Complex IV (CIV) in the mitochondrial respiratory chain is composed of 13 subunits, three of which are encoded by genes in mitochondrial DNA (mtDNA). Cytochrome c oxidase II, MT-CO2, codes for structural subunit 2, which participates in the formation of the catalytic core of CIV. Rare mutations have been described in MT-CO2 in patients with various phenotypes, such as severe lactic acidosis [1], MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) [2], encephalomyopathy [3], or rhabdomyolysis [4]. The 10 nuclear genes encoding the remaining subunits are even more rarely mutated than the mitochondrial ones and only a handful of mutations have been described to date [5–10]. Most of the cases with complex IV deficiency harbor causative mutation in the genes encoding nonstructural factors [11, 12].

We describe a male patient with heteroplasmic de novo deletion, m.8156delG, in MT-CO2 leading to truncation of subunit 2 in cytochrome c oxidase (COX). The patient had a clinically apparent mitochondrial disorder with prominent findings in muscle histology.

The patient is a 30-year-old man with negative family history of neuromuscular disorders. Early childhood development was normal. Sensorineural hearing impairment with tinnitus and mild memory deficit were the first symptoms, which were noticed at the age of 12 years. Neuropsychological examination revealed a moderate cognitive decline at the age of 24 years. Retinitis pigmentosa was diagnosed at the age of 25 years and premature cataract at the age of 29 years. The bilateral cataracts were operated soon after the diagnosis. Generalized epilepsy and psychosis were diagnosed at the age of 29 years, but because of poor tolerance and compliance he does not use medications. Moreover, he has exercise intolerance and mild balance disorder. He lives alone with supervision.

Neurological examination at the age of 29 years revealed cognitive decline, clumsy movements, abnormal balance tests and impaired visual acuity. Hearing was impaired and he had hearing aid in both ears. Ophthalmoplegia was not found. Walking and muscle strength were normal. His height is 170 cm and weight 57 kg.

Blood lactate was first measured at the age of 23 years and was 3.06 mmol/l (laboratory reference, 0.33–1.33 mmol/l). Muscle histology at the age of 23 years revealed a substantial number of COX-deficient fibers and ragged red fibers (Fig. 1). Muscle ultrastructural analysis revealed large intramitochondrial inclusions. In brain MRI ventricular enlargement, moderate cerebellopontine atrophy and hyperintense signals in both basal ganglia were found. Cardiac examination and electrophysiological studies of the muscle and nerve were normal.

Novel mutation in MT-CO2

Sequencing of mtDNA revealed a novel frameshift mutation m.8156delG in the MT-CO2 gene of the proband (Fig. 2). The mutation was heteroplasmic and the proportion of the mutant genome was 26% in blood, 33% in epithelial cells from buccal swab and 95% in the skeletal muscle. The deletion was not detected in the blood or buccal mucosa from the mother or in the blood from the siblings of the patient suggesting de novo mutation. Single-fiber analysis showed that the proportion of the mutation required to cause a biochemical defect was high. Pooled blue COX-negative fibers contained 98% of mutant DNA. The proportion was 96% in COX-deficient fibers with intermediate color and 88% in biochemically normal fibers.

The mutation is predicted to lead to an altered amino acid sequence from position 191 and extending towards the C-terminus of the COX2 subunit. The mutation creates a termination codon resulting in premature truncation of the protein at amino acid 210, while the wild type protein consists of 227 amino acids. Three suggested copper binding sites are located in the affected region. The mtDNA belonged to haplogroup U8a1a1.

Four cell cultures were established from two muscle biopsies obtained on separate occasions. The first two cell cultures turned out to be fibroblasts with less than 20% of the mutant mtDNA. The other two cell cultures were desmin-positive verifying their muscular identity. Surprisingly, the mutation could not be detected in any of the subcultures.

We found a novel mutation m.8156delG that is suggested to result in an altered protein sequence and truncation of subunit COX2 in a patient with mitochondrial disorder. Previously, five mutations that are predicted to lead to truncated COX2 have been reported in patients with phenotypes of variable severity. In addition, six missense mutations in MT-CO2 have been reported in patients with variable phenotypes in MITOMAP database [15]. Furthermore, nine variants have been reported with disease associations, but later studies have classified them as polymorphisms.

