Clinical and genotypic aspects of mitochondrial disease

PhD Thesis. Published works have been removed and the reader should refer to the printed version available from the University Library to view it in its entirety.Mitochondrial myopathies are a clinically multifarious group of genetic disorders that affect the central nervous system and skeletal muscles and other organs heavily dependent on aerobic metabolism. They are typically characterised by multi-system involvement and have extensive phenotypic and disease burden variability. These diseases are often relentlessly progressive with high morbidity and mortality. The biochemical and molecular basis of many of the common mitochondrial myopathies has been elucidated over the last decade, yet the association between mitochondrial gene mutations and clinical symptoms, requires further elucidation. I propose to clearly define the clinico-pathological and molecular features of adults with mitochondrial disease and evaluate if there is a clear correlation between clinical phenotype and the underlying genetic defect. Identifying clear clinical features should help guide genetic diagnosis and enable tailored counselling regarding potential disease progression. Unfortunately, to date, there are few effective treatments and no known cure for patients with mitochondrial myopathies. Exercise has been shown to hold significant positive effects upon skeletal muscle function and perceived health- related quality of life in patients with mitochondrial myopathies. The molecular basis of many of the common mitochondrial disorders has been elucidated over the last decade and although there is a vast spectrum of phenotypic expression throughout different genotypes, common symptoms are reported. Perceived fatigue is often a prominent symptom in patients with mitochondrial disease but to date, its prevalence, severity and aetiology is poorly understood. I wish to determine the prevalence and nature of perceived fatigue in a large, genetically heterogeneous group of patients with mitochondrial disease and systematically assess potential covariates of fatigue compared to healthy controls and patients with Myalgic Encephalopathy /Chronic Fatigue Syndrome. Health-related quality of life is important for understanding the impact and progression of chronic disease and is increasingly recognised as a fundamental patient-based outcome measure in both clinical intervention and research. Generic outcome measures have been extensively validated to assess health-related quality of life across populations and different disease states. However, due to their inclusive construct, it is acknowledged that not all relevant aspects of a specific illness may be captured. Hence there is a need to develop a disease-specific health-related quality of life measures that centre on symptoms characteristic of a specific disease or condition and their impact. SF-36 and its abbreviated version SF-12 are currently the only tools used routinely for measuring patient-reported outcomes in our patients with mitochondrial myopathies. I wish to explore the conceptualisation, development and preliminary psychometric evaluation (validity and reliability) of a mitochondrial disease -specific health-related quality of life measure, which may be used both in research and clinical settings. Indeed, in a condition where the natural history of the disease is poorly understood and therapeutic options are limited, long-term preservation of health-related quality of life in patients with mitochondrial disease poses a real challenge

Disease burden of myotonic dystrophy type 1

Objective: The objective of this cross-sectional, observational study was to investigate the disease burden of myotonic dystrophy type 1 (DM1), a disabling muscle disorder. Methods: Adults with DM1 were recruited as part of the PhenoDM1 study from Newcastle University (Newcastle upon Tyne, UK). Disease burden data were recorded through the Individualized Neuromuscular Quality of Life (INQoL) questionnaire. Results were examined by sex and clinical variables [e.g. the six-minute walk test (6MWT), the Mini Mental State Examination, and estimated progenitor and modal allele CTG repeat length]. Results: Our sample consisted of 60 patients with DM1 (mean age: 45 years; 45% female). Muscle weakness and fatigue constituted the two most common disease manifestations, reported by 93% and 90% of patients, respectively, followed by muscle locking (73%). Most patients (> 55%) reported feeling anxious/worried, depressed, frustrated, and/or having low confidence/self-esteem, 23% and 33% indicated substantial impairment of daily and leisure activities, respectively, and 47% did not work as a consequence of the disease. Estimated progenitor CTG length corrected by age correlated surprisingly well with INQoL scores. Differences by sex were generally minor. Conclusion: We show that DM1 is associated with a substantial disease burden resulting in impairment across many different domains of patients’ lives, emphasizing the need for a holistic approach to medical management. Our results also show that the INQoL records relevant information about patients with DM1, but that further investigation of the psychometric properties of the scale is needed for meaningful interpretation of instrument scores

Distal weakness with respiratory insufficiency caused by the m.8344A>G “MERRF” mutation

