R106C TFG variant causes infantile neuroaxonal dystrophy "plus" syndrome.

TFG (tropomyosin-receptor kinase fused gene) encodes an essential protein in the regulation of vesicular trafficking between endoplasmic reticulum and Golgi apparatus. The homozygous variant c.316C > T within TFG has been previously associated with a complicated hereditary spastic paraplegia (HSP) phenotype in two unrelated Indian families. Here, we describe the first Italian family with two affected siblings harboring the same variant, who in childhood were classified as infantile neuroaxonal dystrophy (INAD) based on clinical and neuropathological findings. Twenty years after the first diagnosis, exome sequencing was instrumental to identify the genetic cause of this disorder and clinical follow-up of patients allowed us to reconstruct the natural history of this clinical entity. Investigations on patient's fibroblasts demonstrate the presence of altered mitochondrial network and inner membrane potential, associated with metabolic impairment. Our study highlights phenotypic heterogeneity characterizing individuals carrying the same pathogenic variant in TFG and provides an insight on tight connection linking mitochondrial efficiency and neuronal health to vesicular trafficking.

Response to medical and a novel dietary treatment in newborn screen identified patients with ethylmalonic encephalopathy.

Ethylmalonic encephalopathy (EE) is a devastating neurodegenerative disease caused by mutations in the ETHE1 gene critical for hydrogen sulfide (H2S) detoxification. Patients present in infancy with hypotonia, developmental delay, diarrhea, orthostatic acrocyanosis and petechiae. Biochemical findings include elevated C4, C5 acylcarnitines and lactic and ethylmalonic acid (EMA) in body fluids. Current treatment modalities include metronidazole and N-acetylcysteine (NAC) to lower the production and promote detoxification of toxic H2S. Patients are typically identified after the onset of clinical symptoms and there is limited information about long term response to treatment. We report the findings of two unrelated patients with EE, identified through newborn screening, who were managed with conventional treatment (NAC, metronidazole alternated with neomycin) and in patient 2, a novel dietary treatment restricting sulfur containing amino acids. Pathogenic mutations were confirmed in the ETHE1 gene (homozygous splice site mutation in patient 1, c.505 + 1G > A; compound heterozygous mutations in patient 2, c.131_132delAG + c.566delG). Both patients were started on metronidazole and NAC by 10 weeks of age and treated for 23 months. Patient 1 did not accept the metabolic formula due to palatability and parental refusal for gastrostomy tube placement. She demonstrated improved biomarkers (EMA, lactic acid and thiosulfate) and an attenuated clinical course. Patient 2 was started on a low methionine and cysteine diet at 8 months of age utilizing SOD Anamix® Early Years, (Nutricia). Baseline EMA levels were (642 mg/g Cr; n = 2) and decreased with medical treatment by 38% to a mean of 399 (n = 4, SD = 71, p 0.0013). With dietary treatment EMA levels were further reduced by 42% to a mean of 233 (n = 8, SD = 52, p 0.0030). Lactic acid, thiosulfates and clinical outcomes were also improved. Our long-term follow-up confirms previous reports of clinical improvement with NAC and metronidazole treatment. Additionally, our studies suggest that a diet restricted in sulfur-containing amino acids results in further improvement in clinical outcomes and biochemical markers.

SURF1 knockout cloned pigs: Early onset of a severe lethal phenotype.

