Mitochondrial DNA mutations, pathogenicity and inheritance

Abstract

Mitochondria contain their own DNA (mtDNA) which codes for 13 proteins (all subunits of the respiratory chain complexes), 22 tRNAs and 2 rRNAs. Several mtDNA point mutations as well as deletions have been shown to be causative in well-defined mitochondrial disorders. A mixture of mutated and wild type mtDNA (heteroplasmy) is found in most of these disorders. Inheritance of mtDNA is maternal, and mothers with heteroplasmic mtDNA transmit different proportions of normal and mutated mtDNA to the children. Mitochondrial tRNA genes have a central role in mitochondrial gene expression at the level of transcription, RNA processing and protein synthesis and they appear to be the mitochondrial genes most frequently affected by mutations causing diseases in man. The aim of this study was to identify new mutations in mtDNA and study pathogenicity and inheritance in patients with features of mitochondrial disorders of unknown cause. Southern-blot analysis, polymerase chain reaction (both standard and Expand long PCR), automatic sequencing, single muscle fibre analysis, cloning and in situ hybridisation methods were used to investigate the patients. Eleven new tRNA point mutations were identified in tRNA genes in nine patients. Nine mutations were homoplasmic and/or did not change nucleotides conserved between species and/or were present in healthy controls. Only two mutations (tRNALeu(UUR) A3251G and tRNALys G8328A) appeared to be pathogenic. Both mutations were heteroplasmic, affected a conserved nucleotide and were present in high percentage in affected muscle. There was genotype to phenotype correlation, and they did not occur in the healthy controls. An A8116G mutation was identified in the cytochrome c oxidase subunit II gene. The mutation was heteroplasmic and was present in high levels. The mutation does not change the aminoacid sequence, and was found in healthy family members. MtDNA with a large-scale deletion in muscle and duplication in lymphocytes was observed in a patient with mild encephalomyopathy. Inheritance of a T8993G, G8328A, A8116G and duplication mutation was studied. The T8993G mutation was identified in the mother but not in the grandmother of the proband. The G8328A mutation was found to be a de novo mutation and was not transmitted to the offspring. The same level of heteroplasmy of the A8116G mutation was found in the mother, two sisters and the proband. Duplicated mtDNA was not observed in the lymphocyte DNA of the mother and brother of the patient. In the tRNA genes, nine polymorphisms and two new pathogenic point mutations were identified in patients with mitochondrial disorders. Deleted but not duplicated mtDNA was found to be associated with expression of disease. It was demonstrated that the heteroplasmy with high levels of mutated mtDNA cannot be used as the sole evidence of pathogenicity. De novo mtDNA point mutations or major mtDNA rearrangements may be the cause of disease in sporadic cases of mitochondrial encephalopathy. Complete segregation of a mitochondrial mutation may occur in two generations or may be inherited at a stable heteroplasmic level.