PNKP Mutations Identified by Whole-Exome Sequencing in a Norwegian Patient with Sporadic Ataxia and Edema.

We identified PNKP mutations in a Norwegian woman with AOA. This patient had the typical findings with cognitive dysfunction, peripheral neuropathy, cerebellar dysarthria, horizontal nystagmus, oculomotor apraxia, and severe truncal and appendicular ataxia. In addition, she had hypoalbuminemia and massive lower limb edema which showed some improvement with treatment. Exome sequencing identified two heterozygous mutations, one in exon 14 (c.1196T>C, p.Leu399Pro) and one in exon 16 (c.1393_1396del, p.Glu465*). This is the first non-Portuguese patient with AOA due to PNKP mutations and provides independent verification that PNKP mutations cause AOA.

ADCK3 mutations with epilepsy, stroke-like episodes and ataxia: a POLG mimic?

BACKGROUND AND PURPOSE: Defects of coenzyme Q10 (CoQ10) metabolism cause a variety of disorders ranging from isolated myopathy to multisystem involvement. ADCK3 is one of several genes associated with CoQ10 deficiency that presents with progressive cerebellar ataxia, epilepsy, migraine and psychiatric disorders. Diagnosis is challenging due to the wide clinical spectrum and overlap with other mitochondrial disorders. METHODS: A detailed description of three new patients and one previously reported patient from three Norwegian families with novel and known ADCK3 mutations is provided focusing on the epileptic semiology and response to treatment. Mutations were identified by whole exome sequencing and in two measurement of skeletal muscle CoQ10 was performed. RESULTS: All four patients presented with childhood-onset epilepsy and progressive cerebellar ataxia. Three patients had epilepsia partialis continua and stroke-like episodes affecting the posterior brain. Electroencephalography showed focal epileptic activity in the occipital and temporal lobes. Genetic investigation revealed ADCK3 mutations in all patients including a novel change in exon 15: c.T1732G, p.F578V. There was no apparent genotype-phenotype correlation. CONCLUSION: ADCK3 mutations can cause a combination of progressive ataxia and acute epileptic encephalopathy with stroke-like episodes. The clinical, radiological and electrophysiological features of this disorder mimic the phenotype of polymerase gamma (POLG) related encephalopathy and it is therefore suggested that ADCK3 mutations be considered in the differential diagnosis of mitochondrial encephalopathy with POLG-like features.

EFNS review on the role of muscle biopsy in the investigation of myalgia.

BACKGROUND: Myalgia, defined as any pain perceived in muscle, is very common in the general population and a frequent cause for referral to neurologists, rheumatologists and internists in general. It is however only rarely due to primary muscle disease and often referred from ligaments, joints, bones, the peripheral and central nervous system. A muscle biopsy should only be performed if this is likely to be diagnostically useful. At present no 'guidelines' exist. METHODS: An EFNS panel of muscle specialists was set to review relevant studies from PubMed dating as far back as 1/1/1990. Only Class IV studies were available and therefore the recommendations arrived at are 'best practice recommendations' based on information harvested from the literature search and expert opinion. RESULTS: Muscle cramps should be recognized while drugs, infections, metabolic/ endocrinological and rheumatological causes of myalgia should be identified from the history and examination and pertinent laboratory tests. A muscle biopsy is more likely to be diagnostically useful if myalgia is exertional and if one or more of the following apply: i) there is myoglobinuria, (ii) there is a second wind phenomenon, (iii) there is muscle weakness, (iv) there is muscle hypertrophy /atrophy, (v) there is hyperCKemia (>2-3× normal), and (vi) there is a myopathic EMG. CONCLUSIONS: Patients presenting with myalgia can be recommended to have a biopsy based on careful history and examination and on simple laboratory screening.

Mutation in the gene encoding ferritin light polypeptide causes dominant adult-onset basal ganglia disease.

