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

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

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

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

Both mitochondrial DNA and mitonuclear gene mutations cause hearing loss through cochlear dysfunction.

PFC is a Wellcome Trust Senior Fellow in Clinical Science (101876/Z/13/Z), and a UK NIHR Senior Investigator, who receives support from the Medical Research Council Mitochondrial Biology Unit (MC_UP_1501/2), the Wellcome Trust Centre for Mitochondrial Research (096919Z/11/Z), the Medical Research Council (UK) Centre for Translational Muscle Disease research (G0601943), EU FP7 TIRCON, and the National Institute for Health Research (NIHR) Biomedical Research Centre based at Cambridge University Hospitals NHS Foundation Trust and the University of Cambridge. PJK is a Wellcome Trust Clinical research fellow (101700/Z/13/Z). PYWM is supported by a Clinician Scientist Fellowship Award (G1002570) from the Medical Research Council (UK), and also receives funding from Fight for Sight (UK) and the UK National Institute of Health Research (NIHR) as part of the Rare Diseases Translational Research Collaboration. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. This study received support from the MRC Mitochondrial Disease Patient Cohort (http://mitocohort.ncl.ac.uk/).This is the final version of the article. It first appeared from Oxford University Press via https://doi.org/10.1093/brain/aww05

Habitual Physical Activity in Mitochondrial Disease

Mitochondrial disease is the most common neuromuscular disease and has a profound impact upon daily life, disease and longevity. Exercise therapy has been shown to improve mitochondrial function in patients with mitochondrial disease. However, no information exists about the level of habitual physical activity of people with mitochondrial disease and its relationship with clinical phenotype.Habitual physical activity, genotype and clinical presentations were assessed in 100 patients with mitochondrial disease. Comparisons were made with a control group individually matched by age, gender and BMI. = −0.49; 95% CI −0.33, −0.63, P<0.01). There were no systematic differences in physical activity between different genotypes mitochondrial disease.These results demonstrate for the first time that low levels of physical activity are prominent in mitochondrial disease. Combined with a high prevalence of obesity, physical activity may constitute a significant and potentially modifiable risk factor in mitochondrial disease

Pathogenic mtDNA mutations causing mitochondrial myopathy: The need for muscle biopsy.

Pathogenic mitochondrial tRNA (mt-tRNA) gene mutations represent a prominent cause of primary mitochondrial DNA (mtDNA)-related disease despite accounting for only 5%-10% of the mitochondrial genome.(1,2) Although some common mt-tRNA mutations, such as the m.3243A>G mutation, exist, the majority are rare and have been reported in only a small number of cases.(3) The MT-TP gene, encoding mt-tRNA(Pro), is one of the less polymorphic mt-tRNA genes, and only 5 MT-TP mutations have been reported as a cause of mitochondrial muscle disease to date (table e-1 at Neurology.org/ng, P6-10). We report 5 patients with myopathic phenotypes, each harboring different pathogenic mutations in the MT-TP gene, highlighting the importance of MT-TP mutations as a cause of mitochondrial muscle disease and the requirement to study clinically relevant tissue

Novel reproductive technologies to prevent mitochondrial disease

BACKGROUND: The use of nuclear transfer (NT) has been proposed as a novel reproductive treatment to overcome the transmission of maternally-inherited mitochondrial DNA (mtDNA) mutations. Pathogenic mutations in mtDNA can cause a wide-spectrum of lifelimiting disorders, collectively known as mtDNA disease, for which there are currently few effective treatments and no known cures. The many unique features of mtDNA make genetic counselling challenging for women harbouring pathogenic mtDNA mutations but reproductive options that involve medical intervention are available that will minimize the risk of mtDNA disease in their offspring. This includes PGD, which is currently offered as a clinical treatment but will not be suitable for all. The potential for NT to reduce transmission of mtDNA mutations has been demonstrated in both animal and human models, and has recently been clinically applied not only to prevent mtDNA disease but also for some infertility cases. In this review, we will interrogate the different NT techniques, including a discussion on the available safety and efficacy data of these technologies for mtDNA disease prevention. In addition, we appraise the evidence for the translational use of NT technologies in infertility. OBJECTIVE AND RATIONALE: We propose to review the current scientific evidence regarding the clinical use of NT to prevent mitochondrial disease. SEARCH METHODS: The scientific literature was investigated by searching PubMed database until Jan 2017. Relevant documents from Human Fertilisation and Embryology Authority as well as reports from both the scientific and popular media were also implemented. The above searches were based on the following key words: 'itochondria', 'mitochondrial DNA'; 'mitochondrial DNA disease', `fertility'; ` preimplantation genetic diagnosis', ` nuclear transfer', ` mitochondrial replacement' and ` mitochondrial donation'. OUTCOMES: While NT techniques have been shown to effectively reduce the transmission of heteroplasmic mtDNA variants in animal models, and increasing evidence supports their use to prevent the transmission of human mtDNA disease, the need for robust, long-term evaluation is still warranted. Moreover, prenatal screening would still be strongly advocated in combination with the use of these IVF-based technologies. Scientific evidence to support the use of NT and other novel reproductive techniques for infertility is currently lacking. WIDER IMPLICATIONS: It is mandatory that any new ART treatments are first adequately assessed in both animal and human models before the cautious implementation of these new therapeutic approaches is clinically undertaken. There is growing evidence to suggest that the translation of these innovative technologies into clinical practice should be cautiously adopted only in highly selected patients. Indeed, given the limited safety and efficacy data, close monitoring of any offspring remains paramount