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Background MT-ATP6 is a mitochondrial gene which encodes for the intramembrane subunit 6 (or A) of the mitochondrial ATP synthase, also known asl complex V, which is involved in the last step of oxidative phosphorylation to produce cellular ATP through aerobic metabolism. Although classically associated with the NARP syndrome, recent evidence highlights an important role of MT-ATP6 pathogenic variants in complicated adult-onset ataxias. Methods We describe two unrelated patients with adult-onset cerebellar ataxia associated with severe optic atrophy and mild cognitive impairment. Whole mitochondrial DNA sequencing was performed in both patients. We employed patients’ primary fibroblasts and cytoplasmic hybrids (cybrids), generated from patients-derived cells, to assess the activity of respiratory chain complexes, oxygen consumption rate (OCR), ATP production and mitochondrial membrane potential. Results In both patients, we identified the same novel m.8777 T > C variant in MT-ATP6 with variable heteroplasmy level in different tissues. We identifed an additional heteroplasmic novel variant in MT-ATP6 , m.8879G > T, in the patients with the most severe phenotype. A significant reduction in complex V activity, OCR and ATP production was observed in cybrid clones homoplasmic for the m.8777 T > C variant, while no functional defect was detected in m.8879G > T homoplasmic clones. In addition, fibroblasts with high heteroplasmic levelsof m.8777 T > C variant showed hyperpolarization of mitochondrial membranes. Conclusions We describe a novel pathogenic mtDNA variant in MT-ATP6 associated with adult-onset ataxia, reinforcing the value of mtDNA screening within the diagnostic workflow of selected patients with late onset ataxias.

Mitochondrial network dynamics, encompassing processes like fission, fusion, and mitophagy, are crucial for mitochondrial function and overall cellular health. Dysregulation of these processes has been linked to various human diseases. Particularly, pathogenic variants in the gene DNM1L can lead to a broad range of clinical phenotypes, ranging from isolated optic atrophy to severe neurological conditions. DNM1L encodes DRP1 (dynamin-1-like protein), which is a key player in mitochondrial and peroxisomal fission. This study describes two twin sisters with a de novo heterozygous variant in DNM1L, due to possible paternal germline mosaicism identified through clinical exome sequencing. The two twins showed a variable clinical presentation, including paraparesis and optic neuropathy. Functional studies of patient-derived fibroblasts revealed altered mitochondrial and peroxisomal morphology, along with dysregulated DNM1L transcript levels, indicating the deleterious effect of the variant. These findings allowed us to reclassify the identified variant from a variant of uncertain significance to a likely pathogenic variant. Our report provides insight into the phenotypic spectrum of DNM1L-related disorders and highlights the need to combine genetic and functional analyses to accurately diagnose rare mitochondrial diseases.

Background KARS encodes lysyl- transfer ribonucleic acid (tRNA) synthetase, which catalyzes the aminoacylation of tRNA-Lys in the cytoplasm and mitochondria. Eleven families/sporadic patients and 16 different mutations in KARS have been reported to date. The associated clinical phenotype is heterogeneous ranging from early onset encephalopathy to isolated peripheral neuropathy or nonsyndromic hearing impairment. Recently additional presentations including leukoencephalopathy as predominant cerebral involvement or cardiomyopathy, isolated or associated with muscular and cerebral involvement, have been reported. A progressive Leukoencephalopathy with brainstem and spinal cord calcifications was previously described in a singleton patient and in two siblings, without the identification of the genetic cause. We reported here about a new severe phenotype associated with biallelic KARS mutations and sharing some common points with the other already reported phenotypes, but with a distinct clinical and neuroimaging picture. Review of KARS mutant patients published to date will be also discussed. Results Herein, we report the clinical, biochemical and molecular findings of 2 unreported Italian patients affected by developmental delay, acquired microcephaly, spastic tetraparesis, epilepsy, sensory-neural hypoacusia, visual impairment, microcytic hypochromic anaemia and signs of hepatic dysfunction. MRI pattern in our patients was characterized by progressive diffuse leukoencephalopathy and calcifications extending in cerebral, brainstem and cerebellar white matter, with spinal cord involvement. Genetic analysis performed on these 2 patients and in one subject previously described with similar MRI pattern revealed the presence of biallelic mutations in KARS in all 3 subjects. Conclusions With our report we define the molecular basis of the previously described Leukoencephalopathy with Brainstem and Spinal cord Calcification widening the spectrum of KARS related disorders, particularly in childhood onset disease suggestive for mitochondrial impairment. The review of previous cases does not suggest a strict and univocal genotype/phenotype correlation for this highly heterogeneous entity. Moreover, our cases confirm the usefulness of search for common brain and spine MR imaging pattern and of broad genetic screening, in syndromes clinically resembling mitochondrial disorders in spite of normal biochemical assay.

