allele carried a novel mutation in the exonuclease domain (c.590T>C; p.F197S), and the other had a previously characterized null mutation in the polymerase domain (c.2740A>C; p.T914P). Biochemical characterization revealed that the novel F197S mutant protein had reduced exonuclease and DNA polymerase activities and confirmed that T914P was inactive. By deep sequencing of mitochondrial DNA (mtDNA) extracted from muscle, multiple large-scale rearrangements were mapped and quantified.ConclusionsThe patient's phenotype was caused by biallelic POLG mutations, resulting in one inactive POLγA protein (T914P) and one with decreased polymerase and exonuclease activity (F197S). The reduction in polymerase activity explains the presence of multiple pathogenic large-scale deletions in the patient's mtDNA.

Mitochondrial DNA Variation and Selfish Propagation Following Experimental Bottlenecking in Two Distantly Related Caenorhabditis briggsae Isolates.

Understanding mitochondrial DNA (mtDNA) evolution and inheritance has broad implications for animal speciation and human disease models. However, few natural models exist that can simultaneously represent mtDNA transmission bias, mutation, and copy number variation. Certain isolates of the nematode Caenorhabditis briggsae harbor large, naturally-occurring mtDNA deletions of several hundred basepairs affecting the NADH dehydrogenase subunit 5 (nduo-5) gene that can be functionally detrimental. These deletion variants can behave as selfish DNA elements under genetic drift conditions, but whether all of these large deletion variants are transmitted in the same preferential manner remains unclear. In addition, the degree to which transgenerational mtDNA evolution profiles are shared between isolates that differ in their propensity to accumulate the nduo-5 deletion is also unclear. We address these knowledge gaps by experimentally bottlenecking two isolates of C. briggsae with different nduo-5 deletion frequencies for up to 50 generations and performing total DNA sequencing to identify mtDNA variation. We observed multiple mutation profile differences and similarities between C. briggsae isolates, a potentially species-specific pattern of copy number dysregulation, and some evidence for genetic hitchhiking in the deletion-bearing isolate. Our results further support C. briggsae as a practical model for characterizing naturally-occurring mtgenome variation and contribute to the understanding of how mtgenome variation persists in animal populations and how it presents in mitochondrial disease states.

Mitochondrial DNA damage in the hair bulb: can it be used as a noninvasive biomarker of local exposure to low LET ionizing radiation?

Purpose: Our aim was to evaluate whether mitochondrial DNA (mtDNA) damage in hair bulbs could be a suitable biomarker for the detection of local exposure to ionizing radiation.Materials and methods: Mouse hair was collected 4 and 24 hours, 3 and 10 days after single whole-body exposure to 0, 0.1, and 2 Gy radiation. Pubic hair (treated area) and scalp hair (control area) were collected from 13 prostate cancer patients before and after fractioned radiotherapy with an average total dose of 2.7 Gy to follicles after five fractions. Unspecified lesion frequency of mtDNA was analyzed with long PCR, large mtDNA deletion levels were tested with real-time PCR.Results: Unspecified lesion frequency of mtDNA significantly increased in mouse hair 24 hours after irradiation with 2 Gy, but variance among samples was high. No increase in lesion frequency could be detected after 0.1 Gy irradiation. In prostate cancer patients, there was no significant change in either the unspecified lesion frequency or in the proportion of 4934-bp deleted mtDNA in pubic hair after radiotherapy. The proportions of murine 3860-bp common deletion, human 4977-bp common deletion and 7455-bp deleted mtDNA were too low to be analyzed reliably.Conclusions: Our results suggest that the unspecified lesion frequency and proportion of large deletions of mtDNA in hair bulbs are not suitable biomarkers of exposure to ionizing radiation.

Roles of the mitochondrial replisome in mitochondrial DNA deletion formation.

Mitochondrial DNA (mtDNA) deletions are a common cause of human mitochondrial diseases. Mutations in the genes encoding components of the mitochondrial replisome, such as DNA polymerase gamma (Pol γ) and the mtDNA helicase Twinkle, have been associated with the accumulation of such deletions and the development of pathological conditions in humans. Recently, we demonstrated that changes in the level of wild-type Twinkle promote mtDNA deletions, which implies that not only mutations in, but also dysregulation of the stoichiometry between the replisome components is potentially pathogenic. The mechanism(s) by which alterations to the replisome function generate mtDNA deletions is(are) currently under debate. It is commonly accepted that stalling of the replication fork at sites likely to form secondary structures precedes the deletion formation. The secondary structural elements can be bypassed by the replication-slippage mechanism. Otherwise, stalling of the replication fork can generate single- and double-strand breaks, which can be repaired through recombination leading to the elimination of segments between the recombination sites. Here, we discuss aberrances of the replisome in the context of the two debated outcomes, and suggest new mechanistic explanations based on replication restart and template switching that could account for all the deletion types reported for patients.

