Multiple mtDNA Deletions Research Articles

Functional relevance of mitochondrial abnormalities in sporadic inclusion body myositis

Cytochrome c oxidase (COX)-deficient fibers and multiple mitochondrial DNA (mtDNA) deletions are frequent findings in sporadic inclusion body myositis (s-IBM). However, the functional impact of these defects is not known. We investigated oxygen desaturation during exercise using the forearm exercise test, accumulation of lactate during exercise using a cycle ergometry test and mitochondrial changes (COX-deficient fibers, biochemical activities of respiratory chain complexes, multiple mtDNA deletions by long-range polymerase chain reaction) in 10 patients with s-IBM and compared the findings with age and sex-matched normal and diseased controls (without mitochondrial disorders) as well as patients with mitochondrial disorder due to nuclear gene defects resulting in multiple mtDNA deletions (MITO group). The mean age of the s-IBM patients was 68.2±5.7years (range: 56–75). Patients with s-IBM had statistically significantly reduced oxygen desaturation (ΔsO2) during the handgrip exercise (p<0.05) and elevated peak serum lactate levels during cycle ergometry compared to normal controls (p<0.05). The percentage of COX-deficient fibers in s-IBM and MITO patients was significantly increased compared to normal controls (p<0.01). Five out of nine s-IBM patients had multiple mtDNA deletions. Thirty-three percent of s-IBM patients showed an increased citrate synthase content and decreased activities of complex IV (COX). The biochemical pattern of respiratory chain complexes in patients with s-IBM and MITO was similar. Histopathological analysis showed similar changes in s-IBM and MITO due to nuclear gene defects. Functional tests reflecting mitochondrial impairment suggest a contribution of mitochondrial defects to disease-related symptoms such as fatigue and exertion-induced symptoms.

G.P.190: Multiple deletions in mitochondrial DNA in myofibrillar myopathy and centronuclear myopathy

Multiple deletions in mitochondrial DNA (mtDNA) are associated with mutations in nuclear genes which encode proteins either directly or indirectly involved in the replication or maintenance of mtDNA. To date, mutations in twelve nuclear genes, POLG, POLG2, C10orf2, SLC25A4, RRM2B, TK2, MPV17, DGUOK, OPA1, MFN2, MGME1, and DNA2 have been identified in patients with multiple deletions in mtDNA, yet the genetic cause of multiple mtDNA deletions still remains undefined in a large number of patients. In addition to patients with a classic mitochondrial myopathy, multiple mtDNA deletions are also found in patients with myopathies such as sporadic IBM. Mitochondrial changes have also been identified in skeletal muscle from patients with myofibrillar myopathy, with histochemical changes such as rubbed out fibres observed with COX and NADH staining, and focal clustering and depletion of mitochondria. Mitochondrial pathology, including COX negative and ragged red fibres, and paracrystalline inclusions have also been described in patients with centronuclear myopathy due to mutations in DNM2, which encodes Dynamin 2, a GTPase which is involved in membrane trafficking. Here we describe our findings in two patients with multiple mtDNA deletions. In one patient, a 70 year old male who presented with a history of exercise induced muscle pain and distal myopathy we identified a novel pArg2364His mutation in the gene FLNC which encodes the actin binding protein filamin-C. In the second patient, a 40 year old male who presented at age 3 years with ataxia and later developed facial myopathy, proximal muscle weakness, and CPEO, histochemical investigation indicated centronuclear myopathy, and a p.Glu560Lys mutation in DNM2 was identified. Our findings suggest that FLNC and DNM2 should be added to the list of genes associated with multiple deletions in mtDNA.

