Deep sequencing of mitochondrial genomes reveals increased mutation load in Friedreich's ataxia

Abstract

ObjectiveFriedreich's ataxia (FRDA) is an autosomal recessive trinucleotide repeat expansion disorder caused by epigenetic silencing of the frataxin gene (FXN). Current research suggests that damage and variation of mitochondrial DNA (mtDNA) contribute to the molecular pathogenesis of FRDA. We sought to establish the extent of the mutation burden across the mitochondrial genome in FRDA cells and investigate the molecular mechanisms connecting FXN downregulation and the acquisition of mtDNA damage.MethodsDamage and mutation load in mtDNA of a panel of FRDA and control fibroblasts were determined using qPCR and next‐generation MiSeq sequencing, respectively. The capacity of FRDA and control cells to repair oxidative lesions in their mtDNA was measured using a quantitative DNA damage assay. Comprehensive RNA sequencing gene expression analyses were conducted to assess the status of DNA repair and metabolism genes in FRDA cells.ResultsAcute or prolonged downregulation of FXN expression resulted in a significant increase in mtDNA damage that translated to a significant elevation of mutation load in mtDNA. The predominant mutations identified throughout the mtDNA were C>T, G>A transitions (P = 0.007). Low FXN expression reduced capacity to repair oxidative damage in mtDNA. Downregulation of FXN expression strongly correlated (r = 0.73) with decreased levels of base excision repair (BER) DNA glycosylase NTHL1.InterpretationDownregulation of FXN expression in FRDA cells elevates mtDNA damage, increases mutation load of the mitochondrial genome, and diminishes DNA repair capacity. Progressive accumulation of mtDNA mutations in vulnerable FRDA patient cells reduces mitochondrial fitness ultimately leading to cell death.

Comprehensive analysis of gene expression patterns in Friedreich's ataxia fibroblasts by RNA sequencing reveals altered levels of protein synthesis factors and solute carriers

“…As previously reported, a number of genes involved in DNA replication and repair are more under-expressed in FRDA cells relative to CTRL cells ( ) ( Bhalla et al, 2016 ; Coppola et al, 2011 ; Haugen et al, 2010 ; Martelli et al, 2007 ). Included in the list of downregulated genes are those encoding the nuclear and mitochondrial DNA glycosylases [ NTHL1 (nth like DNA glycosylase 1), P =5.31E–17; MPG (N-methylpurine DNA glycosylase), P =0.0001; OGG1 (8-oxoguanine DNA glycosylase), P =0.004], DNA endonucleases [ ERCC1 (ERCC excision repair 1, endonuclease non-catalytic subunit), P =0.001; ERCC3 (ERCC excision repair 3, TFIIH core complex helicase subunit), P =0.008], and the DNA damage and replication stress checkpoint protein RAD17 [ RAD17 ( RAD17 checkpoint clamp loader component), P =0.006].…”

Excision of the expanded GAA repeats corrects cardiomyopathy phenotypes of iPSC-derived Friedreich's ataxia cardiomyocytes

“…For mtDNA quantitation, genomic DNA was isolated as above. A qPCR quantitation of mtDNA copy number was performed using primers specific to mtDNA sequence or genomic DNA (Bhalla et al, 2016). Abundance of mtDNA relative to genomic DNA was calculated using the 2 −ΔΔCT method (Bhalla et al, 2016).…”

Mitochondrial damage and senescence phenotype of cells derived from a novel frataxin G127V point mutation mouse model of Friedreich's ataxia

“…Indeed, our previous study in FRDA fibroblasts found that low FXN levels led to increased mtDNA damage and decreased mtDNA repair capacity (Bhalla et al, 2016). To determine whether the integrity of mtDNA was compromised in cells expressing only Fxn G127V , we analyzed mtDNA isolated from WT and MUT MEFs using a quantitative PCR assay based on the principle that DNA lesions can slow down or block the progression of DNA polymerase (Bhalla et al, 2016;Ponti et al, 1991;Redmann et al, 2018). This type of assay detects various forms of DNA damage, from single-or double-strand DNA breaks and gaps to specific chemical lesions (Lehle et al, 2014).…”

“…Frataxin deficiency is also linked with mitochondrial DNA (mtDNA) damage (Haugen et al, 2010;Karthikeyan et al, 2003). Indeed, our previous study in FRDA fibroblasts found that low FXN levels led to increased mtDNA damage and decreased mtDNA repair capacity (Bhalla et al, 2016). To determine whether the integrity of mtDNA was compromised in cells expressing only Fxn G127V , we analyzed mtDNA isolated from WT and MUT MEFs using a quantitative PCR assay based on the principle that DNA lesions can slow down or block the progression of DNA polymerase (Bhalla et al, 2016;Ponti et al, 1991;Redmann et al, 2018).…”