Pathological variants in TOP3A cause distinct disorders of mitochondrial and nuclear genome stability.

Topoisomerase 3α (TOP3A) is an enzyme that removes torsional strain and interlinks between DNA molecules. TOP3A localises to both the nucleus and mitochondria, with the two isoforms playing specialised roles in DNA recombination and replication respectively. Pathogenic variants in TOP3A can cause a disorder similar to Bloom syndrome, which results from bi-allelic pathogenic variants in BLM, encoding a nuclear-binding partner of TOP3A. In this work, we describe 11 individuals from 9 families with an adult-onset mitochondrial disease resulting from bi-allelic TOP3A gene variants. The majority of patients have a consistent clinical phenotype characterised by bilateral ptosis, ophthalmoplegia, myopathy and axonal sensory-motor neuropathy. We present a comprehensive characterisation of the effect of TOP3A variants, from individuals with mitochondrial disease and Bloom-like syndrome, upon mtDNA maintenance and different aspects of enzyme function. Based on these results, we suggest a model whereby the overall severity of the TOP3A catalytic defect determines the clinical outcome, with milder variants causing adult-onset mitochondrial disease and more severe variants causing a Bloom-like syndrome with mitochondrial dysfunction in childhood.

Changing faces of mitochondrial disease: autosomal recessive POLG disease mimicking myasthenia gravis and progressive supranuclear palsy.

Background: Mitochondrial disorders are known to cause diverse neurological phenotypes which cause a diagnostic challenge to most neurologists. Pathogenic polymerase gamma (POLG) variants have been described as a cause of chronic progressive external ophthalmoplegia, which manifests with ptosis, horizontal and vertical eye movement restriction and myopathy. Autosomal dominant progressive external ophthalmoplegia is rarely associated with Parkinsonism responsive to levodopa. Methods: We report a case of a 58-year-old man who presented with an eye movement disorder then Parkinsonism who made his way through the myasthenia then the movement disorder clinic. Results: A diagnostic right tibialis anterior biopsy revealed classical hallmarks of mitochondrial disease, and genetic testing identified compound heterozygous pathogenic gene variants in the POLG gene. The patient was diagnosed with autosomal recessive POLG disease. Conclusions: It is important to maintain a high index of suspicion of pathogenic POLG variants in patients presenting with atypical Parkinsonism and ophthalmoplegia. Patients with POLG-related disease will usually have ptosis, and downgaze is typically preserved until late in the disease. Accurate diagnosis is essential for appropriate prognosis and genetic counselling.

POLG2 deficiency causes adult-onset syndromic sensory neuropathy, ataxia and parkinsonism.

OBJECTIVE: Mitochondrial dysfunction plays a key role in the pathophysiology of neurodegenerative disorders such as ataxia and Parkinson's disease. We describe an extended Belgian pedigree where seven individuals presented with adult-onset cerebellar ataxia, axonal peripheral ataxic neuropathy, and tremor, in variable combination with parkinsonism, seizures, cognitive decline, and ophthalmoplegia. We sought to identify the underlying molecular etiology and characterize the mitochondrial pathophysiology of this neurological syndrome. METHODS: Clinical, neurophysiological, and neuroradiological evaluations were conducted. Patient muscle and cultured fibroblasts underwent extensive analyses to assess mitochondrial function. Genetic studies including genome-wide sequencing were conducted. RESULTS: Hallmarks of mitochondrial dysfunction were present in patients' tissues including ultrastructural anomalies of mitochondria, mosaic cytochrome c oxidase deficiency, and multiple mtDNA deletions. We identified a splice acceptor variant in POLG2, c.970-1G>C, segregating with disease in this family and associated with a concomitant decrease in levels of POLG2 protein in patient cells. INTERPRETATION: This work extends the clinical spectrum of POLG2 deficiency to include an overwhelming, adult-onset neurological syndrome that includes cerebellar syndrome, peripheral neuropathy, tremor, and parkinsonism. We therefore suggest to include POLG2 sequencing in the evaluation of ataxia and sensory neuropathy in adults, especially when it is accompanied by tremor or parkinsonism with white matter disease. The demonstration that deletions of mtDNA resulting from autosomal-dominant POLG2 variant lead to a monogenic neurodegenerative multicomponent syndrome provides further evidence for a major role of mitochondrial dysfunction in the pathomechanism of nonsyndromic forms of the component neurodegenerative disorders.