Broadening the phenotypic spectrum of Pearson syndrome: Five new cases and a review of the literature.

Pearson syndrome (PS) is a multisystem mitochondrial respiratory chain disorder typically characterized by sideroblastic anemia and exocrine pancreatic insufficiency. PS is caused by a single large-scale mitochondrial DNA (mtDNA) deletion. PS classically presents in the first year of life and may be fatal in infancy. Children who survive PS may progress to develop Kearns-Sayre syndrome later in life. The full phenotypic spectrum and prognosis of the condition continue to evolve. Here we report five new patients with PS with unique clinical presentations, including four patients with onset later than previously reported in the literature, and one patient with prenatal onset of symptoms. The timing and unique features of these presentations support an expanded phenotypic spectrum of single large-scale mtDNA deletion syndromes (SLSMDS) and reinforce the importance of including SLSMDS in the differential for children with complex multisystem presentations.

Healthy, mtDNA-mutation\xa0free mesoangioblasts from mtDNA patients qualify for autologous therapy

BackgroundMyopathy and exercise intolerance are prominent clinical features in carriers of a point-mutation or large-scale deletion in the mitochondrial DNA (mtDNA). In the majority of patients, the mtDNA mutation is heteroplasmic with varying mutation loads between tissues of an individual. Exercise-induced muscle regeneration has been shown to be beneficial in some mtDNA mutation carriers, but is often not feasible for this patient group. In this study, we performed in vitro analysis of mesoangioblasts from mtDNA mutation carriers to assess their potential to be used as source for autologous myogenic cell therapy.MethodsWe assessed the heteroplasmy level of patient-derived mesoangioblasts, isolated from skeletal muscle of multiple carriers of different mtDNA point-mutations (n = 25). Mesoangioblast cultures with < 10% mtDNA mutation were further analyzed with respect to immunophenotype, proliferation capacity, in vitro myogenic differentiation potential, mitochondrial function, and mtDNA quantity.ResultsThis study demonstrated that mesoangioblasts in half of the patients contained no or a very low mutation load (< 10%), despite a much higher mutation load in their skeletal muscle. Moreover, none of the large-scale mtDNA deletion carriers displayed the deletion in mesoangioblasts, despite high percentages in skeletal muscle. The mesoangioblasts with no or a very low mutation load (< 10%) displayed normal mitochondrial function, proliferative capacity, and myogenic differentiation capacity.ConclusionsOur data demonstrates that in half of the mtDNA mutation carriers, their mesoangioblasts are (nearly) mutation free and can potentially be used as source for autologous cell therapy for generation of new muscle fibers without mtDNA mutation and normal mitochondrial function.

Broadening the phenotype of the TWNK gene associated Perrault syndrome

BackgroundPerrault syndrome is a genetically heterogenous, very rare disease, characterized clinically by sensorineural hearing loss, ovarian dysfunction and neurological symptoms. We present the case of a 33 years old female patient with TWNK-associated Perrault syndrome. The TWNK gene is coding the mitochondrial protein Twinkle and currently there are only two reports characterizing the phenotype of TWNK-associated Perrault syndrome. None of these publications reported about special brain MRI alterations and neuropathological changes in the muscle and peripheral nerves.Case presentationOur patients with TWNK-dependent Perrault syndrome had severe bilateral hypoacusis, severe ataxia, polyneuropathy, lower limb spastic paraparesis with pyramidal signs, and gonadal dysgenesis. Psychiatric symptoms such as depression and paranoia were present as well. Brain MRI observed progressive cerebellar hyperintensive signs associated with cerebellar, medulla oblongata and cervical spinal cord atrophy. Light microscopy of the muscle biopsy detected severe neurogenic lesions. COX staining was centrally reduced in many muscle fibers. Both muscle and sural nerve electron microscopy detected slightly enlarged mitochondria with abnormal cristae surrounded by lipid vacuoles. In the sural nerve, dystrophic axons had focally uncompacted myelin lamellae present. Genetic investigation revealed multiple mtDNA deletion and compound heterozygous mutations of the TWNK gene (c.1196 A > G, c.1358 G > A).ConclusionThis study demonstrates that TWNK associated Perrault syndrome has a much broader phenotype as originally published. The coexistence of severe hypoacusis, spastic limb weakness, ataxia, polyneuropathy, gonadal dysgensia, hyperintense signals in the cerebellum and the presence of the mtDNA multiple deletion could indicate the impairment of the TWNK gene. This is the first report about pyramidal tract involvement and cerebellar MRI alteration associated with TWNK-related Perrault syndrome.

