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Pathogenic mitochondrial DNA heteroplasmy has mainly been assessed with bulk sequencing in individuals with mitochondrial disease. However, the distribution of heteroplasmy at the single-cell level in skin fibroblasts obtained from individuals, together with detailed clinical and biochemical information, remains to be investigated. We used the mitochondrial DNA single-cell assay for the transposase-accessible chromatin sequencing method. Skin fibroblasts were obtained from six individuals with mitochondrial disease and pathogenic m.3243AG variants of differing severity. Different distributions of heteroplasmy at the single-cell level were identified in skin fibroblasts from all six individuals. Four individuals with different outcomes showed similar averaged heteroplasmy rates with normal mitochondrial respiratory chain enzyme activity, while the distribution of single-cell heteroplasmy patterns differed among the individuals. This study showed different heteroplasmy distribution patterns at the single-cell level in individuals with the m.3243AG variant, who had a similar averaged heteroplasmy rates with normal mitochondrial respiratory chain enzyme activity. Whether such different heteroplasmy distribution patterns explain the different clinical outcomes should be assessed further in future studies. Measuring heteroplasmy of pathogenic mitochondrial DNA variants at the single-cell level could be important in individuals with mitochondrial disease.

Mitochondrial complex I deficiency is caused by pathogenic variants in mitochondrial and nuclear genes associated with complex I structure and assembly. We report the case of a patient with NDUFA8-related mitochondrial disease. The patient presented with developmental delay, microcephaly, and epilepsy. His fibroblasts showed apparent biochemical defects in mitochondrial complex I. Whole-exome sequencing revealed that the patient carried a homozygous variant in NDUFA8. His fibroblasts showed a reduction in the protein expression level of not only NDUFA8, but also the other complex I subunits, consistent with assembly defects. The enzyme activity of complex I and oxygen consumption rate were restored by reintroducing wild-typeNDUFA8 cDNA into patient fibroblasts. The functional properties of the variant in NDUFA8 were also investigated using NDUFA8 knockout cells expressing wild-type or mutated NDUFA8 cDNA. These experiments further supported the pathogenicity of the variant in complex I assembly. This is the first report describing that the loss of NDUFA8, which has not previously been associated with mitochondrial disease, causes severe defect in the assembly of mitochondrial complex I, leading to progressive neurological and developmental abnormalities.

Tetratricopeptide repeat protein 37 (TTC37) is a causative gene of trichohepatoenteric syndrome (THES). However, little is known about the pathogenesis of this disease. Here, we characterize the phenotype of a Drosophila model in which ski3, a homolog of TTC37, is disrupted. The mutant flies are pupal lethal, and the pupal lethality is partially rescued by transgenic expression of wild-type ski3 or human TTC37. The mutant larvae show growth retardation, heart arrhythmia, triacylglycerol accumulation, and aberrant metabolism of glycolysis and the TCA cycle. Moreover, mitochondrial membrane potential and respiratory chain complex activities are significantly reduced in the mutants. Our results demonstrate that ski3 deficiency causes mitochondrial dysfunction, which may underlie the pathogenesis of THES.

Leigh syndrome is a major phenotype of mitochondrial diseases in children. With new therapeutic options being proposed, assessing the mortality and clinical condition of Leigh syndrome patients is crucial for evaluating therapeutics. As data are scarce in Japan, we analysed the mortality rate and clinical condition of Japanese Leigh syndrome patients that we diagnosed since 2007. Data from 166 Japanese patients diagnosed with Leigh syndrome from 2007 to 2017 were reviewed. Patients' present status, method of ventilation and feeding, and degree of disability as of April 2018 was analysed. Overall, 124 (74.7%) were living, 40 (24.1%) were deceased, and 2 (1.2%) were lost to follow‐up. Median age of living patients was 8 years (1‐39 years). Median length of disease course was 91 months for living patients and 23.5 months for deceased patients. Nearly 90% of deaths occurred by age 6. Mortality rate of patients with onset before 6 months of age was significantly higher than that of onset after 6 months. All patients with neonatal onset were either deceased or bedridden. MT‐ATP6 deficiency caused by m.8993T>G mutation and MT‐ND5 deficiency induced a severe form of Leigh syndrome. Patients with NDUFAF6, ECHS1, and SURF1 deficiency had relatively mild symptoms and better survival. The impact of onset age on prognosis varied across the genetic diagnoses. The clinical condition of many patients was poor; however, few did not require mechanical ventilation or tube‐feeding and were not physically dependent. Early disease onset and genetic diagnosis may have prognostic value.