A frameshift deletion m.8042_8043delTA is one of the truncating mutations destroying one third of the COX2 and resulting in severe lactic acidosis and death at the age of few days [1]. The mutation load was 20% in the skeletal muscle and complex IV activity was decreased to half of the normal. The presence of the truncated form of COX2 was not verified in tissues, however. In contrast, the m.7630delT deletion has been found in a patient with MELAS syndrome that was fatal only at the age of 23 years [2]. The predicted length of COX2 was 16 amino acids, mutation heteroplasmy was 93%, and residual activity of CIV was only a few percent of the normal. Moreover, the protein level of COX2 was substantially reduced, but mRNAs of CIV subunits including that of mutant COX2 were significantly increased. The presence of the mutant protein was not investigated. Furthermore, a m.7896G > A nonsense mutation with a heteroplasmy of 60–80% has been reported to result in a 104-amino-acid protein, to lead to 13% activity of complex IV and to cause a severe early-onset multisystem disorder [16]. The amount of wild type COX2 was reported to be decreased, but the truncated protein was not detected. Finally, the mutation p.Glu129* has been found in a patient with adolescence-onset hearing impairment and psychiatric disorder. Mutation heteroplasmy was 90% in skeletal muscle and the remaining activity of complex IV was 12% [3]. Again, the mutant form of the protein was not investigated. At age 25 years he had developed bilateral cataract and within few years, the syndrome progressed to include gait instability, exercise intolerance and cardiac arrhythmia. The patient died from cardiac arrest at the age of 35 years.

These four cases show that in the presence of a truncating mutation the activity of CIV is decreased. Interestingly, there is a lack of correlation between decreased activity and clinical severity suggesting that the truncated proteins may interfere with respiratory chain or other cellular processes by gaining an abnormal function. Unfortunately, the studies have failed to show the presence of truncated COX2 or the existence has not been investigated.

The G stretch spanning between the nucleotides m.8152–8156 in MT-CO2 has previously been found to be mutated in an adult patient with exercise-induced myoglobinuria and rhabdomyolysis [17]. This mutation was a heteroplasmic duplication m.8156dupG with a 56% mutation load in skeletal muscle causing decreased activity of CIV. In our patient, the predicted truncation of C-terminal sequence resulted in a mitochondrial disorder with various classical features of mitochondrial dysfunction. However, similar to the previous reports with frameshift mutations in MT-CO2, the presence of the truncated protein could not be verified.

The m.8156delG mutation was undetectable already in the first passages of the myoblast cultures despite of the high mutation load in the skeletal muscle showing that the mutation was present in highly differentiated myotubes. It is possible that the healthy cells benefitted from the culture conditions optimized for myoblasts leading to the disappearance of the mutant containing cells. Similar findings have previously been reported in a patient with m.9789 T > C in the MT-CO3 with a heteroplasmy of 50% [3] and in a patient with m.9952G > A with a heteroplasmy of 57% [18]. Furthermore, m.7671 T > A in MT-CO2 has been reported to be absent in myoblasts despite the very high heteroplasmy of 90% in the muscle [19]. In contrast, the m.6930G > A mutation in MT-CO1 has been found at 33% heteroplasmy in myoblasts, while the mutation heteroplasmy was 75% in muscle [20]. Furthermore, heteroplasmic disease-causing deletions have been found to be present only in a minority of myoblast subcultures, even though the proportion of the deletion in the satellite cells was shown to be similar to that in the skeletal muscle [21]. In that study, the heteroplasmy decreased during further culturing and differentiation to myotubes. The authors suggested that there might be two periods of selection against the mutated mtDNA, the first occurring during satellite cell activation and second in the replication phase [21].

Frameshift mutations in MT-CO2 are rare and clinically variable. We presented here a patient with a progressive mitochondrial syndrome and a novel frameshift mutation m.8156delG in MT-CO2 as a cause. This case illustrates the importance of mtDNA sequence analysis in patients with a suspected mitochondrial disorder.