Heterozygous p.Y955C mutation in DNA polymerase γ leads to alterations in bioenergetics, complex I subunit expression, and mtDNA replication.

In human cells, ATP is generated using oxidative phosphorylation machinery, which is inoperable without proteins encoded by mitochondrial DNA (mtDNA). The DNA polymerase gamma (Polγ) repairs and replicates the multicopy mtDNA genome in concert with additional factors. The Polγ catalytic subunit is encoded by the POLG gene, and mutations in this gene cause mtDNA genome instability and disease. Barriers to studying the molecular effects of disease mutations include scarcity of patient samples and a lack of available mutant models; therefore, we developed a human SJCRH30 myoblast cell line model with the most common autosomal dominant POLG mutation, c.2864A>G/p.Y955C, as individuals with this mutation can present with progressive skeletal muscle weakness. Using on-target sequencing, we detected a 50% conversion frequency of the mutation, confirming heterozygous Y955C substitution. We found mutated cells grew slowly in a glucose-containing medium and had reduced mitochondrial bioenergetics compared with the parental cell line. Furthermore, growing Y955C cells in a galactose-containing medium to obligate mitochondrial function enhanced these bioenergetic deficits. Also, we show complex I NDUFB8 and ND3 protein levels were decreased in the mutant cell line, and the maintenance of mtDNA was severely impaired (i.e., lower copy number, fewer nucleoids, and an accumulation of Y955C-specific replication intermediates). Finally, we show the mutant cells have increased sensitivity to the mitochondrial toxicant 2'-3'-dideoxycytidine. We expect this POLG Y955C cell line to be a robust system to identify new mitochondrial toxicants and therapeutics to treat mitochondrial dysfunction.

Analysis of Mitochondrial DNA Polymorphisms in the Human Cell Lines HepaRG and SJCRH30.

The mitochondrial DNA (mtDNA) sequences of two commonly used human cell lines, HepaRG and SJCRH30, were determined. HepaRG originates from a liver tumor obtained from a patient with hepatocarcinoma and hepatitis C while SJCRH30 originates from a rhabdomyosarcoma patient tumor. In comparison to the revised Cambridge Reference Sequence, HepaRG and SJCRH30 mtDNA each contain 14 nucleotide variations. In addition to an insertion of a cytosine at position 315 (315insC), the mtDNA sequences from both cell types share six common polymorphisms. Heteroplasmic variants were identified in both cell types and included the identification of the 315insC mtDNA variant at 42 and 75% heteroplasmy in HepaRG and SJCRH30, respectively. Additionally, a novel heteroplasmic G13633A substitution in the HepaRG ND5 gene was detected at 33%. Previously reported cancer-associated mtDNA variants T195C and T16519C were identified in SJCRH30, both at homoplasmy (100%), while HepaRG mtDNA harbors a known prostate cancer-associated T6253C substitution at near homoplasmy, 95%. Based on our sequencing analysis, HepaRG mtDNA is predicted to lie within haplogroup branch H15a1 while SJCRH30 mtDNA is predicted to localize to H27c. The catalog of polymorphisms and heteroplasmy reported here should prove useful for future investigations of mtDNA maintenance in HepaRG and SJCRH30 cell lines.