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Abstract Repeat expansions cause at least 50 hereditary disorders, including Friedreich ataxia and other diseases known to cause mitochondrial dysfunction. We identified a patient with NAXE-related mitochondrial encephalopathy and novel biallelic GGGCC repeat expansion as long as ~200 repeats in the NAXE promoter region using long-read sequencing. In addition to a marked reduction in the RNA and protein, we found a marked reduction in nascent RNA in the promoter using native elongating transcript-cap analysis of gene expression (NET-CAGE), suggesting transcriptional suppression. Accordingly, CpG hypermethylation was observed in the repeat region. Genetic analyses determined that homozygosity in the patient was due to maternal chromosome 1 uniparental disomy (UPD). We assessed short variants within NAXE including the repeat region in the undiagnosed mitochondrial encephalopathy cohort of 242 patients. This study identified the GGGCC repeat expansion causing a mitochondrial disease and suggests that UPD could significantly contribute to homozygosity for rare repeat-expanded alleles.

Abstract Originally, apomorphine was a broad-spectrum dopamine agonist with an affinity for all subtypes of the Dopamine D1 receptor to the D5 receptor. We previously identified apomorphine as a potential therapeutic agent for mitochondrial diseases by screening a chemical library of fibroblasts from patients with mitochondrial diseases. In this study, we showed that apomorphine prevented ferroptosis in fibroblasts from various types of mitochondrial diseases as well as in normal controls. Well-known biomarkers of ferroptosis include protein markers such as prostaglandin endoperoxide synthase 2 (PTGS2), a key gene for ferroptosis-related inflammation PTGS2, lipid peroxidation, and reactive oxygen species. Our findings that apomorphine induced significant downregulation of PTSG2 and suppressed lipid peroxide to the same extent as other inhibitors of ferroptosis also indicate that apomorphine suppresses ferroptosis. To our knowledge, this is the first study to report that the anti-ferroptosis effect of apomorphine is not related to dopamine receptor agonist action and that apomorphine is a potent inhibitor of ferroptotic cell death independent of dopaminergic receptors.

Abstract MRPS23 is a nuclear gene encoding a mitochondrial ribosomal protein. A patient with a mitochondrial disorder was found to carry a variant in MRPS23. More cases are necessary to establish MRPS23 as a mitochondrial disease gene. Of 5134 exomes performed in our center, we identified five independent patients who had similar clinical manifestations and were homozygous for the same germline variant c.119C>T; p.P40L in MRPS23. Detailed clinical findings, mitochondrial enzyme activity assays from cultured skin fibroblasts, PCR-Sanger-sequencing, and variant age estimation were performed. Their available family members were also studied. Eight members homozygous for the MRPS23 p.P40L were identified. All were from Hmong hilltribe. Seven presented with alteration of consciousness and recurrent vomiting, while the eighth who was a younger brother of a proband was found pre-symptomatically. Patients showed delayed growth and development, hearing impairment, hypoglycemia, lactic acidosis, and liver dysfunction. In vitro assays of cultured fibroblasts showed combined respiratory chain complex deficiency with low activities of complexes I and IV. PCR-Sanger-sequencing confirmed the variant, which was estimated to have occurred 1550 years ago. These results establish the MRPS23-associated mitochondrial disorder inherited in an autosomal recessive pattern and provide insight into its clinical and metabolic features.

Decidualization denotes the morphological and biological differentiating process of human endometrial stromal cells (HESCs). Fatty acid pathways are critical for endometrial decidualization. However, the participation of fatty acids as an energy source and their role in endometrial decidualization have received little attention. To identify fatty acids and clarify their role in decidualization, we comprehensively evaluated free fatty acid profiles using liquid chromatography/Fourier transform mass spectrometry (LC/FT-MS). LC/FT-MS analysis detected 26 kinds of fatty acids in the culture medium of decidualized or un-decidualized HESCs. Only the production of octanoic acid, which is an essential energy source for embryonic development, was increased upon decidualization. The expressions of genes related to octanoic acid metabolism including ACADL, ACADM, and ACADS; genes encoding proteins catalyzing the first step of mitochondrial fatty acid beta-oxidation; and ACSL5 and ACSM5; genes encoding fatty acid synthesis proteins were significantly altered upon decidualization. These results suggest that decidualization promotes lipid metabolism, implying that decidualized HESCs require energy metabolism of the mitochondria in embryo implantation.