We describe here a previously unknown, dominantly inherited, late-onset basal ganglia disease, variably presenting with extrapyramidal features similar to those of Huntington's disease (HD) or parkinsonism. We mapped the disorder, by linkage analysis, to 19q13.3, which contains the gene for ferritin light polypeptide (FTL). We found an adenine insertion at position 460-461 that is predicted to alter carboxy-terminal residues of the gene product. Brain histochemistry disclosed abnormal aggregates of ferritin and iron. Low serum ferritin levels also characterized patients. Ferritin, the main iron storage protein, is composed of 24 subunits of two types (heavy, H and light, L) which form a soluble, hollow sphere. Brain iron deposition increases normally with age, especially in the basal ganglia, and is a suspected causative factor in several neurodegenerative diseases in which it correlates with visible pathology, possibly by its involvement in toxic free-radical reactions. We found the same mutation in five apparently unrelated subjects with similar extrapyramidal symptoms. An abnormality in ferritin strongly indicates a primary function for iron in the pathogenesis of this new disease, for which we propose the name 'neuroferritinopathy'.

Multi-system neurological disease is common in patients with OPA1 mutations.

Additional neurological features have recently been described in seven families transmitting pathogenic mutations in OPA1, the most common cause of autosomal dominant optic atrophy. However, the frequency of these syndromal 'dominant optic atrophy plus' variants and the extent of neurological involvement have not been established. In this large multi-centre study of 104 patients from 45 independent families, including 60 new cases, we show that extra-ocular neurological complications are common in OPA1 disease, and affect up to 20% of all mutational carriers. Bilateral sensorineural deafness beginning in late childhood and early adulthood was a prominent manifestation, followed by a combination of ataxia, myopathy, peripheral neuropathy and progressive external ophthalmoplegia from the third decade of life onwards. We also identified novel clinical presentations with spastic paraparesis mimicking hereditary spastic paraplegia, and a multiple sclerosis-like illness. In contrast to initial reports, multi-system neurological disease was associated with all mutational subtypes, although there was an increased risk with missense mutations [odds ratio = 3.06, 95% confidence interval = 1.44-6.49; P = 0.0027], and mutations located within the guanosine triphosphate-ase region (odds ratio = 2.29, 95% confidence interval = 1.08-4.82; P = 0.0271). Histochemical and molecular characterization of skeletal muscle biopsies revealed the presence of cytochrome c oxidase-deficient fibres and multiple mitochondrial DNA deletions in the majority of patients harbouring OPA1 mutations, even in those with isolated optic nerve involvement. However, the cytochrome c oxidase-deficient load was over four times higher in the dominant optic atrophy + group compared to the pure optic neuropathy group, implicating a causal role for these secondary mitochondrial DNA defects in disease pathophysiology. Individuals with dominant optic atrophy plus phenotypes also had significantly worse visual outcomes, and careful surveillance is therefore mandatory to optimize the detection and management of neurological disability in a group of patients who already have significant visual impairment.

A novel mutation in the mitochondrial tRNA for tryptophan causing a late-onset mitochondrial encephalomyopathy.

BACKGROUND: Mitochondrial DNA (mtDNA) mutations are increasingly being recognized as causes of late-onset disease. We report a patient with a late-onset mitochondrial encephalomyopathy caused by a novel G > C transition in mtDNA at position 5556 in the gene encoding the tRNA for tryptophan (MTTW). AIMS: To investigate the cause of disease and assess the pathogenicity of this new mutation. METHODS: Clinical, histopathological and gene sequencing studies. Quantification of the mutation was performed in different tissues from the patient and two relatives and in single muscle fibres. RESULTS: The mutation was heteroplasmic, segregated in biochemically affected muscle fibres and was absent in blood. The level of mutation in skeletal muscle was higher than in brain, although the brain was clinically the most affected tissue. DISCUSSION: The 5556G > C mutation appears sporadic. It was not found in any of the family members tested, although some of them manifested disorders that can be associated with mtDNA disease. In addition to reporting the eighth mutation in MTTW, our case illustrates the challenges posed when assigning pathogenicity to mtDNA mutations.