G.P.188: Autosomal recessive Kearns-Sayre syndrome in a girl with altered mitochondrial DNA transcription caused by RRM2B gene defect

Kearns-Sayre syndrome (KSS) is a mitochondrial DNA (mtDNA) disorder characterized by ophthalmoplegia, ptosis, retinopathy, onset before age 20 years plus one: cardiac conduction defects, cerebellar syndrome, elevated CSF protein. KSS may affect many organs causing encephalomyopathy, sensorineural hearing loss, endocrinopathy and/or heart and renal involvement. De novo single large-scale mtDNA rearrangements are the genetic hallmark, detectable at high heteroplasmic levels. We present the first child with KSS described so far with a nuclear gene defect of RRM2B, encoding the small subunit of p53-inducible ribonucleotide reductase. In the 15 year-old girl, sensorineural hearing loss was noticed at the age of 11/2 year leading to cochlear implants at ages 2 and 6 years. Her psychomotor development was slightly retarded until the age of 10 years when muscle weakness and an atactic gait became increasingly evident. At the age of 8 years, the girl developed edema caused by a protein-loosing nephropathy. She now presents with ptosis, external ophthalmoplegia and retinitis pigmentosa. Muscle biopsy showed COX-positive and -negative ragged-red fibers, but no biochemical abnormalities of the respiratory chain complexes. Muscle mtDNA analysis revealed no single large-scale rearrangements, but multiple low-level mtDNA deletions. Nuclear gene analysis identified compound heterozygous mutations in the nuclear RRM2B gene: c.328C > T, p.Arg110Cys; c.846G > C, p.Met282Ile. Ribonucleotide reductase has a crucial role in the de novo conversion of ribonucleoside diphosphates into the corresponding deoxyribonucleoside diphosphates, and thus is essential for proper mtDNA synthesis. RRM2B mutations have been described so far only in a single adult patient with KSS. This second case, the first pediatric patient, underlines that KSS is not exclusively a sporadic mtDNA deletion/duplication disorder but rarely an autosomal recessive disease caused by nuclear encoded mtDNA transcription alteration. Kearns-Sayre syndrome (KSS) is a mitochondrial DNA (mtDNA) disorder characterized by ophthalmoplegia, ptosis, retinopathy, onset before age 20 years plus one: cardiac conduction defects, cerebellar syndrome, elevated CSF protein. KSS may affect many organs causing encephalomyopathy, sensorineural hearing loss, endocrinopathy and/or heart and renal involvement. De novo single large-scale mtDNA rearrangements are the genetic hallmark, detectable at high heteroplasmic levels. We present the first child with KSS described so far with a nuclear gene defect of RRM2B, encoding the small subunit of p53-inducible ribonucleotide reductase. In the 15 year-old girl, sensorineural hearing loss was noticed at the age of 11/2 year leading to cochlear implants at ages 2 and 6 years. Her psychomotor development was slightly retarded until the age of 10 years when muscle weakness and an atactic gait became increasingly evident. At the age of 8 years, the girl developed edema caused by a protein-loosing nephropathy. She now presents with ptosis, external ophthalmoplegia and retinitis pigmentosa. Muscle biopsy showed COX-positive and -negative ragged-red fibers, but no biochemical abnormalities of the respiratory chain complexes. Muscle mtDNA analysis revealed no single large-scale rearrangements, but multiple low-level mtDNA deletions. Nuclear gene analysis identified compound heterozygous mutations in the nuclear RRM2B gene: c.328C > T, p.Arg110Cys; c.846G > C, p.Met282Ile. Ribonucleotide reductase has a crucial role in the de novo conversion of ribonucleoside diphosphates into the corresponding deoxyribonucleoside diphosphates, and thus is essential for proper mtDNA synthesis. RRM2B mutations have been described so far only in a single adult patient with KSS. This second case, the first pediatric patient, underlines that KSS is not exclusively a sporadic mtDNA deletion/duplication disorder but rarely an autosomal recessive disease caused by nuclear encoded mtDNA transcription alteration.

SANDO syndrome in a cohort of 107 patients with CPEO and mitochondrial DNA deletions

ObjectiveThe sensory ataxic neuropathy with dysarthria and ophthalmoparesis (SANDO) syndrome is a subgroup of mitochondrial chronic progressive external ophthalmoplegia (CPEO)-plus disorders associated with multiple mitochondrial DNA (mtDNA) deletions. There is...

