Detecting mitochondrial electron transport chain enzyme defects in low-heteroplasmy single large-scale mtDNA deletion syndromes (SLSMDSs).

Selenium and zinc protect brain mitochondrial antioxidants and electron transport chain enzymes following postnatal protein malnutrition

The failure to trigger mitophagy is implicated in the pathogenesis of familial Parkinson disease that is caused by PINK1 or Parkin mutations. According to the prevailing PINK1-Parkin signaling model, mitophagy is promoted by the mitochondrial translocation of Parkin, an essential PINK1-dependent step that occurs via a previously unknown mechanism. Here we determined that critical concentrations of NO was sufficient to induce the mitochondrial translocation of Parkin even in PINK1 deficiency, with apparent increased interaction of full-length PINK1 accumulated during mitophagy, with neuronal nitric oxide synthase (nNOS). Specifically, optimum levels of NO enabled PINK1-null dopaminergic neuronal cells to regain the mitochondrial translocation of Parkin, which appeared to be significantly suppressed by nNOS-null mutation. Moreover, nNOS-null mutation resulted in the same mitochondrial electron transport chain (ETC) enzyme deficits as PINK1-null mutation. The involvement of mitochondrial nNOS activation in mitophagy was further confirmed by the greatly increased interactions of full-length PINK1 with nNOS, accompanied by mitochondrial accumulation of phospho-nNOS (Ser(1412)) during mitophagy. Of great interest is that the L347P PINK1 mutant failed to bind to nNOS. The loss of nNOS phosphorylation and Parkin accumulation on PINK1-deficient mitochondria could be reversed in a PINK1-dependent manner. Finally, non-toxic levels of NO treatment aided in the recovery of PINK1-null dopaminergic neuronal cells from mitochondrial ETC enzyme deficits. In summary, we demonstrated the full-length PINK1-dependent recruitment of nNOS, its activation in the induction of Parkin translocation, and the feasibility of NO-based pharmacotherapy for defective mitophagy and ETC enzyme deficits in Parkinson disease.

Progressive external ophthalmoplegia (PEO) is a common subtype of mitochondrial encephalomyopathy. The study aimed to investigate the relationship between mitochondrial DNA (mtDNA) abnormalities, muscle pathology, and clinical manifestations in Chinese patients with single large-scale mtDNA deletion presenting with PEO. This is a retrospective single-center study. Patients with PEO who had a single large deletion in mitochondrial DNA were included in this study. The associations were analyzed between mtDNA deletion patterns, myopathological changes, and clinical characteristics. In total, 155 patients with mitochondrial PEO carrying single large-scale mtDNA mutations were enrolled, including 137 chronic progressive external ophthalmoplegia (CPEO) and 18 Kearns-Sayre syndrome (KSS) patients. The onset ages were 9.61 ± 4.12 in KSS and 20.15 ± 9.06 in CPEO. The mtDNA deletions ranged from 2225 bp to 9131 bp, with m.8470_13446del being the most common. The KSS group showed longer deletions than the CPEO group (p = 0.004). Additionally, a higher number of deleted genes encoding respiratory chain complex subunits (p = 0.001) and tRNA genes (p = 0.009) were also observed in the KSS group. A weak negative correlation between the mtDNA deletion size and ages of onset (p < 0.001, r = -0.369) was observed. The proportion of ragged red fibers, ragged blue fibers, and cytochrome c negative fibers did not correlate significantly with onset ages (p > 0.05). However, a higher percentage of abnormal muscle fibers corresponds to an increased prevalence of exercise intolerance, limb muscle weakness, dysphagia, and cerebellar ataxia. We reported a large Chinese cohort consisting of mitochondrial PEO patients with single large-scale mtDNA deletions. Our results demonstrated that the length and locations of mtDNA deletions may influence onset ages and clinical phenotypes. The severity of muscle pathology could not only indicate diagnosis but also may be associated with clinical manifestations beyond the extraocular muscles.

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

Nature of Mitochondrial DNA Deletions in Substantia Nigra Neurons

Risk of developing a mitochondrial DNA deletion disorder

Defects of mitochondrial DNA (mtDNA) are an important cause of genetic disease in humans.1 The defect may take the form of a deletion or point mutation, and clinical features range...