Leigh syndrome (LS) associated with cytochrome c oxidase (COX) deficiency is an early onset, fatal mitochondrial encephalopathy, leading to multiple neurological failure and eventually death, usually in the first decade of life. Mutations in SURF1, a nuclear gene encoding a mitochondrial protein involved in COX assembly, are among the most common causes of LS. LSSURF1 patients display severe, isolated COX deficiency in all tissues, including cultured fibroblasts and skeletal muscle. Recombinant, constitutive SURF1-/- mice show diffuse COX deficiency, but fail to recapitulate the severity of the human clinical phenotype. Pigs are an attractive alternative model for human diseases, because of their size, as well as metabolic, physiological and genetic similarity to humans. Here, we determined the complete sequence of the swine SURF1 gene, disrupted it in pig primary fibroblast cell lines using both TALENs and CRISPR/Cas9 genome editing systems, before finally generating SURF1-/- and SURF1-/+ pigs by Somatic Cell Nuclear Transfer (SCNT). SURF1-/- pigs were characterized by failure to thrive, muscle weakness and highly reduced life span with elevated perinatal mortality, compared to heterozygous SURF1-/+ and wild type littermates. Surprisingly, no obvious COX deficiency was detected in SURF1-/- tissues, although histochemical analysis revealed the presence of COX deficiency in jejunum villi and total mRNA sequencing (RNAseq) showed that several COX subunit-encoding genes were significantly down-regulated in SURF1-/- skeletal muscles. In addition, neuropathological findings, indicated a delay in central nervous system development of newborn SURF1-/- piglets. Our results suggest a broader role of sSURF1 in mitochondrial bioenergetics.

Reduced mitochondrial Ca(2+) transients stimulate autophagy in human fibroblasts carrying the 13514A>G mutation of the ND5 subunit of NADH dehydrogenase.

Mitochondrial disorders are a group of pathologies characterized by impairment of mitochondrial function mainly due to defects of the respiratory chain and consequent organellar energetics. This affects organs and tissues that require an efficient energy supply, such as brain and skeletal muscle. They are caused by mutations in both nuclear- and mitochondrial DNA (mtDNA)-encoded genes and their clinical manifestations show a great heterogeneity in terms of age of onset and severity, suggesting that patient-specific features are key determinants of the pathogenic process. In order to correlate the genetic defect to the clinical phenotype, we used a cell culture model consisting of fibroblasts derived from patients with different mutations in the mtDNA-encoded ND5 complex I subunit and with different severities of the illness. Interestingly, we found that cells from patients with the 13514A>G mutation, who manifested a relatively late onset and slower progression of the disease, display an increased autophagic flux when compared with fibroblasts from other patients or healthy donors. We characterized their mitochondrial phenotype by investigating organelle turnover, morphology, membrane potential and Ca(2+) homeostasis, demonstrating that mitochondrial quality control through mitophagy is upregulated in 13514A>G cells. This is due to a specific downregulation of mitochondrial Ca(2+) uptake that causes the stimulation of the autophagic machinery through the AMPK signaling axis. Genetic and pharmacological manipulation of mitochondrial Ca(2+) homeostasis can revert this phenotype, but concurrently decreases cell viability. This indicates that the higher mitochondrial turnover in complex I deficient cells with this specific mutation is a pro-survival compensatory mechanism that could contribute to the mild clinical phenotype of this patient.

A multi-center comparison of diagnostic methods for the biochemical evaluation of suspected mitochondrial disorders.

A multicenter comparison of mitochondrial respiratory chain and complex V enzyme activity tests was performed. The average reproducibility of the enzyme assays is 16% in human muscle samples. In a blinded diagnostic accuracy test in patient fibroblasts and SURF1 knock-out mouse muscle, each lab made the correct diagnosis except for two complex I results. We recommend that enzyme activities be evaluated based on ratios, e.g. with complex IV or citrate synthase activity. In spite of large variations in observed enzyme activities, we show that inter-laboratory comparison of patient sample test results is possible by using normalization against a control sample.

Microscale oxygraphy reveals OXPHOS impairment in MRC mutant cells.

Given the complexity of the respiratory chain structure, assembly and regulation, the diagnostic workout for the identification of defects of oxidative phosphorylation (OXPHOS) is a major challenge. Spectrophotometric assays, that measure the activity of individual respiratory complexes in tissue and cell homogenates or isolated mitochondria, are highly specific, but their utilization is limited by the availability of sufficient biological material and intrinsic sensitivity. A further limitation is tissue specificity, which usually determines attenuation, or disappearance, in cultured fibroblasts, of defects detected in muscle or liver. We used numerous fibroblast cell lines derived from patients with OXPHOS deficiencies to set up experimental protocols required for the direct readout of cellular respiration using the Seahorse XF96 apparatus, which measures oxygen consumption rate (OCR) and extra-cellular acidification rate (ECAR) in 96 well plates. Results demonstrate that first level screening based on microscale oxygraphy is more sensitive, cheaper and rapid than spectrophotometry for the biochemical evaluation of cells from patients with suspected mitochondrial disorders.