Myopathic involvement and mitochondrial pathology in Kennedy disease and in other motor neuron diseases.

Kennedy disease (spinal and bulbar muscular atrophy, or SBMA) is a motor neuron disease caused by a CAG expansion in the androgen-receptor (AR) gene. Increasing evidence shows that SBMA may have a primary myopathic component and that mitochondrial dysfunction may have some role in the pathogenesis of this disease. In this article, we review the role of mitochondrial dysfunction and of the mitochondrial genome (mtDNA) in SBMA, and we present the illustrative case of a patient who presented with increased CK levels and exercise intolerance. Molecular analysis led to definitive diagnosis of SBMA, whereas muscle biopsy showed a mixed myopathic and neurogenic process with "mitochondrial features" and multiple mtDNA deletions, supporting some role of mitochondria in the pathogenesis of the myopathic component of Kennedy disease. Furthermore, we briefly review the role of mitochondrial dysfunction in two other motor neuron diseases (namely spinal muscular atrophy and amyotrophic lateral sclerosis). Most likely, in most cases mtDNA does not play a primary role and it is involved subsequently. MtDNA deletions may contribute to the neurodegenerative process, but the exact mechanisms are still unclear. It will be important to develop a better understanding of the role of mitochondrial dysfunction in motoneuron diseases, since it may lead to the development of more effective strategies for the treatment of this devastating disorder.

Inherited peripheral neuropathies due to mitochondrial disorders

Mitochondrial disorders (MIDs) are frequently responsible for neuropathies with variable severity. Mitochondrial diseases causing peripheral neuropathies (PNP) may be due to mutations of mitochondrial DNA (mtDNA), as is the case in MERRF and MELAS syndromes, or to mutations of nuclear genes. Secondary abnormalities of mtDNA (such as multiple deletions of muscle mtDNA) may result from mitochondrial disorders due to mutations in nuclear genes involved in mtDNA maintenance. This is the case in several syndromes caused by impaired mtDNA maintenance, such as Sensory Ataxic Neuropathy, Dysarthria and Ophthalmoplegia (SANDO) due to recessive mutations in the POLG gene, which encodes the catalytic subunit of mtDNA polymerase (DNA polymerase gamma), or Mitochondrial Neuro-Gastro-Intestinal Encephalomyopathy (MNGIE), due to recessive mutations in the TYMP gene, which encodes thymidine phosphorylase. The last years have seen a growing list of evidence demonstrating that mitochondrial bioenergetics and dynamics might be dysfunctional in axonal Charcot-Marie-Tooth disease (CMT2), and these mechanisms might present a common link between dissimilar CMT2-causing genes.

Dissecting the mechanisms underlying the accumulation of mitochondrial DNA deletions in human skeletal muscle.

Large-scale mitochondrial DNA (mtDNA) deletions are an important cause of mitochondrial disease, while somatic mtDNA deletions cause focal respiratory chain deficiency associated with ageing and neurodegenerative disorders. As mtDNA deletions only cause cellular pathology at high levels of mtDNA heteroplasmy, an mtDNA deletion must accumulate to levels which can result in biochemical dysfunction—a process known as clonal expansion. A number of hypotheses have been proposed for clonal expansion of mtDNA deletions, including a replicative advantage for deleted mitochondrial genomes inferred by their smaller size—implying that the largest mtDNA deletions would also display a replicative advantage over smaller mtDNA deletions. We proposed that in muscle fibres from patients with mtDNA maintenance disorders, which lead to the accumulation of multiple mtDNA deletions, we would observe the largest mtDNA deletions spreading the furthest longitudinally through individual muscle fibres by means of a greater rate of clonal expansion. We characterized mtDNA deletions in patients with mtDNA maintenance disorders from a range of ‘large’ and ‘small’ cytochrome c oxidase (COX)-deficient regions in skeletal muscle fibres. We measured the size of clonally expanded deletions in 62 small and 60 large individual COX-deficient f regions. No significant difference was observed in individual patients or in the total dataset (small fibre regions mean 6.59 kb—large fibre regions mean 6.51 kb). Thus no difference existed in the rate of clonal expansion throughout muscle fibres between mtDNA deletions of different sizes; smaller mitochondrial genomes therefore do not appear to have an inherent replicative advantage in human muscle.

