Respiratory chain deficiency in nonmitochondrial disease.

OBJECTIVE: In this study, we report 5 patients with heterogeneous phenotypes and biochemical evidence of respiratory chain (RC) deficiency; however, the molecular diagnosis is not mitochondrial disease. METHODS: The reported patients were identified from a cohort of 60 patients in whom RC enzyme deficiency suggested mitochondrial disease and underwent whole-exome sequencing. RESULTS: Five patients had disease-causing variants in nonmitochondrial disease genes ORAI1, CAPN3, COLQ, EXOSC8, and ANO10, which would have been missed on targeted next-generation panels or on MitoExome analysis. CONCLUSIONS: Our data demonstrate that RC abnormalities may be secondary to various cellular processes, including calcium metabolism, neuromuscular transmission, and abnormal messenger RNA degradation.

Use of whole-exome sequencing to determine the genetic basis of multiple mitochondrial respiratory chain complex deficiencies.

IMPORTANCE: Mitochondrial disorders have emerged as a common cause of inherited disease, but their diagnosis remains challenging. Multiple respiratory chain complex defects are particularly difficult to diagnose at the molecular level because of the massive number of nuclear genes potentially involved in intramitochondrial protein synthesis, with many not yet linked to human disease. OBJECTIVE: To determine the molecular basis of multiple respiratory chain complex deficiencies. DESIGN, SETTING, AND PARTICIPANTS: We studied 53 patients referred to 2 national centers in the United Kingdom and Germany between 2005 and 2012. All had biochemical evidence of multiple respiratory chain complex defects but no primary pathogenic mitochondrial DNA mutation. Whole-exome sequencing was performed using 62-Mb exome enrichment, followed by variant prioritization using bioinformatic prediction tools, variant validation by Sanger sequencing, and segregation of the variant with the disease phenotype in the family. RESULTS: Presumptive causal variants were identified in 28 patients (53%; 95% CI, 39%-67%) and possible causal variants were identified in 4 (8%; 95% CI, 2%-18%). Together these accounted for 32 patients (60% 95% CI, 46%-74%) and involved 18 different genes. These included recurrent mutations in RMND1, AARS2, and MTO1, each on a haplotype background consistent with a shared founder allele, and potential novel mutations in 4 possible mitochondrial disease genes (VARS2, GARS, FLAD1, and PTCD1). Distinguishing clinical features included deafness and renal involvement associated with RMND1 and cardiomyopathy with AARS2 and MTO1. However, atypical clinical features were present in some patients, including normal liver function and Leigh syndrome (subacute necrotizing encephalomyelopathy) seen in association with TRMU mutations and no cardiomyopathy with founder SCO2 mutations. It was not possible to confidently identify the underlying genetic basis in 21 patients (40%; 95% CI, 26%-54%). CONCLUSIONS AND RELEVANCE: Exome sequencing enhances the ability to identify potential nuclear gene mutations in patients with biochemically defined defects affecting multiple mitochondrial respiratory chain complexes. Additional study is required in independent patient populations to determine the utility of this approach in comparison with traditional diagnostic methods.

NDUFS8-related Complex I Deficiency Extends Phenotype from "PEO Plus" to Leigh Syndrome.

With over 1,000 nuclear genes that could potentially cause a mitochondrial disorder, the current diagnostic approach requires targeted molecular analysis, guided by a combination of clinical and biochemical features. However, the expanding molecular and clinical spectrum means that this approach does not always yield a result. Here we report the unusual clinical presentation of "Progressive External Ophthalmoplegia (PEO) plus" Leigh syndrome in three children from a consanguineous family where exome sequencing identified mutations in NDUFS8. NDUFS8 is a nuclear-encoded structural core protein of complex I, and mutations are expected to cause infantile onset and severe disease. Our patients had a later onset, milder and a clinically distinct phenotype, and this gene would not normally be considered in this context. Being untargeted to specific genes, whole exome analysis has the potential to re-write the phenotype and reveal an unexpected molecular aetiology, as illustrated by this family.

Early muscle and brain ultrastructural changes in polymerase gamma 1-related encephalomyopathy.