Ethylmalonic encephalopathy: application of improved biochemical and molecular diagnostic approaches.

Ethylmalonic encephalopathy (EE, OMIM # 602473) is an autosomal recessive metabolic disorder of infancy affecting the brain, the gastrointestinal tract and peripheral vessels. It is caused by a defect in the ETHE1 gene product, which was recently shown to be part of a metabolic pathway devoted to sulphide detoxification. We report the application of improved biochemical and molecular approaches to the diagnosis of three cases of EE from two unrelated Cypriot families. The children presented all the typical biochemical hallmarks of the disease including elevated lactate and butyrylcarnitine in blood and elevated urinary excretion of ethylmalonic acid, 2-methylsuccinate, isobutyrylglycine and isovalerylglycine. We also detected an elevated level of thiosulphate in urine, which we propose as an additional biochemical marker of the disease. The proband of the first family was shown to be a compound heterozygote for a missense mutation in exon 5, L185R, and a deletion of exon 4. The deletion was identified using quantitative real-time polymerase chain reaction (qRT-PCR). Using the same technique, the proband of the second family was found to be homozygous for the exon 4 deletion. A prenatal diagnosis was performed for the second family using qRT-PCR, thus establishing the usefulness of RT-PCR in prenatal diagnosis.

Identification of new mutations in the ETHE1 gene in a cohort of 14 patients presenting with ethylmalonic encephalopathy.

BACKGROUND: Ethylmalonic encephalopathy (EE) is a rare autosomal recessive metabolic disorder characterised by progressive encephalopathy, recurrent petechiae, acrocyanosis and chronic diarrhoea, with a fatal outcome in early in life. METHODS: 14 patients with EE were investigated for mutations in the ETHE1 gene. RESULTS: Of the 14 patients, 5 were found to carry novel mutations. CONCLUSIONS: This work expands our knowledge of the causative mutations of EE.

ETHE1 mutations are specific to ethylmalonic encephalopathy.

Mutations in ETHE1, a gene located at chromosome 19q13, have recently been identified in patients affected by ethylmalonic encephalopathy (EE). EE is a devastating infantile metabolic disorder, characterised by widespread lesions in the brain, hyperlactic acidaemia, petechiae, orthostatic acrocyanosis, and high levels of ethylmalonic acid in body fluids. To investigate to what extent ETHE1 is responsible for EE, we analysed this gene in 29 patients with typical EE and in 11 patients presenting with early onset progressive encephalopathy with ethylmalonic aciduria (non-EE EMA). Frameshift, stop, splice site, and missense mutations of ETHE1 were detected in all the typical EE patients analysed. Western blot analysis of the ETHE1 protein indicated that some of the missense mutations are associated with the presence of the protein, suggesting that the corresponding wild type amino acid residues have a catalytic function. No ETHE1 mutations were identified in non-EE EMA patients. Experiments based on two dimensional blue native electrophoresis indicated that ETHE1 protein works as a supramolecular, presumably homodimeric, complex, and a three dimensional model of the protein suggests that it is likely to be a mitochondrial matrix thioesterase acting on a still unknown substrate. Finally, the 625G-->A single nucleotide polymorphism in the gene encoding the short chain acyl-coenzyme A dehydrogenase (SCAD) was previously proposed as a co-factor in the aetiology of EE and other EMA syndromes. SNP analysis in our patients ruled out a pathogenic role of SCAD variants in EE, but did show a highly significant prevalence of the 625A alleles in non-EE EMA patients.

Novel mutations in COX15 in a long surviving Leigh syndrome patient with cytochrome c oxidase deficiency.