AbstractThe m.8344A>G mutation in the mt-tRNALys gene, first described in myoclonic epilepsy and ragged red fibers (MERRF), accounts for approximately 80% of mutations in individuals with MERRF syndrome. Although originally described in families with a classical syndrome of myoclonus, ataxia, epilepsy and ragged red fibers in muscle biopsy, the m.8344A>G mutation is increasingly recognised to exhibit marked phenotypic heterogeneity. This paper describes the clinical, morphological and laboratory features of an unusual phenotype in a patient harboring the m.8344A>G ‘MERRF’ mutation. We present the case of a middle-aged woman with distal weakness since childhood who also had ptosis and facial weakness and who developed mid-life respiratory insufficiency necessitating non-invasive nocturnal ventilator support. Neurophysiological and acetylcholine receptor antibody analyses excluded myasthenia gravis whilst molecular genetic testing excluded myotonic dystrophy, prompting a diagnostic needle muscle biopsy. Mitochondrial histochemical abnormalities including subsarcolemmal mitochondrial accumulation (ragged-red fibers) and in excess of 90% COX-deficient fibers, was seen leading to sequencing of the mitochondrial genome in muscle. This identified the m.8344A>G mutation commonly associated with the MERRF phenotype. This case extends the evolving phenotypic spectrum of the m.8344A>G mutation and emphasizes that it may cause indolent distal weakness with respiratory insufficiency, with marked histochemical defects in muscle. Our findings support consideration of screening of this gene in cases of indolent myopathy resembling distal limb-girdle muscular dystrophy in which screening of the common genes prove negative

Epilepsy in adults with mitochondrial disease: A cohort study.

OBJECTIVE: The aim of this work was to determine the prevalence and progression of epilepsy in adult patients with mitochondrial disease. METHODS: We prospectively recruited a cohort of 182 consecutive adult patients attending a specialized mitochondrial disease clinic in Newcastle upon Tyne between January 1, 2005 and January 1, 2008. We then followed this cohort over a 7-year period, recording primary outcome measures of occurrence of first seizure, status epilepticus, stroke-like episode, and death. RESULTS: Overall prevalence of epilepsy in the cohort was 23.1%. Mean age of epilepsy onset was 29.4 years. Prevalence varied widely between genotypes, with several genotypes having no cases of epilepsy, a prevalence of 34.9% in the most common genotype (m.3243A>G mutation), and 92.3% in the m.8344A>G mutation. Among the cohort as a whole, focal seizures, with or without progression to bilateral convulsive seizures, was the most common seizure type. Conversely, all of the patients with the m.8344A>G mutation and epilepsy experienced myoclonic seizures. Patients with the m.3243A>G mutation remain at high risk of developing stroke-like episodes (1.16% per year). However, although the standardized mortality ratio for the entire cohort was high (2.86), this ratio did not differ significantly between patients with epilepsy (2.96) and those without (2.83). INTERPRETATION: Epilepsy is a common manifestation of mitochondrial disease. It develops early in the disease and, in the case of the m.3243A>G mutation, often presents in the context of a stroke-like episode or status epilepticus. However, epilepsy does not itself appear to contribute to the increased mortality in mitochondrial disease

Pathophysiology of exercise intolerance in chronic diseases: the role of diminished cardiac performance in mitochondrial and heart failure patients

Objective: Exercise intolerance is a clinical hallmark of chronic conditions. The present study determined pathophysiological mechanisms of exercise intolerance in cardiovascular, neuromuscular, and metabolic disorders. Methods: In a prospective cross-sectional observational study 152 patients (heart failure reduced ejection fraction, n=32; stroke, n=34; mitochondrial disease, n=28; type two diabetes, n=28; and healthy controls, n=30) performed cardiopulmonary exercise testing with metabolic and haemodynamic measurements. Peak exercise O2 consumption and cardiac power output were measures of exercise tolerance and cardiac performance. Results: Exercise tolerance was significantly diminished in patients compared with controls (ie, by 45% stroke, 39% mitochondria disease, and 33% diabetes and heart failure, p<0.05). Cardiac performance was only significantly reduced in heart failure (due to reduced heart rate, stroke volume, and blood pressure) and mitochondrial patients (due reduced stroke volume) compared with controls (ie, by 53% and 26%, p<0.05). Ability of skeletal muscles to extract oxygen (ie, arterial-venous O2 difference) was diminished in mitochondrial, stroke, and diabetes patients (by 24%, 22%, and 18%, p<0.05), but increased by 21% in heart failure (p<0.05) compared with controls. Cardiac output explained 65% and 51% of the variance in peak O2 consumption (p<0.01) in heart failure and mitochondrial patients, whereas arterial-venous O2 difference explained 69% (p<0.01) of variance in peak O2 consumption in diabetes, and 65% and 48% in stroke and mitochondrial patients (p<0.01). Conclusions: Different mechanisms explain exercise intolerance in patients with heart failure, mitochondrial dysfunction, stroke and diabetes. Their better understanding may improve management of patients, their stress tolerance and quality of life

Multifocal demyelinating motor neuropathy and hamartoma syndrome associated with a de novo PTEN mutation.