Mitochondrial abnormalities in myofibrillar myopathies

Histological mitochondrial changes are generally found to be associated with late onset myofibrillar myopathies (MFMs). How these changes contribute to the pathogenesis of MFMs is unknown. Mitochondrial changes, including COX-deficient fibers (n = 8), biochemical activities of respiratory chain complexes (n = 7), and multiple mtDNA deletions by long-range PCR (n = 9) were examined in patients with genetically confirmed MFMs [MYOT (n = 2), DES (n = 1), ZASP (n = 2), FLNC (n = 4)] and compared with age and sex matched normal controls (n = 27) and patients with a mitochondrial disorder with multiple mtDNA deletions due to nuclear genetic defects (n = 8). In 2 MFM patients, micro dissected fibers were analyzed for multiple mtDNA deletions by nested long-range PCR. The COX-deficient fibers only partly corresponded with fibers containing myofibrillar accumulations. In total, there was no difference in the percentage of COX-deficient fibers in MFM patients and normal controls. However, the percentage of COX-deficient fibers was significantly higher in 3 MFM patients. Two MFM patients but none of the controls had multiple mtDNA deletions. Nested long-range PCR detected multiple mtDNA deletions only in COX-deficient fibers. Citrate synthase activities in MFM patients were 1.5-fold increased by compared to those in controls, suggesting initiation of mitochondrial alterations. However, it is unclear whether this is a direct consequence of MFM pathology. *both authors contributed equally to the manuscript.

P32 Mutations in SPG7 cause chronic progressive external ophthalmoplegia through disordered mtDNA maintenance

P32 Mutations in SPG7 cause chronic progressive external ophthalmoplegia through disordered mtDNA maintenance

P31 Genotypic and phenotypic heterogeneity in adult-onset progressive external ophthalmoplegia (PEO) with mitochondrial DNA instability: a systematic review

P31 Genotypic and phenotypic heterogeneity in adult-onset progressive external ophthalmoplegia (PEO) with mitochondrial DNA instability: a systematic review

Adult-onset Mendelian PEO Associated with Mitochondrial Disease

Progressive external ophthalmoplegia (PEO) is an eye movement disorder characterised by paresis of the extra ocular muscles and muscle restricted multiple mitochondrial DNA (mtDNA) deletions. Classification of patients is particularly difficult due to overlapping phenotypes and a poor genotype-phenotype relationship. Despite the identification of several nuclear encoded genes causing PEO, over half of patients with clinically confirmed PEO do not have a genetic diagnosis. To systematically review genotypic and phenotypic correlates of published cases of adult-onset PEO. Patients were identified from interrogation of articles from Scopus, Medline via PubMed, and Genetic Abstracts databases using electronic searches (1st January 1970 to 8th November 2013). Reference lists and UniProt entries were also manually checked for additional articles. Twelve nuclear encoded genes were identified (TYMP, SLC25A4, POLG, C10ORF2, OPA1, POLG2, RRM2B, TK2, DGUOK, MPV17, MGME1, and DNA2) systematically from 583 patients. At the time of writing, mutations in SPG7 and AFG3L2 genes were reported to be associated with ophthalmoparesis and multiple mtDNA deletions in fourteen additional adult-onset PEO patients, bringing the total number of known genes to fourteen. Diagnostic yield is still critically dependent on the meticulous clinical and biochemical characterisation of patients. Understanding the intimate relationship between genotype and phenotype remains a fundamental challenge. The results of this systematic review provide guidance to both patients and clinician about future prognosis, and will serve, in future, to assess methods of disease prevention and evaluation of targeted therapeutic strategies.