This comprehensive review provides insights and suggested strategies for the analysis of germline variants using second- and third-generation sequencing technologies (SGS and TGS). It addresses the critical stages of data processing, starting from alignment and preprocessing to quality control, variant calling, and the removal of artifacts. The document emphasized the importance of meticulous data handling, highlighting advanced methodologies for annotating variants and identifying structural variations and methylated DNA sites. Special attention is given to the inspection of problematic variants, a step that is crucial for ensuring the accuracy of the analysis, particularly in clinical settings where genetic diagnostics can inform patient care. Additionally, the document covers the use of various bioinformatics tools and software that enhance the precision and reliability of these analyses. It outlines best practices for the annotation of variants, including considerations for problematic genetic alterations such as those in the human leukocyte antigen region, runs of homozygosity, and mitochondrial DNA alterations. The document also explores the complexities associated with identifying structural variants and copy number variations, underscoring the challenges posed by these large-scale genomic alterations. The objective is to offer a comprehensive framework for researchers and clinicians, ensuring that genetic analyses conducted with SGS and TGS are both accurate and reproducible. By following these best practices, the document aims to increase the diagnostic accuracy for hereditary diseases, facilitating early diagnosis, prevention, and personalized treatment strategies. This review serves as a valuable resource for both novices and experts in the field, providing insights into the latest advancements and methodologies in genetic analysis. It also aims to encourage the adoption of these practices in diverse research and clinical contexts, promoting consistency and reliability across studies.

The unique features of mitochondrial DNA (mtDNA), including its circular and multicopy nature, the possible coexistence of wild-type and mutant molecules (i.e., heteroplasmy) and the presence of nuclear mitochondrial DNA segments (NUMTs), make the diagnosis of mtDNA diseases particularly challenging. The extensive deployment of next-generation sequencing (NGS) technologies has significantly advanced the diagnosis of mtDNA-related diseases. However, the vast amounts and diverse types of sequencing data complicate the interpretation of these variants. From sequence alignment to variant calling, NGS-based mtDNA sequencing requires specialized bioinformatics tools, adapted for the mitochondrial genome. This study presents the use of new bioinformatics approaches, optimized for short- and long-read sequencing data, to enhance the accuracy of mtDNA analysis in diagnostics. Two recent and emerging free bioinformatics tools, Mitopore and MitoSAlt, were evaluated on patients previously diagnosed with single nucleotide variants or large-scale deletions. Analyses were performed in Linux-based environments and web servers implemented in Python, Perl, Java, and R. The results indicated that each tool demonstrated high sensitivity and specific accuracy in identifying and quantifying various types of pathogenic variants. The study suggests that the integrated and parallel use of these tools offers a significant advantage over traditional methods in interpreting mtDNA genetic variants, reducing the computational demands, and provides an accurate diagnostic solution.

