Single Large-scale Deletions Research Articles

Mitochondrial DNA Mutation Load

Mitochondrial DNA Mutation Load

Prevalence and severity of voice and swallowing difficulties in mitochondrial disease

Mutations of mitochondrial DNA (mtDNA) cause a broad spectrum of clinical phenotypes. Anecdotal evidence suggests that voice and swallow problems are a common feature of these diseases. To characterize accurately the prevalence and severity of voice and swallow problems in a large cohort of patients with mitochondrial disease. Patients with proven mitochondrial disease were sent validated questionnaires to assess both voice and swallow function. The presence of voice and swallow symptoms was correlated with other clinical features of mitochondrial disease in affected patients. From the original 177 patients contacted, 98 swallowing status questionnaires and 96 Voice Handicap Index questionnaires were returned, response rates of 55% and 54%, respectively. Swallow: 48% of patients reported more difficulties with swallowing than control participants. Patients with single mtDNA deletions were most likely to report problems (65.2%), with patients with an m.8344A>G point mutation least likely (33.3%). All genotypes had a mean severity score in excess of the normal range, the highest mean score being found in the single large-scale mtDNA deletion group (10.12), the lowest in the m.3243A>G group (5.56) Voice; 48% of patients reported some difficulty with voice. Patients with single large-scale deletions showed the highest prevalence (65.2%), patients with the m.3243A>G mutation the lowest (33%). The most severe voice difficulties were reported by patients with an m.8344A>G point mutation. Patients with an m.3243A>G point mutation had the mildest and lowest incidence of voice problems. All genotypes scored outside of the normal range expected on the VHI overall (≥11.5 in control trials). Patients with an m.8344A>G point mutation reported a significantly higher degree of physical voice handicap than m.3243A>G patients (13.13 versus 4.40, p = 0.02). In patients with either single or multiple mtDNA deletions the likely pathophysiological mechanism is of proximal muscle weakness, whereas in patients with the m.8344A>G mutation cerebellar ataxia is the likely cause. Dysphagia and dysarthria have been identified as symptoms in previous research, however the prevalence and pathophysiology of these symptoms have not been explored. This paper indicates that voice and swallow problems are a common, though predominantly mild feature of mitochondrial disease and that there is a core group of pathophysiological symptoms linked to the presence of voice and swallowing problems. This paper recommends early referral to speech and language therapists to identify emerging dysphonia and dysphagia and to provide appropriate intervention.

Whole-Genome Comparison Reveals Novel Genetic Elements That Characterize the Genome of Industrial Strains of Saccharomyces cerevisiae

Human intervention has subjected the yeast Saccharomyces cerevisiae to multiple rounds of independent domestication and thousands of generations of artificial selection. As a result, this species comprises a genetically diverse collection of natural isolates as well as domesticated strains that are used in specific industrial applications. However the scope of genetic diversity that was captured during the domesticated evolution of the industrial representatives of this important organism remains to be determined. To begin to address this, we have produced whole-genome assemblies of six commercial strains of S. cerevisiae (four wine and two brewing strains). These represent the first genome assemblies produced from S. cerevisiae strains in their industrially-used forms and the first high-quality assemblies for S. cerevisiae strains used in brewing. By comparing these sequences to six existing high-coverage S. cerevisiae genome assemblies, clear signatures were found that defined each industrial class of yeast. This genetic variation was comprised of both single nucleotide polymorphisms and large-scale insertions and deletions, with the latter often being associated with ORF heterogeneity between strains. This included the discovery of more than twenty probable genes that had not been identified previously in the S. cerevisiae genome. Comparison of this large number of S. cerevisiae strains also enabled the characterization of a cluster of five ORFs that have integrated into the genomes of the wine and bioethanol strains on multiple occasions and at diverse genomic locations via what appears to involve the resolution of a circular DNA intermediate. This work suggests that, despite the scrutiny that has been directed at the yeast genome, there remains a significant reservoir of ORFs and novel modes of genetic transmission that may have significant phenotypic impact in this important model and industrial species.