Clinical evolution of Kearns-Sayre syndrome with polyendocrinopathy and respiratory failure.

BACKGROUND: The triad of progressive external ophthalmoplegia, atypical retinal pigmentation and cardiac conduction defects characterizes Kearns-Sayre syndrome (KSS), which is most often caused by a single, large deletion of mitochondrial DNA. Endocrine disease appears to be more common in KSS than in other mitochondrial diseases. MATERIALS, METHODS AND RESULTS: A patient presenting with KSS developed Addison's disease, hypothyroidism and glucose intolerance. Thyroid peroxidase antibodies and adrenal 21-hydroxylase antibodies were identified. She developed acute respiratory failure requiring invasive ventilatory support, but improved and currently requires only non-invasive, nocturnal BiPAP treatment. DISCUSSION AND CONCLUSION: This case confirms the association of KSS and endocrine dysfunction. Our finding of autoantibodies to thyroid and adrenal glands distinguishes this patient from most other published cases and suggests a potential synergy between the two disease mechanisms. In addition, we demonstrate that respiratory failure can be a treatable event in this disease.

Combined enzyme defect of mitochondrial fatty acid oxidation.

A young girl presented with recurrent episodes of muscle weakness culminating in a severe attack of generalized muscle weakness. In the muscle mitochondria from the patient there was an abnormal pattern of intermediates of beta-oxidation with an accumulation of 3-hydroxyacyl- and 2-enoyl-CoA and carnitine esters, and 3-oxoacylcarnitines. There was low activity of long-chain 3-hydroxyacyl-CoA dehydrogenase in mitochondria from all tissues. The activity of long-chain 2-enoyl-CoA hydratase was low in muscle mitochondria and 3-oxoacyl-CoA thiolase activity measured with 3-oxohexadecanoyl-CoA as substrate was low in fibroblast, muscle, and cardiac mitochondria but only partial deficiency was present when the activity was measured with 3-oxooctanoyl-CoA. The activity of the long-chain 3-hydroxyacyl-CoA dehydrogenase and long-chain 3-oxoacyl-CoA thiolase in fibroblasts from the patient's parents was intermediate between those of controls and the patient. The patient has a combined defect of the long-chain 3-hydroxyacyl-CoA dehydrogenase, long-chain 3-oxoacyl-CoA thiolase, and long-chain 2-enoyl-CoA hydratase which appears to be inherited in an autosomal recessive manner. This suggests there is a multifunctional enzyme catalyzing these activities in human mitochondria and that this enzyme is deficient in our patient.

Impaired mitochondrial beta-oxidation in a patient with an abnormality of the respiratory chain. Studies in skeletal muscle mitochondria.

Defects of complex I of the mitochondrial respiratory chain are important causes of neurological disease. We report studies that demonstrate a severe deficiency of complex I activity with less severe abnormalities of complexes III and IV (less than 5, 63, and 30% of control values, respectively) in a skeletal muscle mitochondrial fraction from a 22-yr-old female with weakness, lactic acidemia, and the deposition of intramuscular neutral lipid. The observation that lipid accumulates in this and other patients with complex I deficiency suggests impaired mitochondrial fatty acid oxidation. To investigate this mechanism we have shown impaired flux through beta-oxidation [( U-14C]hexadecanoate oxidation was 66% of control rate) and accumulation of specific acyl-CoA ester intermediates. The changes in fatty acid metabolism in complex I deficiency are secondary to the reduced state within the mitochondrial matrix with low NAD+/NADH ratios.

Biochemical and molecular studies of mitochondrial function in diabetes insipidus, diabetes mellitus, optic atrophy, and deafness.