Background Deafness, dystonia, and cerebral hypomyelination (DDCH) is an X‐linked disorder due to hemizygous mutations of BCAP31. Methods We report an 8‐year‐old boy with DDCH who possibly accompanied mitochondrial dysfunction. Clinical evaluation, respiratory chain enzyme assay, and whole exome sequencing analysis were performed. Results Mitochondrial dysfunction was suspected by respiratory chain enzyme assay on his cultured skin fibroblasts which showed significantly decreased complex I enzyme activity. Whole exome sequencing analysis revealed a recurrent BCAP31 mutation (c.97C>T:p.Gln33*) which confirmed the diagnosis of DDCH for the patient. Conclusion We speculate that mitochondrial dysfunction may be a feature in patients with DDCH., Deafness, dystonia, and cerebral hypomyelination (DDCH) is an X‐linked disorder due to hemizygous mutations of BCAP31. Mitochondrial dysfunction may be associated with DDCH.

Aims To evaluate comprehensively the use of the glycated albumin to HbA1c ratio for estimation of glycaemic control in the previous month. Methods A total of 306 children with Type 1 diabetes mellitus underwent ≥10 simultaneous measurements of glycated albumin and HbA1c . Correlation and concordance rates were examined between HbA1c measurements taken 1 month apart (ΔHbA1c ) and glycated albumin/HbA1c ratio fluctuations were calculated as Z-scores from the cohort value at enrolment of this study cohort (method A) or the percent difference from the individual mean over time (method B). Results Fluctuations in glycated albumin/HbA1c ratio (using both methods) were weakly but significantly correlated with ΔHbA1c , whereas concordance rates were significant for glycaemic deterioration but not for glycaemic improvement. Concordance rates were higher using method B than method A. Conclusions The glycated albumin/HbA1c ratio was able to estimate glycaemic deterioration in the previous month, while estimation of glycaemic improvement in the preceding month was limited. Because method B provided a better estimate of recent glycaemic control than method A, the individual mean of several measurements of the glycated albumin/HbA1c ratio over time may also identify individuals with high or low haemoglobin glycation phenotypes in a given population, such as Japanese children with Type 1 diabetes, thereby allowing more effective diabetes management.

Recent studies suggest that impaired transcription or mitochondrial translation of small RNAs can cause abnormal myelination. A polynucleotide phosphorylase (PNPase) encoded by PNPT1 facilitates the import of small RNAs into mitochondria. PNPT1 mutations have been reported in patients with neurodevelopmental diseases with mitochondrial dysfunction. We report here 2 siblings with PNPT1 mutations who presented delayed myelination as well as mitochondrial dysfunction. We identified compound heterozygous mutations (c.227G>A; p.Gly76Asp and c.574C>T; p.Arg192*) in PNPT1 by quartet whole-exome sequencing. Analyses of skin fibroblasts from the patient showed that PNPase expression was markedly decreased and that import of the small RNA RNaseP into mitochondria was impaired. Exogenous expression of wild-type PNPT1, but not mutants, rescued ATP production in patient skin fibroblasts, suggesting the pathogenicity of the identified mutations. Our cases expand the phenotypic spectrum of PNPT1 mutations that can cause delayed myelination.

A female infant born at 36 weeks gestational age with birthweight 2135 g, and who developed respiratory disorder, hyperlactacidemia and hypertrophic cardiomyopathy after birth, was admitted to hospital at 3 days of age. After admission, bilious emesis, abdominal distention, and passage disorder of the gastrointestinal tract were resistant to various drugs. Exploratory laparotomy was performed at 93 days of age, but no organic lesions were identified and normal Meissner/Auerbach nerve plexus was confirmed, which led to a clinical diagnosis of chronic intestinal pseudo-obstruction (CIPO). She was diagnosed with mitochondrial respiratory chain complex IV deficiency on histopathology of the abdominal rectus muscle and enzyme activity measurement. This is the first report of a neonate with mitochondrial respiratory chain complex deficiency with intractable CIPO. CIPO can occur in neonates with mitochondrial respiratory chain disorder, necessitating differential diagnosis from Hirschsprung disease.

