*4.1. Mitochondrial Diseases*

Mitochondrial diseases are generally provoked by genetic mutations in complexes' subunits or assembly factors, as extensively discussed in numerous other reviews [276–280]. However, defects in the machinery involved in the import of these subunits have also been identified as the cause of mitochondrial pathologies, leading to different clinical features.

Mutations in *TIMM50,* which encodes a subunit of TIM23 complex, have been associated with severe lactic acidosis and seizures, linked to defects in import of ETC proteins, alterations in SC formation and a general respiratory deficiency [281–283]. Biochemical analysis of a patient with impaired TIM23 complex due to compound heterozygous mutations in *TIMM50* revealed reduced steady state levels of CI, II, and IV, but interestingly not CIII and V [282]. These results suggest the possibility of alternative import routes for certain ETC subunits or different interactions with the import machinery for subunits transported into the matrix or inserted into the IMM. Another study with patients with two homozygous missense mutations in the *TIMM50* gene, however, produced opposite results, showing normal activities of Complex I–IV and defective activity of CV [281]. A

possible explanation for this phenotype is that, as shown in yeast, the import of subunits 9 and β of CV is highly dependent on TIM50, whilst import of CIII subunit CYC1 and CIV subunit COX5A import is only mildly affected by TIM50 defects [66]. Finally, another subject carrying compound heterozygous mutations within the IMS domain of *TIMM50* exhibited 3-ethylglutaconic aciduria, symptoms of Leigh syndrome, and dilated hypercardiomyopathy, associated with altered mitochondrial morphology [283]. Additionally, patients also experienced a general decrease in the levels of fully assembled complexes and their activity alongside a reduction in SC formation and a drop in the maximum respiratory capacity [283].

Interestingly, defects in other TIM23 complex subunits do not result in impaired mitochondrial respiration. For example, alterations in the formation of the DDP1/TIMM8a– TIMM13 complex found in a patient with deafness/dystonia and with a *de novo* mutation in DDP1(C66W) did not lead to defects in the activity of any of the respiratory complexes [284]. More recently, another reported case of neuromuscular presentation of mitochondrial disease was found to be associated with compound heterozygous mutations in *TIMM22* [285]. However, while cellular respiration was reduced in the patient cells, no evident defect in respiratory complexes or SC assembly was found. In a different report, where TIM22 assembly and activity was impaired by the removal of AGK, a mild CI assembly defect and respiration impairment was observed [111]. In contrast to the compound heterozygous variant, this observation suggests a direct involvement of the TIM22 complex in the import of CI subunits and/or factors required for CI assembly that requires further investigation.

Regarding defects at the TOM complex level, a patient with severe anaemia, lactic acidosis, and developmental delay were identified with compound heterozygous variants in the *TOMM70* gene [286]. Interestingly, this patient presented with respiratory complex deficiencies with a primarily marked defect in CIV, including decreased steady state levels of fully assembled enzyme, activity, and a reduction in CIV-containing SC, while SCI:CIII<sup>2</sup> species appeared unaffected.

Finally, defects in OXA1L, essential for the re-localisation of newly imported nuclearencoded proteins in the matrix into the IMM, could have an impact on complexes' assembly and activity. One patient was identified with mutations in *OXA1L* and tissue-specific combined respiratory complex deficiencies, which led to severe encephalopathy, hypotonia, and developmental delay [196]. Interestingly, skeletal muscle biopsy from this patient showed defects primarily at the CIV and CV level with only milder defects in CI despite the fact that neuropathology experiments indicated an isolated CI deficiency in the central nervous system. Although the tissue-specificity observed in this patient remains unclear, it suggests a possible differential expression of OXA1L isoforms in different tissues or the presence of alternative insertases in human mitochondria.
