Mutations of mitochondrial DNA polymerase gamma: an important cause of neurological disorders

The central themes of this thesis are mutations of the POLG1 gene encoding the catalytic subunit of mitochondrial DNA polymerase gamma protein, pol , their association with the wide spectrum of clinical phenotypes and tissue specificity and the biochemical characterization of particular defective mutant variants in the protein’s spacer region. PolG-holoenzyme is the sole DNA polymerase found in mitochondria. It is involved in replication and repair of the mitochondrial genome, mtDNA. Holoenzyme also includes the accessory subunit pol , which is required for the processivity of pol . Defective pol causes accumulation of secondary mutations on mtDNA, which leads to a defective oxidative phosphorylation system. The clinical consequences of such mutations are variable, affecting nervous system, skeletal muscles, liver and other post-mitotic tissues. In 2001, three PEO (Progressive External Ophthalmoplegia) -families were reported to carry either recessive or dominant mutations in the POLG1 gene. Shortly thereafter, an Italian group found novel POLG1 mutations in several Italian PEO-families. These findings prompted us to study the role of POLG1 in patients with putative mitochondrial diseases. The aims of the studies were: 1) Determination of the role of POLG1 mutations in neurological syndromes with features of mitochondrial dysfunction and an unknown molecular cause. 2) Development and set up of diagnostic tests for routine clinical purposes. 3) Biochemical characterization of the functional consequences of the identified pol variants. Clinical, molecular genetic and biochemical studies were conducted. Clinical examinations consisted of detailed neurological assessments including MRI and PET scans. Most of the genetic studies were done with PCR-based methods, including DNA sequencing, solid-phase minisequencing and denaturing highpressure liquid chromatography (dHPLC). Biochemical characterization was accomplished with the aim of baculovirus-based recombinant protein expression and subsequent purification. In vitro assays included polymerase activity, DNA binding, and processivity measurements. Studies describe new neurological phenotypes in addition to PEO caused by POLG1 mutations, including parkinsonism, premature amenorrhea, ataxia and Parkinson’s disease (PD). POLG1 mutations and polymorphisms are both common and potential genetic risk factors at least among the Finnish population. The major findings and applications reported here are: 1) POLG1 mutations cause parkinsonism and premature menopause in PEO families in either a recessive or a dominant manner. (Study I) 2) The common recessive POLG1 mutations (A467T and W748S) in the homozygous state causes severe adult or juvenile-onset spinocerebellar ataxia without muscular symptoms or histological or mtDNA abnormalities in muscles. (Study III) 3) A common recessive pathogenic change A467T can also cause a mild dominant disease in heterozygote carriers. (Study IV) 4) The A467T variant shows reduced polymerase activity due to defective template binding. (Study IV) 5) Rare polyglutamine tract length variants of POLG1 are significantly enriched in Finnish idiopathic Parkinson’s disease patients. (Study II) 6) Dominant mutations are clearly restricted to the highly conserved polymerase domain motifs, whereas recessive ones are more evenly distributed along the pol protein. (Studies I,III,IV) The present results highlight and confirm the new role of mitochondria in parkinsonism/Parkinson’s disease and describe a new mitochondrial ataxia. Based on these results, a POLG1 diagnostic routine has been set up in Helsinki University Central Hospital (HUSLAB).