Mucopolysaccharidosis type II (MPS II, OMIM 309900) is an X-linked disorder caused by a deficiency of lysosomal enzyme iduronate-2-sulfatase (IDS). The clinical manifestations of MPS II involve cognitive decline, bone deformity, and visceral disorders. These manifestations are closely associated with IDS enzyme activity, which catalyzes the stepwise degradation of heparan sulfate and dermatan sulfate. In this study, we established a novel Ids-deficient mice and further assessed the enzyme's physiological role. Using DNA sequencing, we found a genomic modification of the Ids genome, which involved the deletion of a 138-bp fragment spanning from intron 2 to exon 3, along with the insertion of an adenine at the 5′ end of exon 3 in the mutated allele. Consistent with previous data, our Ids-deficient mice showed an attenuated enzyme activity and an enhanced accumulation of glycosaminoglycans. Interestingly, we noticed a distinct enlargement of the calvarial bone in both neonatal and young adult mice. Our examination revealed that Ids deficiency led to an enhanced osteoblastogenesis in the parietal bone, a posterior part of the calvarial bone originating from the paraxial mesoderm and associated with an enhanced expression of osteoblastic makers, such as Col1a and Runx2. In sharp contrast, cell proliferation of the parietal bone in these mice appeared similar to that of wild-type controls. These results suggest that the deficiency of Ids could be involved in an augmented differentiation of calvarial bone, which is often noticed as an enlarged head circumference in MPS II-affected individuals.

The identification of the m.12207G > A variant in MT-TS2, (NC_012920.1:m.12207G > A) was first reported in 2006. The affected individual presented with developmental delay, feeding difficulty, proximal muscle weakness, and lesions within her basal ganglia, with heteroplasmy levels of 92% in muscle and no evidence of maternal inheritance. Herein, we report a case involving a 16-year-old boy with the same pathogenic variation and different phenotype, including sensorineural deafness, epilepsy, and intellectual disability, without diabetes mellitus (DM). His mother and maternal grandmother had similar but milder symptoms with DM. Heteroplasmy levels of the proband in blood, saliva, and urinary sediments were 31.3%, 52.6%, and 73.9%, respectively, while those of his mother were 13.8%, 22.1%, and 29.4%, respectively. The differences in the symptoms might be explained by the different levels of heteroplasmy. To our knowledge, this is the first familial report of the m.12207G > A variant in MT-TS2 that causes DM. The present case showed milder neurological symptoms than did the former report, and suggests the presence of a good phenotype–genotype correlation within this family.

Coenzyme Q10 (CoQ10) is involved in ATP production through electron transfer in the mitochondrial respiratory chain complex. CoQ10 receives electrons from respiratory chain complex I and II to become the reduced form, and then transfers electrons at complex III to become the oxidized form. The redox state of CoQ10 has been reported to be a marker of the mitochondrial metabolic state, but to our knowledge, no reports have focused on the individual quantification of reduced and oxidized CoQ10 or the ratio of reduced to total CoQ10 (reduced/total CoQ10) in patients with mitochondrial diseases.We measured reduced and oxidized CoQ10 in skin fibroblasts from 24 mitochondrial disease patients, including 5 primary CoQ10 deficiency patients and 10 respiratory chain complex deficiency patients, and determined the reduced/total CoQ10 ratio.In primary CoQ10 deficiency patients, total CoQ10 levels were significantly decreased, however, the reduced/total CoQ10 ratio was not changed. On the other hand, in mitochondrial disease patients other than primary CoQ10 deficiency patients, total CoQ10 levels did not decrease. However, the reduced/total CoQ10 ratio in patients with respiratory chain complex IV and V deficiency was higher in comparison to those with respiratory chain complex I deficiency.Measurement of CoQ10 in fibroblasts proved useful for the diagnosis of primary CoQ10 deficiency. In addition, the reduced/total CoQ10 ratio may reflect the metabolic status of mitochondrial disease.

