Mitochondrial Point Mutations Research Articles

Long‐term response to idebenone treatment in a patient with advanced Leber hereditary optic neuropathy

Aims/Purpose: To present a case with advanced LHON and its response to 2‐year‐treatment with idebenone, which was prescribed 13 years after the onset of the disease.Methods: A 40‐year‐old Caucasian male patient with a history of bilateral painless visual loss since the age of 28 was examined at our Ophthalmology department. His brother had also suffered similar visual loss. At presentation ETDRS best corrected visual acuity (BCVA) was counting fingers (CF) in both eyes (BE). Fundoscopy revealed pale optic discs bilaterally. Visual fields (30‐2) showed central scotomas in BE. During OCT/OCTA imaging of the optic disc RNFL and GCC thickness were decreased bilaterally. In electrophysiological assessment with flash visual evoked potentials a reduction in amplitude was recorded in BE but with normal latency. Full‐field ERG was within normal limits in BE. At the genetic test, a mitochondrial point mutation m. 3460 G>A at MT‐ND1 gene was detected, which was compatible with LHON. The patient was prescribed 300 mg idebenone per os three times/day for 24 months. Follow‐up visits were scheduled at 6, 12, 18 and 24 months.Results: At 6‐ and 12‐month follow‐up visits visual acuity, fundoscopic findings and OCT‐RNFL imaging remained unchanged. However, the patient reported subjective visual improvement bilaterally, with greater improvement in the visual field of the left eye. At 18 months following initiation of treatment an improvement of the visual field was documented in BE. No change of the GCC thickness was noted. At 2‐year follow‐up his condition remained improved.Conclusions: Delayed treatment with idebenone in advanced LHON can lead to subjective visual improvement. Moreover, an improvement in visual fields can also be documented even after 18 to 24 months of the initiation of the treatment.

PSUN248 Mitochondrial diabetes - a rare but important cause of diabetes

Abstract Background Mitochondrial Encephalopathy, Lactic Acidosis, and Stroke-like episodes (MELAS) syndrome is a rare mitochondrial disorder which presents with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes. Mitochondrial diabetes is the cause in 1% of all diabetic patients. Here we present a case of a patient with insulin dependent diabetes mellitus (DM) who experienced sensorineural deafness and was diagnosed with MELAS. Case 33-year-old female presented with stroke like symptoms in December 2018. Medical history was significant for Diabetes Mellitus (DM), bilateral deafness and end stage renal disease requiring peritoneal dialysis with subsequent kidney transplant. On presentation, she had left sided weakness, lab work revealed lactic acidosis. MRI brain showed large areas of restricted diffusion in right hemisphere and cerebral edema. CSF analysis was positive for lactate and pyruvate. Left lower extremity muscle biopsy and mitochondrial genome analysis were positive for m.3243A>G in the MT-TL1 with 80% heteroplasmy, confirming the diagnosis of MELAS. Patient was started on Arginine, CoQ10, Levetiracetam, phenytoin, IVIG and her symptoms improved. DM was diagnosed in 2013 when patient was on peritoneal dialysis. Family history significant for type 2 DM in maternal uncle. Discussion Mitochondrial diseases are rare, maternally inherited, heterogeneous conditions caused by mutations in mitochondrial DNA and present with a variety of clinical symptoms. Mitochondrial diabetes results from the 3243 A>G gene mutation, responsible for both MELAS and Mitochondrial inherited diabetes and deafness (MIDD). MELAS presents with encephalopathy, stroke like symptoms and lactic acidosis (1). MIDD presents with nerve deafness, which usually occurs at an early age (2). In our patient, she presented with neurological symptoms suggesting MELAS and bilateral deafness suggesting MIDD. Her presentation is unusual and constitutes an overlap between both disorders. Conclusion Early recognition and diagnosis of mitochondrial diabetes is important. The diagnosis has important genetic implications for other family members. This case report highlights the presentation of MELAS and MIDD and importance of genetic studies in the confirmation of diagnosis 1) D. M. Sproule and P. Kaufmann, "Mitochondrial encephalopathy, lactic acidosis, and strokelike episodes: basic concepts, clinical phenotype, and therapeutic management of MELAS syndrome," Annals of the New York Academy of Sciences, vol. 1142, pp. 133–158, 20082) Murphy R, Turnbull DM, Walker M, Hattersley AT. Clinical features, diagnosis and management of maternally inherited diabetes and deafness (MIDD) associated with the 3243A>G mitochondrial point mutation. Diabetic Medicine 2008. Apr;25(4): 383-99. doi: 10.1111/j.1464-5491.2008.02359.x.Epub 2008 Feb 18. PMID: 18294221. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Neuropathie optique héréditaire de Leber : tableau clinique et données du bilan initial