Incidence of Primary Mitochondrial Disease in Children Younger Than 2 Years Presenting With Acute Liver Failure.

BACKGROUND: Mitochondrial liver disease (MLD), and in particular mitochondrial DNA (mtDNA) depletion syndrome (MDS) is an important cause of acute liver failure (ALF) in infancy. Early and accurate diagnosis is important because liver transplantation (LT) is often contraindicated. It is unclear which methods are the best to diagnose MLD in the setting of ALF. OBJECTIVE: The aim of the study was to determine the incidence of MLD in children younger than 2 years with ALF and the utility of routine investigations to detect MLD. METHODS: Thirty-nine consecutive infants with ALF were admitted to a single unit from 2009 to 2011. All were extensively investigated using an established protocol. Genes implicated in mitochondrial DNA depletion syndrome were sequenced in all cases and tissue mtDNA copy number measured where available. RESULTS: Five infants (17%) had genetically proven MLD: DGUOK (n = 2), POLG (n = 2), and MPV17 (1). Four of these died, whereas 1 recovered. Two had normal muscle mtDNA copy number and 3 had normal muscle respiratory chain enzymes. An additional 8 children had low hepatic mtDNA copy number but pathogenic mutations were not detected. One of these developed fatal multisystemic disease after LT, whereas 5 who survived remain well without evidence of multisystemic disease up to 6 years later. Magnetic resonance spectroscopy did not distinguish between those with and without MLD. CONCLUSIONS: Low liver mtDNA copy number may be a secondary phenomenon in ALF.Screening for mtDNA maintenance gene mutations may be the most efficient way to confirm MLD in ALF in the first 2 years of life.

Ultrasonography in the Assessment of Lateral Ankle Ligament Injury, Instability, and Anterior Ankle Impingement: A Diagnostic Case Report.

OBJECTIVE: The purpose of this case report is to describe the diagnostic value of ultrasonography (US) in a patient with injury to the lateral ligaments of the ankle with concomitant ankle joint osteoarthritis and anterior impingement. CLINICAL FEATURES: A 28-year-old male had a history of an inversion injury of the left ankle. Diagnostic US of the left ankle using an 8- to 15-MHz linear array transducer demonstrated a full thickness tear of the anterior talofibular ligament, partial thickness tearing of the calcaneofibular ligament, and laxity of the ankle with varus stress testing. In addition, US was able to demonstrate degeneration of the ankle and talonavicular joints and anterior impingement with dorsiflexion. Osteoarthritic changes were confirmed with radiography. Other US findings included remote deltoid ligamentous complex injury, multiple sites of tenosynovitis, and a large ankle joint effusion with synovial hypertrophy and synovitis. INTERVENTION AND OUTCOME: Using US, an accurate diagnosis was established with respect to the pathology and functional impairments of the patient's ankle. CONCLUSION: This case report exemplifies the value and utility of US in diagnosing derangement in ligamentous, tendinous, articular, and osseous injuries of the ankle.

Clinical and molecular characteristics of mitochondrial DNA depletion syndrome associated with neonatal cholestasis and liver failure.

OBJECTIVE: To determine the frequency of mitochondrial DNA depletion syndrome (MDS) in infants with cholestasis and liver failure and to further clarify the clinical, biochemical, radiologic, histopathologic, and molecular features associated with MDS due to deoxyguanosine kinase (DGUOK) and MPV17 gene mutations. STUDY DESIGN: We studied 20 infants with suspected hepatocerebral MDS referred to our tertiary care center between 2007 and 2013. Genomic DNA was isolated from blood leukocytes, liver, and/or skeletal muscle samples by standard methods. Mitochondrial DNA copy number relative to nuclear DNA levels was determined in muscle and/or liver DNA using real-time quantitative polymerase chain reaction and compared with age-matched controls. Nuclear candidate genes, including polymerase γ, MPV17, and DGUOK were sequenced using standard analyses. RESULTS: We identified pathogenic MPV17 and DGUOK mutations in 11 infants (6 females) representing 2.5% of the 450 cases of infantile cholestasis and 22% of the 50 cases of infantile liver failure referred to our center during the study period. All of the 11 patients manifested cholestasis that was followed by a rapidly progressive liver failure and death before 2 years of life. Mitochondrial DNA depletion was demonstrated in liver or muscle for 8 out of the 11 cases where tissue was available. Seven patients had mutations in the MPV17 gene (3 novel mutations), 4 patients had DGUOK mutations (of which 2 were novel mutations). CONCLUSION: Mutations in the MPV17 and DGUOK genes are present in a significant percentage of infants with liver failure and are associated with poor prognosis.