Mutations affecting the mitochondrial DNA-polymerase gamma 1 (POLG1) gene have been shown to cause Alpers-Huttenlocher disease. Ultrastructural data on brain and muscle tissue are rare. We report on ultrastructural changes in brain and muscle tissue of two sisters who were compound heterozygous for the c.2243G>C and c.1879C>T POLG1 mutations. Patient 1 (16 years) presented with epilepsia partialis continua that did not respond to antiepileptic treatment. Neuroimaging showed right occipital and bithalamic changes. Light microscopy from a brain biopsy performed after 3 weeks suggested chronic encephalitis showing astro- and microgliosis as well as perivascular CD8-positive T-cells. However, immunosuppressive therapy failed to improve her condition. When her 17-year-old sister (patient 2) also developed epilepsy, an intensified search for metabolic diseases led to the diagnosis. On electron microscopy mitochondrial abnormalities mainly affecting neurons were detected in the brain biopsy of patient 1, including an increase in number and size, structural changes and globoid inclusions. In patient 2, light and electron microscopy on a muscle biopsy confirmed a mitochondrial myopathy, also revealing an increase in mitochondrial size and number, as well as globoid inclusions. Neurons may be the primary target of mitochondrial dysfunction in brains of patients with Alpers disease related to POLG1 mutations. During early disease stages, brain histopathology may be misleading, showing reactive inflammatory changes.

What is influencing the phenotype of the common homozygous polymerase-γ mutation p.Ala467Thr?

Polymerase-γ (POLG) is a major human disease gene and may account for up to 25% of all mitochondrial diseases in the UK and in Italy. To date, >150 different pathogenic mutations have been described in POLG. Some mutations behave as both dominant and recessive alleles, but an autosomal recessive inheritance pattern is much more common. The most frequently detected pathogenic POLG mutation in the Caucasian population is c.1399G>A leading to a p.Ala467Thr missense mutation in the linker domain of the protein. Although many patients are homozygous for this mutation, clinical presentation is highly variable, ranging from childhood-onset Alpers-Huttenlocher syndrome to adult-onset sensory ataxic neuropathy dysarthria and ophthalmoparesis. The reasons for this are not clear, but familial clustering of phenotypes suggests that modifying factors may influence the clinical manifestation. In this study, we collected clinical, histological and biochemical data from 68 patients carrying the homozygous p.Ala467Thr mutation from eight diagnostic centres in Europe and the USA. We performed DNA analysis in 44 of these patients to search for a genetic modifier within POLG and flanking regions potentially involved in the regulation of gene expression, and extended our analysis to other genes affecting mitochondrial DNA maintenance (POLG2, PEO1 and ANT1). The clinical presentation included almost the entire phenotypic spectrum of all known POLG mutations. Interestingly, the clinical presentation was similar in siblings, implying a genetic basis for the phenotypic variability amongst homozygotes. However, the p.Ala467Thr allele was present on a shared haplotype in each affected individual, and there was no correlation between the clinical presentation and genetic variants in any of the analysed nuclear genes. Patients with mitochondrial DNA haplogroup U developed epilepsy significantly less frequently than patients with any other mitochondrial DNA haplotype. Epilepsy was reported significantly more frequently in females than in males, and also showed an association with one of the chromosomal markers defining the POLG haplotype. In conclusion, our clinical results show that the homozygous p.Ala467Thr POLG mutation does not cause discrete phenotypes, as previously suggested, but rather there is a continuum of clinical symptoms. Our results suggest that the mitochondrial DNA background plays an important role in modifying the disease phenotype but nuclear modifiers, epigenetic and environmental factors may also influence the severity of disease.

Adult-onset cerebellar ataxia due to mutations in CABC1/ADCK3.

