The POLG Variant c.678G>C; p.(Gln226His) Is Associated with Mitochondrial Dysfunction in Patient-Derived Fibroblasts

Background: The nuclear encoded enzyme Polymerase gamma (Pol-γ) is crucial for the replication of the mitochondrial genome (mtDNA), which in turn is vital for mitochondria and hence nu-merous metabolic processes and energy production in eukaryotic cells. Variants in the POLG gene, which encodes the catalytic subunit of Pol-γ, can significantly impair Pol-γ enzyme function. Pol-γ-associated disorders are referred to as POLG-spectrum disorders (POLG-SD) and are mainly autosomal-recessively inherited. Clinical manifestations include seizures, muscle weakness and fatigue, severe forms of the disease can lead to premature death in infancy, childhood, and early adulthood, often associated with liver failure or intractable epilepsy. Here, we analyze fibroblasts from a compound heterozygous patient with the established pathogenic variant c.2419C>T; p.(Arg807Cys) and a previously undescribed variant c.678G>C; p.(Gln226His) with a clinical manifestation compatible with POLG-SD, sensory ataxic neuropathy and infantile muscular at-rophy. We conducted a battery of functional studies for Pol-γ and mitochondrial dysfunction on the patient’s fibroblasts, to test whether the novel variant c.678G>C; p.(Gln226His) may be causative for human disease. Aims/Methods: We analyzed skin-derived fibroblasts in comparison to a first degree relative, an asymptomatic carrier harboring only the established c.2419C>T; p.(Arg807Cys) mutation. As-sessments of mitochondrial function included measurements of mtDNA content, mRNA levels of mitochondrial genes, mitochondrial mass and mitochondrial morphology. Case Presentation and Results: A 13-year-old male presented with symptoms starting at three years of age, including muscle weakness and atrophy in the lower extremities and facial muscles, which later extended to the upper limbs, voice, and back muscles without further progression. The patient also reported fatigue and muscle pain after physical activity with no sensory deficits. Ex-tensive diagnostic tests such as electromyography, nerve conduction studies, muscle biopsy, and MRI were unremarkable. Exome sequencing revealed that he carries the compound heterozygous variants in POLG c.678G>C; p.(Gln226His) and c.2419C>T; p.(Arg807Cys), but no other potential genetic pathogenic causes. In comparison to a first degree relative who only carried the c.2419C>T; p.(Arg807Cys) pathogenic mutation, in vitro analyses revealed a significant reduction mtDNA content (~50%), and mRNA levels of mtDNA-encoded proteins. Mitochondrial mass was decreased by approximately 20%, and mitochondrial interconnectivity within cells was impaired, as de-termined by (immune-) fluorescence microscopy and mitochondrial stainings. Conclusion: Our findings suggest that the c.678G>C; p.(Gln226His) mutation, in conjunction with the c.2419C>T; p.(Arg807Cys) mutation, may compromise mtDNA replication and mitochondrial function and could result in clinically significant mitochondriopathy. As this study is based on one patient compared to a first- degree relative, future studies and cases must confirm pathogenicity of c.678G>C; p.(Gln226His), in particular in conjunction with other POLG-variants.