Hemifacial presentation of mitochondrial myopathy

Chronic progressive external ophthalmoplegia (CPEO) is a common presentation of mitochondrial disease and is typically bilaterally symmetric. We present an unusual case of CPEO that remained unilateral and, therefore, eluded diagnosis for nearly 30 years. A 39-year-old woman presented with horizontal diplopia. She had a long standing right partial ptosis from her early twenties. There were no other neurological symptoms or relevant features in the history. Examination showed a right mild, non-fatigable ptosis, with limited adduction, elevation and depression of the right eye. The left eye and remaining ophthalmic and neurological examination were normal. Computer tomography (CT) imaging of brain and orbits was unremarkable and conservative management was pursued. Seven years later (age 46 years) the patient developed a droop of the right lower eyelid and mouth. Examination confirmed new facial weakness and the previous ptosis and ophthalmoplegia, all confined to the right side of the face (Fig. 1). There were no other neurological findings and no response to intravenous edrophonium test. Cranial CT and magnetic resonance imaging (MRI) were once again normal and the diagnosis remained elusive. Two years later, however (age 48), a limitation of depression and abduction developed in the left eye. Bilateral ophthalmoplegia now suggested the possibility of CPEO. Left triceps muscle biopsy was performed. Although there were no ragged red fibres, histochemistry demonstrated occasional cytochrome-C oxidase (COX)deficient fibres. Electron microscopy showed peripheral collections of glycogen-rich cytoplasm, containing small aggregates of mitochondria. Screening of the mitochondrial DNA for deletions using long range PCR followed by Southern blotting revealed the presence of a single large scale deletion of the mitochondrial genome. Sequencing across the deletion breakpoint confirmed the presence of the ‘‘common’’ deletion of 4,977 base pairs, with break points in the flanking repeats between nucleotide (nt) 8,470 and 8,482, and nt 13,447 and 13,459. CPEO is characterised by bilateral ptosis and global restriction of eye movements [1]. It is the commonest ocular manifestation of the mitochondrial myopathies and can occur in isolation or with additional non-ocular features [2, 3]. Diagnosis is often delayed for many years [4, 5]. In our case, two atypical features failed to suggest the diagnosis (1) the strict unilaterality of the ptosis and ophthalmoplegia for decades, and (2) the additional feature of unilateral facial weakness. Bilateral ptosis is a hallmark of CPEO and is the presenting complaint in up to 90% of cases [1]. The ptosis can be asymmetric, however [2]. Unilateral or asymmetric ptosis was noted in 11 patients out of 27 in a recent CPEO series [6]. Although unilateral or asymmetric ptosis may occur in early disease, bilateral ptosis becomes the norm as the disease progresses. Our case was very atypical because the ptosis remained unilateral throughout. Ophthalmoplegia in CPEO is usually symmetric and, in advanced cases, often complete [1]. Although asymmetric ophthalmoplegia does occur in a minority of CPEO patients [1, 7], accurate measurements of ocular motility have shown that the extent of asymmetry is very small [8]. The marked asymmetry of ophthalmoplegia in our case is therefore surprising. N. Ali (&) J. Acheson Department of Neuro-Ophthalmology, National Hospital for Neurology and Neurosurgery, Queen Square, London WC1N 3BG, UK e-mail: nadeem.ali@nhs.net

A single large-scale deletion of mtDNA in a child with recurrent encephalopathy and tubulopathy

A 26-month-old child presented with an unusual combination of growth retardation, renal proximal tubulopathy, hypoparathyroidism, and episodic encephalopathy with fever and lethargy. Muscle biopsy revealed defects of mitochondrial respiratory chain enzyme complexes I, III, and IV, but no ragged-red fibers or cytochrome c oxidase deficient fibers. Analysis of muscle mitochondrial DNA (mtDNA) showed a heteroplasmic 7663 base pair (bp) single deletion with a perfect 10 bp direct sequence repeat at the boundaries. At age 3 years and 9 months, the child developed sepsis and acute deterioration of her encephalopathy leading to death. This case expands the phenotypic diversity of mitochondrial disorders in pediatric patients and reinforces the importance of biochemical analyses of muscle biopsies in patients suspected of having a mitochondrial disorder.