Leber hereditary optic neuropathy (LHON) is a rare mitochondrial disease, responsible for blindness by bilateral involvement of central vision. It usually affects young men but can occur at any age and in women. Its diagnosis is suspected on the family history and the initial clinical picture, and the definitive diagnosis of LHON is obtained by genetic testing and the molecular identification of the mitochondrial genetic point mutation. The initial workup should include an assessment of visual structure and function. Its visual prognosis is severe, but depends on the causative mutation. Support should include genetic counseling, a therapeutic proposal and a support for visual impairment.

Crosslink between mutations in mitochondrial genes and brain disorders: implications for mitochondrial-targeted therapeutic interventions

At the present, association of mitochondrial dysfunction and progression of neurological disorders has gained significant attention. Defects in mitochondrial network dynamics, point mutations, deletions, and interaction of pathogenomic proteins with mitochondria are some of the possible underlying mechanisms involved in these neurological disorders. Mitochondrial genetics, defects in mitochondrial oxidative phosphorylation machinery, and reactive oxygen species production might share common crosstalk in the progression of these neurological disorders. It is of significant interests to explore and develop therapeutic strategies aimed at correcting mitochondrial abnormalities. This review provided insights on mitochondrial dysfunction/mutations involved in the progression of Alzheimer’s disease, Huntington’s disease, and epilepsy with a special focus on Parkinson’s disease pathology. Along with the deleterious effects of mitochondrial mutations in aforesaid neurological disorders, this paper unraveled the available therapeutic strategy, specifically aiming to improve mitochondrial dysfunction, drugs targeting mitochondrial proteins, gene therapies aimed at correcting mutant mtDNA, peptide-based approaches, and lipophilic cations.

IDF21-0347 Treatment response in the first Emirati patient with diabetes associated with the A3243G mitochondrial point mutation

Background: Maternally inherited diabetes and deafness (MIDD) is estimated as the cause of 1% of cases of diabetes. Due to the small patient population, it is not clear which diabetes treatments are most effective in MIDD. Aim: We discuss the response to different classes of hypoglycaemic agents in the first Emirati patient reported with mitochondrial diabetes. Method: A 23-year old male presented with hyperglycaemia and features atypical for Type 1 Diabetes including retinal changes at diagnosis. MIDD was confirmed by presence of an A to G point mutation in the tRNALeu(UUR) gene at base pair 3243 (A3243G) of the mitochondrial genome using targeted next generation sequencing. We report his management and his clinical features at 3.8 years of follow-up. Results: At diagnosis, anti-GAD and anti-IA2 antibody titres were not elevated and insulin secretory reserve was maintained, with C-peptide of 0.631nmol/L and simultaneous plasma glucose of 11.6mmol/L. No objective hearing loss was detected at presentation. Microvascular disease was detected at 39 days after onset of diabetes with background retinopathy (R1) and maculopathy (M1), and elevated albumin-creatinine ratio (ACR) (3.7 mg/mmoL, with subsequent microalbuminuria of 3.1, 4.1, 3.1, and 5.6 mg/mmoL at 12, 14, 17, and 19 months, respectively). Pinhole visual acuity (VA) was 0.8 (left) and 0.7 (right). BMI, HbA1c, and triglycerides were 27.3 kg/m2, 8.5%, and 1.34mmol/L, respectively. MIDD was confirmed by genetic analysis in the patient’s mother, who had an established diagnosis of diabetes mellitus; his brother declined screening. He was initially managed with insulin and metformin (HbA1c from 8.5% to 6.9%). He was switched to sulphonylurea (gliclazide) after diagnosis of MIDD (HbA1c from 6.9% to 7.7%), with canagliflozin added at 12 weeks (HbA1c from 7.7% to 6.2%, subsequently to 9%) and sitagliptin combined at 48 weeks (HbA1c from 9% to 8.7%). Dulaglutide was added at 73 weeks, leading to a dramatic improvement in his HbA1c from 8.7% to 6.9% and BMI from 28.4 to 25.4kg/m2 within 4 months and cessation of insulin, canagliflozin and sulphonylurea treatment. Dulaglutide was discontinued due to intolerance with subsequent deterioration of glycaemic control (HbA1c 9.5%). At 3.8 years of follow-up, he progressed to pre-proliferative retinopathy (R2, M1); his ACR was 7.0mg/mmoL. Pinhole VA reduced to 0.4 (left) and 0.3 (right). He remained overweight (BMI, 27.6kg/m2). LDL was elevated at 3.5mmol/L, and triglycerides were 1.3mmol/L while taking atorvastatin 20 mg. Discussion: The use of glucagon-like peptide-1 (GLP-1) analogues in patients with MIDD has been shown to effectively improve HbA1c within 6 months in three recent case reports. In this case, a GLP-1 induced a dramatic improvement in HbA1c within 4 months with cessation of several concomitant medications, sustained until the GLP-1 was discontinued due to lack of tolerability, with subsequent deterioration in glycaemic control. Although a full case series is lacking, accumulating evidence from case reports suggests that GLP-1 treatment may be particularly effective in people with MIDD.