Objective Mitochondrial respiratory chain disorder (MRCD) is an intractable disease of infants with variable clinical symptoms. Our goal was to identify the causative mutations in MRCD patients. Methods The subjects were 90 children diagnosed with MRCD by enzyme assay. We analyzed whole mitochondrial DNA (mtDNA) sequences. A cybrid study was performed in two patients. Whole exome sequencing was performed for one of these two patients whose mtDNA variant was confirmed as non-pathogenic. Results Whole mtDNA sequences identified 29 mtDNA variants in 29 patients (13 were previously reported, the other 13 variants and three deletions were novel). The remaining 61 patients had no pathogenic mutations in their mtDNA. Of the 13 patients harboring unreported mtDNA variants, we excluded seven variants by manual curation. Of the remaining six variants, we selected two Leigh syndrome patients whose mitochondrial enzyme activity was decreased in their fibroblasts and performed a cybrid study. We confirmed that m.14439G>A (MT-ND6) was pathogenic, while m.1356A>G (mitochondrial 12S rRNA) was shown to be a non-pathogenic polymorphism. Exome sequencing and a complementation study of the latter patient identified a novel c.55C>T hemizygous missense mutation in the nuclear-encoded gene NDUFA1. Interpretation Our results demonstrate that it is important to perform whole mtDNA sequencing rather than only typing reported mutations. Cybrid assays are also useful to diagnose the pathogenicity of mtDNA variants, and whole exome sequencing is a powerful tool to diagnose nuclear gene mutations as molecular diagnosis can provide a lead to appropriate genetic counseling.

The patient had hepatomegaly with liver dysfunction at the age of 1 month. Magnetic resonance imaging performed at the age of 1 year showed multiple nodules of varying size in his liver. We were able to examine the mitochondrial respiratory chain function in the liver biopsy samples because all other differential diagnoses for hepatic cirrhosis had been ruled out. Complex I and IV activities were below the normal level (

In two Japanese infants with neonatal cholestasis, 3-oxo-Δ(4)-steroid 5β-reductase deficiency was diagnosed based on mutations of the SRD5B1 gene. Unusual bile acids such as elevated 3-oxo-Δ(4) bile acids were detected in their serum and urine by gas chromatography-mass spectrometry. We studied effects of oral chenodeoxycholic acid treatment.SRD5B1 gene analysis used peripheral lymphocyte genomic DNA. Diagnosis and treatment of these two patients were investigated retrospectively and prospectively investigated.With respect to SRD5B1, one patient was heterozygous (R266Q, a novel mutation) while the other was a compound heterozygote (G223E/R261C). Chenodeoxycholic acid treatment was effective in improving liver function and decreasing unusual bile acids such as 7α-hydroxy- and 7α,12α-dihydroxy-3-oxo-4-cholen-24-oic acids in serum and urine.Primary bile acid treatment using chenodeoxycholic acid was effective for these patients treated in early infancy before the late stage of chronic cholestatic liver dysfunction.

Background: Succinyl-coenzyme A ligase (SUCL) is a mitochondrial enzyme that catalyses the reversible conversion of succinyl-coenzyme A to succinate. SUCL consists of an α subunit, encoded by SUCLG1, and a β subunit, encoded by either SUCLA2 or SUCLG2. Recently, mutations in SUCLG1 or SUCLA2 have been identified in patients with infantile lactic acidosis showing elevated urinary excretion of methylmalonate, mitochondrial respiratory chain (MRC) deficiency, and mitochondrial DNA depletion. Methods: Case description of a Japanese female patient who manifested a neonatal-onset lactic acidosis with urinary excretion of methylmalonic acid. Enzymatic analyses (MRC enzyme assay and Western blotting) and direct sequencing analysis of SUCLA2 and SUCLG1 were performed. Results: MRC enzyme assay and Western blotting showed that MRC complex I was deficient. SUCLG1 mutation analysis showed that the patient was a compound heterozygote for disease-causing mutations (p.M14T and p.S200F). Conclusion: For patients showing neonatal lactic acidosis and prolonged mild methylmalonic aciduria, MRC activities and mutations of SUCLG1 or SUCLA2 should be screened for.

Carnitine palmitoyltransferase II (CPT II) deficiency is an inherited disorder involving beta-oxidation of long-chain fatty acids. CPT II deficiency is a wide-spectrum disorder that includes a lethal neonatal form, an infantile form, and an adult-onset form. However, the ethnic characteristics and the relationship between genotype and clinical manifestation are not well understood. We investigated three non-consanguineous Japanese patients with CPT II deficiency and examined cell lines from 4 unrelated patients and 50 healthy donors. The CPT 2 gene was typed by direct DNA sequencing of polymerase chain reaction-amplified gene products. Case 1 (infantile form) was heterozygous for a phenylalanine to tyrosine substitution at position 383 (p.F383Y) and a novel valine to leucine substitution at 605 (p.V605L). Cases 2, 4, and 5 (infantile form) and case 3 (adult-onset form) were heterozygous for a single mutation at F383Y. Case 6 (adult-onset form) was compound heterozygous at the CPT 2 locus, with deletion of cytosine and thymine at residue 408, resulting in a stop signal at 420 (p.Y408fsX420), and an arginine to cysteine substitution at position 631 (p.R631C). Case 7 (adult-onset form) was homozygous for the p.F383Y mutation. In conclusion, we identified p.F383Y mutations in six of seven patients with CPT II deficiency and two novel variants of the coding gene: p.Y408fsX420 and p.V605L. These mutations differ from those in Caucasian patients, who commonly harbor p.S113L, p.P50H, and p.Q413fsX449 mutations; therefore, our data and those of other Japanese groups suggest that the p.F383Y mutation is significant in Japanese patients with CPT II deficiency.