Response To Prolonged Endurance Training and Detraining In Mitochondrial Myopathy

Patients with mitochondrial myopathies (MM) due to mtDNA mutations in skeletal muscle often suffer disabling exercise intolerance. Our previous studies suggest that short duration (14 weeks) endurance training improves exercise capacity and increases the activity of rate-limiting enzymes. The effect of a longer duration exercise training has not been studied. PURPOSE: To assess the effect of 6 months of regular endurance training on exercise capacity and to determine if training adaptations are maintained after a prolonged period of self-selected activity in a patient with severe muscle oxidative impairment due to a mitochondrial myopathy. METHODS: A 26-year male with a heteroplasmic single large-scale deletion of mtDNA underwent 26 weeks of cycle exercise training followed by a 52 week period of self-selected activity level. Peak work, oxygen uptake (VO2), and submaximal heart rate and blood lactate were determined at baseline, after training and following 52 weeks of self-determined activity. RESULTS: Compared to baseline, training increased peak work from 110 to 130 watts and VO2 from 1.527 to 1.837 L/min. During submaximal (50 watt) exercise, peak heart rate decreased from 153 to 120 bpm and venous lactate from 6.41 to 3.25 mM. After 52 weeks of self-selected activity following 6 months of endurance training, peak work and VO2 fell to 110 watts and 1.684 L/min respectively. During submaximal exercise, after the self-selected activity period compared to post training, heart rate (133 vs. 120 bpm) and lactate levels (4.36 vs. 3.25 mM) were higher. CONCLUSION: Endurance training in MM improved work and oxidative capacity supporting the efficacy of this therapy in patients with mitochondrial myopathy due to single large-scale mtDNA deletions. Despite the substantial benefits of exercise training, the patient did not choose to maintain regular exercise training and after the 52 week period of self-selected activity, exercise capacity declined to near pre-training levels indicating that deconditioning contributes to exercise limitations in these patients and that the applicability of exercise training as therapy is limited by patient motivation.

Differential diagnosis in ptosis and ophthalmoplegia: mitochondrial disease or myasthenia?