Abstract Background Lack of functional evidence hampers variant interpretation, leaving a large proportion of individuals with a suspected Mendelian disorder without genetic diagnosis after whole genome or whole exome sequencing (WES). Research studies advocate to further sequence transcriptomes to directly and systematically probe gene expression defects. However, collection of additional biopsies and establishment of lab workflows, analytical pipelines, and defined concepts in clinical interpretation of aberrant gene expression are still needed for adopting RNA sequencing (RNA-seq) in routine diagnostics. Methods We implemented an automated RNA-seq protocol and a computational workflow with which we analyzed skin fibroblasts of 303 individuals with a suspected mitochondrial disease that previously underwent WES. We also assessed through simulations how aberrant expression and mono-allelic expression tests depend on RNA-seq coverage. Results We detected on average 12,500 genes per sample including around 60% of all disease genes—a coverage substantially higher than with whole blood, supporting the use of skin biopsies. We prioritized genes demonstrating aberrant expression, aberrant splicing, or mono-allelic expression. The pipeline required less than 1 week from sample preparation to result reporting and provided a median of eight disease-associated genes per patient for inspection. A genetic diagnosis was established for 16% of the 205 WES-inconclusive cases. Detection of aberrant expression was a major contributor to diagnosis including instances of 50% reduction, which, together with mono-allelic expression, allowed for the diagnosis of dominant disorders caused by haploinsufficiency. Moreover, calling aberrant splicing and variants from RNA-seq data enabled detecting and validating splice-disrupting variants, of which the majority fell outside WES-covered regions. Conclusion Together, these results show that streamlined experimental and computational processes can accelerate the implementation of RNA-seq in routine diagnostics.

Biallelic deletions extending into the ATPase family AAA-domain containing protein 3A (ATAD3A) gene lead to infantile lethality with severe pontocerebellar hypoplasia (PCH). However, only 12 such cases have been reported worldwide to date, and the genotype–phenotype correlations are not well understood. We describe cases associated with the same novel biallelic deletions of the ATAD3A and ATAD3B/3A regions in Japanese siblings with severe spinal cord hypoplasia and multiple malformations, including PCH, leading to neonatal death. The ATAD3A protein is essential for normal interaction between mitochondria and endoplasmic reticulum and is important for mitochondrial biosynthesis. The cases were evaluated using whole-genome sequencing for genetic diagnosis of mitochondrial disease. Spinal cord lesions associated with biallelic compound heterozygous deletion extending into the ATAD3A gene have not been reported. In addition, the ATAD3A deletion was 19 base pairs long, which is short compared with those reported previously. This deletion introduced a frameshift, resulting in a premature termination codon, and was expected to be a null allele. The pathological findings of the atrophic spinal cord showed gliosis and tissue destruction of the gray and white matter. We describe spinal cord lesions as a new central nervous system phenotype associated with a biallelic compound heterozygous deletion extending into the ATAD3A gene. Biallelic ATAD3A deletions should be considered in cases of mitochondrial disease with spinal cord hypoplasia and PCH.

This article presents a case study of a 37-year-old woman who was diagnosed with osteogenesis imperfecta (OI) after experiencing subchondral insufficiency fractures (SIF) in both femoral heads. The patient had a history of multiple fractures, blue sclera, and mild bilateral sensorineural hearing loss, which led to the diagnosis of OI. Genetic testing revealed a mutation in the COL1A1 gene. After conservative therapy, the patient's symptoms improved, and after four years, both hips were pain-free with no evidence of osteoarthritis progression. The article highlights the association between OI and SIF and emphasizes that bilateral SIF is rare in cases of OI. [Extracted from the article]

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.

