Mitochondrial disorders are clinically and genetically heterogeneous, with variants in mitochondrial or nuclear genes leading to varied clinical phenotypes. TAMM41 encodes a mitochondrial protein with cytidine diphosphate-diacylglycerol synthase activity: an essential early step in the biosynthesis of phosphatidylglycerol and cardiolipin. Cardiolipin is a mitochondria-specific phospholipid that is important for many mitochondrial processes. We report three unrelated individuals with mitochondrial disease that share clinical features, including lethargy at birth, hypotonia, developmental delay, myopathy, and ptosis. Whole exome and genome sequencing identified compound heterozygous variants in TAMM41 in each proband. Western blot analysis in fibroblasts showed a mild oxidative phosphorylation (OXPHOS) defect in only one of the three affected individuals. In skeletal muscle samples, however, there was severe loss of subunits of complexes I-IV and a decrease in fully assembled OXPHOS complexes I-V in two subjects as well as decreased TAMM41 protein levels. Similar to the tissue-specific observations on OXPHOS, cardiolipin levels were unchanged in subject fibroblasts but significantly decreased in the skeletal muscle of affected individuals. To assess the functional impact of the TAMM41 missense variants, the equivalent mutations were modeled in yeast. All three mutants failed to rescue the growth defect of the Δtam41 strains on non-fermentable (respiratory) medium compared with wild-type TAM41, confirming the pathogenicity of the variants. We establish that TAMM41 is an additional gene involved in mitochondrial phospholipid biosynthesis and modification and that its deficiency results in a mitochondrial disorder, though unlike families with pathogenic AGK (Sengers syndrome) and TAFAZZIN (Barth syndrome) variants, there was no evidence of cardiomyopathy.

We describe the presentation and follow-up of a three-year-old girl with nemaline myopathy due to a de-novo variant in ACTA1 (encoding skeletal alpha actin) and moderately low enzyme level of Complex I of the mitochondrial respiratory chain. She presented in the neonatal period with hypotonia, followed by weakness in the facial, bulbar, respiratory and neck flexors muscles. A biopsy of her quadriceps muscle at the age of one year showed nemaline rods. Based on her clinical presentation of a congenital myopathy and histopathological features on a muscle biopsy, ACTA1 was sequenced, and this revealed a novel sequence variant, c.760 A>C p. (Asn254His). In addition, mitochondrial respiratory chain enzymatic activity of skeletal muscle biopsy showed a moderately low activity of complex I (nicotinamide adenine dinucleotide (NADH): ubiquinone oxidoreductase). Disturbances of Complex I of the respiratory chain have been reported in patients with nemaline myopathy, although the mechanism remains unclear.

Recent studies have identified both recessive and dominant forms of mitochondrial disease that result from ATAD3A variants. The recessive form includes subjects with biallelic deletions mediated by non-allelic homologous recombination. We report five unrelated neonates with a lethal metabolic disorder characterized by cardiomyopathy, corneal opacities, encephalopathy, hypotonia, and seizures in whom a monoallelic reciprocal duplication at the ATAD3 locus was identified. Analysis of the breakpoint junction fragment indicated that these 67 kb heterozygous duplications were likely mediated by non-allelic homologous recombination at regions of high sequence identity in ATAD3A exon 11 and ATAD3C exon 7. At the recombinant junction, the duplication allele produces a fusion gene derived from ATAD3A and ATAD3C, the protein product of which lacks key functional residues. Analysis of fibroblasts derived from two affected individuals shows that the fusion gene product is expressed and stable. These cells display perturbed cholesterol and mitochondrial DNA organization similar to that observed for individuals with severe ATAD3A deficiency. We hypothesize that the fusion protein acts through a dominant-negative mechanism to cause this fatal mitochondrial disorder. Our data delineate a molecular diagnosis for this disorder, extend the clinical spectrum associated with structural variation at the ATAD3 locus, and identify a third mutational mechanism for ATAD3 gene cluster variants. These results further affirm structural variant mutagenesis mechanisms in sporadic disease traits, emphasize the importance of copy number analysis in molecular genomic diagnosis, and highlight some of the challenges of detecting and interpreting clinically relevant rare gene rearrangements from next-generation sequencing data.