OBJECTIVE: To determine if diabetes insipidus, diabetes mellitus, optic atrophy, and deafness (DIDMOAD) combined with a cerebellar syndrome is associated with a systemic disorder of respiratory chain function as found in similar genetic syndromes. CASE: A muscle biopsy was taken from a patient with DIDMOAD, and a mitochondrial fraction was prepared. Respiratory chain function was assessed by analysis of intermediary metabolites, histochemical analysis of muscle biopsy, measurement of the activity of individual respiratory chain complexes, oxidative flux through the respiratory chain, and cytochrome concentration and compared with a population with normal respiratory chain function. Mitochondrial DNA from skeletal muscle, brain, and pancreas was examined for major rearrangements and specific point mutations. Brain tissue was examined neuropathologically for abnormalities, particularly those previously described in association with DIDMOAD. RESULTS: No abnormality was found in mitochondrial oxidation, individual complex activity, or cytochrome concentration. Histochemical analysis and electron microscopy showed no abnormality known to be associated with mitochondrial dysfunction. A single-base substitution at position 12308 of the mitochondrial genome was found, but no major rearrangement of mitochondrial DNA was demonstrated. Neuropathological examination revealed severe demyelination and gliosis in the optic nerves and loss of Purkinje cells associated with gliosis in the white matter in the cerebellum. CONCLUSIONS: We have found no evidence that DIDMOAD is associated with a systemic abnormality of respiratory chain function. The mitochondrial DNA single-base substitution noted is likely to be a polymorphism rather than a pathogenic point mutation. We have confirmed that DIDMOAD may be associated with a neurodegenerative disorder, but the cause of this remains undetermined.

Conversion of a reporter gene for mitochondrial gene expression using iterative mega-prime PCR.

Mammalian mitochondria possess their own multicopy genome, mtDNA. Although much is known about mtDNA replication and transcription, our knowledge of the mechanisms governing mt-RNA processing, stability and translation remains rudimentary. We have taken a step towards addressing these issues by altering the luciferase reporter gene to accommodate the variation in mitochondrial codon recognition. 19 essential substitutions have been generated by an iterative mega-primer PCR technique. To mimic mt-mRNA species and to optimise intramitochondrial translation, further engineering has produced a template which, when transcribed in vitro, generates an RNA species with only two nucleotides upstream from the initiation codon, an absence of a 3' untranslated region and a polyadenylated tail of 40 residues. It is intended that mt-luciferase (mt-luc) RNA will be an excellent reporter for revealing cis-acting elements essential for in organello RNA processing, maturation and expression. Additionally, the mt-luc gene can be readily incorporated into any novel mitochondrial transducing vectors to assess intra-organellar transcription and translation.

Longitudinal analysis of the segregation of mtDNA mutations in heteroplasmic individuals.

The mutation load of the pathogenic LHON (Leber hereditary optic neuropathy) mtDNA mutation at nucleotide 3460 has been followed over time in the WBC/platelet fraction from members of a matrilineal pedigree. Longitudinal analysis over a sampling period of five to six years indicates that, in all five heteroplasmic family members, the mutation load decreases at a mean overall rate of approximately 1% per year. There was no change in mutation load in homoplasmic wildtype or in homoplasmic mutant individuals. For the purposes of comparison, a longitudinal analysis of a silent mtDNA polymorphism at nucleotide 14560 was also carried out for members of a second matrilineal pedigree. In contrast to the results for the pathogenic mtDNA mutation, there was no change in the proportion of the silent polymorphism in the WBC/platelet fraction of four family members over a period of seven years. These results indicate that the pathogenic 3460 LHON mutation segregates under negative selection in these cell populations. One possible mechanism through which selection may operate is that, in heteroplasmic individuals, the hematopoietic stem cells are generally homoplasmic, either for the wildtype or for the mutant allele. The homoplasmic mutant stem cells, because of their mitochondrial respiratory chain defect, produce fewer mature WBCs and platelets over time than do the wildtype stem cells. Alternatively, the stem cells may be heteroplasmic and selection may act to favor proliferation of mitochondria with lower levels of the pathogenic mutation in the WBC/platelet cell populations.

