Isolated complex I deficiency is a common biochemical phenotype observed in pediatric mitochondrial disease and often arises as a consequence of pathogenic variants affecting one of the ∼65 genes encoding the complex I structural subunits or assembly factors. Such genetic heterogeneity means that application of next-generation sequencing technologies to undiagnosed cohorts has been a catalyst for genetic diagnosis and gene-disease associations. We describe the clinical and molecular genetic investigations of four unrelated children who presented with neuroradiological findings and/or elevated lactate levels, highly suggestive of an underlying mitochondrial diagnosis. Next-generation sequencing identified bi-allelic variants in NDUFA6, encoding a 15 kDa LYR-motif-containing complex I subunit that forms part of the Q-module. Functional investigations using subjects' fibroblast cell lines demonstrated complex I assembly defects, which were characterized in detail by mass-spectrometry-based complexome profiling. This confirmed a marked reduction in incorporated NDUFA6 and a concomitant reduction in other Q-module subunits, including NDUFAB1, NDUFA7, and NDUFA12. Lentiviral transduction of subjects' fibroblasts showed normalization of complex I. These data also support supercomplex formation, whereby the ∼830 kDa complex I intermediate (consisting of the P- and Q-modules) is in complex with assembled complex III and IV holoenzymes despite lacking the N-module. Interestingly, RNA-sequencing data provided evidence that the consensus RefSeq accession number does not correspond to the predominant transcript in clinically relevant tissues, prompting revision of the NDUFA6 RefSeq transcript and highlighting not only the importance of thorough variant interpretation but also the assessment of appropriate transcripts for analysis.

• MeSH
• alely MeSH
• fenotyp MeSH
• fibroblasty patologie   MeSH
• genetická heterogenita MeSH
• kojenec MeSH
• lidé MeSH
• mitochondriální nemoci genetika   MeSH
• mitochondriální proteiny genetika   MeSH
• mitochondrie genetika   MeSH
• mutace genetika   MeSH
• respirační komplex I nedostatek   genetika   MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• Check Tag
• kojenec MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The oxidative phosphorylation (OXPHOS) system localized in the inner mitochondrial membrane secures production of the majority of ATP in mammalian organisms. Individual OXPHOS complexes form supramolecular assemblies termed supercomplexes. The complexes are linked not only by their function but also by interdependency of individual complex biogenesis or maintenance. For instance, cytochrome c oxidase (cIV) or cytochrome bc1 complex (cIII) deficiencies affect the level of fully assembled NADH dehydrogenase (cI) in monomeric as well as supercomplex forms. It was hypothesized that cI is affected at the level of enzyme assembly as well as at the level of cI stability and maintenance. However, the true nature of interdependency between cI and cIV is not fully understood yet. We used a HEK293 cellular model where the COX4 subunit was completely knocked out, serving as an ideal system to study interdependency of cI and cIV, as early phases of cIV assembly process were disrupted. Total absence of cIV was accompanied by profound deficiency of cI, documented by decrease in the levels of cI subunits and significantly reduced amount of assembled cI. Supercomplexes assembled from cI, cIII, and cIV were missing in COX4I1 knock-out (KO) due to loss of cIV and decrease in cI amount. Pulse-chase metabolic labeling of mitochondrial DNA (mtDNA)-encoded proteins uncovered a decrease in the translation of cIV and cI subunits. Moreover, partial impairment of mitochondrial protein synthesis correlated with decreased content of mitochondrial ribosomal proteins. In addition, complexome profiling revealed accumulation of cI assembly intermediates, indicating that cI biogenesis, rather than stability, was affected. We propose that attenuation of mitochondrial protein synthesis caused by cIV deficiency represents one of the mechanisms, which may impair biogenesis of cI.

• MeSH
• glykolýza MeSH
• HEK293 buňky MeSH
• lidé MeSH
• mitochondriální nemoci metabolismus   MeSH
• mitochondriální proteiny biosyntéza   MeSH
• oxidativní fosforylace MeSH
• podjednotky proteinů metabolismus   MeSH
• proteosyntéza * MeSH
• respirační komplex IV metabolismus   MeSH
• spotřeba kyslíku MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH