Kyle Thompson

Recurrent De Novo Dominant Mutations in SLC25A4 Cause Severe Early-Onset Mitochondrial Disease and Loss of Mitochondrial DNA Copy Number

Mutations in SLC25A4 encoding the mitochondrial ADP/ATP carrier AAC1 are well-recognized causes of mitochondrial disease. Several heterozygous SLC25A4 mutations cause adult-onset autosomal-dominant progressive external ophthalmoplegia associated with multiple mitochondrial DNA deletions, whereas recessive SLC25A4 mutations cause childhood-onset mitochondrial myopathy and cardiomyopathy. Here, we describe the identification by whole-exome sequencing of seven probands harboring dominant, de novo SLC25A4 mutations. All affected individuals presented at birth, were ventilator dependent and, where tested, revealed severe combined mitochondrial respiratory chain deficiencies associated with a marked loss of mitochondrial DNA copy number in skeletal muscle. Strikingly, an identical c.239G>A (p.Arg80His) mutation was present in four of the seven subjects, and the other three case subjects harbored the same c.703C>G (p.Arg235Gly) mutation. Analysis of skeletal muscle revealed a marked decrease of AAC1 protein levels and loss of respiratory chain complexes containing mitochondrial DNA-encoded subunits. We show that both recombinant AAC1 mutant proteins are severely impaired in ADP/ATP transport, affecting most likely the substrate binding and mechanics of the carrier, respectively. This highly reduced capacity for transport probably affects mitochondrial DNA maintenance and in turn respiration, causing a severe energy crisis. The confirmation of the pathogenicity of these de novo SLC25A4 mutations highlights a third distinct clinical phenotype associated with mutation of this gene and demonstrates that early-onset mitochondrial disease can be caused by recurrent de novo mutations, which has significant implications for the application and analysis of whole-exome sequencing data in mitochondrial disease.

Recessive Mutations in TRMT10C Cause Defects in Mitochondrial RNA Processing and Multiple Respiratory Chain Deficiencies

Mitochondrial disorders are clinically and genetically diverse, with mutations in mitochondrial or nuclear genes able to cause defects in mitochondrial gene expression. Recently, mutations in several genes encoding factors involved in mt-tRNA processing have been identified to cause mitochondrial disease. Using whole-exome sequencing, we identified mutations in TRMT10C (encoding the mitochondrial RNase P protein 1 [MRPP1]) in two unrelated individuals who presented at birth with lactic acidosis, hypotonia, feeding difficulties, and deafness. Both individuals died at 5 months after respiratory failure. MRPP1, along with MRPP2 and MRPP3, form the mitochondrial ribonuclease P (mt-RNase P) complex that cleaves the 5′ ends of mt-tRNAs from polycistronic precursor transcripts. Additionally, a stable complex of MRPP1 and MRPP2 has m1R9 methyltransferase activity, which methylates mt-tRNAs at position 9 and is vital for folding mt-tRNAs into their correct tertiary structures. Analyses of fibroblasts from affected individuals harboring TRMT10C missense variants revealed decreased protein levels of MRPP1 and an increase in mt-RNA precursors indicative of impaired mt-RNA processing and defective mitochondrial protein synthesis. The pathogenicity of the detected variants—compound heterozygous c.542G>T (p.Arg181Leu) and c.814A>G (p.Thr272Ala) changes in subject 1 and a homozygous c.542G>T (p.Arg181Leu) variant in subject 2—was validated by the functional rescue of mt-RNA processing and mitochondrial protein synthesis defects after lentiviral transduction of wild-type TRMT10C. Our study suggests that these variants affect MRPP1 protein stability and mt-tRNA processing without affecting m1R9 methyltransferase activity, identifying mutations in TRMT10C as a cause of mitochondrial disease and highlighting the importance of RNA processing for correct mitochondrial function.

Long-term survival in a child with severe encephalopathy, multiple respiratory chain deficiency and GFM1 mutations.

Background: Mitochondrial diseases due to deficiencies in the mitochondrial oxidative phosphorylation system (OXPHOS) can be associated with nuclear genes involved in mitochondrial translation, causing heterogeneous early onset and often fatal phenotypes. Case report: The authors describe the clinical features and diagnostic workup of an infant who presented with an early onset severe encephalopathy, spastic-dystonic tetraparesis, failure to thrive, seizures and persistent lactic acidemia. Brain imaging revealed thinning of the corpus callosum and diffuse alteration of white matter signal. Genetic investigation confirmed two novel mutations in the GFM1 gene, encoding the mitochondrial translation elongation factor G1 (mtEFG1), resulting in combined deficiencies of OXPHOS. Discussion: The patient shares multiple clinical, laboratory and radiological similarities with the 11 reported patients with mutations involving this gene, but presents with a stable clinical course without metabolic decompensations, rather than a rapidly progressive fatal course. Defects in GFM1 gene confer high susceptibility to neurologic or hepatic dysfunction and this is, to the best of our knowledge, the first described patient who has survived beyond early childhood. Reporting of such cases is essential so as to delineate the key clinical and neuroradiological features of this disease and provide a more comprehensive view of its prognosis.

Pathogenic variants in HTRA2 cause an early-onset mitochondrial syndrome associated with 3-methylglutaconic aciduria

Mitochondrial diseases collectively represent one of the most heterogeneous group of metabolic disorders. Symptoms can manifest at any age, presenting with isolated or multiple-organ involvement. Advances in next-generation sequencing strategies have greatly enhanced the diagnosis of patients with mitochondrial disease, particularly where a mitochondrial aetiology is strongly suspected yet OXPHOS activities in biopsied tissue samples appear normal. We used whole exome sequencing (WES) to identify the molecular basis of an early-onset mitochondrial syndrome—pathogenic biallelic variants in the HTRA2 gene, encoding a mitochondria-localised serine protease—in five subjects from two unrelated families characterised by seizures, neutropenia, hypotonia and cardio-respiratory problems. A unifying feature in all affected children was 3-methylglutaconic aciduria (3-MGA-uria), a common biochemical marker observed in some patients with mitochondrial dysfunction. Although functional studies of HTRA2 subjects’ fibroblasts and skeletal muscle homogenates showed severely decreased levels of mutant HTRA2 protein, the structural subunits and complexes of the mitochondrial respiratory chain appeared normal. We did detect a profound defect in OPA1 processing in HTRA2-deficient fibroblasts, suggesting a role for HTRA2 in the regulation of mitochondrial dynamics and OPA1 proteolysis. In addition, investigated subject fibroblasts were more susceptible to apoptotic insults. Our data support recent studies that described important functions for HTRA2 in programmed cell death and confirm that patients with genetically-unresolved 3-MGA-uria should be screened by WES with pathogenic variants in the HTRA2 gene prioritised for further analysis.

Lethal Neonatal LTBL Associated with Biallelic EARS2 Variants: Case Report and Review of the Reported Neuroradiological Features

Mitochondrial translation defects are important causes of early onset mitochondrial disease. Although the biochemical (combined respiratory chain deficiency) signature and neuroimaging are usually distinctive, they are not diagnostic as the genetic origin of mitochondrial translation defects is heterogeneous. We report a female child, born at term to non-consanguineous parents, who exhibited global hypotonia, failure to thrive, persistent and progressive hyperlactacidaemia with lactic acidosis, liver dysfunction and encephalopathy and died at the age of 5 months. Brain MRI revealed hypogenesis of the corpus callosum, T2 signal abnormalities in the medulla oblongata, pons, midbrain, thalami, cerebellar white matter, and a lactate peak on MRS. Muscle histochemistry showed cytochrome c oxidase (COX)-deficient and ragged-red fibres, while muscle biochemical studies showed decreased activities of mitochondrial respiratory chain complexes I and IV. Whole exome sequencing (WES) identified biallelic EARS2 (NM_001083614) variants, a previously reported start-loss (c.1>G, p.Met1?) variant and a novel missense (c.184A>T, p.Ile62Phe) variant. Patient fibroblasts and muscle homogenate displayed markedly decreased EARS2 protein levels, although decreased steady-state levels of complex I (NDUFB8) and complex IV (MT-CO1 and MT-CO2) subunits were only observed in muscle. Pathogenic variants in EARS2, encoding mitochondrial glutamyl-tRNA synthetase (mtGluR), are associated with Leukoencephalopathy involving the Thalamus and Brainstem with high Lactate (LTBL), a mitochondrial disorder characterised by a distinctive brain MRI pattern and a biphasic clinical course. We further outline the unique phenotypic spectrum of LTBL and review the neuroradiological features reported in all patients documented in the literature.

Biallelic mutations in mitochondrial tryptophanyl-tRNA synthetase cause Levodopa-responsive infantile-onset Parkinsonism

Mitochondrial aminoacyl‐tRNA synthetases (mtARSs) are essential, ubiquitously expressed enzymes that covalently attach amino acids to their corresponding tRNA molecules during translation of mitochondrial genes. Deleterious variants in the mtARS genes cause a diverse array of phenotypes, many of which involve the nervous system. Moreover, distinct mutations in mtARSs often cause different clinical manifestations. Recently, the gene encoding mitochondrial tryptophanyl tRNA synthetase (WARS2) was reported to cause 2 different neurological phenotypes, a form of autosomal recessive intellectual disability and a syndrome of severe infantile‐onset leukoencephalopathy. Here, we report the case of a 17‐year‐old boy with compound heterozygous mutations in WARS2 (p.Trp13Gly, p.Ser228Trp) who presented with infantile‐onset, Levodopa‐responsive Parkinsonism at the age of 2 years. Analysis of patient‐derived dermal fibroblasts revealed decreased steady‐state WARS2 protein and normal OXPHOS content. Muscle mitochondrial studies suggested mitochondrial proliferation without obvious respiratory chain deficiencies at the age of 9 years. This case expands the phenotypic spectrum of WARS2 deficiency and emphasizes the importance of mitochondrial protein synthesis in the pathogenesis of Parkinsonism.

A homozygous variant in mitochondrial RNase P subunit PRORP is associated with Perrault syndrome characterized by hearing loss and primary ovarian insufficiency

Perrault syndrome is a rare autosomal recessive condition characterised by sensorineural hearing loss in both sexes and primary ovarian insufficiency in 46 XX, females. It is genetically heterogeneous with biallelic variants in six genes identified to date (HSD17B4, HARS2, LARS2, CLPP, C10orf2 and ERAL1). Most genes possessing variants associated with Perrault syndrome are involved in mitochondrial translation. We describe a consanguineous family with three affected individuals homozygous for a novel missense variant c.1454C>T; p.(Ala485Val) in KIAA0391, encoding proteinaceous RNase P (PRORP), the metallonuclease subunit of the mitochondrial RNase P complex, responsible for the 5’-end processing of mitochondrial precursor tRNAs. In RNase P activity assays, RNase P complexes containing the PRORP disease variant produced ~35-45% less 5’-processed tRNA than wild type PRORP. Consistently, the accumulation of unprocessed polycistronic mitochondrial transcripts was observed in patient dermal fibroblasts, leading to an observable loss of steady-state levels of mitochondrial oxidative phosphorylation components. Expression of wild type KIAA0391 in patient fibroblasts rescued tRNA processing. Immunohistochemistry analyses of the auditory sensory epithelium from postnatal and adult mouse inner ear showed a high level of PRORP in the efferent synapses and nerve fibres of hair cells, indicating a possible mechanism for the sensorineural hearing loss observed in affected individuals. We have identified a variant in an additional gene associated with Perrault syndrome. With the identification of this disease-causing variant in KIAA0391, reduced function of each of the three subunits of mitochondrial RNase P have now been associated with distinct clinical presentations. Author Summary Perrault syndrome is a rare genetic condition which results in hearing loss in both sexes and ovarian dysfunction in females. Perrault syndrome may also cause neurological symptoms in some patients. Here, we present the features and genetic basis of the condition in three sisters affected by Perrault syndrome. The sisters did not have pathogenic variants in any of the genes previously associated with Perrault syndrome. We identified a change in the gene KIAA0391, encoding PRORP, a subunit of the mitochondrial RNase P complex. Mitochondrial RNase P is a key enzyme in RNA processing in mitochondria. Impaired RNA processing reduces protein production in mitochondria, which we observed in patient cells along with high levels of unprocessed RNA. When we expressed wild type PRORP in patient cells, the RNA processing improved. We also investigated PRORP localisation in the mouse inner ear and found high levels in the synapses and nerve fibers that transmit sound. It may be that disruption of RNA processing in the mitochondria of these cells causes hearing loss in this family.

Periventricular Calcification, Abnormal Pterins and Dry Thickened Skin: Expanding the Clinical Spectrum of RMND1?