Progressive external ophthalmoplegia (PEO), Kearns-Sayre syndrome (KSS) and Pearson syndrome are the three sporadic clinical syndromes classically associated with single large-scale deletions of mitochondrial DNA (mtDNA). PEO plus is a term frequently utilized in the clinical setting to identify patients with PEO and some degree of multisystem involvement, but a precise definition is not available. The purpose of the present study is to better define the clinical phenotypes associated with a single mtDNA deletion, by a retrospective study on a large cohort of 228 patients from the database of the "Nation-wide Italian Collaborative Network of Mitochondrial Diseases". In our database, single deletions account for about a third of all patients with mtDNA-related disease, more than previously recognized. We elaborated new criteria for the definition of PEO and "KSS spectrum" (a category of which classic KSS represents the most severe extreme). The criteria for "KSS spectrum" include the resulting multisystem clinical features associated with the KSS features, and which therefore can predict their presence or subsequent development. With the new criteria, we were able to classify nearly all our single-deletion patients: 64.5% PEO, 31.6% KSS spectrum (including classic KSS 6.6%) and 2.6% Pearson syndrome. The deletion length was greater in KSS spectrum than in PEO, whereas heteroplasmy was inversely related with age at onset. We believe that the new phenotype definitions implemented here may contribute to a more homogeneous patient categorization, which will be useful in future cohort studies of natural history and clinical trials.

We estimated the frequency of multiple mtDNA rearrangements by Southern blot in 32 patients affected by mitochondrial disorders associated with single deletions in order to assess genotype-phenotype correlations and elucidate the pathogenic significance of mtDNA duplications. Muscle in situ hybridization studies were performed in patients showing mtDNA duplications at Southern blot. We found multiple rearrangements in 12/32 (37.5%) patients; in particular, mtDNA duplications were detected in 4/4 Kearns-Sayre syndrome (KSS), in 1 Pearson's syndrome, in 1/3 encephalomyopathies with progressive external ophthalmoplegia (PEO), and in 2/23 PEO. In situ studies documented an exclusive accumulation of deleted mtDNAs in cytochrome c oxidase negative fibers of patients with mtDNA duplications. The presence of mtDNA duplications significantly correlated with onset of symptoms before age 15 and occurrence of clinical multisystem involvement. Analysis of biochemical data documented a predominant reduction of complex III in patients without duplications compared to patients with mtDNA duplications. Our data indicate that multiple mtDNA rearrangements are detectable in a considerable proportion of patients with single deletions and that mtDNA duplications do not cause any oxidative impairment. They more likely play a pathogenic role in the determination of clinical expression of mitochondrial diseases associated with single mtDNA deletions, possibly generating deleted mtDNAs in embryonic tissues by homologous recombination.

Large-scale mitochondrial DNA (mtDNA) deletions are associated with clinical conditions such as Kearns-Sayre syndrome and chronic progressive external ophthalmoplegia in adults and Pearson syndrome in children. Reported case series have suggested that deletions are not uncommon in the population, but their prevalence has not been documented. The authors ascertained patients with clinical features associated with mtDNA deletions in a defined adult population in northern Finland. Buccal epithelial samples were requested from each patient fulfilling the selection criteria, and full-length mtDNA was amplified using the long PCR method. Deletion breakpoints were identified using sequencing. Patients with deletions were examined clinically. The authors identified four patients with single large-scale mtDNA deletions. The prevalence of deletions was calculated to be 1.6/100,000 in the adult population in the province of Northern Ostrobothnia (0.0 to 3.2; 95% CI). Analysis of incident cases from a neighboring province revealed two patients with deletions and yielded a similar population frequency. The frequency of large-scale mitochondrial DNA deletions is similar among populations, suggesting that there is a constant rate of new deletions.

Recognizing the evolution of clinical syndrome spectrum progression in individuals with single large-scale mitochondrial DNA deletion syndromes (SLSMDS).