OBJECTIVE: Inherited ataxias are heterogeneous disorders affecting both children and adults. The primary cause can be identified in about half of the patients and only very few can receive causative therapy. METHODS: The authors performed sequencing of known Coenzyme Q10 (CoQ10) deficiency genes in 22 patients with unexplained recessive or sporadic ataxia. RESULTS: CABC1/ADCK3 mutations were detected in four patients and two siblings presenting with cerebellar ataxia, epilepsy and muscle symptoms. Spasticity, dystonia, tremor and migraine were variably present; cognitive impairment was severe in early childhood cases, but was absent in adults. In contrast to previous reports, two of the patients had a later-onset, very mild phenotype and remained ambulatory in their late forties. Muscle biopsy revealed lipid accumulation, mitochondrial proliferation and cytochrome c oxidase-deficient fibres, but no typical ragged red fibres. Respiratory-chain enzyme activities and CoQ10 were decreased in severely affected patients but remained normal in a mildly affected patient at 46 years of age. CONCLUSIONS: These observations highlight the importance of screening for a potentially treatable cause, CABC1/ADCK3 mutations, not only in severe childhood-onset ataxia, but also in patients with mild cerebellar ataxia in adult life.

Deficiency of the mitochondrial phosphate carrier presenting as myopathy and cardiomyopathy in a family with three affected children.

In a family three children presented with severe neonatal lactic acidosis, hypertrophic cardiomyopathy and generalised muscular hypotonia. One child died in infancy, two survived a clinically severe neonatal period. At an age of 9 and 17years, respectively, they present with exercise intolerance, proximal muscle weakness, non-progressive hypertrophic cardiomyopathy and normal mental development. In a muscle biopsy normal activity of respiratory chain enzymes was found; however the amount of the mitochondrial phosphate carrier was decreased. This protein is expressed in two tissue-specific isoforms generated by mutually exclusive alternative splicing of the SLC25A3 gene transcript. We identified a homozygous mutation c.158-9A>G located in the 5'-intron next to exon 3A specific for heart and skeletal muscle. This creates a novel splice site resulting in a more than 95% decrease of the wild type allele.

The phenotypic spectrum of neutral lipid storage myopathy due to mutations in the PNPLA2 gene.

Neutral lipid storage disease is caused by mutations in the CGI-58 or the PNPLA2 genes. Lipid storage can be detected in various cell types including blood granulocytes. While CGI-58 mutations are associated with Chanarin-Dorfman syndrome, a condition characterized by lipid storage and skin involvement (ichthyosis), mutations in the patatin-like phospholipase domain-containing protein 2 gene (PNPLA2) were reported with skeletal and cardiac muscle disease only. We describe clinical, myopathological, magnetic resonance imaging (MRI), and genetic findings of six patients carrying different recessive PNPLA2 mutations. Pulse-chase labeling of control and patient cells with supplementation of clenbuterol, salmeterol, and dexamethasone was performed in vitro. The patients share a recognizable phenotype with prominent shoulder girdle weakness and mild pelvic girdle and distal muscle weakness, with highly elevated creatine kinase (CK) and cardiomyopathy developing at later stages. Muscle histology invariably reveals massive accumulation of lipid droplets. New muscle or whole-body MRI techniques may assist diagnosis and may become a useful tool to quantify intramuscular lipid storage. Four novel and two previously reported mutations were detected, affecting different parts of the PNPLA2 gene. Activation of hormone-sensitive lipase by beta-adrenergic substances such as clenbuterol appears to bypass the enzymatic block in PNPLA2-deficient patient cells in vitro. PNPLA2 deficiency is a slowly progressive myopathy with onset around the third decade. Cardiac involvement is relatively common at a later stage. Muscle MRI may detect increased lipid in a characteristic distribution, which could be used for monitoring disease progression. Beta-adrenergic agents may be beneficial in improving triacylglycerol breakdown in patients with PNPLA2 mutations.

Acute liver failure with subsequent cirrhosis as the primary manifestation of TRMU mutations.

Combined respiratory chain deficiency accounts for about 30% of mitochondrial respiratory chain deficiencies and is frequently associated with mtDNA depletion, deletions or point mutations. However combined respiratory chain deficiency may also be caused by mutations in nuclear genes affecting mitochondrial translation. Here we describe a 2-year-old girl, who developed an acute, isolated, severe liver failure with mitochondrial pathology and decreased respiratory chain enzyme activities both in liver and skeletal muscle at 4 months of age. Her liver function improved significantly within a month, liver function tests returned to normal. Liver cirrhosis remained without any further complications so far. Pathogenic compound heterozygous mutations were identified in the TRMU gene. This condition is one of the few mitochondrial disorders with a life-threatening onset showing recovery later in life, therefore a prompt diagnosis and treatment of these patients has great importance in clinical practice. We suggest that TRMU deficiency should be considered in infants with acute liver disease.