パーキンソン症候群と多発ニューロパチーを呈し，&lt;i&gt;POLG1&lt;/i&gt;遺伝子変異をみとめた家族性進行性外眼筋麻痺の1家系

Multiple mitochondrial DNA (mtDNA) deletions usually occur secondarily to a mutation in one of the enzymes involved in mtDNA maintenance, such as polymerase γ, which is encoded by the nuclear polymerase γ1 gene (POLG1) and POLG2. Patients with multiple mtDNA deletion disorders show clinical heterogeneity of symptoms, in addition to usually seen progressive external ophthalmoplegia (PEO). We conducted clinical, histological and genetic analyses of two affected sisters in a family with the autosomal dominant inheritance pattern of PEO. A 73-year-old woman (patient 1) with congenital hypogonadism and PEO developed L-dopa responsive parkinsonism about the age of 60. Neurological examination revealed mild proximal muscle weakness and polyneuropathy too. Her 69-year-old sister (patient 2) also showed PEO, parkinsonism and polyneuropathy. Histopathological studies of biopsied muscle specimens from patient 1 revealed numerous ragged red fibers as well as fibers with increased succinate dehydrogenase activity and decreased cytochrome c oxidase activity. Multiple mtDNA deletions were detected, both by Southern blot and long-range PCR assays of total DNA from the biopsied muscle specimens. A systemic mutational analysis in both sisters revealed a heterozygous p.Y955C (c.2864A>G) mutation in POLG1. This is the first Japanese family identified with this mutation. We reviewed cases with this mutation highlighting a wide phenotypic spectrum of this disorder.

Analysis of mtDNA multiple deletions in 209 muscle specimens using massively parallel sequencing (MPS)

Analysis of mtDNA multiple deletions in 209 muscle specimens using massively parallel sequencing (MPS)

Twinkle mutations in two Chinese families with autosomal dominant progressive external ophthalmoplegia

Autosomal dominant progressive external ophthalmoplegia (adPEO) is a common adult onset mitochondrial disease caused by mutations in nuclear DNA (nDNA). Twinkle is one of the nuclear genes associated with adPEO. Clinical, histochemical, and molecular genetics findings of 6 patients from two Chinese families with adPEO were reported. Two point mutations (c.1423G>C, p.A475P and c.1061G>C, p.R354P) of Twinkle gene have been found. Multiple mtDNA deletions were also detected in patient's muscle and fibroblasts. This study confirms two mutations in Chinese adPEO families, which were first reported in the Chinese population.

Extraocular Muscle Atrophy and Central Nervous System Involvement in Chronic Progressive External Ophthalmoplegia

BackgroundChronic progressive external ophthalmoplegia (CPEO) is a classical mitochondrial ocular disorder characterised by bilateral progressive ptosis and ophthalmoplegia. These ocular features can develop either in isolation or in association with other prominent neurological deficits (CPEO+). Molecularly, CPEO can be classified into two distinct genetic subgroups depending on whether patients harbour single, large-scale mitochondrial DNA (mtDNA) deletions or multiple mtDNA deletions secondary to a nuclear mutation disrupting mtDNA replication or repair. The aim of this magnetic resonance imaging (MRI) study was to investigate whether the ophthalmoplegia in CPEO is primarily myopathic in origin or whether there is evidence of contributory supranuclear pathway dysfunction.MethodsTen age-matched normal controls and twenty patients with CPEO were recruited nine patients with single, large-scale mtDNA deletions and eleven patients with multiple mtDNA deletions secondary to mutations in POLG, PEO1, OPA1, and RRM2B. All subjects underwent a standardised brain and orbital MRI protocol, together with proton magnetic resonance spectroscopy in two voxels located within the parietal white matter and the brainstem.ResultsThere was evidence of significant extraocular muscle atrophy in patients with single or multiple mtDNA deletions compared with controls. There was no significant difference in metabolite concentrations between the patient and control groups in both the parietal white matter and brainstem voxels. Volumetric brain measurements revealed marked cortical and cerebellar atrophy among patients with CPEO+ phenotypes.ConclusionThe results of this study support a primary myopathic aetiology for the progressive limitation of eye movements that develops in CPEO.