Mitochondrial DNA (mtDNA) deletions are a common cause of mitochondrial disorders. Large mtDNA deletions can lead to a broad spectrum of clinical features with different age of onset, ranging from mild mitochondrial myopathies (MM), progressive external ophthalmoplegia (PEO), and Kearns-Sayre syndrome (KSS), to severe Pearson syndrome. The aim of this study is to investigate the molecular signatures surrounding the deletion breakpoints and their association with the clinical phenotype and age at onset. MtDNA deletions in 67 patients were characterized using array comparative genomic hybridization (aCGH) followed by PCR-sequencing of the deletion junctions. Sequence homology including both perfect and imperfect short repeats flanking the deletion regions were analyzed and correlated with clinical features and patients' age group. In all age groups, there was a significant increase in sequence homology flanking the deletion compared to mtDNA background. The youngest patient group (<6 years old) showed a diffused pattern of deletion distribution in size and locations, with a significantly lower sequence homology flanking the deletion, and the highest percentage of deletion mutant heteroplasmy. The older age groups showed rather discrete pattern of deletions with 44% of all patients over 6 years old carrying the most common 5 kb mtDNA deletion, which was found mostly in muscle specimens (22/41). Only 15% (3/20) of the young patients (<6 years old) carry the 5 kb common deletion, which is usually present in blood rather than muscle. This group of patients predominantly (16 out of 17) exhibit multisystem disorder and/or Pearson syndrome, while older patients had predominantly neuromuscular manifestations including KSS, PEO, and MM. In conclusion, sequence homology at the deletion flanking regions is a consistent feature of mtDNA deletions. Decreased levels of sequence homology and increased levels of deletion mutant heteroplasmy appear to correlate with earlier onset and more severe disease with multisystem involvement.

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.

Over 20years ago single clonal deletions were the first mitochondrial DNA (mtDNA) genetic defects described in association with human disease. Since then very large numbers of children and adults harbouring such deletions have been described and it is clear they are an important cause of human mitochondrial disease. However, there still remain many important challenges in relation to our understanding of mechanisms leading to deletion formation and propagation and in relation to the factors determining the complex and varying relationship between genotype and clinical phenotype. Although multidisciplinary team care is essential and can improve quality of life and outcomes for patients, a definitive molecular treatment for single mtDNA deletions remains an important translational research goal. Patients with mtDNA deletions exhibit a very wide range of different clinical phenotypes with marked variation in age at onset and disease severity. Single mtDNA deletions may enter into the differential diagnosis of many different paediatric and adult presentations across a wide range of medical specialties, although neurological presentations are amongst the most common. In this review, we examine the molecular mechanisms underpinning mtDNA replication and we consider the hypotheses proposed to explain the formation and propagation of single large-scale mtDNA deletions. We also describe the range of clinical features associated with single mtDNA deletions, outline a molecular diagnostic approach and discuss current management including the role of aerobic and resistance exercise training programmes.

Objective: To correlate clinical findings with muscle histological and molecular data in 19 patients with POLG-related disorder. Background POLG encodes for the catalytic subunit of the mitochondrial DNA (mtDNA) polymerase gamma. Mutations in POLG result in a spectrum of clinical phenotypes varying from Alpers syndrome to ataxia-peripheral neuropathy (PN) syndrome to progressive external ophthalmoplegia (PEO). Design/Methods: Review of clinical and muscle histological findings, quantitation of mtDNA in blood and/or muscle by qPCR, search for mtDNA multiple deletions in muscle and/or blood by Southern blot and/or PCR. Results: 1. Four patients with Alpers syndrome had normal muscle biopsy. All had normal mtDNA content in blood; 1/4 had mtDNA depletion in muscle; none of them had mtDNA deletions in muscle. All 4 patients were compound heterozygous for two missense mutations. 2. Twelve patients with PEO and myopathy accompanied by PN in 9 and stroke and epilepsy in 2, had normal mtDNA content in blood. Eleven patients had muscle biopsy showing mitochondrial myopathy only in 9. One patient had mtDNA depletion and deletions in muscle; another had multiple deletions in both muscle and blood, and 3 others had multiple mtDNA deletions in muscle but not in blood. Two patients were homozygous for missense mutations and 10 were compound heterozygous for missense or splice-site mutations. 3. Two patients with PN and ataxia had no histological signs of mitochondrial myopathy. One patient was homozygous for p.A467T and the other was a compound heterozygous for two missense mutations. 4. A patient with intractable epilepsy and stroke-like events had COX (-) fibers, no mtDNA deletions in blood and normal muscle mtDNA. This patient carried two missense mutations. Conclusions: Sequencing of POLG was the most reliable diagnostic test in Alpers syndrome. Histological signs of mitochondrial dysfunction did not correlate with mtDNA depletion or deletions. Supported by: Mayo Clinic CTSA through grant number UL1 RR024150 from NIH/NCRR. Disclosure: Dr. Milone has nothing to disclose. Dr. Tang has nothing to disclose. Dr. Sorenson has nothing to disclose. Dr. Zhiyv has nothing to disclose. Dr. Wong has nothing to disclose.