Identification of a novel MT-ND3 variant and restoring mitochondrial function by allotopic expression of MT-ND3 gene.

Mitochondrial diseases are caused by nuclear, or mitochondrial DNA (mtDNA) variants and related co-factors. Here, we report a novel m.10197G > C variant in MT-ND3 in a patient, and two other patients with m.10191 T > C. MT-ND3 variants are known to cause Leigh syndrome or mitochondrial complex I deficiency. We performed the functional analyses of the novel m.10197G > C variant that significantly lowered MT-ND3 protein levels, causing complex I assembly and activity deficiency, and reduction of ATP synthesis. We adapted a previously described re-engineering technique of delivering mitochondrial genes into mitochondria through codon optimization for nuclear expression and translation by cytoplasmic ribosomes to rescue defects arising from the MT-ND3 variants. We constructed mitochondrial targeting sequences along with the codon-optimized MT-ND3 and imported them into the mitochondria. To achieve the goal, we imported codon-optimized MT-ND3 into mitochondria in three patients with m.10197G > C and m.10191 T > C missense variants in the MT-ND3. Nuclear expression of the MT-ND3 gene partially restored protein levels, complex I deficiency, and significant improvement of ATP production indicating a functional rescue of the mutant phenotype. The codon-optimized nuclear expression of mitochondrial protein and import inside the mitochondria can supplement the requirements for ATP in energy-deficient mitochondrial disease patients.

Focal segmental glomerulosclerosis with a mutation in the mitochondrially encoded NADH dehydrogenase 5 gene: A case report.

NADH dehydrogenase 5 (ND5) is one of 44 subunits composed of Complex I in mitochondrial respiratory chain. Therefore, a mitochondrially encoded ND5 (MT-ND5) gene mutation causes mitochondrial oxidative phosphorylation (OXPHOS) disorder, resulting in the development of mitochondrial diseases. Focal segmental glomerulosclerosis (FSGS) which had podocytes filled with abnormal mitochondria is induced by mitochondrial diseases. An MT-ND5 mutation also causes FSGS. We herein report a Japanese woman who was found to have proteinuria and renal dysfunction in an annual health check-up at 29 years old. Because her proteinuria and renal dysfunction were persistent, she had a kidney biopsy at 33 years of age. The renal histology showed FSGS with podocytes filled with abnormal mitochondria. The podocytes also had foot process effacement and cytoplasmic vacuolization. In addition, the renal pathological findings showed granular swollen epithelial cells (GSECs) in tubular cells, age-inappropriately disarranged and irregularly sized vascular smooth muscle cells (AiDIVs), and red-coloured podocytes (ReCPos) by acidic dye. A genetic analysis using peripheral mononuclear blood cells and urine sediment cells detected the m.13513 G > A variant in the MT-ND5 gene. Therefore, this patient was diagnosed with FSGS due to an MT-ND5 gene mutation. Although this is not the first case report to show that an MT-ND5 gene mutation causes FSGS, this is the first to demonstrate podocyte injuries accompanied with accumulation of abnormal mitochondria in the cytoplasm.

Valine metabolites analysis in ECHS1 deficiency.

Short-chain enoyl-CoA hydratase (ECHS1) is involved in amino acid and fatty acid catabolism in mitochondria and its deficiency causes Leigh syndrome or exercise-induced dystonia. More than 60 patients with this condition have been reported till date. The accumulation of intermediate metabolites of valine is assumed to be responsible for the cytotoxicity. Since protein restriction, including valine reportedly improves neurological symptoms, it is essential to consider the possible incidence of and diagnose ECHS1 syndrome in the earlier stages. This study reported the liquid chromatography with tandem mass spectrometry (LC-MS/MS) urine and plasma metabolite analysis in six cases, including four new cases with ECHS1 deficiency. The values of urine cysteine/cysteamine conjugates from valine metabolites, S-(2-carboxypropyl) cysteine/cysteamine from methacrylyl-CoA, and S-(2-carboxyethyl) cysteine/cysteamine from acryloyl-CoA were separated between six patients and six normal controls. The LC-MS/MS analysis revealed that these metabolites can be used for the early diagnosis and evaluation of diet therapy.

Trigenic ADH5/ALDH2/ADGRV1 mutations in myelodysplasia with Usher syndrome.

Trio-next generation sequencing is useful to identify undiagnosed inherited diseases. We have attended a patient with trigenic ADH5/ALDH2/ADGRV1 pathogenic variants, which caused two distinct diseases, myelodysplastic syndrome and Usher syndrome. Whole genome sequencing of peripheral blood from the patient and his parents were applied to identify disease-causing genes. Sanger sequencing was performed to validate the identified ADH5/ALDH2/ADGRV1 variants. Our results identified disease-associated variants in ADGRV1 (disease inheritance autosomal recessive) and in ADH5 (disease inheritance also autosomal recessive) and a variant in ALDH2 (disease inheritance autosomal dominant). Although the variants identified in ADH5 and ALDH2 have been reported, their co-existence in association with disease-causing variation in a third gene has not. They broaden the spectrum of ADGRV1 in Usher syndrome. Findings on next generation sequencing guided rapid and accurate diagnosis, resulting in patient-tailored therapeutic intervention.

Effects of 5-aminolevulinic acid and sodium ferrous citrate on fibroblasts from individuals with mitochondrial diseases.