OXA1, the mitochondrial member of the YidC/Alb3/Oxa1 membrane protein insertase family, is required for the assembly of oxidative phosphorylation complexes IV and V in yeast. However, depletion of human OXA1 (OXA1L) was previously reported to impair assembly of complexes I and V only. We report a patient presenting with severe encephalopathy, hypotonia and developmental delay who died at 5 years showing complex IV deficiency in skeletal muscle. Whole exome sequencing identified biallelic OXA1L variants (c.500_507dup, p.(Ser170Glnfs*18) and c.620G>T, p.(Cys207Phe)) that segregated with disease. Patient muscle and fibroblasts showed decreased OXA1L and subunits of complexes IV and V. Crucially, expression of wild-type human OXA1L in patient fibroblasts rescued the complex IV and V defects. Targeted depletion of OXA1L in human cells or Drosophila melanogaster caused defects in the assembly of complexes I, IV and V, consistent with patient data. Immunoprecipitation of OXA1L revealed the enrichment of mtDNA-encoded subunits of complexes I, IV and V. Our data verify the pathogenicity of these OXA1L variants and demonstrate that OXA1L is required for the assembly of multiple respiratory chain complexes.

While >300 disease-causing variants have been identified in the mitochondrial DNA (mtDNA) polymerase γ, no mitochondrial phenotypes have been associated with POLRMT, the RNA polymerase responsible for transcription of the mitochondrial genome. Here, we characterise the clinical and molecular nature of POLRMT variants in eight individuals from seven unrelated families. Patients present with global developmental delay, hypotonia, short stature, and speech/intellectual disability in childhood; one subject displayed an indolent progressive external ophthalmoplegia phenotype. Massive parallel sequencing of all subjects identifies recessive and dominant variants in the POLRMT gene. Patient fibroblasts have a defect in mitochondrial mRNA synthesis, but no mtDNA deletions or copy number abnormalities. The in vitro characterisation of the recombinant POLRMT mutants reveals variable, but deleterious effects on mitochondrial transcription. Together, our in vivo and in vitro functional studies of POLRMT variants establish defective mitochondrial transcription as an important disease mechanism.

In recent years, an increasing number of mitochondrial disorders have been associated with mutations in mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs), which are key enzymes of mitochondrial protein synthesis. Bi-allelic functional variants in VARS2, encoding the mitochondrial valyl tRNA-synthetase, were first reported in a patient with psychomotor delay and epilepsia partialis continua associated with an oxidative phosphorylation (OXPHOS) Complex I defect, before being described in a patient with a neonatal form of encephalocardiomyopathy. Here we provide a detailed genetic, clinical, and biochemical description of 13 patients, from nine unrelated families, harboring VARS2 mutations. All patients except one, who manifested with a less severe disease course, presented at birth exhibiting severe encephalomyopathy and cardiomyopathy. Features included hypotonia, psychomotor delay, seizures, feeding difficulty, abnormal cranial MRI, and elevated lactate. The biochemical phenotype comprised a combined Complex I and Complex IV OXPHOS defect in muscle, with patient fibroblasts displaying normal OXPHOS activity. Homology modeling supported the pathogenicity of VARS2 missense variants. The detailed description of this cohort further delineates our understanding of the clinical presentation associated with pathogenic VARS2 variants and we recommend that this gene should be considered in early-onset mitochondrial encephalomyopathies or encephalocardiomyopathies.

Mitochondrial diseases collectively represent one of the most heterogeneous group of metabolic disorders. Symptoms can manifest at any age, presenting with isolated or multiple-organ involvement. Advances in next-generation sequencing strategies have greatly enhanced the diagnosis of patients with mitochondrial disease, particularly where a mitochondrial aetiology is strongly suspected yet OXPHOS activities in biopsied tissue samples appear normal. We used whole exome sequencing (WES) to identify the molecular basis of an early-onset mitochondrial syndrome-pathogenic biallelic variants in the HTRA2 gene, encoding a mitochondria-localised serine protease-in five subjects from two unrelated families characterised by seizures, neutropenia, hypotonia and cardio-respiratory problems. A unifying feature in all affected children was 3-methylglutaconic aciduria (3-MGA-uria), a common biochemical marker observed in some patients with mitochondrial dysfunction. Although functional studies of HTRA2 subjects' fibroblasts and skeletal muscle homogenates showed severely decreased levels of mutant HTRA2 protein, the structural subunits and complexes of the mitochondrial respiratory chain appeared normal. We did detect a profound defect in OPA1 processing in HTRA2-deficient fibroblasts, suggesting a role for HTRA2 in the regulation of mitochondrial dynamics and OPA1 proteolysis. In addition, investigated subject fibroblasts were more susceptible to apoptotic insults. Our data support recent studies that described important functions for HTRA2 in programmed cell death and confirm that patients with genetically-unresolved 3-MGA-uria should be screened by WES with pathogenic variants in the HTRA2 gene prioritised for further analysis.

Sengers syndrome is an autosomal recessive condition characterized by congenital cataract, hypertrophic cardiomyopathy, skeletal myopathy and lactic acidosis. Mutations in the acylglycerol kinase (AGK) gene have been recently described as the cause of Sengers syndrome in nine families.