O.24 Loss of function of MGME1, a novel player in mitochondrial DNA replication, causes a distinct autosomal recessive mitochondrial disorder

Mutations in genes involved in mitochondrial DNA (mtDNA) replication result in two molecular phenotypes of mitochondrial disorders, multiple mtDNA deletion and/or depletion syndromes, known collectively as mtDNA maintenance disorders. Disease mechanisms alter either the mtDNA replication machinery or the biosynthesis pathways of deoxyribonucleotides. We identified loss-of-function mutations in the orphan gene C20orf72, renamed as MGME1 (mitochondrial genome maintenance exonuclease 1), in 6 patients from 3 families originating from Lebanon, Italy, and Germany. The clinical phenotypes were almost identical and included upper eyelid ptosis, mild progressive external ophthalmoplegia, exercise intolerance, proximal and axial muscular weakness, muscle wasting, and profound emaciation. Intriguingly, all patients developed dyspnea, and respiratory failure usually required non-invasive ventilation. Cerebellar atrophy, gastrointestinal dysfunction, mental retardation, and cardiomyopathy were additional common symptoms. Muscle biopsies showed few ragged red and COX-negative fibers, respiratory chain dysfunction, multiple mtDNA deletions and mtDNA depletion. Furthermore, MGME1 mutations resulted in increased 7S DNA levels in patients‘ muscle and fibroblasts, which was also evident in MGME1-depleted cells. Patient fibroblasts failed to repopulate upon induced mtDNA depletion and accumulated replication intermediates similar to MGME1-depleted cells. MGME1 encodes a mitochondrial RecB-type exonuclease belonging to the PD-(D/E) XK nuclease superfamily. MGME1 preferentially cleaves single-stranded DNA with free 5′ DNA ends and processes flap-like substrates that contain RNA at the displaced 5′ arm thus resembling Okazaki fragments. In conclusion, MGME1 mutations affect mtDNA maintenance and result in a distinct mitochondrial disorder. MGME1 is the first identified mitochondrial exonuclease shown to be involved in replication and might play an additional role in mtDNA repair.

P17.19 Deoxypyrimidine monophosphates treatment for thymidine kinase 2 deficiency

Autosomal recessive TK2 mutations have been associated with severe depletion of mitochondrial DNA (mtDNA) and devastating neuromuscular diseases in infants and children, and with mtDNA multiple deletions and progressive external ophthalmoplegia in adults. Similar to other mitochondrial disorders, only supportive treatments are available for TK2 deficiency. We generated a Tk2 H126N knock-in mouse model that manifests a phenotype strikingly similar to the human infantile encephalomyopathy. We demonstrated that lack of Tk2 activity cause nucleotide pools unbalance with severe reductions of deoxypyrimidine triphosphates (dTTP and dCTP) in brain and liver, leading to reduction of mtDNA copy number. To bypass Tk2 deficiency, we administered deoxypyrimidine monophosphates (dCMP + dTMP) to Tk2 knock-in mice by oral gavage from postnatal day 4, when mutant mice are biochemically affected but phenotypically normal. Assessment of 13-day old Tk2−/− mice treated with dCMP + dTMP 200 mg/kg/day each demonstrated that in mutant animals, the compounds: raise dCMP + dTMP concentrations; increase levels of mtDNA, augment quantity and activities of mitochondrial respiratory chain enzyme; and significantly prolong their lifespan (34 days with treatment vs 13 days untreated). A second trial of dCMP + dTMP at 400 mg/kg/day showed even greater phenotypic and biochemical improvements. No adverse effects were observed with both doses of dCMP + dTMP. Oral dCMP + dTMP supplementation is the first effective and safe treatment for TK2 deficiency in mice. This treatment can potentially be applied to patients.