Nuclear factors involved in mitochondrial translation cause a subgroup of combined respiratory chain deficiency.

Mutations in several mitochondrial DNA and nuclear genes involved in mitochondrial protein synthesis have recently been reported in combined respiratory chain deficiency, indicating a generalized defect in mitochondrial translation. However, the number of patients with pathogenic mutations is small, implying that nuclear defects of mitochondrial translation are either underdiagnosed or intrauterine lethal. No comprehensive studies have been reported on large cohorts of patients with combined respiratory chain deficiency addressing the role of nuclear genes affecting mitochondrial protein synthesis to date. We investigated a cohort of 52 patients with combined respiratory chain deficiency without causative mitochondrial DNA mutations, rearrangements or depletion, to determine whether a defect in mitochondrial translation defines the pathomechanism of their clinical disease. We followed a combined approach of sequencing known nuclear genes involved in mitochondrial protein synthesis (EFG1, EFTu, EFTs, MRPS16, TRMU), as well as performing in vitro functional studies in 22 patient cell lines. The majority of our patients were children (<15 years), with an early onset of symptoms <1 year of age (65%). The most frequent clinical presentation was mitochondrial encephalomyopathy (63%); however, a number of patients showed cardiomyopathy (33%), isolated myopathy (15%) or hepatopathy (13%). Genomic sequencing revealed compound heterozygous mutations in the mitochondrial transfer ribonucleic acid modifying factor (TRMU) in a single patient only, presenting with early onset, reversible liver disease. No pathogenic mutation was detected in any of the remaining 51 patients in the other genes analysed. In vivo labelling of mitochondrial polypeptides in 22 patient cell lines showed overall (three patients) or selective (four patients) defects of mitochondrial translation. Immunoblotting for mitochondrial proteins revealed decreased steady state levels of proteins in some patients, but normal or increased levels in others, indicating a possible compensatory mechanism. In summary, candidate gene sequencing in this group of patients has a very low detection rate (1/52), although in vivo labelling of mitochondrial translation in 22 patient cell lines indicate that a nuclear defect affecting mitochondrial protein synthesis is responsible for about one-third of combined respiratory chain deficiencies (7/22). In the remaining patients, the impaired respiratory chain activity is most likely the consequence of several different events downstream of mitochondrial translation. Clinical classification of patients with biochemical analysis, genetic testing and, more importantly, in vivo labelling and immunoblotting of mitochondrial proteins show incoherent results, but a systematic review of these data in more patients may reveal underlying mechanisms, and facilitate the identification of novel factors involved in combined respiratory chain deficiency.

Predicting the contribution of novel POLG mutations to human disease through analysis in yeast model.

The yeast Saccharomyces cerevisiae was used to validate the pathogenic significance of eight human mutations in the gene encoding for the mitochondrial DNA polymerase gamma, namely G303R, S305R, R386H, R574W, P625R, D930N, K947R and P1073L, among which three are novel and four are of unclear pathological significance. Mitochondrial DNA extended and point mutability as well as dominance/recessivity of each mutation has been evaluated. The analysis in yeast revealed that two mutations, S305R and R386H, cannot be the sole cause of pathology observed in patients. These data led us to search for a second mutation in compound with S305R and we found a mutation, P1073L, missed in the first genetic analysis. Finally, a significant rescue of extended mutability has been observed for several dominant mutations by treatment with mitochondrial antioxidants.

Clinical and neuropathological findings in patients with TACO1 mutations.

We have recently identified mutations in the translation activator of cytochrome c oxidase 1 (TACO1) gene, leading to cytochrome c oxidase (COX) deficiency. Here, we report the clinical and neuroimaging findings of five members of a big consanguinous family homozygous for c.472insC in TACO1. All 5 patients had an uneventful early childhood and a subtle onset, slowly progressive cognitive dysfunction, dystonia or visual impairment between ages 4 and 16years. Affected girls had a milder phenotype and preserved ambulation into the late twenties. Brain MRI revealed bilateral, symmetric lesions of the basal ganglia in all affected family members, but less prominent in girls. TACO1 analysis showed no mutations in 17 patients with juvenile-onset Leigh syndrome and isolated COX or combined respiratory chain deficiency, indicating that TACO1 mutations are a rare cause of Leigh syndrome.