Neuropathic Pain as Main Manifestation of POLG-Related Disease: A Case Report

Mutations in nuclear-encoded genes that are involved in mitochondrial DNA replication and maintenance (e.g., POLG) have been associated with chronic progressive external ophthalmoplegia (CPEO) phenotype. These nuclear genome mutations may lead to multiple mitochondrial DNA deletions or mitochondrial DNA depletion. On the other hand, primary genetic defects of mitochondrial DNA (such as single large-scale deletion or point mutations) have also been associated with the CPEO phenotype. Chronic progressive external ophthalmoplegia (CPEO) may be a manifestation of specific syndromes that, when clinically recognized, prompt clinicians to investigate specific genetic defects. Thus, CPEO, as part of Kearns Sayre syndrome, suggests the presence of a large-scale deletion of mitochondrial DNA. However, in pure CPEO or CPEO plus phenotypes, it is more difficult to know whether causative genetic defects affect the nuclear or mitochondrial DNA. Here, we present a patient with a long-standing history of CPEO plus phenotype, in whom the sequencing of mitochondrial DNA from skeletal muscle was normal, and no other genetic defect was suspected at first. At the time of our evaluation, the presence of polyneuropathy and neuropathic pain prompted us to investigate nuclear genetic defects and, specifically, mutations in the POLG gene. Thus, the sequencing of the POLG gene revealed p.Thr251Ile and p.Pro587Leu mutations in one allele, and p.Ala467Thr mutation in another allele. Although one would expect that mutations in POLG lead to multiple mitochondrial DNA deletions or depletion (loss of copies), the absence of mitochondrial DNA abnormalities in tissue may be explained by heteroplasmy, a lack or no significant involvement of biopsied tissue, or a sampling bias. So, the absence of secondary mitochondrial DNA alterations should not discourage clinicians from further investigating mutations in nuclear-encoded genes. Lastly, mitochondrial point mutations and single mitochondrial DNA deletions very rarely cause CPEO associated with polyneuropathy and neuropathic pain, and POLG-related disease should be considered in this scenario, instead.

Hydrogen bonding rearrangement by a mitochondrial disease mutation in cytochrome bc1 perturbs heme bH redox potential and spin state

Hemes are common elements of biological redox cofactor chains involved in rapid electron transfer. While the redox properties of hemes and the stability of the spin state are recognized as key determinants of their function, understanding the molecular basis of control of these properties is challenging. Here, benefiting from the effects of one mitochondrial disease-related point mutation in cytochrome b, we identify a dual role of hydrogen bonding (H-bond) to the propionate group of heme bH of cytochrome bc1, a common component of energy-conserving systems. We found that replacing conserved glycine with serine in the vicinity of heme bH caused stabilization of this bond, which not only increased the redox potential of the heme but also induced structural and energetic changes in interactions between Fe ion and axial histidine ligands. The latter led to a reversible spin conversion of the oxidized Fe from 1/2 to 5/2, an effect that potentially reduces the electron transfer rate between the heme and its redox partners. We thus propose that H-bond to the propionate group and heme-protein packing contribute to the fine-tuning of the redox potential of heme and maintaining its proper spin state. A subtle balance is needed between these two contributions: While increasing the H-bond stability raises the heme potential, the extent of increase must be limited to maintain the low spin and diamagnetic form of heme. This principle might apply to other native heme proteins and can be exploited in engineering of artificial heme-containing protein maquettes.

Detection of Mitochondrial Mutations Through Isothermal Nucleic Acid Amplification Coupled With Clustered Regularly Interspaced Short Palindromic Repeat-Associated Endonuclease Cas13a.