Mitochondrial respiratory chain complexes II, III, and IV and cytochrome c contain haem, which is generated by the insertion of Fe2+ into protoporphyrin IX. 5-Aminolevulinic acid (ALA) combined with sodium ferrous citrate (SFC) was reported to enhance haem production, leading to respiratory complex and haem oxygenase-1 (HO-1) upregulation. Here, we investigated the effects of different concentrations of ALA and SFC alone or in combination (ALA/SFC) on fibroblasts from 8 individuals with mitochondrial diseases and healthy controls. In normal fibroblasts, expression levels of oxidative phosphorylation (OXPHOS) complex subunits and corresponding genes were upregulated only by ALA/SFC. Additionally, the increased oxygen consumption rate (OCR) and ATP levels in normal fibroblasts were more obvious after treatment with ALA/SFC than after treatment with ALA or SFC. OXPHOS complex proteins were enhanced by ALA/SFC, whereas OCR and ATP levels were increased in 6 of the 8 patient-derived fibroblasts. Further, HO-1 protein and mRNA levels were enhanced by ALA/SFC in all fibroblasts. The relative mtDNA copy number was increased by ALA/SFC. Thus, our findings indicate that ALA/SFC is effective in elevating OXPHOS, HO-1 protein, and mtDNA copy number, resulting in an increase in OCR and ATP levels, which represents a promising therapeutic option for mitochondrial diseases.

Mutations in TOP3A Cause a Bloom Syndrome-like Disorder.

Bloom syndrome, caused by biallelic mutations in BLM, is characterized by prenatal-onset growth deficiency, short stature, an erythematous photosensitive malar rash, and increased cancer predisposition. Diagnostically, a hallmark feature is the presence of increased sister chromatid exchanges (SCEs) on cytogenetic testing. Here, we describe biallelic mutations in TOP3A in ten individuals with prenatal-onset growth restriction and microcephaly. TOP3A encodes topoisomerase III alpha (TopIIIα), which binds to BLM as part of the BTRR complex, and promotes dissolution of double Holliday junctions arising during homologous recombination. We also identify a homozygous truncating variant in RMI1, which encodes another component of the BTRR complex, in two individuals with microcephalic dwarfism. The TOP3A mutations substantially reduce cellular levels of TopIIIα, and consequently subjects' cells demonstrate elevated rates of SCE. Unresolved DNA recombination and/or replication intermediates persist into mitosis, leading to chromosome segregation defects and genome instability that most likely explain the growth restriction seen in these subjects and in Bloom syndrome. Clinical features of mitochondrial dysfunction are evident in several individuals with biallelic TOP3A mutations, consistent with the recently reported additional function of TopIIIα in mitochondrial DNA decatenation. In summary, our findings establish TOP3A mutations as an additional cause of prenatal-onset short stature with increased cytogenetic SCEs and implicate the decatenation activity of the BTRR complex in their pathogenesis.

Clinical validity of biochemical and molecular analysis in diagnosing Leigh syndrome: a study of 106 Japanese patients.

Leigh syndrome (LS) is a progressive neurodegenerative disorder of infancy and early childhood. It is clinically diagnosed by typical manifestations and characteristic computed tomography (CT) or magnetic resonance imaging (MRI) studies. Unravelling mitochondrial respiratory chain (MRC) dysfunction behind LS is essential for deeper understanding of the disease, which may lead to the development of new therapies and cure. The aim of this study was to evaluate the clinical validity of various diagnostic tools in confirming MRC disorder in LS and Leigh-like syndrome (LL). The results of enzyme assays, molecular analysis, and cellular oxygen consumption rate (OCR) measurements were examined. Of 106 patients, 41 were biochemically and genetically verified, and 34 had reduced MRC activity but no causative mutations. Seven patients with normal MRC complex activities had mutations in the MT-ATP6 gene. Five further patients with normal activity in MRC were identified with causative mutations. Conversely, 12 out of 60 enzyme assays performed for genetically verified patients returned normal results. No biochemical or genetic background was confirmed for 19 patients. OCR was reduced in ten out of 19 patients with negative enzyme assay results. Inconsistent enzyme assay results between fibroblast and skeletal muscle biopsy samples were observed in 33% of 37 simultaneously analyzed cases. These data suggest that highest diagnostic rate is reached using a combined enzymatic and genetic approach, analyzing more than one type of biological materials where suitable. Microscale oxygraphy detected MRC impairment in 50% cases with no defect in MRC complex activities.

Intra-mitochondrial Methylation Deficiency Due to Mutations in SLC25A26.

S-adenosylmethionine (SAM) is the predominant methyl group donor and has a large spectrum of target substrates. As such, it is essential for nearly all biological methylation reactions. SAM is synthesized by methionine adenosyltransferase from methionine and ATP in the cytoplasm and subsequently distributed throughout the different cellular compartments, including mitochondria, where methylation is mostly required for nucleic-acid modifications and respiratory-chain function. We report a syndrome in three families affected by reduced intra-mitochondrial methylation caused by recessive mutations in the gene encoding the only known mitochondrial SAM transporter, SLC25A26. Clinical findings ranged from neonatal mortality resulting from respiratory insufficiency and hydrops to childhood acute episodes of cardiopulmonary failure and slowly progressive muscle weakness. We show that SLC25A26 mutations cause various mitochondrial defects, including those affecting RNA stability, protein modification, mitochondrial translation, and the biosynthesis of CoQ10 and lipoic acid.