Characterization of the human homozygous R182W POLG2 mutation in mitochondrial DNA depletion syndrome

Abstract

Mutations in mitochondrial DNA (mtDNA) have been linked to a variety of metabolic, neurological and muscular diseases which can present at any time throughout life. MtDNA is replicated by DNA polymerase gamma (Pol γ), twinkle helicase and mitochondrial single-stranded binding protein (mtSSB). The Pol γ holoenzyme is a heterotrimer consisting of the p140 catalytic subunit and a p55 homodimeric accessory subunit encoded by the nuclear genes POLG and POLG2, respectively. The accessory subunits enhance DNA binding and promote processive DNA synthesis of the holoenzyme. Mutations in either POLG or POLG2 are linked to disease and adversely affect maintenance of the mitochondrial genome, resulting in depletion, deletions and/or point mutations in mtDNA. A homozygous mutation located at Chr17: 62492543G>A in POLG2, resulting in R182W substitution in p55, was previously identified to cause mtDNA depletion and fatal hepatic liver failure. Here we characterize this homozygous R182W p55 mutation using in vivo cultured cell models and in vitro biochemical assessments. Compared to control fibroblasts, homozygous R182W p55 primary dermal fibroblasts exhibit a two-fold slower doubling time, reduced mtDNA copy number and reduced levels of POLG and POLG2 transcripts correlating with the reported disease state. Expression of R182W p55 in HEK293 cells impairs oxidative-phosphorylation. Biochemically, R182W p55 displays DNA binding and association with p140 similar to WT p55. R182W p55 mimics the ability of WT p55 to stimulate primer extension, support steady-state nucleotide incorporation, and suppress the exonuclease function of Pol γ in vitro. However, R182W p55 has severe defects in protein stability as determined by differential scanning fluorimetry and in stimulating function as determined by thermal inactivation. These data demonstrate that the Chr17: 62492543G>A mutation in POLG2, R182W p55, severely impairs stability of the accessory subunit and is the likely cause of the disease phenotype.

Therapy Prospects for Mitochondrial DNA Maintenance Disorders

“…Moreover, the symptoms are associated with the age of onset, with feeding difficulties and seizures being the most common symptoms in people under 12 years, and ataxia, peripheral neuropathy and seizures in patients between 12 and 40 years of age and ptosis, PEO and ataxia in patients with onset above 40 years [ 53 ]. Mutations in POLG2 , encoding the ancillary subunit of polymerase gamma, are much rarer than those found in POLG , but since the number of patients with new pathogenic mutations in POLG2 is increasing, it has become evident that the range of phenotypes is also variable [ 17 , 54 ], including fulminant cases [ 55 , 56 ].…”

Mitochondrial Dysfunction in Aging and Cancer

“…Another example linking mtDNA to the aging phenotype comes from human subjects carrying mutations in POLγ. More than 300 pathogenic mutations in human POLγ have been identified so far (), and they have been linked to several disorders, including age-related pathologies such as Parkinson’s disease [10,48,49].…”

POLG2-Linked Mitochondrial Disease: Functional Insights from New Mutation Carriers and Review of the Literature

“…The first patient was a 3-month-old boy carrying the homozygous missense variant c.544C > T (p.Arg182Trp). He suffered from a mitochondrial DNA depletion syndrome manifesting as a fulminant liver failure with consecutive death [ 6 , 26 ]. Other biallelic POLG2 mutations in two independent individuals have been linked to epilepsy [ 27 ] and a combination of childhood-onset optic atrophy, cerebellar ataxia, peripheral neuropathy, psychiatric comorbidities, and premature ovarian failure [ 28 ], respectively (Table S 3 ).…”