Clinical, biochemical, cellular and molecular characterization of mitochondrial DNA depletion syndrome due to novel mutations in the MPV17 gene.

Mitochondrial DNA (mtDNA) depletion syndromes (MDS) are severe autosomal recessive disorders associated with decreased mtDNA copy number in clinically affected tissues. The hepatocerebral form (mtDNA depletion in liver and brain) has been associated with mutations in the POLG, PEO1 (Twinkle), DGUOK and MPV17 genes, the latter encoding a mitochondrial inner membrane protein of unknown function. The aims of this study were to clarify further the clinical, biochemical, cellular and molecular genetic features associated with MDS due to MPV17 gene mutations. We identified 12 pathogenic mutations in the MPV17 gene, of which 11 are novel, in 17 patients from 12 families. All patients manifested liver disease. Poor feeding, hypoglycaemia, raised serum lactate, hypotonia and faltering growth were common presenting features. mtDNA depletion in liver was demonstrated in all seven cases where liver tissue was available. Mosaic mtDNA depletion was found in primary fibroblasts by PicoGreen staining. These results confirm that MPV17 mutations are an important cause of hepatocerebral mtDNA depletion syndrome, and provide the first demonstration of mosaic mtDNA depletion in human MPV17 mutant fibroblast cultures. We found that a severe clinical phenotype was associated with profound tissue-specific mtDNA depletion in liver, and, in some cases, mosaic mtDNA depletion in fibroblasts.

Late-onset respiratory failure due to TK2 mutations causing multiple mtDNA deletions.

Mutations in nuclear genes involved in the maintenance of mitochondrial DNA (mtDNA) are associated with an extensive spectrum of clinical phenotypes, manifesting as either mtDNA depletion syndromes or multiple mtDNA deletion disorders.(1.)

Pathogenic mitochondrial tRNA point mutations: nine novel mutations affirm their importance as a cause of mitochondrial disease.

Mutations in the mitochondrial genome, and in particular the mt-tRNAs, are an important cause of human disease. Accurate classification of the pathogenicity of novel variants is vital to allow accurate genetic counseling for patients and their families. The use of weighted criteria based on functional studies-outlined in a validated pathogenicity scoring system--is therefore invaluable in determining whether novel or rare mt-tRNA variants are pathogenic. Here, we describe the identification of nine novel mt--tRNA variants in nine families, in which the probands presented with a diverse range of clinical phenotypes including mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes, isolated progressive external ophthalmoplegia, epilepsy, deafness and diabetes. Each of the variants identified (m.4289T>C, MT-TI; m.5541C>T, MT-TW; m.5690A>G, MT-TN; m.7451A>T, MT-TS1; m.7554G>A, MT-TD; m.8304G>A, MT-TK; m.12206C>T, MT-TH; m.12317T>C, MT-TL2; m.16023G>A, MT-TP) was present in a different tRNA, with evidence in support of pathogenicity, and where possible, details of mutation transmission documented. Through the application of the pathogenicity scoring system, we have classified six of these variants as "definitely pathogenic" mutations (m.5541C>T, m.5690A>G, m.7451A>T, m.12206C>T, m.12317T>C, and m.16023G>A), whereas the remaining three currently lack sufficient evidence and are therefore classed as 'possibly pathogenic' (m.4289T>C, m.7554G>A, and m.8304G>A).

Adults with RRM2B-related mitochondrial disease have distinct clinical and molecular characteristics.