The clustered regularly interspaced short palindromic repeat (CRISPR)-associated endonuclease Cas13a can specifically bind and cleave RNA. After nucleic acid pre-amplification, bacterial Cas13a has been used to detect genetic mutations. In our study, using a transcription-mediated amplification together with Cas13a, we can isothermally amplify and detect mitochondrial point mutations under non-denaturing conditions from human genomic DNA. Unlike previous reports, we prepared CRISPR DNA with T7 promoter sequences and generated CRISPR RNA via transcription-mediated amplification instead of synthesizing and adding CRISPR RNA in a separate step. As a proof-of-concept, we showed that both m.1494C > T and m.1555A > G mutations were detected within 90 min. In addition, we explored various designs of CRISPR DNA to improve assay specificity, including the location and number of nucleotide mismatches, length of protospacer sequence, and different buffering conditions. We also confirmed the possibility of a “one-step single-tube” reaction for mutation detection. This assay can robustly distinguish circular DNA templates that differ by a single nucleotide. It has the potential to be adapted for automated applications, such as the screening of mitochondrial diseases.

Somatic Mitochondrial DNA Point Mutations Used as Biomarkers to Demonstrate Genomic Heterogeneity in Primary Prostate Cancer.

Primary prostate tumor heterogeneity is poorly understood, leaving research efforts with challenges regarding the initiation and advancement of the disease. The growth of tumor cells is accompanied by mutations in nuclear and in mitochondrial genomes. Thus, mitochondrial DNA mutations may be used as tumor cell markers. By the use of laser capture microdissection coupled with assays for mitochondrial point mutation detection, mtDNA mutations were used to trace mutated cells at a histological level. Point mutations in mtDNA were determined in 12 primary prostate cancers. The tumors represent different pathology-prognostic grade groups. Known mutational hotspots of the mtDNA were scanned for heteroplasmy. All specimens with mtDNA heteroplasmy were subsequently subsampled by laser capture microdissection. From a total number of 1728 microsamples, mitochondrial DNA target sequences were amplified and base substitutions detected by cycling temperature capillary electrophoresis. Real-time PCR was used as a quantitative assay to determine the relative mtDNA copy number of 12 tumors studied, represented by two samples from each (N = 24); a high degree (75%) demonstrated tumor specimen heterogeneity. A grid of 96 spots isolated by laser capture microdissection demonstrated interfocal sample heterogeneity and increased the limit of detection. The spots demonstrated a wide range of mutant fractions from 0 to 100% mutant copies. The mitochondrial DNA copy number in the samples was determined by real-time PCR. No correlation between copy number and pathology-prognostic grade groups was observed. Somatic mitochondrial DNA point mutations represent traceable biomarkers demonstrating heterogeneity in primary prostate cancer. Mutations can be detected in areas before changes in tissue histopathology are evident to the pathologist.

Novel Alterations of Some Mitochondrial tRNA Genes in Vienamese Colorectal Cancer Patients

