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.
Molecular methods and cell culture
DNA extraction from blood, buccal swab and myoblasts was done by using QIAamp DNA Blood Mini Kit (QIAGEN, Hilden, Germany). From muscle biopsy, the purification was done with Wizard® Genomic DNA purification kit (Promega Corporation, Madison, WI). The mitochondrial DNA was amplified and sequenced in 12 overlapping fragments.
Mutation heteroplasmy in tissues from the patient, his mother and the four siblings was determined by cloning as described previously [13]. One hundred colonies per cloned sample were screened for the mutation with the conformation sensitive gel electrophoresis.
A muscle sample was obtained from vastus lateralis and myoblast culture was established as described previously [14]. Cells were immunostained with myoblast-specific antibody for desmin (BioGenex, Fremont, CA, U.S.A.).
Laser-capture microdissection of COX-SDH stained frozen sections was done using Zeiss P.A.L.M. microscope (Microlaser Technologies GmbH, Bernried, Germany) in Turku Centre for Biotechnology. Ten COX-negative, COX-positive and COX-deficient fibers were collected in tubes with adhesive caps (Carl Zeiss MicroImaging GmbH, Munich, Germany). DNA was released from the ten pooled fibers of each COX phenotype using lysis buffer (200 mM KOH, 50 mM DTT) and 30 min incubation at 65 °C. The lysis reaction was then neutralized with Tris-HCl (900 mM, pH 8.3) and 4 μl of the solution was used for PCR. PCR was carried out using Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific, Waltham, MA, U.S.A.) in a 50 μl reaction that was established according to the provided protocol. The reaction conditions are available on request. The heteroplasmy was determined by cloning.
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.