A novel 8.7-kb mitochondrial genome deletion accurately detects endometriosis in the plasma of symptomatic women.

Aim: To evaluate an 8.7-kb mitochondrial DNA (mtDNA) deletion as a potential biomarker of endometriosis. Materials& methods: We tested the diagnostic accuracy of the 8.7-kb deletion real-time PCR assay using 182 prospectively collected blood samples from females presenting with symptoms of endometriosis in a case-control format. Results: The assay differentiated between endometriosis and controls (area under curve: 0.74-0.89) with a statistically significant difference (p<0.05) in 8.7-kb deletion levels measured for all disease subtypes and stages. No correlation was seen between 8.7-kb deletion levels and participant or specimen age, hormone status or menstrual phase. Conclusion: The diagnostic accuracy of the 8.7-kb deletion for endometriosis suggests potential utility in the clinic to improve patient management.

4,977-bp human mitochondrial DNA deletion is associated with asthenozoospermic infertility in Jordan.

Male infertility is commonly associated with sperm abnormalities including asthenozoospermia. The molecular basis of asthenozoospermia was linked to mitochondrial DNA (mtDNA) mutations. The 4,977-bp human mtDNA deletion is one of the most common mutations of spermatozoa and results in loss of about 33% of the mitochondrial genome. In this preliminary study, we aimed to investigate the presence of 4,977-bp mtDNA deletion in asthenozoospermic infertile men in Jordan. Semen specimens of 120 asthenozoospermic infertile men and 80 normozoospermic individuals were collected at the in vitro fertilization unit. MtDNA was extracted after the enrichment of spermatozoa; then, polymerase chain reaction was performed using 4,977-bp mtDNA deletion-specific primers. The deletion of 4,977-bp mtDNA was detected in 79.2% of asthenozoospermic patients compared to 10% in normozoospermic controls. The results showed a significant association between the presence of 4,977-bp mtDNA deletion and the asthenozoospermia and infertility (OR=34.2000, 95% CI=14.57-80.26, p-value<.001). In conclusion, our findings underscored a strong association between 4,977-bp mtDNA deletion and asthenozoospermia in the Jordanian population.

Independent and Combined Effects of Telomere Shortening and mtDNA4977 Deletion on Long-term Outcomes of Patients with Coronary Artery Disease.

Aging is one of the main risk factors for cardiovascular disease, resulting in a progressive organ and cell decline. This study evaluated a possible joint impact of two emerging hallmarks of aging, leucocyte telomere length (LTL) and common mitochondrial DNA deletion (mtDNA4977), on major adverse cardiovascular events (MACEs) and all-cause mortality in patients with coronary artery disease (CAD). We studied 770 patients (673 males, 64.8 ± 8.3 years) with known or suspected stable CAD. LTL and mtDNA4977 deletion were assessed in peripheral blood using qRT-PCR. During a median follow-up of 5.4 ± 1.2 years, MACEs were 140 while 86 deaths were recorded. After adjustments for confounding risk factors, short LTLs and high mtDNA4977 deletion levels acted independently as predictors of MACEs (HR: 2.2, 95% CI: 1.2–3.9, p = 0.01 and HR: 1.7, 95% CI: 1.1–2.9, p = 0.04; respectively) and all-cause mortality events (HR: 2.1, 95% CI: 1.1–4.6, p = 0.04 and HR: 2.3, 95% CI: 1.1–4.9, p = 0.02; respectively). Patients with both short LTLs and high mtDNA4977 deletion levels had an increased risk for MACEs (HR: 4.3; 95% CI: 1.9–9.6; p = 0.0006) and all-cause mortality (HR: 6.0; 95% CI: 2.0–18.4; p = 0.001). The addition of mtDNA4977 deletion to a clinical reference model was associated with a significant net reclassification improvement (NRI = 0.18, p = 0.01). Short LTL and high mtDNA4977 deletion showed independent and joint predictive value on adverse cardiovascular outcomes and all-cause mortality in patients with CAD. These findings strongly support the importance of evaluating biomarkers of physiological/biological age, which can predict disease risk and mortality more accurately than chronological age.