BACKGROUND: Isolated cytochrome c oxidase (COX) deficiency is usually associated with mutations in several factors involved in the biogenesis of COX. METHODS: We describe a patient with atypical, long surviving Leigh syndrome carrying two novel mutations in the COX15 gene, which encodes an enzyme involved in the biosynthesis of heme A. RESULTS: Only two COX15 mutated patients, one with severe neonatal cardiomyopathy, the other with rapidly fatal Leigh syndrome, have been described to date. In contrast, our patient had a slowly progressive course with no heart involvement. COX deficiency was mild in muscle and a normal amount of fully assembled COX was present in cultured fibroblasts. CONCLUSIONS: The clinical and biochemical phenotypes in COX15 defects are more heterogeneous than in other conditions associated with COX deficiency, such as mutations in SURF1.

Mutations of ANT1, Twinkle, and POLG1 in sporadic progressive external ophthalmoplegia (PEO).

To verify the impact of mutations in ANT1, Twinkle, and POLG1 genes in sporadic progressive external ophthalmoplegia associated with multiple mitochondrial DNA (mtDNA) deletions, DNA samples from 15 Italian and 12 British patients were screened. Mutations in ANT1 were found in one patient, in Twinkle in two patients, and in POLG1 in seven patients. Irrespective of the inheritance mode, screening of these genes should be performed in all patients with progressive external ophthalmoplegia with multiple mtDNA deletions.

Unusual association of sporadic olivopontocerebellar atrophy and motor neuron disease.

Sporadic olivopontocerebellar atrophy (OPCA) is a neurodegenerative disorder that presents a wide clinical spectrum. Motor neuron disease (MND) is characterized by a selective degeneration of motor neurons. A 60-year-old man developed slurred speech and unsteadiness of gait. He had also noticed difficulty in holding his head upright and shoulder weakness. The disease had a rapid progression. At the age of 63 years, magnetic resonance imaging supported a diagnosis of OPCA, and a diagnosis of MND was suggested by clinical and electrophysiological findings. He also had upward gaze palsy. A muscular biopsy showed sporadic ragged red and Cox deficient fibers. The present case could define a unique disorder, as the occasional occurrence of two degenerative disorders appears unlikely.

Human mitochondrial DNA deletions associated with mutations in the gene encoding Twinkle, a phage T7 gene 4-like protein localized in mitochondria.

The gene products involved in mammalian mitochondrial DNA (mtDNA) maintenance and organization remain largely unknown. We report here a novel mitochondrial protein, Twinkle, with structural similarity to phage T7 gene 4 primase/helicase and other hexameric ring helicases. Twinkle colocalizes with mtDNA in mitochondrial nucleoids. Screening of the gene encoding Twinkle in individuals with autosomal dominant progressive external ophthalmoplegia (adPEO), associated with multiple mtDNA deletions, identified 11 different coding-region mutations co-segregating with the disorder in 12 adPEO pedigrees of various ethnic origins. The mutations cluster in a region of the protein proposed to be involved in subunit interactions. The function of Twinkle is inferred to be critical for lifetime maintenance of human mtDNA integrity.

Multiple origins of the mtDNA 7472insC mutation associated with hearing loss and neurological dysfunction.

Several mtDNA mutations have been reported in families with both syndromic and non-syndromic hearing loss. One such mutation is the heteroplasmic 7472insC in the tRNA(Ser(UCN)) gene which has been found in six families, all from Western Europe. However, it was not clear if this distribution was due to a common founder effect or chance sampling of several unrelated families, the 7472insC mutation having occurred multiple times. Haplotype analysis of all six families supports the latter notion. This confirms the pathogenicity of the 7472insC mutation and suggests it may exist in other populations where it may prove to be a small but significant cause of hearing loss, particularly when neurological symptoms are also present.

Mutations in the SURF1 gene associated with Leigh syndrome and cytochrome C oxidase deficiency.

Cytochrome c oxidase (COX) deficiency is one of the major causes of Leigh Syndrome (LS), a fatal encephalopathy of infancy or childhood, characterized by symmetrical lesions in the basal ganglia and brainstem. Mutations in the nuclear genes encoding COX subunits have not been found in patients with LS and COX deficiency, but mutations have been identified in SURF1. SURF1 encodes a factor involved in COX biogenesis. To date, 30 different mutations have been reported in 40 unrelated patients. We aim to provide an overview of all known mutations in SURF1, and to propose a common nomenclature. Twelve of the mutations were insertion/deletion mutations in exons 1, 4, 6, 8, and 9; 10 were missense/nonsense mutations in exons 2, 4, 5, 7, and 8; and eight were detected at splicing sites in introns 3 to 7. The most frequent mutation was 312_321del 311_312insAT which was found in 12 patients out of 40. Twenty mutations have been described only once. We also list all polymorphisms discovered to date.