Respiratory chain complex I deficiency is a common cause of Leigh's disease (LD) and can be caused by mutations in genes encoded by either nuclear or mitochondrial DNA (mtDNA). Most pathogenic mtDNA mutations act recessively and only cause disease when present at high mutant loads (typically >90%) in tissues such as muscle and brain. Two mitochondrial DNA mutations in complex I subunit genes, G14459A in ND6, and T12706C in ND5, have been associated with complex I deficiency and LD. We report another ND5 mutation, G13513A, in three unrelated patients with complex I deficiency and LD. The G13513A mutation was present at mutant loads of approximately 50% or less in all tissues tested, including multiple brain regions. The threshold mutant load for causing a complex I defect in cultured cells was approximately 30%. Blue Native polyacrylamide gel electrophoresis showed that fibroblasts with 45% G13513A mutant load had approximately 50% of the normal amount of fully assembled complex I. Fibroblasts with greater than 97% of the ND6 G14459A mutation had only 20% fully assembled complex I, suggesting that both mutations disrupt complex I assembly or turnover. We conclude that the G13513A mutation causes a complex I defect when present at unusually low mutant load and may act dominantly.

We report an ornithine transcarbamylase(OTC)-deficient male patient who had no detectable immunoreactive materials but did have active mRNA for OTC-related protein. The total absence of OTC activity in the liver of the patient was caused by a complete lack of immunoreactive material, as determined by Ouchterlony double immunodiffusion, single radial immunodiffusion, and sodium dodecylsulphate-polyacrylamide gel electrophoresis of immunoprecipitate and of liver homogenate. However, mRNA coding for the precursor of OTC was clearly detected in autopsy specimens of the patient's liver as well as of controls in a cell-free translation system consisting of rabbit reticulocyte lysates and [35S]methionine. The labelled precursor of OTC synthesized in vitro with mRNA from the patient could be transported into rat liver and kidney mitochondria and processed to form a protein with a molecular weight indistinguishable from mature OTC, suggesting that there was no defect in the protein structure necessary for its transport into mitochondria. These results suggest that the primary defect of the OTC deficiency was located in the structural gene and that the labile OTC-related protein, after being synthesized with its mRNA, was degraded too rapidly to be detected by the method used.

We report a case of Niemann-Pick disease (NPD) with accumulation of sphingomyelin in reticuloendothelial system (RES), hepatocellular giant cell transformation (GCT), cirrhosis, and multiple hepatocellular adenomata in a 19-month-old girl. GCT, but no NP-cells, were seen at age 3 months by biopsy. Cirrhosis and hepatocellular adenomata were demonstrated in the liver at 19 months of age. Cytoplasmic, probably locally synthesized, globules of alpha-1-antitrypsin (A-1-AT) were accumulated in the hepatocellular adenomata. A-1-AT and alpha-fetoprotein (AFP) were present in the serum.

A female infant, who showed hyperammonemia in the neonatal period, died on 43rd postnatal day. Her female sibling also died on 42nd day after birth with an identical clinical picture and hyperammonemia. Urinary organic acids were negative in both cases. Their blood amino acids showed no specific pattern, and urinary orotic acid excretion was normal. The first two urea cycle enzymes and N-acetyl L-glutamate synthetase of the liver tissues of these two infants obtained at autopsy were assayed. They revealed a selective deficiency of carbamyl phosphate synthetase I.

Ornithine transcarbamylase (OTC, EC 2.1.3.3), located in the mitochondriat matrix, is synthesized on membrane-free polysomes in the form of a larger precursor and is then proteolytically processed to the mature form. OTC deficiency, which is a disease with X-linked dominant inheritance, is caused by various kinds of mutation (Briand et al., 1982). We report a heterozygous patient with OTC deficiency showing a decrease of OTC protein and a truncated enzyme precursor.