Various causes have been found for sporadic CPEO, including both large rearrangements and point mutations in mtDNA. In a patient presenting with CPEO, we identified a novel point mutation in mitochondrial tRNAIle
which could interfere with formation of the T stem and stabilize an alternate secondary structure. The following features substantiate the pathogenic nature of the m.4308G>A mutation. The lack of large rearrangements in the mtDNA suggests that primary mutations in a nuclear gene (e.g. POLG, POLG2, Twinkle and ANT1) is unlikely (Spinazzola and Zeviani, 2009
; Tuppen et al., 2010
). The mutation was associated with clinical symptoms and was present in a heteroplasmic form in the muscle tissue, consistent with a general criterion for mtDNA. Electron microscopy showed the typical features of a mitochondrial cytopathy (Mierau et al., 2004
). Morphologically, there were ultrastructural mitochondrial abnormalities, including deranged internal architecture and crystalline inclusions which have not only been observed in PEO patients (Mitsumoto et al., 1983
) but also in other diseases linked to defective mitochondrial function such as MERRF (Suomalainen, 1997
), Kearns-Sayre Syndrome (McKechnie et al., 1985
), myopathy (Frey and Mannella, 2000
; Zanssen et al., 1997
), and encephalopathy (Kaido et al., 1995
). Biochemically, we found a multiple partial respiratory chain enzyme deficiency, affecting the activities of complexes I, III and IV. The m.4308G>A variant was not found in analysis of more than 2700 individuals (Ingman and Gyllensten, 2006
), strongly suggesting that it is not a neutral polymorphism. A general approach to demonstrate the pathogenicity of a newly identified point mutation in the mtDNA includes single fibre PCR to show a co-segregation of the mutation with COX-negative fibres. However, in this particular case, the amount of native muscle biopsy remaining after electron microscopy, biochemical analysis and genetic testing, was insufficient to obtain good quality sections for accurate qualitative assessment of COX-negative fibers, prompting us to investigate the pathogenicity of the newly identified variant at a functional level. Mitochondrial tRNAs are embedded in long primary transcripts, punctuating the two ribosomal RNAs and 11 mRNAs (Anderson et al., 1981
). Production of mature, functional mitochondrial RNAs requires endonucleolytic cleavage of the precursors at the 5’ and/or 3’ ends of the tRNAs (Montoya et al., 1981
; Ojala et al., 1981
5’ end and ND1 3’ end have an overlap of 1 nt, which therefore requires the addition of an A after processing to complete the ND1 translation termination codon. At its 3’ end, tRNAIle
has a 70 nt spacer followed by tRNAMet
, making the reductions in tRNase Z processing efficiency due to pathogenesis-related mutations still more remarkable. This is also observed for tRNASer(UCN)
which is followed by an even longer 2.5 kb spacer at its 3’ end (Levinger et al., 2001
). In these instances, the molecular defect is probably the reduced quantity and impaired function of the affected aminoacyl tRNA that causes a deficiency in translation of mitochondrial messages, leading to the pathology.
For functional studies of the m.4308G>A substitution, we initially investigated the effect of the m.4308G>A substitution on tRNase Z processing and observed a five fold reduction in processing efficiency. On the basis of this reduced processing efficiency, we performed Michaelis-Menten kinetics on wild-type and two mutants (m.4308G>A and m.4309G>A). The latter has previously been described to be associated with CPEO (Franceschina et al., 1998
) and resulted in a pathology-related reduction in 3’ end processing (Levinger et al., 2003
). Both mutants display substantially reduced 3’ end processing efficiency, due principally to a lower kcat
. Whereas the m.4309G>A mutant decreases 3’end processing about 3 fold, the G4308A mutant has 5 fold reduced 3’ end processing efficiency.
Wild-type and m.4308G>A mutant structures were compared to search for structural reasons for reduced processing efficiency. The mutation disrupts a strictly conserved GC base pair in the T stem of tRNAIle close to the V-Loop – T-stem boundary. The effect of the G4308A substitution on tRNAIle structure (, 6) and tRNase Z processing combine to suggest an overall reduction in availability of Ile-tRNAIle for translation. Computer analysis and visual inspection of mutant tRNAIle suggest that the m.4308G>A mutation destabilises the already weak T stem. While no single secondary structure completely explains the changes in V1 nuclease susceptibility, the three-headed arrow between Figure 6A, B and C indicates a shift in the equilibrium away from the coaxially stacked acceptor stem and T stem that is required of a tRNase Z substrate in favour of altered structures. The m.4308G>A mutation thus severely affects secondary and tertiary structure of tRNAIle, thereby reducing its function.
End processing reactions by RNase P, tRNase Z and CCA-adding enzyme require an intact, coaxially stacked acceptor stem and T domain (Levinger et al., 2003
). Thus, the location of the m.4308G>A mutation and its implication in structural, functional and physiological effects are consistent with interference of the mutated tRNAIle
with cleavage by tRNase Z, contributing to pathology.
Interestingly, while our manuscript was in the process of revision, we became aware of a publication (Sihem et al., 2010
) in which the authors found the m.4308G>A in a patient with sporadic CPEO, suggesting an association of the m.4308G>A mutation with this clinical phenotype.
Genetic counseling for a mtDNA mutation is generally a difficult task as factors determining the transmission of mtDNA mutations are largely unknown. Thus, the likelihood that the m.4308G>A mutation would be transmitted to the patient’s two children is difficult to predict (Elson et al., 2009
). The mutation found in the patient is confined to muscle and the absence of the mutation in blood suggests that it is not a germline mutation. However, there is no guarantee that the patient’s oocytes are mutation-free. To fully exclude transmission of the mutation, a muscle biopsy should be performed on the presently unaffected children. However, a muscle biopsy in unaffected individuals is not standard practice and therefore, the status of her children remains unknown.
In summary, this study adds m.4308G>A to the growing list of point mutations associated with CPEO and further emphasizes the importance of analyzing all the 22 tRNAs in sporadic CPEO patients after exclusion of large deletions. It also demonstrates the need of a muscle biopsy when no mutations can be detected in DNA extracted from blood. Our data provide a further example in support of the thesis that inefficient 3’ end processing of mutant tRNAs can contribute to pathology (Levinger et al., 2004
). CPEO may be associated with mtDNA deletions or related to point mutations within one or another of the tRNA genes. A link between these two mitochondrial genome-based causes could be presumed to arise from a decrease in mitochondrial protein synthesis which would cause the multiple partial defects in respiratory chain activity.