A novel mtDNA mutation in the ND5 subunit of complex I in two MELAS patients.

We identified a novel heteroplasmic mutation in the mitochodrial DNA gene encoding the ND5 subunit of complex I. This mutation (13514A-->G) hits the same codon affected by a previously reported mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS)-associated mutation (13513G-->A), but the amino acid replacement is different (D393G vs D393N). The 13514A-->G mutation was found in two unrelated MELAS-like patients. However, in contrast to typical MELAS, lactic acidosis was absent or mild and the muscle biopsy was morphologically normal. Strongly positive correlation between the percentage of heteroplasmy and defective activity of complex I was found in cybrids. We found an additional 13513G-->A-positive case, affected by a progressive mitochondrial encephalomyopathy. Our results clearly demonstrate that the amino acid position D393 is crucial for the function of complex I. Search for D393 mutations should be part of the routine screening for mitochondrial disorders.

A novel missense adenine nucleotide translocator-1 gene mutation in a Greek adPEO family.

Autosomal dominant progressive external ophthalmoplegia (adPEO) is caused by mutations in at least three different genes: ANT1 (chromosome 4q34-35), TWINKLE, and POLG. The ANT1 gene encodes the adenine nucleotide translocator-1 (ANT1). We identified a heterozygous T293C mutation of the ANT1 gene in a Greek family with adPEO. The resulting leucine to proline substitution likely modifies the secondary structure of the ANT1 protein. ANT1 gene mutations may account for adPEO in families with different ethnic backgrounds.

A novel frameshift mutation of the mtDNA COIII gene leads to impaired assembly of cytochrome c oxidase in a patient affected by Leigh-like syndrome.

We report on a novel frameshift mutation in the mtDNA gene encoding cytochrome c oxidase (COX) subunit III. The proband is an 11-year-old girl with a negative family history and an apparently healthy younger brother. Since 4 years of age, she has developed a progressive spastic paraparesis associated with ophthalmoparesis and moderate mental retardation. The presence of severe lactic acidosis and Leigh-like lesions of putamina prompted us to perform muscle and skin biopsies. In both, a profound, isolated defect of COX was found by histochemical and biochemical assays. Sequence analysis of muscle mtDNA resulted in the identification of a virtually homoplasmic frameshift mutation in the COIII gene, due to the insertion of an extra C at nucleotide position 9537 of mtDNA. Although the 9537C(ins) does not impair transcription of COIII, no full-length COX III protein was detected in mtDNA translation assays in vivo. Western blot analysis of two-dimensional blue-native electrophoresis showed a reduction of specific crossreacting material and the accumulation of early-assembly intermediates of COX, whereas the fully assembled complex was absent. One of these intermediates had an electrophoretic mobility different from those seen in controls, suggesting the presence of a qualitative abnormality of COX assembly. Immunostaining with specific antibodies failed to detect the presence of several smaller subunits in the complex lacking COX III, in spite of the demonstration that these subunits were present in the crude mitochondrial fraction of patient's cultured fibroblasts. Taken together, the data indicate a role for COX III in the incorporation and maintenance of smaller COX subunits within the complex.

Role of adenine nucleotide translocator 1 in mtDNA maintenance.

Autosomal dominant progressive external ophthalmoplegia is a rare human disease that shows a Mendelian inheritance pattern, but is characterized by large-scale mitochondrial DNA (mtDNA) deletions. We have identified two heterozygous missense mutations in the nuclear gene encoding the heart/skeletal muscle isoform of the adenine nucleotide translocator (ANT1) in five families and one sporadic patient. The familial mutation substitutes a proline for a highly conserved alanine at position 114 in the ANT1 protein. The analogous mutation in yeast caused a respiratory defect. These results indicate that ANT has a role in mtDNA maintenance and that a mitochondrial disease can be caused by a dominant mechanism.