OBJECTIVE: To describe a patient with a multifocal demyelinating motor neuropathy with onset in childhood and a mutation in phosphatase and tensin homolog (PTEN), a tumor suppressor gene associated with inherited tumor susceptibility conditions, macrocephaly, autism, ataxia, tremor, and epilepsy. Functional implications of this protein have been investigated in Parkinson and Alzheimer diseases. METHODS: We performed whole-exome sequencing in the patient's genomic DNA validated by Sanger sequencing. Immunoblotting, in vitro enzymatic assay, and label-free shotgun proteomic profiling were performed in the patient's fibroblasts. RESULTS: The predominant clinical presentation of the patient was a childhood onset, asymmetric progressive multifocal motor neuropathy. In addition, he presented with macrocephaly, autism spectrum disorder, and skin hamartomas, considered as clinical criteria for PTEN-related hamartoma tumor syndrome. Extensive tumor screening did not detect any malignancies. We detected a novel de novo heterozygous c.269T>C, p.(Phe90Ser) PTEN variant, which was absent in both parents. The pathogenicity of the variant is supported by altered expression of several PTEN-associated proteins involved in tumorigenesis. Moreover, fibroblasts showed a defect in catalytic activity of PTEN against the secondary substrate, phosphatidylinositol 3,4-trisphosphate. In support of our findings, focal hypermyelination leading to peripheral neuropathy has been reported in PTEN-deficient mice. CONCLUSION: We describe a novel phenotype, PTEN-associated multifocal demyelinating motor neuropathy with a skin hamartoma syndrome. A similar mechanism may potentially underlie other forms of Charcot-Marie-Tooth disease with involvement of the phosphatidylinositol pathway

Activities of daily living in myotonic dystrophy type 1

Objectives: The objective of this cross‐sectional, observational study was to investigate performance of activities of daily living in patients with myotonic dystrophy type 1 (DM1). Materials and Methods: Adults with genetically confirmed DM1 were recruited from Newcastle University (Newcastle upon Tyne, UK) and University College London Hospitals NHS Foundation Trust (London, UK). Data on activities of daily living were recorded through the DM1‐ActivC (scale scores range between 0 and 100, where a higher/lower score indicates a higher/lower ability). Results: Our sample comprised 192 patients with DM1 (mean age: 46 years; 51% female). Patients reported most difficulties with running, carrying and putting down heavy objects, and standing on one leg, and least difficulties with eating soup, washing upper body, and taking a shower. Irrespective of the disease duration (mean: 20 years), most patients were able to perform basic and instrumental activities of daily living (eg personal hygiene and grooming, showering, eating, cleaning and shopping), with the exception of functional mobility/transfer tasks (eg walking uphill and running). The mean DM1‐ActivC total score was estimated at 71 (95% CI: 68‐74). Estimated progenitor cytosine‐thymine‐guanine repeat length and age explained 27% of the variance in DM1‐ActivC total scores (P < .001). Conclusions: We show that DM1 impairs performance of activities of daily living, in particular those requiring a high degree of muscle strength, stability and coordination. Yet, across the evolution of the disease, the majority of patients will still be able to independently perform most basic and instrumental activities of daily living

Extensive respiratory chain defects in inhibitory interneurones in patients with mitochondrial disease