Respiratory chain abnormalities in skeletal muscle from patients with Parkinson's disease.

Parkinson's disease is one of the commonest neurodegenerative disorders in Western society. Although the neuropathological changes have been well documented, the underlying biochemical defect is unknown. Toxins may play a part in the aetiology of this disorder. It has been shown that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produces a Parkinson-like syndrome in both man and primates and 1-methyl-4-phenylpyridine (MPP+), a metabolite of MPTP, inhibits NADH-ubiquinone oxidoreductase (complex I) of the mitochondrial respiratory chain. We studied mitochondrial respiratory chain function in skeletal muscle from patients with Parkinson's disease because, like brain, it has a high dependence on oxidative metabolism. Our results show low activity in all complexes studied (I, II and IV). The implications of these findings are discussed in relation to the aetiology of Parkinson's disease.

Familial intermittent ataxia due to a defect of the E1 component of pyruvate dehydrogenase complex.

Disturbances of pyruvate metabolism have been implicated in the aetiology of several neurological disorders including Leigh's disease and familial ataxia. We have re-investigated a patient whose initial description documented intermittent ataxia, a presumed disorder of pyruvate metabolism and an X-linked pattern of inheritance. Recent studies showed he had slow oxidation of pyruvate, low pyruvate dehydrogenase complex (PDC) activity and immunochemical evidence of E1 deficiency in skeletal muscle mitochondria. This is consistent with the recent finding that the gene for E1 alpha is on the X chromosome.

Multiple defects of the mitochondrial respiratory chain in a mitochondrial encephalopathy (MERRF): a clinical, biochemical and molecular study.

We describe a young man with a progressive neurological disorder including myoclonus, mental retardation, muscle weakness and a mitochondrial myopathy (myoclonus epilepsy and ragged red fibres--MERRF). Multiple abnormalities of the mitochondrial respiratory chain in skeletal muscle are shown by direct measurement of the flux through the individual complexes, low-temperature redox spectroscopy and decreased immunodetectable subunits of complexes I and IV by immunoblotting. No abnormality of mitochondrial DNA was found. This is the first report of combined defects of complexes I, III and IV as a cause of this clinical syndrome. However, we propose that the occurrence of multiple respiratory chain defects may be more common than previously recognised and that this particular combination of defects, involving complexes I, III and IV, may be the predominant biochemical abnormality in MERRF.

Spontaneous intracranial hypotension: clinical and magnetic resonance imaging characteristics.

The syndrome of spontaneous intracranial hypotension (SIH) is an uncommon cause of postural headache. We describe three patients with classical low pressure headache associated with low CSF pressure, one of whom presented with sudden deafness and another with a unilateral VIth nerve palsy. Initial magnetic resonance imaging (MRI) scans revealed bilateral diffuse subdural fluid collections in all three cases. Follow up MRI scans performed on two patients at 6 months demonstrated partial resolution of the subdural collections but persistent striking meningeal enhancement despite clinical recovery. These findings differ from previous reported cases wherein clinical resolution of postural symptoms was preceded or closely followed by resolution of the MRI changes.

Deficiency of the pyruvate dehydrogenase complex and of mitochondrial fatty acid oxidation.

Defects of the pyruvate dehydrogenase complex and of mitochondrial fatty acid oxidation are important causes of disease. Defects of pyruvate dehydrogenase may present in early childhood with severe CNS changes or, as lactic acidosis or later with ataxia. Defects of fatty acid oxidation may present with hypoglycaemic coma, myopathy, liver disease with encephalopathy, cardiomyopathy or sudden infant death. The investigation of both these groups of disorders is difficult and depends upon a combination of biochemical and molecular biology techniques.