Large-single scale mitochondrial DNA deletions in different tissues in Kearns-Sayre syndrome

Objective To investigate the clinical significance of different samples (the peripheral blood, urine and skeletal muscle) that could be detected the large-scale single deletions directly by using next-generation sequencing in the diagnosis of Kearns-Sayre syndrome (KSS) by concluding the clinical and genetic features of KSS, in order to explore a non-invasive method for diagnosis. Methods The clinical data, skeletal muscle′s pathology and enzymology and genetic results of individuals with KSS, who were hospitalized from October 2016 to October 2017 in Department of Neurology, Beijing Children′s Hospital, Capital Medical University, were collected.The gene tests were performed by using next generation sequencing technology and long-PCR technology of mitochondrial DNA(mtDNA) and the whole exon in the peripheral blood, urine and skeletal muscle. Results Four patients were all consistent with the diagnosis criteria of KSS, among whom the age of onset was 8.2 years old on average, and the initial symptoms were statue, ptosis, headache and vomiting, and visual impairment.The common symptoms of the 4 cases were ophthalmoplegia, exercise intolerance, development delay, loss of appetite, hypotonia, muscle weakness, with cerebrospinal fluid protein concentration over 1 000 mg/L, the cerebral magnetic resonance imaging showed that abnormal signals in the brainstem, in addition, white matter, thalamus, basal ganglia, cerebrum and cerebellum atrophy could be found.Moreover, 3 cases had cardiac conduction block.Two cases had maternal family history.Molecular analysis of the 4 cases revealed the large-scale single deletions of mtDNA from the peripheral blood, the urine, the skeletal muscle through the next-generation sequencing, which were m. 6460-15590(9 131 bp del), m.8482-13446(4 964 bp del), m.6831-14981(8 151 bp del), m.7983-15495(7 513 bp del), respectively.Among 3 cases who did pedigree analysis, only the mother of case 4 was detected with the same variation of the proband. Conclusions KSS is a rare mitochondrial disease, which could be detected with the single large scale mtDNA deletions in the peripheral blood, urine and skeletal muscle.With the development of the methodology, the diagnosis of KSS maybe no longer than depends on the muscle biopsy with the next-generation sequencing.And the possibility to get the positive results in the peripheral blood and urine by the non-invasive method could improve the molecular diagnosis of KSS. Key words: Kearns-Sayre syndrome; Large-single scale mtDNA deletions; Next generation sequencing; Peripheral blood; Urine

NEXT GENERATION SEQUENCING: P.292A new method for mtDNA deletion detection using massively parallel sequencing

Human mitochondrial genome (mtDNA) is a circular DNA of 16 Mb in size. Mutations on mtDNA can cause several inherited diseases, many of which are neuromuscular. The mutational rate compared to nuclear DNA is very high. Clinically relevant mtDNA mutations are not only single nucleotide variants but also large deletions that can compass several thousand nucleotides. Because every cell contains several mitochondria, mtDNA mutations are usually heteroplasmic (1-99%). MtDNA mutations have been a methodological challenge because there has not been a method that could detect all mutation types. Especially estimating the heteroplasmic rate of mtDNA deletions has been challenging. We have analyzed 60 samples for mtDNA deletions. DNA was isolated from muscle. MtDNA was amplified using long range PCR followed by massively parallel sequencing (MPS). In addition, for five positive control samples mtDNA was enriched and the samples were sequenced without PCR amplification. The results were analyzed for all mutation types. The mtDNA deletions were detected using a new algorithm generated for the purpose. The results of mtDNA deletion analysis were confirmed by MLPA. Our method was able to detect all single deletions with great precision, with exact breakpoints of the deletions. Our new algorithm calculated the heteroplasmic rate correctly for smallest deletions but with very large deletions the MPS results were not fully concordant with MLPA. Our new method is an easy and comprehensive method for detecting all kinds of mtDNA mutations and calculating their heteroplasmic rate. The PCR amplification seems to create a bias that exaggerates the amount of large deletions. However, we are confident that this problem can be circumvented by replacing the PCR step with mtDNA enrichment.