Long-term survival of neonatal mitochondrial complex III deficiency associated with a novel BCS1L gene mutation.

Mutations of the BCS1L gene are a recognised cause of isolated respiratory chain complex III deficiency and underlie several fatal, neonatal mitochondrial diseases. Here we describe a 20-year-old Kenyan woman who initially presented as a floppy infant but whose condition progressed during childhood and adolescence with increasing muscle weakness, focal motor seizures and optic atrophy. Muscle biopsy demonstrated complex III deficiency and the pathogenicity of a novel, homozygous BCS1L mutation was confirmed by yeast complementation studies. Our data indicate that BCS1L mutations can cause a variable, neurological course which is not always fatal in childhood.

Late-onset ptosis and myopathy in a patient with a heterozygous insertion in POLG2.

Polymerase gamma 1 (POLG) mutations are a frequent cause of both autosomal dominant and recessive complex neurological phenotypes. In contrast, only a single pathogenic mutation in one patient was reported in POLG2 so far. Here we describe a 62-year-old woman, carrying a novel heterozygous sequence variant in the POLG2 gene. She developed bilateral ptosis at 30 years of age, followed by exercise intolerance, muscle weakness and mild CK increase in her late forties. Muscle histology and respiratory chain activities were normal. Southern blot and long range PCR detected multiple mtDNA deletions, but no depletion in muscle DNA. Sequencing of POLG, PEO1, ANT1, OPA1 and RRM2B showed normal results. A novel heteroallelic 24 bp insertion (c.1207_1208ins24) was detected in POLG2. This 24 bp insertion into exon 7 causes missplicing and loss of exon 7 in myoblast cDNA. We did not detect POLG2 mutations in 62 patients with multiple mtDNA deletions in muscle DNA, suggesting that POLG2 mutations may represent a rare cause of autosomal dominant PEO.

The prevalence and natural history of dominant optic atrophy due to OPA1 mutations.

PURPOSE: Autosomal dominant optic atrophy (DOA) is a major cause of visual impairment in young adults that is characterized by selective retinal ganglion cell loss. To define the prevalence and natural history of this optic nerve disorder, we performed a population-based epidemiologic and molecular study of presumed DOA cases in the north of England. DESIGN: Case series. PARTICIPANTS: Seventy-six affected probands with a clinical diagnosis of DOA were identified from our neuro-ophthalmology and neurogenetics database. METHODS: OPA1 genetic testing was performed using a polymerase chain reaction-based sequencing strategy. OPA1-negative cases were then screened for large-scale OPA1 rearrangements and OPA3 mutations. Additional affected family members identified through contact tracing were examined, and longitudinal visual data were analyzed. MAIN OUTCOME MEASURES: The prevalence and molecular characteristics of DOA in the north of England. Visual function and disease progression among patients with OPA1-positive mutations. RESULTS: The detection rate of OPA1 mutations was 57.6% among probands with a positive family history of optic atrophy (19/33) and 14.0% among singleton cases (6/43). Approximately two thirds of our families with DOA harbored OPA1 mutations (14/22, 63.6%), and 5 novel OPA1 mutations were identified. Only 1 family carried a large-scale OPA1 rearrangement, and no OPA3 mutations were found in our optic atrophy cohort. The minimum point prevalence of DOA in the north of England was 2.87 per 100,000 (95% confidence interval [CI], 2.54-3.20), or 2.09 per 100,000 (95% CI, 1.95-2.23) when only OPA1-positive cases were considered. Snellen visual acuity varied markedly between OPA1-positive cases with a mean of 20/173 (range 20/20 to hand movements), and visual function worsened in 67.4% of patients during follow-up. The mean rate of visual loss was 0.032 logarithm of the minimum angle of resolution per year, but some patients experienced faster visual decline (range = 0-0.171 logarithm of the minimum angle of resolution/year). OPA1 missense mutations were associated with a significantly worse visual outcome compared with other mutational subtypes (P=0.0001). CONCLUSIONS: Dominant optic atrophy causes significant visual morbidity and affects at least 1 in 35,000 of the general population.

