Across a variety of Mendelian disorders, ∼50–75% of patients do not receive a genetic diagnosis by exome sequencing indicating disease-causing variants in non-coding regions. Although genome sequencing in principle reveals all genetic variants, their sizeable number and poorer annotation make prioritization challenging. Here, we demonstrate the power of transcriptome sequencing to molecularly diagnose 10% (5 of 48) of mitochondriopathy patients and identify candidate genes for the remainder. We find a median of one aberrantly expressed gene, five aberrant splicing events and six mono-allelically expressed rare variants in patient-derived fibroblasts and establish disease-causing roles for each kind. Private exons often arise from cryptic splice sites providing an important clue for variant prioritization. One such event is found in the complex I assembly factor TIMMDC1 establishing a novel disease-associated gene. In conclusion, our study expands the diagnostic tools for detecting non-exonic variants and provides examples of intronic loss-of-function variants with pathological relevance.

Addition of hydrogen peroxide (H₂O₂) is a method commonly used to trigger cellular oxidative stress. However, the doses used (often hundreds of micromolar) are disproportionally high with regard to physiological oxygen concentration (low micromolar). In this study using polarographic measurement of oxygen concentration in cellular suspensions we show that H₂O₂ addition results in O₂ release as expected from catalase reaction. This reaction is fast enough to, within seconds, decrease drastically H₂O₂ concentration and to annihilate it within a few minutes. Firstly, this is likely to explain why recording of oxidative damage requires the high concentrations found in the literature. Secondly, it illustrates the potency of intracellular antioxidant (H₂O₂) defense. Thirdly, it complicates the interpretation of experiments as subsequent observations might result from high/transient H₂O₂ exposure and/or from the diverse possible consequences of the O₂ release.

Mitochondrial acyl-CoA dehydrogenase family member 9 (ACAD9) is essential for the assembly of mitochondrial respiratory chain complex I. Disease causing biallelic variants in ACAD9 have been reported in individuals presenting with lactic acidosis and cardiomyopathy. We describe the genetic, clinical and biochemical findings in a cohort of 70 patients, of whom 29 previously unpublished. We found 34 known and 18 previously unreported variants in ACAD9. No patients harbored biallelic loss of function mutations, indicating that this combination is unlikely to be compatible with life. Causal pathogenic variants were distributed throughout the entire gene, and there was no obvious genotype-phenotype correlation. Most of the patients presented in the first year of life. For this subgroup the survival was poor (50% not surviving the first 2 years) comparing to patients with a later presentation (more than 90% surviving 10 years). The most common clinical findings were cardiomyopathy (85%), muscular weakness (75%) and exercise intolerance (72%). Interestingly, severe intellectual deficits were only reported in one patient and severe developmental delays in four patients. More than 70% of the patients were able to perform the same activities of daily living when compared to peers. Our data show that riboflavin treatment improves complex I activity in the majority of patient-derived fibroblasts tested. This effect was also reported for most of the treated patients and is mirrored in the survival data. In the patient group with disease-onset below 1 year of age, we observed a statistically-significant better survival for patients treated with riboflavin.

Many different pathogenic mutations in the mitochondrial (mt) transfer RNA (tRNA) genes have been reported for patients with mitochondrial encephalomyopathy. Although some of them are recurrent, most have only been described once and appear to be restricted to one patient or to one family. The incidence of mt tRNA gene alterations is not known, even though the frequency of some recurrent mutations has been analysed both in patients and in the general population. In this study, we describe the results of stepwise screening for sequence variations in the mt tRNA genes of 166 patients selected according to several criteria. Extensive sequence analysis of the tRNA genes was performed using denaturing gradient gel electrophoresis. A total of 31 patients (19%) were found to harbour significant levels of a pathogenic mutation, thus confirming the importance of mt tRNA mutations in human pathology. Forty-three different sequence variations were found, illustrating the great diversity of the mtDNA sequence in humans. The functional assessment of all these sequence variations represented a difficult task; it was mostly based on indirect data, such as the phylogenetic conservation of modified nucleotides and the proportions of variant species in different tissues of the index case or in blood of maternal relatives. Direct demonstration of a correlation between the proportion of heteroplasmic sequence variations and the cytochrome c oxidase defect was performed at the single muscle-fibre level. Eleven heteroplasmic sequence variations were found, six of which are new mutations. One is a known Caucasian polymorphism but the other 10 are pathogenic. Two of them are the well-known pathogenic MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) (A3243G) and MERRF (myoclonic epilepsy with ragged-red fibres) (A8344G) point mutations. They were found in 23 patients. The eight other mutations were restricted to one patient. The pathogenic nature of these mutations was demonstrated directly for five of them and hypothesized from indirect arguments for the other three. Thirty-two homoplasmic sequence variations were found. Twenty-nine were considered to be polymorphisms, even though 15 of these were found for the first time in our patients and two others had been reported previously as pathogenic. The pathogenic nature of three homoplasmic variants remains questionable.

Increased susceptibility to apoptosis has been shown in many models of mitochondrial defects but its relevance to human diseases is still discussed. We addressed the presence of apoptosis in muscle from patients with mitochondrial DNA (mtDNA) disorders. Taking advantage of the mosaic pattern of muscle morphological anomalies associated with heteroplasmic mtDNA alterations, we have used an in situ approach to address the relationship between apoptosis and respiratory defect, mitochondrial proliferation and mutation load. Different patterns of mitochondrial morphological alterations were provided by the analysis of muscles with large mtDNA deletion (16 cases) or with the MELAS mutation (4 cases). The patient's age at biopsy ranged from 0.4 to 66 years and the muscle mutant mtDNA proportion from 32 to 82%. Apoptotic muscle fibres were observed in a small proportion of muscle fibres of 16 out of the 20 biopsies by three different detection methods for different steps of apoptosis: caspase 3 activation, fragmentation of nuclear DNA [terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay] or overexpression of the pro-apoptotic factor Bax. Analysis of apoptotic features in parallel to cytochrome c oxidase (COX) and succinate dehydrogenase activity of more than 34 000 individual muscle fibres showed that apoptosis occurred only in muscle fibres with mitochondrial proliferation (ragged red fibres, RRF) irrespective of their COX activity. Molecular analyses of single muscle fibres evidenced that, as expected, the presence of COX defect was associated with higher proportion of mutant mtDNA and lower amount of normal mtDNA. Within COX-defective fibres, the presence of mitochondrial proliferation was associated with increase of the mtDNA content but without change in the ratio between normal and mutant mtDNA molecules, thus showing that mitochondrial proliferation was accompanied by similar amplification of normal and mutant mtDNA molecules. Within RRF, apoptosis was associated with higher mutation proportion, suggesting that it was provoked by severe respiratory defect in the same time as increased mitochondrial mass. In conclusion, apoptosis most probably contributes to mitochondrial pathology. It is tightly linked to mitochondrial proliferation and high mutation load. When considering training therapeutics, one will have to take into account the possibility to induce apoptosis in parallel to mitochondrial proliferation.