Sirs: One of the commonest phenotypes of mitochondrial disease is generalised fatigue with ptosis and a chronic progressive external ophthalmoplegia. The main differential diagnosis in these patients is with myasthenia gravis (MG). Ocular symptoms are the most common presentation of MG [1] and in up to 48% of patients are the only clinical manifestation [2]. Correct diagnosis is vital both to provide the best treatments and to avoid side effects from inappropriate ones [3]. We reviewed the records of 430 patients with proven mitochondrial disease on the basis of muscle histochemistry or mitochondrial DNA analysis to determine how many had originally been diagnosed with MG. Eighty-one of our patients had first presented with ptosis or ophthalmoplegia. Of these, a relatively small number, 15, had been investigated for possible MG as part of their diagnostic workup, and 5 (6.2%) had been given a primary diagnosis of MG. Table 1 shows the extra-ocular clinical features of the patients both at the time of diagnosis with MG and at subsequent diagnosis with mitochondrial disease. The average time between the initial diagnosis of MG and the subsequent diagnosis of mitochondrial disease was 3.6 years (range 1–9). Three of these patients had a single largescale deletion of mitochondrial DNA, and in two multiple deletions of the mitochondrial genome were found. All of the five patients initially diagnosed with MG had negative anti-acetylcholine receptor antibodies Table 2. Three patients were reported as having had a positive response to tensilon, although in one patient a repeat test was negative. Only three patients had undergone the more sensitive test of single fibre electromyography of orbicularis oculi. In one patient the initial result was reported as being consistent with myasthenia gravis on the basis of increased jitter (but with no blocking); this was subsequently repeated and reported as normal. All five patients had been treated with oral anticholinesterases. In one, an initial but shortlived symptomatic improvement was recorded. Three patients had received prolonged courses of oral steroids because of poor response to anticholinesterase treatment and one patient had undergone thymectomy. The differential diagnosis between mitochondrial disease presenting as ptosis and external ophthalmoplegia and myasthenia gravis can be difficult. The presenting clinical features of all the patients were entirely compatible with MG. Indeed, the majority reported variability in symptoms, often regarded as a hallmark of MG. Even those symptoms, which became apparent years later, such as proximal weakness and dysphagia, are common in MG. The investigations performed were similarly ambiguous; although all patients had negative anti-acetylcholine receptor antibodies, these are negative in up to 50% of ocular MG. Three had a positive tensilon test and one a suggestive single fibre EMG. Whilst all of these were probably false positives, an intriguing possibility is that these patients had disturbed neuromuscular transmission as a direct result of their mitochondrial disease. Blocking mitochondrial function in vitro leads to reduced phasic acetyl choline vesicle release [4] and changes in neuromuscular transmission similar to antibody mediated MG. Families with proven mitochondrial disease show minor but definite abnormalities on single fibre electromyography [5–7]. There are also anecdotal reports of symptomatic improvement in patients with mitochondrial disease given anticholinesterases [8], including one patient in this study. Although these drugs are as likely as any other to produce a placebo effect, these remain interesting observations. Our study emphasises the need to consider mitochondrial disease in the differential diagnosis of ocular MG, especially in those patients in whom symptoms do not respond well to treatment. R. G. Whittaker (&) AE A. M. Schaefer R. W. Taylor AE D. M. Turnbull Mitochondrial Research Group School of Neurology Neurobiology and Psychiatry University of Newcastle upon Tyne Newcastle upon Tyne NE2 4HH, UK Tel.: +1-91-2228334 Fax: +1-91-2228553 E-Mail: r.whittaker@ncl.ac.uk LETTER TO THE EDITORS J Neurol (2007) 254:1138– 1139 DOI 10.1007/s00415-006-0484-5

Aerobic training is safe and improves exercise capacity in patients with mitochondrial myopathy

Exercise intolerance is a prominent symptom in patients with mitochondrial myopathy (MM), but it is still unsettled whether exercise training is safe and beneficial for patients with MM. To address this, we studied the effect of 12 weeks cycle training on exercise capacity, quality of life and underlying molecular and cellular events in five patients with single large-scale deletions, one with a microdeletion and 14 with point mutations of mitochondrial DNA (mtDNA), and 13 healthy subjects. Each training session lasted 30 min, and was performed at an intensity of 70% of VO2max (maximal oxygen uptake). Each subject performed 50 training sessions in 12 weeks. All subjects were evaluated before and after training, and 13 MM patients were studied after 8 weeks of deconditioning. Evaluation included VO2max and mutation load and mtDNA quantity, mitochondrial enzymatic activity, and number of centrally nucleated, apoptotic, ragged red and cytochrome oxidase (COX)-negative fibres in muscle biopsies from the quadriceps muscle. After 12 weeks of training, VO2max and muscle citrate synthase increased in MM (26 and 67%) and healthy (17 and 65%) subjects, while mtDNA quantity in muscle only increased in the MM patients (81%). In the MM patients, training did not change mtDNA mutation load in muscle, mitochondrial enzyme complex activities, muscle morphology and plasma creatine kinase. After deconditioning, VO2max and citrate synthase activity returned to values before training, while muscle mtDNA mutation load decreased. These findings show that aerobic training efficiently improves oxidative capacity in MM patients. Based on unchanged levels of mutant load in muscle, morphological findings on muscle biopsy and plasma creatine kinase levels during training, the treatment appears to be safe. Regular, supervised aerobic exercise is therefore recommended in MM patients with the studied mutations.

Executive and visuospatial deficits in patients with chronic progressive external ophthalmoplegia and Kearns-Sayre syndrome.