Mutations in the nuclear-encoded mitochondrial maintenance gene RRM2B are an important cause of familial mitochondrial disease in both adults and children and represent the third most common cause of multiple mitochondrial DNA deletions in adults, following POLG [polymerase (DNA directed), gamma] and PEO1 (now called C10ORF2, encoding the Twinkle helicase) mutations. However, the clinico-pathological and molecular features of adults with RRM2B-related disease have not been clearly defined. In this multicentre study of 26 adult patients from 22 independent families, including five additional cases published in the literature, we show that extra-ocular neurological complications are common in adults with genetically confirmed RRM2B mutations. We also demonstrate a clear correlation between the clinical phenotype and the underlying genetic defect. Myopathy was a prominent manifestation, followed by bulbar dysfunction and fatigue. Sensorineural hearing loss and gastrointestinal disturbance were also important findings. Severe multisystem neurological disease was associated with recessively inherited compound heterozygous mutations with a mean age of disease onset at 7 years. Dominantly inherited heterozygous mutations were associated with a milder predominantly myopathic phenotype with a later mean age of disease onset at 46 years. Skeletal muscle biopsies revealed subsarcolemmal accumulation of mitochondria and/or cytochrome c oxidase-deficient fibres. Multiple mitochondrial DNA deletions were universally present in patients who underwent a muscle biopsy. We identified 18 different heterozygous RRM2B mutations within our cohort of patients, including five novel mutations that have not previously been reported. Despite marked clinical overlap between the mitochondrial maintenance genes, key clinical features such as bulbar dysfunction, hearing loss and gastrointestinal disturbance should help prioritize genetic testing towards RRM2B analysis, and sequencing of the gene may preclude performance of a muscle biopsy.

Mitochondrial DNA m.3242G > A mutation, an under diagnosed cause of hypertrophic cardiomyopathy and renal tubular dysfunction?

We present two new patients with the recently described mitochondrial m.3242G > A mutation. Although the mutation is situated next to the well known m.3243A > G mutation, the most common alteration associated with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome, the clinical presentation is quite different, but characteristic. All three m.3242G > A patients presented in the neonatal period with hypertrophic and dilated cardiomyopathy, generalized muscle hypotonia and lactic acidosis. Two additionally had creatine kinase elevation, renal tubular acidosis/dysfunction and showed a mild clinical course with a favourable psychomotor development. The third patient had more neurological involvement and died in infancy. The mutation occurred de novo in the two patients where maternal investigations were performed. The combination of hypertrophic cardiomyopathy and renal tubular acidosis/renal tubular dysfunction is clinically distinctive and may represent a separate entity.

A p.R369G POLG2 mutation associated with adPEO and multiple mtDNA deletions causes decreased affinity between polymerase γ subunits.

Human mitochondrial DNA (mtDNA) polymerase γ (pol γ) is the sole enzyme required to replicate and maintain the integrity of the mitochondrial genome. It comprises two subunits, a catalytic p140 subunit and a smaller p55 accessory subunit encoded by the POLG2 gene. We describe the molecular characterization of a potential dominant POLG2 mutation (p.R369G) in a patient with adPEO and multiple mtDNA deletions. Biochemical studies of the recombinant mutant p55 protein showed a reduced affinity to the pol γ p140 subunit, leading to impaired processivity of the holoenzyme complex but did not show sensitivity to N-ethylmalaimide (NEM) inhibition, inferring a novel disease mechanism.

Pathogenic expansions of the SCA6 locus are associated with a common CACNA1A haplotype across the globe: founder effect or predisposing chromosome?

Spinocerebellar ataxia type 6 (SCA6) is a common cause of dominantly inherited ataxia due to an expansion of the CAG repeat in the CACNA1A gene. Affected individuals from the same population share a common haplotype, raising the possibility that most SCA6 cases have descended from a small number of common founders across the globe. To test this hypothesis, we carried out haplotype analysis on SCA6 families from Europe, South America and the Far East, including an established de novo SCA6 expansion. A core CACNA1A disease haplotype was found in affected individuals across the globe. This was also present in the unaffected father of the de novo case, suggesting that the shared chromosome predisposes to the CAG repeat expansion at the SCA6 locus. The SCA6 expansion lies within a CpG island, which could act as a cis-acting element predisposing to repeat expansion as for other CAG/CTG repeat diseases. Polymorphic variation in this region may explain the high-risk haplotype found in SCA6 families.