Some mutations of mt-DNA which encode tRNA (mt-tRNA) were previously reported to be associated with clinical manifestations of neuromuscular disorders syndrome. In addition, alterations of the mitochondrial genome have been suggested to contribute to mitochondrial dysfunction and tumorigenesis. Alterations in some mt-tRNA genes have also been identified in breast cancer, lung cancer and colorectal cancer. However, so far, we have not found any report on mt-tRNA gene alteration in the Vietnamese colorectal cancer patients. So that, in this study, we analyzed the alterations of some mt-tRNA genes in a group of Vietnamese colorectal cancer patients and predicted influence of the alterations to secondary structure of tRNA based on bioinformatic tools. PCR-RFLP and DNA sequencing methods were used to screening alterations, secondary structure of tRNA was predicted in silico by using a tool of the Vienna RNA Websuite. Results: both A12309G and A12310G of tRNALeu were identified together in two out of 98 patients, and both T12150G and C12154G of tRNAHis were indentified in one out of 19 patients. All of these alterations were heteroplasmic and have not been reported in cancer patients. In particular, the C12154G alteration in the DHR loop led to change of secondary structure of tRNAHis and may affect to function of this tRNA molecule.
 Keywords
 mt-tRNA, Vietnamese colorectal cancer, PCR-RFLP, DNA sequencing
 References
 [1] http://globocan.iarc.fr/Pages/online.aspx (30/11/2017).[2] M. Brandon, P. Baldi, D.C Wallace, Mitochodrial mutations in cancer, Oncogen 25(34) (2006) 4647.[3] G. Li, YX. Duan, XB. Zhang, F Wu, Mitochondrial RNA mutations may be infrequent in hepatocellular carcinoma patients, Genet Mol Res15(2) (2016) doi: 10.4238/gmr.15027665.[4] F. Mohammed, AR. Rezaee, E. Mosaieby, M. Houshmand, Mitochondrial A12308G alteration in RNA Leu (CUN)) in colorectal cancer samples, Diagn Pathol (2015) doi: 10.1186/s13000-015-0337-6.[5] S. Datta, M. Majumder, NK. Biswas, N. Sikdar, B. Roy, Increased risk of oral cancer in relation to common Indian mitochondrial polymorphisms and Autosomal GSTP1 locus, Cancer, 110 (2007) 1999.[6] L .Wang , ZJ. Chen , YK. Zhang , HB. Le, The role of mitochondrial RNA mutations in lung cancer, Int J Clin Exp Med 8(8) (2015) 13341.[7] AR. Gruber, R. Lorenz, SH. Bernhart, R. Neubock, IL Hofacker The Vienna ARN Websuite, Nucleic Acids Res 36 (2008) 70.[8] https://blast.ncbi.nlm.nih.gov/Blast.cgi (11/2017).[9] Y. Goto, I Nonaka, S Horai, A mutation in the RNALeu (UUR) gene associated with the MELAS subgroup of mitochondrial encephalomyopathies, Nature 348(6302 (1990) 651[10] http://www.mitomap.org/MITOMAP (11/2017).[11] A. Lorenc, J. Bryk Golik P, Homoplasmic MELAS A3243G mtDNA mutation in a colon cancer sample, Mitochondrion 3(2) (2003) 119.[12] EL. Blakely, J.W Yarham , C.L. Alston , K Craig , J. Poulton , C. Brierley , SM. Park , A. Dean , JH. Xuereb , KN. Anderson , A. Compston , C. Allen , S. Sharif , P. Enevoldson , M. Wilson , SR. Hammans , DM. Turnbull , R. McFarland , RW.Taylor , Pathogenic mitochondrial tRNA point mutations: nine novel mutations affirm their importance as a cause of mitochondrial disease, Hum Mutat 34(9) (2013) 1260.[13] S. DiMauro, Mitochondrial ADN medicine, Biosci Rep 27(3) (2007) 5.[14] DC. Wallace, D. Chalkia, Mitochondrial ADN genetics and the heteroplasmy conundrum in evolution and disease”, Cold Spring Harb Perspect Biol 5(11) (2013), doi:10.1101/cshperspect. a021220

MITOCHONDRIAL DISEASES (Posters): P.183MELAS and reversible cerebral vasoconstriction

A 22-year-old woman was admitted due to fever and severe headache for 10 days and altered mental status and generalized tonic-clonic seizures for 8 days. She had a short stature and hairy body surface, and her mother passed away about ten years ago due to ``encephalitis''. Laboratory data showed elevated lactate in both peripheral blood and CSF. Brain MRI using diffusion weighted imaging revealed hyperintense lesions located in the whole right temporal and occipital lobes and pulvinar of thalamus. MR angiography revealed segmental stenoses at the right P1 and M2-3 junction. Muscle biopsy showed typical RRF and SSV. A genetic study revealed a mitochondrial DNA A3243G point mutation. The patient's clinical symptoms and MRI/MR angiography (MRA) findings improved after the administration of L-arginine, L-carnitine, lipoic acid, vitamin B and dexamethasone. Therefore we speculate the possible pathophysiology just like RCVS (reversible cerebaral vasoconstriction syndrome) involving in the stroke-like episodes of MELAS.

Cerebrospinal fluid monoamines, pterins, and folate in patients with mitochondrial diseases: systematic review and hospital experience.