Background Heterozygous mutations in OPA1 are a common cause of autosomal dominant optic atrophy, sometimes associated with extra-ocular manifestations. Few cases harboring compound heterozygous OPA1 mutations have been described manifesting complex neurodegenerative disorders in addition to optic atrophy. Results We report here three patients: one boy showing an early-onset mitochondrial disorder with hypotonia, ataxia and neuropathy that was severely progressive, leading to early death because of multiorgan failure; two unrelated sporadic girls manifesting a spastic ataxic syndrome associated with peripheral neuropathy and, only in one, optic atrophy. Using a targeted resequencing of 132 genes associated with mitochondrial disorders, in two probands we found compound heterozygous mutations in OPA1 : in the first a 5 nucleotide deletion, causing a frameshift and insertion of a premature stop codon (p.Ser64Asnfs*7), and a missense change (p.Ile437Met), which has recently been reported to have clinical impact; in the second, a novel missense change (p.Val988Phe) co-occurred with the p.Ile437Met substitution. In the third patient a homozygous mutation, c.1180G > A (p.Ala394Thr) in OPA1 was detected by a trio-based whole exome sequencing approach. One of the patients presented also variants in mitochondrial DNA that may have contributed to the peculiar phenotype. The deleterious effect of the identified missense changes was experimentally validated in yeast model. OPA1 level was reduced in available patients’ biological samples, and a clearly fragmented mitochondrial network was observed in patients’ fibroblasts. Conclusions This report provides evidence that bi-allelic OPA1 mutations may lead to complex and severe multi-system recessive mitochondrial disorders, where optic atrophy might not represent the main feature.

Leigh syndrome is a progressive neurodegenerative disorder, most commonly observed in paediatric mitochondrial disease, and is often associated with pathogenic variants in complex I structural subunits or assembly factors resulting in isolated respiratory chain complex I deficiency. Clinical heterogeneity has been reported, but key diagnostic findings are developmental regression, elevated lactate and characteristic neuroimaging abnormalities. Here, we describe three affected children from two unrelated families who presented with Leigh syndrome due to homozygous variants (c.346_*7del and c.173A>T p.His58Leu) in NDUFC2 , encoding a complex I subunit. Biochemical and functional investigation of subjects’ fibroblasts confirmed a severe defect in complex I activity, subunit expression and assembly. Lentiviral transduction of subjects’ fibroblasts with wild‐type NDUFC2 cDNA increased complex I assembly supporting the association of the identified NDUFC2 variants with mitochondrial pathology. Complexome profiling confirmed a loss of NDUFC2 and defective complex I assembly, revealing aberrant assembly intermediates suggestive of stalled biogenesis of the complex I holoenzyme and indicating a crucial role for NDUFC2 in the assembly of the membrane arm of complex I, particularly the ND2 module. Synopsis This work describes the first confirmed pathogenic variants in NDUFC2 in a case of mitochondrial disease presenting with symptoms of Leigh syndrome and a severe deficiency in OXPHOS complex I. NDUFC2 was shown to be important for the assembly of the membrane arm of complex I. Bi‐allelic variants in NDUFC2 (NM_004549.6) were identified in 2 families by whole exome sequencing. Two affected siblings in one family were homozygous fora c.346_*7del, p.(His116_Arg119delins21) variant and an affected individual from another family was homozygous for a c.173A>T, p.(His58Leu) NDUFC2 variant. Fibroblasts from one case from each family showed decreased protein levels of NDUFC2 and other subunits of OXPHOS complex I. Introduction of wild‐type NDUFC2 into fibroblasts samples from affected individuals by lentiviral transduction ameliorated the complex I defects, confirming pathogenicity of these NDUFC2 variants. Complexome analysis revealed an increase in specific aberrant complex I assembly intermediates consistent with a stalling of complex I assembly, particularly the assembly of the ND2 module. Graphical Abstract This work describes the first confirmed pathogenic variants in NDUFC2 in a case of mitochondrial disease presenting with symptoms of Leigh syndrome and a severe deficiency in OXPHOS complex I. NDUFC2 was shown to be important for the assembly of the membrane arm of complex I.