Aims: Mitochondrial disorders are among the most frequently inherited cause of neurological disease and arise due to mutations in mitochondrial or nuclear DNA. Currently, we do not understand the specific involvement of certain brain regions or selective neuronal vulnerability in mitochondrial disease. Recent studies suggest γ-aminobutyric acid (GABA)-ergic interneurones are particularly susceptible to respiratory chain dysfunction. In this neuropathological study, we assess the impact of mitochondrial DNA defects on inhibitory interneurones in patients with mitochondrial disease. Methods: Histochemical, immunohistochemical and immunofluorescent assays were performed on post-mortem brain tissue from 10 patients and 10 age-matched control individuals. We applied a quantitative immunofluorescent method to interrogate complex I and IV protein expression in mitochondria within GABAergic interneurone populations in the frontal, temporal and occipital cortices. We also evaluated the density of inhibitory interneurones in serial sections to determine if cell loss was occurring. Results: We observed significant, global reductions in complex I expression within GABAergic interneurones in frontal, temporal and occipital cortices in the majority of patients. While complex IV expression is more variable, there is reduced expression in patients harbouring m.8344A>G point mutations and POLG mutations. In addition to the severe respiratory chain deficiencies observed in remaining interneurones, quantification of GABAergic cell density showed a dramatic reduction in cell density suggesting interneurone loss. Conclusions: We propose that the combined loss of interneurones and severe respiratory deficiency in remaining interneurones contributes to impaired neuronal network oscillations and could underlie development of neurological deficits, such as cognitive impairment and epilepsy, in mitochondrial disease

Instability of the mitochondrial alanyl-tRNA synthetase underlies fatal infantile-onset cardiomyopathy

Recessively inherited variants in AARS2 (NM_020745.2) encoding mitochondrial alanyl-tRNA synthetase (mt-AlaRS) were first described in patients presenting with fatal infantile cardiomyopathy and multiple oxidative phosphorylation defects. To date, all described patients with AARS2-related fatal infantile cardiomyopathy are united by either a homozygous or compound heterozygous c.1774C>T (p.Arg592Trp) missense founder mutation that is absent in patients with other AARS2-related phenotypes. We describe the clinical, biochemical and molecular investigations of two unrelated boys presenting with fatal infantile cardiomyopathy, lactic acidosis and respiratory failure. Oxidative histochemistry showed cytochrome c oxidase-deficient fibres in skeletal and cardiac muscle. Biochemical studies showed markedly decreased activities of mitochondrial respiratory chain complexes I and IV with a mild decrease of complex III activity in skeletal and cardiac muscle. Using next-generation sequencing, we identified a c.1738C>T (p.Arg580Trp) AARS2 variant shared by both patients that was in trans with a loss-of-function heterozygous AARS2 variant; a c.1008dupT (p.Asp337*) nonsense variant or an intragenic deletion encompassing AARS2 exons 5-7. Interestingly, our patients did not harbour the p.Arg592Trp AARS2 founder mutation. In silico modelling of the p.Arg580Trp substitution suggested a deleterious impact on protein stability and folding. We confirmed markedly decreased mt-AlaRS protein levels in patient fibroblasts, skeletal and cardiac muscle, although mitochondrial protein synthesis defects were confined to skeletal and cardiac muscle. In vitro data showed that the p.Arg580Trp variant had a minimal effect on activation, aminoacylation or misaminoacylation activities relative to wild-type mt-AlaRS, demonstrating that instability of mt-AlaRS is the biological mechanism underlying the fatal cardiomyopathy phenotype in our patients.Peer reviewe

Management of seizures in patients with primary mitochondrial diseases: consensus statement from the InterERNs Mitochondrial Working Group

Background and purposePrimary mitochondrial diseases (PMDs) are common inborn errors of energy metabolism, with an estimated prevalence of one in 4300. These disorders typically affect tissues with high energy requirements, including heart, muscle and brain. Epilepsy may be the presenting feature of PMD, can be difficult to treat and often represents a poor prognostic feature. The aim of this study was to develop guidelines and consensus recommendations on safe medication use and seizure management in mitochondrial epilepsy. MethodsA panel of 24 experts in mitochondrial medicine, pharmacology and epilepsy management of adults and/or children and two patient representatives from seven countries was established. Experts were members of five different European Reference Networks, known as the Mito InterERN Working Group. A Delphi technique was used to allow the panellists to consider draft recommendations on safe medication use and seizure management in mitochondrial epilepsy, using two rounds with predetermined levels of agreement. ResultsA high level of consensus was reached regarding the safety of 14 out of all 25 drugs reviewed, resulting in endorsement of National Institute for Health and Care Excellence guidelines for seizure management, with some modifications. Exceptions including valproic acid in POLG disease, vigabatrin in patients with gamma-aminobutyric acid transaminase deficiency and topiramate in patients at risk for renal tubular acidosis were highlighted. ConclusionsThese consensus recommendations describe our intent to improve seizure control and reduce the risk of drug-related adverse events in individuals living with PMD-related epilepsy