Heteroplasmic mutation in the anticodon-stem of mitochondrial tRNA(Val) causing MNGIE-like gastrointestinal dysmotility and cachexia.

While mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is typically associated with mutations in the nuclear gene encoding for thymidine phosphorylase (ECGF1, TYMP), a similar clinical phenotype was described in patients carrying mutations in the nuclear-encoded polymerase gamma (POLG1) as well as a few mitochondrial tRNA genes. Here we report a novel mutation in the mitochondrial tRNA(Val) (MTTV) gene in a girl presenting with clinical symptoms of MNGIE-like gastrointestinal dysmotility and cachexia. Clinical, histological, biochemical and single cell investigations were performed. The heteroplasmic m.1630A>G mutation was detected in the mitochondrial tRNA(Val) (MTTV) gene in the patient's muscle, blood leukocytes and myoblasts, as well as in blood DNA of the unaffected mother. We provide clinical, biochemical, histological, and molecular genetic evidence on the single cell level for the pathogenicity of this mutation. Our finding adds to the genetic heterogeneity of MNGIE-like gastrointestinal symptoms and highlights the importance of a thorough genetic workup in case of suspected mitochondrial disease.

ESC, ESCL and their roles in Polycomb Group mechanisms.

The Drosophila esc gene is a Polycomb Group (PcG) gene whose product is essential for histone H3 K27 methylation and PcG silencing yet genetic analysis indicated that its product was needed only in the very early embryo. We know now that escl, a close homologue of esc exists in the Drosophila genome. In contrast with earlier studies, we find that both esc and escl are expressed at all stages of development. We show that three major differences between the two genes are in the transcriptional control, which allows esc to make a much stronger maternal contribution; in the splicing efficiency, which makes a major difference in the early escl function; and in the lower participation of ESCL in the PRC2 complex and lower enzymatic activity of the resulting complex. Both genes can sustain normal development in the absence of the other except for the critical role provided by maternal esc product in early embryonic development. Finally, using zygotic mutations in both genes, we show that the gradual loss of function of PRC2 activity leads first to a loss of histone H3 K27 methylation and only at a later stage to a gradual loss of PRC1 binding to chromatin.

The N-terminus of Drosophila SU(VAR)3-9 mediates dimerization and regulates its methyltransferase activity.

In most eukaryotes, the histone methyltransferase SU(VAR)3-9 and its orthologues play a major role in the function of centromeric heterochromatin. Although the methyltransferase domain is required for the formation of a fully functional centromere, mutations within other regions of the gene such as the N-terminus also have a strong impact on its in vivo function. To analyze the contribution of the N-terminus on the methyltransferase activity, we have expressed the full-length Drosophila SU(VAR)3-9 (dSU(VAR)3-9) together with various N-terminal deletions in Escherichia coli and analyzed the structural and enzymatic properties of the purified recombinant enzymes. Full-length dSU(VAR)3-9 specifically methylates lysine 9 within histone H3 on peptides, on intact histones, and, to a lesser extent, on nucleosomes. A detailed analysis of the reaction products shows that dSU(VAR)3-9 adds two methyl groups to an unmethylated H3 tail peptide in a nonprocessive manner. The full-length enzyme elutes with an apparent molecular weight of 160 kDa from a gel filtration column, which indicates the formation of a dimer. This property is dependent on an intact N-terminus. In contrast to the full-length enzymes, proteins lacking the N-terminus fail to dimerize, and show a 10-fold lower specific activity and a linear dependence of methyltransferase activity on enzyme concentration. A N-terminal peptide containing amino acids 1-152 of dSU(VAR)3-9 is sufficient to mediate this interaction in vitro. The dimerization of dSU(VAR)3-9 and the subsequent increase of its methyltransferase activity provide a starting point to understand the molecular details of the formation of heterochromatic structures in vivo.