Biallelic 4-hydroxyphenylpyruvate dioxygenase-like protein (HPDL) variants were recently reported as a cause of progressive and incurable neurodegenerative diseases ranging from neonatal-onset leukoencephalopathy with severe neurodevelopmental delay to spastic paraplegia. Although the physiological function of HPDL remains unknown, its subcellular localization in the mitochondria has been reported. Here, we report a case of HPDL-related neurological disease that was clinically and neuroimaging compatible with Leigh syndrome, previously unreported, and was treated with a ketogenic diet.

Background Enoyl-CoA hydratase short-chain 1 (ECHS1) is an enzyme involved in the metabolism of branched chain amino acids and fatty acids. Mutations in the ECHS1 gene lead to mitochondrial short-chain enoyl-CoA hydratase 1 deficiency, resulting in the accumulation of intermediates of valine. This is one of the most common causative genes in mitochondrial diseases. While genetic analysis studies have diagnosed numerous cases with ECHS1 variants, the increasing number of variants of uncertain significance (VUS) in genetic diagnosis is a major problem. Methods Here, we constructed an assay system to verify VUS function for ECHS1 gene. A high-throughput assay using ECHS1 knockout cells was performed to index these phenotypes by expressing cDNAs containing VUS. In parallel with the VUS validation system, a genetic analysis of samples from patients with mitochondrial disease was performed. The effect on gene expression in cases was verified by RNA-seq and proteome analysis. Results The functional validation of VUS identified novel variants causing loss of ECHS1 function. The VUS validation system also revealed the effect of the VUS in the compound heterozygous state and provided a new methodology for variant interpretation. Moreover, we performed multiomics analysis and identified a synonymous substitution p.P163= that results in splicing abnormality. The multiomics analysis complemented the diagnosis of some cases that could not be diagnosed by the VUS validation system. Conclusions In summary, this study uncovered new ECHS1 cases based on VUS validation and omics analysis; these analyses are applicable to the functional evaluation of other genes associated with mitochondrial disease. [ABSTRACT FROM AUTHOR]

Objective: Neonatal-onset mitochondrial disease has not been fully characterised owing to its heterogeneity. We analysed neonatal-onset mitochondrial disease in Japan to clarify its clinical features, molecular diagnosis and prognosis.Design: Retrospective observational study from January 2004 to March 2020.Setting: Population based.Patients: Patients (281) with neonatal-onset mitochondrial disease diagnosed by biochemical and genetic approaches.Interventions: None.Main Outcome Measures: Disease types, initial symptoms, biochemical findings, molecular diagnosis and prognosis.Results: Of the 281 patients, multisystem mitochondrial disease was found in 194, Leigh syndrome in 26, cardiomyopathy in 38 and hepatopathy in 23 patients. Of the 321 initial symptoms, 236 occurred within 2 days of birth. Using biochemical approaches, 182 patients were diagnosed by mitochondrial respiratory chain enzyme activity rate and 89 by oxygen consumption rate. The remaining 10 patients were diagnosed using a genetic approach. Genetic analysis revealed 69 patients had nuclear DNA variants in 36 genes, 11 of 15 patients had mitochondrial DNA variants in five genes and four patients had single large deletion. The Cox proportional hazards regression analysis showed the effects of Leigh syndrome (HR=0.15, 95% CI 0.04 to 0.63, p=0.010) and molecular diagnosis (HR=1.87, 95% CI 1.18 to 2.96, p=0.008) on survival.Conclusions: Neonatal-onset mitochondrial disease has a heterogenous aetiology. The number of diagnoses can be increased, and clarity regarding prognosis can be achieved by comprehensive biochemical and molecular analyses using appropriate tissue samples. [ABSTRACT FROM AUTHOR]