EP.02Deep sequencing of mitochondrial DNA and functional characterization of a novel POLG mutation in a patient with autosomal recessive progressive external ophthalmoplegia

Progressive external ophthalmoplegia (PEO) can be caused by mutations in the POLG gene, encoding the alpha subunit of polymerase gamma (pol γ). Pol γ consists of the 140 kDa catalytic subunit, POLγA, containing polymerase- and exonuclease-activities and a dimer of POLγB (POLG2) the 55 kDa processivity factor. This trimeric protein is a DNA polymerase and is essential for replication of mitochondrial DNA (mtDNA). We describe a 69 years old male patient who at age 55 he noted slowly progressive bilateral ptosis and also a limitation in both horizontal and vertical gaze. Since the age of 65 he has had complete horizontal opthalmoplegia, no upward gaze whatsoever, but limited downward gaze. A muscle biopsy from the deltoid muscle at age 66 showed slight variability in muscle fiber size, scattered ragged red fibers and 10-20% fibers showing cytochrome c oxidase deficiency. He was compound heterozygous for POLG mutations: a novel c.590T>C; p.(F197S) mutation in the exonuclease domain and a previously described c.2740A>C; p.(T914P) mutation in the polymerase domain. By deep sequencing it was possible to identify and characterize multiple large-scale mtDNA rearrangements and a small but significant increase in somatic point mutations. Biochemical in vitro analyses of the mutant proteins showed that p.(T914P) had no detectable activity, consistent with its previous characterization. The novel p.(F197S) substitution resulted in an active protein with reduced exonuclease as well as polymerase activities. The reduced polymerase activity of both mutant proteins may explain the multiple rearrangements by stalling and slipped mispairing during replication. The reduced exonuclease activity of p.(F197S) may explain the occurrence of multiple somatic point mutations at low frequency. Our study confirms the pathogenicity and recessive nature of the mutations and expands the molecular understanding of the patient's phenotype.