Biallelic mutations in NDUFAF6 have been identified as responsible for cases of autosomal recessive Leigh syndrome associated with mitochondrial complex I deficiency. Here we report two siblings and two unrelated subjects with Leigh syndrome, in which we found the same compound heterozygous missense (c.532G>C:p.A178P) and deep intronic (c.420+784C>T) variants in NDUFAF6. We demonstrated that the identified intronic variant creates an alternative splice site, leading to the production of an aberrant transcript. A detailed analysis of whole-exome sequencing data together with the functional validation based on mRNA analysis may reveal pathogenic variants even in non-exonic regions.

Objective COASY, the gene encoding the bifunctional enzyme CoA synthase, which catalyzes the last two reactions of cellular de novo coenzyme A (CoA) biosynthesis, has been linked to two exceedingly rare autosomal recessive disorders, such as COASY protein‐associated neurodegeneration (CoPAN), a form of neurodegeneration with brain iron accumulation (NBIA), and pontocerebellar hypoplasia type 12 (PCH12). We aimed to expand the phenotypic spectrum and gain insights into the pathogenesis of COASY‐related disorders. Methods Patients were identified through targeted or exome sequencing. To unravel the molecular mechanisms of disease, RNA sequencing, bioenergetic analysis, and quantification of critical proteins were performed on fibroblasts. Results We identified five new individuals harboring novel COASY variants. While one case exhibited classical CoPAN features, the others displayed atypical symptoms such as deafness, language and autism spectrum disorders, brain atrophy, and microcephaly. All patients experienced epilepsy, highlighting its potential frequency in COASY‐related disorders. Fibroblast transcriptomic profiling unveiled dysregulated expression in genes associated with mitochondrial respiration, responses to oxidative stress, transmembrane transport, various cellular signaling pathways, and protein translation, modification, and trafficking. Bioenergetic analysis revealed impaired mitochondrial oxygen consumption in COASY fibroblasts. Despite comparable total CoA levels to control cells, the amounts of mitochondrial 4′‐phosphopantetheinylated proteins were significantly reduced in COASY patients. Interpretation These results not only extend the clinical phenotype associated with COASY variants but also suggest a continuum between CoPAN and PCH12. The intricate interplay of altered cellular processes and signaling pathways provides valuable insights for further research into the pathogenesis of COASY‐associated diseases.

Endonuclease G (ENDOG) is a nuclear-encoded mitochondrial-localized nuclease. Although its precise biological function remains unclear, its proximity to mitochondrial DNA (mtDNA) makes it an excellent candidate to participate in mtDNA replication, metabolism and maintenance. Indeed, several roles for ENDOG have been hypothesized, including maturation of RNA primers during mtDNA replication, splicing of polycistronic transcripts and mtDNA repair. To date, ENDOG has been deemed as a determinant of cardiac hypertrophy, but no pathogenic variants or genetically defined patients linked to this gene have been described. Here, we report biallelic ENDOG variants identified by NGS in a patient with progressive external ophthalmoplegia, mitochondrial myopathy and multiple mtDNA deletions in muscle. The absence of the ENDOG protein in the patient’s muscle and fibroblasts indicates that the identified variants are pathogenic. The presence of multiple mtDNA deletions supports the role of ENDOG in mtDNA maintenance; moreover, the patient’s clinical presentation is very similar to mitochondrial diseases caused by mutations in other genes involved in mtDNA homeostasis. Although the patient’s fibroblasts did not present multiple mtDNA deletions or delay in the replication process, interestingly, we detected an accumulation of low-level heteroplasmy mtDNA point mutations compared with age-matched controls. This may indicate a possible role of ENDOG in mtDNA replication or repair. Our report provides evidence of the association of ENDOG variants with mitochondrial myopathy.