Nuclear factors involved in mitochondrial translation cause a subgroup of combined respiratory chain deficiency

Abstract

Mutations in several mitochondrial DNA and nuclear genes involved in mitochondrial protein synthesis have recently been reported in combined respiratory chain deficiency, indicating a generalized defect in mitochondrial translation. However, the number of patients with pathogenic mutations is small, implying that nuclear defects of mitochondrial translation are either underdiagnosed or intrauterine lethal. No comprehensive studies have been reported on large cohorts of patients with combined respiratory chain deficiency addressing the role of nuclear genes affecting mitochondrial protein synthesis to date. We investigated a cohort of 52 patients with combined respiratory chain deficiency without causative mitochondrial DNA mutations, rearrangements or depletion, to determine whether a defect in mitochondrial translation defines the pathomechanism of their clinical disease. We followed a combined approach of sequencing known nuclear genes involved in mitochondrial protein synthesis (EFG1, EFTu, EFTs, MRPS16, TRMU), as well as performing in vitro functional studies in 22 patient cell lines. The majority of our patients were children (<15 years), with an early onset of symptoms <1 year of age (65%). The most frequent clinical presentation was mitochondrial encephalomyopathy (63%); however, a number of patients showed cardiomyopathy (33%), isolated myopathy (15%) or hepatopathy (13%). Genomic sequencing revealed compound heterozygous mutations in the mitochondrial transfer ribonucleic acid modifying factor (TRMU) in a single patient only, presenting with early onset, reversible liver disease. No pathogenic mutation was detected in any of the remaining 51 patients in the other genes analysed. In vivo labelling of mitochondrial polypeptides in 22 patient cell lines showed overall (three patients) or selective (four patients) defects of mitochondrial translation. Immunoblotting for mitochondrial proteins revealed decreased steady state levels of proteins in some patients, but normal or increased levels in others, indicating a possible compensatory mechanism. In summary, candidate gene sequencing in this group of patients has a very low detection rate (1/52), although in vivo labelling of mitochondrial translation in 22 patient cell lines indicate that a nuclear defect affecting mitochondrial protein synthesis is responsible for about one-third of combined respiratory chain deficiencies (7/22). In the remaining patients, the impaired respiratory chain activity is most likely the consequence of several different events downstream of mitochondrial translation. Clinical classification of patients with biochemical analysis, genetic testing and, more importantly, in vivo labelling and immunoblotting of mitochondrial proteins show incoherent results, but a systematic review of these data in more patients may reveal underlying mechanisms, and facilitate the identification of novel factors involved in combined respiratory chain deficiency.

Figure 1

Sequence electropherograms of the compound heterozygous TRMU mutations in Patient 37 (top). Both wild type and mutant sequences are detailed. Inserted bases are highlighted (boxes). Alignment of C-terminal sequence of human TRMU with its homologs (bottom). Mutant sequence with the three amino acid insertion in blue and the highly conserved shifted glutamine residue in red are shown.

Figure 2

³⁵S-methionine pulse labelling of myoblast (A and B) and fibroblast (C) cell lines. Patients (P) 1, 19 and 36 showed a significant overall reduction in mitochondrial oxidative phosphorylation complex subunits. Patients 32 and 16 showed an isolated decrease in the translation of ND4. Patient 12 had reduced steady-state levels of ND1, ND4 and ND5, with normal ND2, and COXI and ND4 were decreased in Patient 34. Patient 9 contained a structurally altered, smaller COXI mitochondrial protein. Deficient bands and lanes are marked with white arrows and stars.

Figure 3

Immunoblotting for mitochondrial proteins COXI, COXII, NDUFB8 (representing ND subunits) and porin as a control protein in patients’ primary cell lines (P) compared with controls (C). Patients 12 and 19 showed an overall decrease of all three mitochondrial respiratory chain proteins COXI, COXII and NDUFB8. In Patients 10, 32 and 36 we detected normal and even stronger than normal signals for COXI, COXII and NDUFB8. In Patient 9, we could confirm the aberrant migration of COXI whilst the other mitochondrial-encoded proteins were normal.