Mitochondrial diseases are a group of genetic disorders leading to the dysfunction of mitochondrial energy metabolism pathways. We aimed to assess the clinical phenotype and the biochemical cerebrospinal fluid (CSF) biogenic amine profiles of patients with different diagnoses of genetic mitochondrial diseases. We recruited 29 patients with genetically confirmed mitochondrial diseases harboring mutations in either nuclear or mitochondrial DNA (mtDNA) genes. Signs and symptoms of impaired neurotransmission and neuroradiological data were recorded. CSF monoamines, pterins, and 5-methyltetrahydrofolate (5MTHF) concentrations were analyzed using high-performance liquid chromatography with electrochemical and fluorescence detection procedures. The mtDNA mutations were studied by Sanger sequencing, Southern blot, and real-time PCR, and nuclear DNA was assessed either by Sanger or next-generation sequencing. Five out of 29 cases showed predominant dopaminergic signs not attributable to basal ganglia involvement, harboring mutations in different nuclear genes. A chi-square test showed a statistically significant association between high homovanillic acid (HVA) values and low CSF 5-MTHF values (chi-square = 10.916; p = 0.001). Seven out of the eight patients with high CSF HVA values showed cerebral folate deficiency. Five of them harbored mtDNA deletions associated with Kearns-Sayre syndrome (KSS), one had a mitochondrial point mutation at the mtDNA ATPase6 gene, and one had a POLG mutation. In conclusion, dopamine deficiency clinical signs were present in some patients with mitochondrial diseases with different genetic backgrounds. High CSF HVA values, together with a severe cerebral folate deficiency, were observed in KSS patients and in other mtDNA mutation syndromes.

Pigmentary retinopathy, rod–cone dysfunction and sensorineural deafness associated with a rare mitochondrial tRNALys (m.8340G>A) gene variant

Background/AimThe rare mitochondrial DNA (mtDNA) variant m.8340G>A has been previously reported in the literature in a single, sporadic case of mitochondrial myopathy. In this report, we aim to investigate the...

Activation of Dun1 in response to nuclear DNA instability accounts for the increase in mitochondrial point mutations in Rad27/FEN1 deficient S. cerevisiae.

Rad27/FEN1 nuclease that plays important roles in the maintenance of DNA stability in the nucleus has recently been shown to reside in mitochondria. Accordingly, it has been established that Rad27 deficiency causes increased mutagenesis, but decreased microsatellite instability and homologous recombination in mitochondria. Our current analysis of mutations leading to erythromycin resistance indicates that only some of them arise in mitochondrial DNA and that the GC→AT transition is a hallmark of the mitochondrial mutagenesis in rad27 null background. We also show that the mitochondrial mutator phenotype resulting from Rad27 deficiency entirely depends on the DNA damage checkpoint kinase Dun1. DUN1 inactivation suppresses the mitochondrial mutator phenotype caused by Rad27 deficiency and this suppression is eliminated at least in part by subsequent deletion of SML1 encoding a repressor of ribonucleotide reductase. We conclude that Rad27 deficiency causes a mitochondrial mutator phenotype via activation of DNA damage checkpoint kinase Dun1 and that a Dun1-mediated increase of dNTP pools contributes to this phenomenon. These results point to the nuclear DNA instability as the source of mitochondrial mutagenesis. Consistently, we show that mitochondrial mutations occurring more frequently in yeast devoid of Rrm3, a DNA helicase involved in rDNA replication, are also dependent on Dun1. In addition, we have established that overproduction of Exo1, which suppresses DNA damage sensitivity and replication stress in nuclei of Rad27 deficient cells, but does not enter mitochondria, suppresses the mitochondrial mutagenesis. Exo1 overproduction restores also a great part of allelic recombination and microsatellite instability in mitochondria of Rad27 deficient cells. In contrast, the overproduction of Exo1 does not influence mitochondrial direct-repeat mediated deletions in rad27 null background, pointing to this homologous recombination pathway as the direct target of Rad27 activity in mitochondria.

Mitochondrial Point Mutation m.3243A>G Associates With Lower Bone Mineral Density, Thinner Cortices, and Reduced Bone Strength: A Case-Control Study.

Mitochondrial dysfunction is associated with several clinical manifestations including diabetes mellitus (DM), neurological disorders, renal and hepatic diseases, and myopathy. Although mitochondrial dysfunction is associated with increased bone resorption and decreased bone formation in mouse models, effects of alterations in mitochondrial function on bone remodeling and mass have not been investigated in humans. We recruited 45 carriers (29 females, 16 males) with the m.3243A>G mutation and healthy controls matched for gender, age, height, and menopausal status. DXA and HRpQCT scans were performed, and bone turnover markers (BTMs) P1NP and CTX were measured. Cases and controls were well matched except for body weight, which was lower in cases (63.6 ± 18.1 kg versus 74.6 ± 14.8 kg, p < 0.01), and manifest DM was present in 25 of 45 cases (none in controls). Bone scans showed lower BMD at the lumbar spine, total hip, and femoral neck in cases. Mean lumbar spine, total hip, and femoral neck T-scores were -1.5, -1.3, and -1.6 in cases, respectively, and -0.8, -0.3, and -0.7 in controls (all p < 0.05). The m.3243A>G mutation was associated with lower BMD, cortical but not trabecular density, cortical thickness, and estimated bone strength. Furthermore, BTMs were lower in the m.3243A>G group before but not after adjustment for DM. The mitochondrial point mutation m.3243A>G was associated with decreased bone mass and strength. Although the coexistence of DM may have influenced bone turnover, the bone phenotype observed in m.3243A>G cases appeared to mirror age-related deterioration in bone, suggesting that mitochondrial dysfunction may cause a premature aging of bone. © 2017 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.

Is detection of intraperitoneal exfoliated tumor cells after surgical resection of rectal cancer a prognostic factor of survival?

BackgroundThe prognostic significance of free cancer cells detected in peritoneal fluid at the time of rectal surgery remains unclear. A substantial number of patients will develop metastatic disease even with successful local treatment. This prospective non-randomized study investigated the prognostic value of intraperitoneal free cancer cells harvested in peritoneal lavage after surgery for rectal cancer. Mutational hotspots in mitochondrial DNA were examined as potential molecular signatures to detect circulating intraperitoneal free cancer cells when present in primary tumor and in lavage.MethodsPoint mutations in mitochondrial DNA amplifications were determined in primary tumors and corresponding exfoliated intraperitoneal free cancer cells in lavage from 191 patients with locally advanced rectal cancer scheduled for radical treatment. Mitochondrial DNA target sequences were amplified by polymerase chain reaction and base substitutions were detected by denaturant, cycling temperature capillary electrophoresis. Detection of intraperitoneal free cancer cells was correlated to survival.ResultsOf 191patients analyzed, 138 (72%) were identified with somatic mitochondrial point mutations in rectal cancer tumors. From this fraction, 45 patients (33%) had positive lavage fluid with corresponding somatic mtDNA point mutations in lavage representing circulating intraperitoneal free cancer cells. There was no significant survival difference between patients identified with or without somatic mitochondrial DNA point mutations in the corresponding lavage.ConclusionSomatic mitochondrial DNA point mutations identified in primary rectal tumors enable detection of circulating intraperitoneal free cancer cells in lavage fluid.Intraperitoneal free cancer cells harvested from lavage immediately after surgery for rectal cancer does not represent an independent prognostic factor on survival.

Mapping mitochondrial heteroplasmy in a Leydig tumor by laser capture micro-dissection and cycling temperature capillary electrophoresis

BackgroundThe growth of tumor cells is accompanied by mutations in nuclear and mitochondrial genomes creating marked genetic heterogeneity. Tumors also contain non-tumor cells of various origins. An observed somatic mitochondrial mutation would have occurred in a founding cell and spread through cell division. Micro-anatomical dissection of a tumor coupled with assays for mitochondrial point mutations permits new insights into this growth process. More generally, the ability to detect and trace, at a histological level, somatic mitochondrial mutations in human tissues and tumors, makes these mutations into markers for lineage tracing.MethodA tumor was first sampled by a large punch biopsy and scanned for any significant degree of heteroplasmy in a set of sequences containing known mutational hotspots of the mitochondrial genome. A heteroplasmic tumor was sliced at a 12 μm thickness and placed on membranes. Laser capture micro-dissection was used to take 25000 μm2 subsamples or spots. After DNA amplification, cycling temperature capillary electrophoresis (CTCE) was used on the laser captured samples to quantify mitochondrial mutant fractions.ResultsOf six testicular tumors studied, one, a Leydig tumor, was discovered to carry a detectable degree of heteroplasmy for two separate point mutations: a C → T mutation at bp 64 and a T → C mutation found at bp 152. From this tumor, 381 spots were sampled with laser capture micro-dissection. The ordered distribution of spots exhibited a wide range of fractions of the mutant sequences from 0 to 100% mutant copies. The two mutations co-distributed in the growing tumor indicating they were present on the same genome copies in the founding cell.ConclusionLaser capture microdissection of sliced tumor samples coupled with CTCE-based point mutation assays provides an effective and practical means to obtain maps of mitochondrial mutational heteroplasmy within human tumors.

Mitochondrial DNA exhibits resistance to induced point and deletion mutations.