Differential selection of acridine resistance mutations in human DNA topoisomerase IIbeta is dependent on the acridine structure.

Type II DNA topoisomerases are targets of acridine drugs. Nine mutations conferring resistance to acridines were obtained by forced molecular evolution, using methyl N-(4'-(9-acridinylamino)-3-methoxy-phenyl) methane sulfonamide (mAMSA), methyl N-(4'-(9-acridinylamino)-2-methoxy-phenyl) carbamate hydrochloride (mAMCA), methyl N-(4'-(9-acridinylamino)-phenyl) carbamate hydrochloride (AMCA), and N-[2-(dimethylamino)ethyl]acridines-4-carboxamide (DACA) as selection agents. Mutations betaH514Y, betaE522K, betaG550R, betaA596T, betaY606C, betaR651C, and betaD661N were in the B' domain, and betaG465D and betaP732L were not. With AMCA, four mutations were selected (betaE522K, betaG550R, betaA596T, and betaD661N). Two mutations were selected with mAMCA (betaY606C and betaR651C) and two with mAMSA (betaG465D and betaP732L). It is interesting that there was no overlap between mutation selection with AMCA and mAMSA or mAMCA. AMCA lacks the methoxy substituent present in mAMCA and mAMSA, suggesting that this motif determines the mutations selected. With the fourth acridine DACA, five mutations were selected for resistance (betaG465D, betaH514Y, betaG550R, betaA596T, and betaD661N). betaG465D was selected with both DACA and mAMSA, and betaG550R, betaA596T, and betaD661N were selected with both DACA and AMCA. DACA lacks the anilino motif of the other three drugs but retains the acridine ring motif. The overlap in selection with DACA and mAMSA or AMCA suggests that altered recognition of the acridine moiety may be involved in these mutations. We used restriction fragment length polymorphisms and heteroduplex analysis to demonstrate that some mutations were selected multiple times (betaG465D, betaE522K, betaG550R, betaA596T, and betaD661N), whereas others were selected only once (betaH514Y, betaY606C, betaR651C, and betaP732L). Here, we compare the drug resistance profile of all nine mutations and report the biochemical characterization of three, betaG550R, betaY606C, and betaD661N.

Patterning lines by capillary flows.

We report that capillary flows in an evaporating thin film create line patterns, with widths ranging from a few micrometers to less than 100 nm. Deliberate patterning of such lines requires contact-line pinning and the presence of foaming surfactants. Large-scale photolithography can guide and control these structures by creating pinning points and steering evaporation. We provide demonstrations of this process by making self-assembling lines of colloidal quantum dots and microspheres.

Minimum prevalence of spinocerebellar ataxia 17 in the north east of England.

OBJECTIVE: To determine the minimum prevalence of spinocerebellar ataxia type 17 (SCA17) in the north east of England. PATIENTS AND METHODS: A defined region containing 2,516,500 individuals with 192 families with undiagnosed ataxia, 90 patients with a Huntington's disease-like phenotype and 292 controls. The number of (CAG/CAA)(n) repeats in the SCA17/TBP gene was determined by fluorescent PCR and sequenced in affected individuals. RESULTS: The mean repeat size for 584 control alleles was 34 (S.D.=3.58), ranging from 25 to 40. Two index cases had larger alleles with repeat lengths greater than the control range. Affected family members presented in adult life with ataxia followed by extrapyramidal features and cognitive impairment. In one family 44 repeats were associated with a younger age of onset than has been previously described. CONCLUSIONS: The minimum prevalence of SCA17 in the north east of England was 0.16/100,000 (upper 95% confidence interval 0.31/100,000).

Molecular epidemiology of spinocerebellar ataxia type 6.

We performed a population-based clinical and molecular genetic study of spinocerebellar ataxia type 6 (SCA6) in the northeast of England. The minimum point prevalence of SCA6 was 1.59 in 100,000 (95% confidence interval [CI], 1.04-2.14), and the number of individuals who either had SCA6 or are at risk of developing SCA6 was at least 5.21 in 100,000 (95% CI, 4.31-6.10), or 1 in 19,210. Microsatellite analysis of the CACNA1A gene indicated a founder effect for SCA6 within this region.