P.17.14 Myohistological mitochondrial changes in patients with mtDNA singular deletions, multiple deletions and A3243G point mutation

Chronic progredient external opthalmoplegia (CPEO) and the MELAS syndrome are the most common disorders that are associated with mitochondrial changes. CPEO is either associated with singular or multiple mtDNA deletions, whereas, MELAS is usually caused by the A3243G point mutation on mtDNA. The basic diagnostic criteria for these diseases are histopathological changes in muscle biopsy. So far, a systematic study comparing myopathological changes in these three groups has not yet been reported. We analysed muscle biopsies from 29 patients (16 males, 13 females) with mitochondrial diseases to investigate whether the genetic background influences the incidence of histopathological changes. 10 patients had singular mtDNA deletions and 11 patients had multiple deletions due to POLG1 nuclear mutations. Additionally, 8 patients harboured the MELAS A3243G point mutation. Freeze-dried sections of muscle biopsies were stained for histochemical reactions with Gomori trichrome, COX/SDH and SDH stainings. Furthermore, immunohistological reaction with COX antibody was also carried out. In all three groups, the COX-negative fibers were observed most frequently. The frequency of RRF was about a third of the COX-negative fibers. Dark blue COX-negative fibers were significantly higher than the hyperreactive fibers in SDH staining. The parallel analysis of individual pathological fibres showed that, in all three groups, all COX negative fibers had pathological changes also in other stainings and immunohistology. The results confirm that the COX/SDH double staining is the most sensitive method in the diagnosis compared to the other two stainings. Nevertheless, these changes could have been occurred due to the high overlap between the three groups in each individual cases.

P.10.17 Morphological changes in skeletal muscle associated with chronic ischemia

Chronic ischemia of the lower limbs is frequent in elderly individuals and is usually caused by arteriosclerosis and/or diabetes. In elderly people changes caused by chronic ischemia may influence the interpretation of a muscle biopsy analysis. It is therefore important to be aware of morphological alterations that may be caused by chronic ischemia. We investigated muscle biopsy specimens from the tibialis anterior muscle in 26 individuals (54–90 years of age, 13 with diabetes) amputated because of lower leg ischemia and compared them with 10 controls (72–78 years of age). None of the patients had a diagnosis of a primary neuromuscular disease. The specimens were investigated by routine histological techniques and by LX-PCR to identify mtDNA deletions. Morphological alterations in the 26 patients with chronic ischemia that were not present in controls included rimmed vacuoles (12 patients), interstitial fibrosis (14 patients), fiber type grouping (22 patients), group atrophy (19 patients) fibers with glycogen depletion (7 patients), necrotic fibers (6 patients), internalized nuclei (26 pateints), MHC class I upregulation (3 patients) and microangiopathy (13 patients). Cytochrome c oxidase deficient muscle fibres were identified in 25 patients (0.1–1.8% of the fibers) and in 7 controls (0.2–0.5% of the fibers). Multiple mtDNA deletions were identified in 16 patients and one control. None of the alterations were predominant in patients with diabetes. The results demonstrate that chronic ischemia is associated with marked morphological alterations in muscle that may mimic a primary myopathy. Neurogenic changes were frequent both among diabetic and non-diabetic patients.

Large scale deletions of the mitochondrial DNA in astheno, asthenoterato and oligoasthenoterato-spermic men

The purpose of this study was to investigate the association of large-scale deletions of mtDNA between idiopathic astheno, asthenoterato and oligoasthenoterato-spermic as patient group and normospermic as control group. Forty semen samples including: 10 asthenospermic (A), 10 asthenoteratospermic (AT), 10 oligoasthenoteratospermic (OAT) and 10 normospermic samples as control group, were collected from IVF center. Our analysis of long-range polymerase chain reaction were shown multiple deletions; 4977-bp, 7599-bp and 7491-bp of mtDNA in spermatozoa of patients (A, AT and OAT) and control groups. However, the frequency of multiple mtDNA deletions in astheno (60%), asthenoterato (60%), oligoasthenoterato (70%) spermic groups were significantly higher than normal (40%) group. These results suggest that mtDNA mutations cause infertility through an effect on sperm motility. Therefore, identification of mtDNA mutations and large scale deletions in the pathophysiology of human spermatozoa dysfunction is considered to be important to better understanding of the etiology of idiopathic infertility.