BACKGROUND We report a consanguineous Sudanese family whose two affected sons presented with a lethal disorder characterised by severe neonatal lactic acidosis, hypertonia, microcephaly and intractable seizures. One child had additional unique features of periventricular calcification, abnormal pterins and dry thickened skin. METHODS Exome enrichment was performed on pooled genomic libraries from the two affected children and sequenced on an Illumina HiSeq2000. After quality control and variant identification, rare homozygous variants were prioritised. Respiratory chain complex activities were measured and normalised to citrate synthase activity in cultured patient fibroblasts. RMND1 protein levels were analysed by standard Western blotting. RESULTS Exome sequencing identified a previously reported homozygous missense variant in RMND1 (c.1250G>A; p.Arg417Gln), the gene associated with combined oxidation phosphorylation deficiency 11 (COXPD11), as the most likely cause of this disorder. This finding suggests the presence of a mutation hotspot at cDNA position 1250. Patient fibroblasts showed a severe decrease in mitochondrial respiratory chain complex I, III and IV activities and protein expression, albeit with normal RMND1 levels, supporting a generalised disorder of mitochondrial translation caused by loss of function. CONCLUSIONS The current study implicates RMND1 in the development of calcification and dermatological abnormalities, likely due to defective ATP-dependent processes in vascular smooth muscle cells and skin. Review of reported patients with RMND1 mutations shows intra-familial variability and evidence of an evolving phenotype, which may account for the clinical variability. We suggest that COXPD11 should be considered in the differential for patients with calcification and evidence of a mitochondrial disorder.

Biallelic C1QBP Mutations Cause Severe Neonatal-, Childhood-, or Later-Onset Cardiomyopathy Associated with Combined Respiratory-Chain Deficiencies

Complement component 1 Q subcomponent-binding protein (C1QBP; also known as p32) is a multi-compartmental protein whose precise function remains unknown. It is an evolutionary conserved multifunctional protein localized primarily in the mitochondrial matrix and has roles in inflammation and infection processes, mitochondrial ribosome biogenesis, and regulation of apoptosis and nuclear transcription. It has an N-terminal mitochondrial targeting peptide that is proteolytically processed after import into the mitochondrial matrix, where it forms a homotrimeric complex organized in a doughnut-shaped structure. Although C1QBP has been reported to exert pleiotropic effects on many cellular processes, we report here four individuals from unrelated families where biallelic mutations in C1QBP cause a defect in mitochondrial energy metabolism. Infants presented with cardiomyopathy accompanied by multisystemic involvement (liver, kidney, and brain), and children and adults presented with myopathy and progressive external ophthalmoplegia. Multiple mitochondrial respiratory-chain defects, associated with the accumulation of multiple deletions of mitochondrial DNA in the later-onset myopathic cases, were identified in all affected individuals. Steady-state C1QBP levels were decreased in all individuals’ samples, leading to combined respiratory-chain enzyme deficiency of complexes I, III, and IV. C1qbp−/− mouse embryonic fibroblasts (MEFs) resembled the human disease phenotype by showing multiple defects in oxidative phosphorylation (OXPHOS). Complementation with wild-type, but not mutagenized, C1qbp restored OXPHOS protein levels and mitochondrial enzyme activities in C1qbp−/− MEFs. C1QBP deficiency represents an important mitochondrial disorder associated with a clinical spectrum ranging from infantile lactic acidosis to childhood (cardio)myopathy and late-onset progressive external ophthalmoplegia.

Expanding the phenotype of de novo SLC25A4-linked mitochondrial disease to include mild myopathy

Objective To determine the disease relevance of a novel de novo dominant variant in the SLC25A4 gene, encoding the muscle mitochondrial adenosine diphosphate (ADP)/adenosine triphosphate (ATP) carrier, identified in a child presenting with a previously unreported phenotype of mild childhood-onset myopathy. Methods Immunohistochemical and western blot analysis of the patients muscle tissue were used to assay for the evidence of mitochondrial myopathy and for complex I–V protein levels. To determine the effect of a putative pathogenic p.Lys33Gln variant on ADP/ATP transport, the mutant protein was expressed in Lactococcus lactis and its transport activity was assessed with fused membrane vesicles. Results Our data demonstrate that the heterozygous c.97A>T (p.Lys33Gln) SLC25A4 variant is associated with classic muscle biopsy findings of mitochondrial myopathy (cytochrome c oxidase [COX]-deficient and ragged blue fibers), significantly impaired ADP/ATP transport in Lactococcus lactis and decreased complex I, III, and IV protein levels in patients skeletal muscle. Nonetheless, the expression levels of the total ADP/ATP carrier (AAC) content in the muscle biopsy was largely unaffected. Conclusions This report further expands the clinical phenotype of de novo dominant SLC25A4 mutations to a childhood-onset, mild skeletal myopathy, without evidence of previously reported clinical features associated with SLC25A4-associated disease, such as cardiomyopathy, encephalopathy or ophthalmoplegia. The most likely reason for the milder disease phenotype is that the overall AAC expression levels were not affected, meaning that expression of the wild-type allele and other isoforms may in part have compensated for the impaired mutant variant.