Phenotypic considerations

The surprisingly mild phenotype associated with the T7671A mutation in patient P26 probably reflects the tissue distribution of the mutation. It is likely that skeletal

muscle, the only tissue affected clinically, is also the only tissue with a mutant load above the threshold required to reduce OXPHOS capacity. This threshold is

generally about 85% for mitochondrial tRNA mutations (Bentlage and Attardi, 1996; Schon et al. 1997). However the threshold for phenotypic expression of mutations in mtDNA polypeptide-coding genes appears to be lower than that reported in tRNA mutations (Hanna et al. 1998b). For example, transmitochondrial cybrids harbouring 35-65% mutant load of the G930A nonsense mutation in COX subunit I were COX deficient (Bruno et al. 1999). Furthermore a dinucleotide deletion 8042delAT

resulting in frameshift and truncation of the COX II polypeptide was present at <20% of total mtDNA in several tissues of a premature infant who died of severe lactic acidosis associated with apnoea and bradycardia (Wong et al. 2001). These findings might be explained by tight posttranscriptional or translational regulation of respiratory chain polypeptide synthesis, compared to relative overproduction of the mitochondrial tRNAs (Bruno et al. 1999). Another possibility is that unassembled

respiratory chain subunits might be degraded rapidly by mitochondrial proteases (Rep and Grivell, 1996). Finally, since functional COX exists as a dimer, a truncated COX subunit might exert a dominant negative effect by binding to a normal COX monomer to form a nonfunctional dimer. This means that a mutant load of 15%, for example, would lead to 28% defective COX holoenzyme (Wong et al. 2001).

Mutations involving mtDNA polypeptide-coding genes seem to present fairly frequently with isolated myopathy or even exercise intolerance alone. Such

mutations include two other mutations in COX subunit genes (Keightley et al. 1996; Karadimas et al. 2000), two mutations in ND genes of complex I (Andreu et al. 1999b; Musumeci et al. 2000) and 9 mutations in the cytochrome b subunit of complex III (reviewed by DiMauro and Andreu, 2000). Mutations in tRNA genes, on the other hand, are usually associated with multisystem features such as MELAS and MERRF syndromes (Goto et al. 1990; Shoffner et al. 1990). Exceptions to these generalisations include a CO III mutation associated with MELAS (Manfredi et al. 1995a) and two mutations in CO II: T7587C, which presented as an

encephalomyopathy with ataxia, dementia and optic atrophy (Clark et al. 1999); and G7896A, which was associated with an early onset multisystem disorder including encephalopathy and hypertrophic cardiomyopathy (Campos et al. 2001). Therefore there does not appear to be any correlation between which COX subunit is mutated and the clinical phenotype. The tissue distribution of the mutation is likely to be more important.

The lack of RRF in patient P26 is also of interest. The first mutations described in mtDNA polypeptide-coding genes were associated with LHON and NARP

(neurogenic muscle weakness, ataxia and retinitis pigmentosa) syndrome (Wallace et al. 1988; Holt et al. 1990). These patients did not have RRF and it was initially thought that lack of mitochondrial proliferation might be a general characteristic of polypeptide-coding mutations (Schon et al. 1997). Accordingly, there were no or very few RRF in some of the patients reported to have COX mutations (Manfredi et al. 1995a; Hanna et al. 1998b; Bruno et al. 1999). However, although P26 does not have RRF, he does have evidence of muscle mitochondrial proliferation with the SDH stain. Furthermore RRF were observed in two patients with mutations involving COX subunit genes (Keightley et al. 1996; Comi et al. 1998). Both these patients had microdeletions but the nature of the mutation cannot explain the difference in

muscle morphology since both groups contain mutations resulting in premature termination of translation (Hanna et al. 1998b; Comi et al. 1998; Bruno et al. 1999). The overall mutant load in skeletal muscle in patient P26 has not changed over a period of three years, and this is mirrored by clinical stability over this same period. Stability of mutant load was also observed in the patient with the stop mutation at nucleotide position 9952 in the mitochondrial CO III gene (Hanna et al. 1998b; see Chapter 4), and may be a common feature of mtDNA COX gene mutations. In contrast mutant load of mtDNA tRNA mutations has frequently been observed to increase with time in skeletal muscle (Fu et al. 1996; Weber et al. 1997). The explanation for this is not clear. Demonstration of low levels of mutation in peripheral blood cells from our patient may reflect clearance of mutation in this rapidly dividing tissue (Rahman et al. 2001). This is supported by the finding of lower mutant load (4.5%) in blood at 14 years compared to 6% at 11 years.

Absence of the T7671A mutation in maternal blood but presence in both muscle and blood of P26 suggests that the mutation may have arisen sporadically in early embryogenesis of this patient. However the possibility of presence of the mutation in the maternal germline cannot be excluded. The apparently sporadic nature of the T7671A mutation concurs with almost all other reported COX subunit mutations. An exception is the T7587C mutation in COX subunit II that was maternally inherited (Clark et al. 1999). Other maternally inherited polypeptide-coding mtDNA mutations include homoplasmic ND mutations associated with LHON and heteroplasmic mutations in the ATPase gene associated with NARP and maternally inherited Leigh syndrome. It is important to note the lack of maternal inheritance of most COX mutations, as defined by muscle mtDNA analysis, because this may lead to a delay in achieving a molecular diagnosis, and because it has significant implications for genetic counselling.

The T7671A mutation was not detected in cultured myoblasts, despite the high mutant load in mature skeletal muscle. Although it is possible that there was selection against satellite cells (undifferentiated muscle precursor cells) and

myoblasts containing high levels of mutation during the tissue culture process if they had a growth disadvantage, lack of mutation in satellite muscle cells has previously been reported with another COX point mutation (Hanna et al. 1998b) and also with tRNA point mutations (Fu et al. 1996; Weber et al. 1997). Absence of the mutation

from satellite cells has potential therapeutic implications. Previous studies have demonstrated that in patients with strong segregation of mutation between satellite and mature muscle cells, induction of muscle necrosis, either by bupivicaine local anaesthesia (Clark et al. 1997) or by local trauma (Shoubridge et al. 1997), was followed by repopulation of the muscle with cells containing only wild type mtDNA. It remains to be seen if induction of widespread muscle necrosis will be a viable therapeutic option for such patients. Eccentric exercise has been suggested as a therapeutic rationale to attempt to induce widespread muscle necrosis (Taivassalo et al. 1999) but so far there have not been any clinical trials of this treatment.

5.8.4 Conclusions

The T7671A mutation was one of the first mutations to be identified in the mtDNA gene for COX subunit II (Rahman et al. 1999). The data derived from this study and specifically the coexisting severe deficiency of COX subunit II and haem 8 3 indicate

that COX subunit II is required for stability of haem as attachment to the

holoenzyme. Further evidence for this role of COX subunit II in assembling the metal centres of the enzyme has come from studies of the aas-type COX in the prokaryote Rhodobacter sphaeroides (Bratton et al. 2000). Thus the clinical, morphological and biochemical consequences of the T7671A mutation provide valuable information about structure/function relationships within the COX holoenzyme.