Although neuropsychological deficits have been reported in mitochondrial cytopathies, patients with chronic progressive external ophthalmoplegia (CPEO) or Kearns-Sayre syndrome (KSS) have not been studied systematically using a comprehensive test battery. The aim of our study was to assess the range and extent of putative cognitive dysfunction in 22 patients with CPEO or KSS, and to compare cognitive performance of patients with healthy control subjects matched for age, sex and years of education. Genetic analysis of skeletal muscle tissue from 22 patients with CPEO or KSS included screening for mitochondrial DNA (mtDNA) point mutations (3243/8344) and mtDNA deletions. All patients were examined by a neuropsychological test battery covering verbal skills, verbal and visual memory, visuo-spatial perception, visual construction, attention, abstraction and flexibility, and Quality of Life. Molecular genetic analysis of mtDNA revealed single large-scale deletions in 15 out of 22 patients and the tRNA (Leu) A3243G point mutation in two out of 22 patients. In five out of 22 patients none of the frequently encountered mtDNA mutations could be detected. Neuropsychological testing did not reveal general intellectual deterioration, but specific cognitive deficits, particularly in visual construction, attention and abstraction/flexibility. Subgroup analysis of 15 patients with mtDNA deletions showed similar results when compared with the full group. In our series of patients with CPEO or KSS neuropsychological testing did not reveal signs that would suggest general intellectual decline or dementia, but provided evidence of specific focal neuropsychological deficits, suggesting particular impairment of visuospatial perception associated to parieto-occipital lobes and executive deficits associated to the prefrontal cortex.

Identical Mitochondrial DNA Deletion in a Woman with Ocular Myopathy and in Her Son with Pearson Syndrome

Single deletions of mitochondrial DNA (mtDNA) are associated with three major clinical conditions: Kearns-Sayre syndrome, a multisystem disorder; Pearson syndrome (PS), a disorder of the hematopoietic system; and progressive external ophthalmoplegia (PEO), primarily affecting the ocular muscles. Typically, single mtDNA deletions are sporadic events, since the mothers, siblings, and offspring of affected individuals are unaffected. We studied a woman who presented with PEO, ptosis, and weakness of pharyngeal, facial, neck, and limb muscles. She had two unaffected children, but another of her children, an infant son, had sideroblastic anemia, was diagnosed with PS, and died at age 1 year. Morphological analysis of a muscle biopsy sample from the mother showed cytochrome c oxidase-negative ragged-red fibers-a typical pattern in patients with mtDNA deletions. Southern blot analysis using multiple restriction endonucleases and probed with multiple mtDNA fragments showed that both the mother and her infant son harbored an identical 5,355-bp single deletion in mtDNA, without flanking direct repeats. The deletion was the only abnormal species of mtDNA identified in both patients, and there was no evidence for duplications. We conclude that, although the vast majority of single large-scale deletions in mtDNA are sporadic, in rare cases, single deletions can be transmitted through the germline.

New insights into the metabolic consequences of large-scale mtDNA deletions: a quantitative analysis of biochemical, morphological, and genetic findings in human skeletal muscle.

In order to study putative genotype phenotype correlations in mitochondrial disorders due to large-scale mtDNA deletions we performed a quantitative analysis of biochemical, morphological, and genetic findings in 20 patients. The size of the mtDNA deletions varied from 2 to 7.5 kb with a degree of heteroplasmy ranging from 16% to 78%. Applying improved methods for measuring respiratory chain enzyme activities, we found highly significant inverse correlations between the percentage of cytochrome c oxidase (COX)- negative fibers and citrate synthase (CS) normalized COX ratios. Significant correlations were also established between CS normalized complex I and complex IV ratios as well as between the degree of heteroplasmy of mtDNA deletions and the percentage of ragged red fibers, COX-negative fibers, and CS normalized complex I and complex IV ratios. Our results indicate that the degree of heteroplasmy of mtDNA deletions is mirrored on the histological as well as the biochemical level. Furthermore, our findings suggest that single large-scale deletions equally influence the activities of all mitochondrially encoded respiratory chain enzymes. Even low degrees of heteroplasmy of mtDNA deletions were found to result in biochemical abnormalities indicating the absence of any well-defined mtDNA deletion threshold in skeletal muscle.