The accumulation of somatic mitochondrial DNA (mtDNA) mutations contributes to the pathogenesis of human disease. Currently, mitochondrial mutations are largely considered results of inaccurate processing of its heavily damaged genome. However, mainly from a lack of methods to monitor mtDNA mutations with sufficient sensitivity and accuracy, a link between mtDNA damage and mutation has not been established. To test the hypothesis that mtDNA-damaging agents induce mtDNA mutations, we exposed MutaTMMouse mice to benzo[a]pyrene (B[a]P) or N-ethyl-N-nitrosourea (ENU), daily for 28 consecutive days, and quantified mtDNA point and deletion mutations in bone marrow and liver using our newly developed Digital Random Mutation Capture (dRMC) and Digital Deletion Detection (3D) assays. Surprisingly, our results demonstrate mutagen treatment did not increase mitochondrial point or deletion mutation frequencies, despite evidence both compounds increase nuclear DNA mutations and demonstrated B[a]P adduct formation in mtDNA. These findings contradict models of mtDNA mutagenesis that assert the elevated rate of mtDNA mutation stems from damage sensitivity and abridged repair capacity. Rather, our results demonstrate induced mtDNA damage does not readily convert into mutation. These findings suggest robust mitochondrial damage responses repress induced mutations after mutagen exposure.

Short peptides from leucyl-tRNA synthetase rescue disease-causing mitochondrial tRNA point mutations.

Mutations in mitochondrial (mt) genes coding for mt-tRNAs are responsible for a range of syndromes, for which no effective treatment is available. We recently showed that the carboxy-terminal domain (Cterm) of human mt-leucyl tRNA synthetase rescues the pathologic phenotype associated either with the m.3243A>G mutation in mt-tRNALeu(UUR) or with mutations in the mt-tRNAIle, both of which are aminoacylated by Class I mt-aminoacyl-tRNA synthetases (mt-aaRSs). Here we show, by using the human transmitochondrial cybrid model, that the Cterm is also able to improve the phenotype caused by the m.8344A>G mutation in mt-tRNALys, aminoacylated by a Class II aaRS. Importantly, we demonstrate that the same rescuing ability is retained by two Cterm-derived short peptides, β30_31 and β32_33, which are effective towards both the m.8344A>G and the m.3243A>G mutations. Furthermore, we provide in vitro evidence that these peptides bind with high affinity wild-type and mutant human mt-tRNALeu(UUR) and mt-tRNALys, and stabilize mutant mt-tRNALeu(UUR). In conclusion, we demonstrate that small Cterm-derived peptides can be effective tools to rescue cellular defects caused by mutations in a wide range of mt-tRNAs.

Magnetic resonance imaging correlates of genetically characterized patients with mitochondrial disorders: A study from south India

BackgroundLarge studies analyzing magnetic resonance imaging correlates in different genotypes of mitochondrial disorders are far and few. This study sought to analyze the pattern of magnetic resonance imaging findings in a cohort of genetically characterized patients with mitochondrial disorders. MethodsThe study cohort included 33 patients (age range 18months–50years, M:F — 0.9:1) with definite mitochondrial disorders seen over a period of 8yrs. (2006–2013). Their MR imaging findings were analyzed retrospectively. ResultsThe patients were classified into three groups according to the genotype, Mitochondrial point mutations and deletions (n=21), SURF1 mutations (n=7) and POLG1 (n=5). The major findings included cerebellar atrophy (51.4%), cerebral atrophy (24.2%), signal changes in basal ganglia (45.7%), brainstem (34.2%) & white matter (18.1%) and stroke like lesions (25.7%). Spinal cord imaging showed signal changes in 4/6 patients. Analysis of the special sequences revealed, basal ganglia mineralization (7/22), lactate peak on magnetic resonance spectrometry (10/15), and diffusion restriction (6/22). Follow-up images in six patients showed that the findings are dynamic. Comparison of the magnetic resonance imaging findings in the three groups showed that cerebral atrophy and cerebellar atrophy, cortical signal changes and basal ganglia mineralization were seen mostly in patients with mitochondrial mutation. Brainstem signal changes with or without striatal lesions were characteristically noted in SURF1 group. There was no consistent imaging pattern in POLG1 group. ConclusionMagnetic resonance imaging findings in mitochondrial disorders are heterogeneous. Definite differences were noted in the frequency of anatomical involvement in the three groups. Familiarity with the imaging findings in different genotypes of mitochondrial disorders along with careful analysis of the family history, clinical presentation, biochemical findings, histochemical and structural analysis will help the physician for targeted metabolic and genetic testing.