High throughput single cell analysis of mitochondrial heteroplasmy in mitochondrial diseases

Kami

et al. 2021

Sci Rep

Self Cite

Mitochondrial diseases currently have no cure regardless of whether the cause is a nuclear or mitochondrial genome mutation. Mitochondrial dysfunction notably affects a wide range of disorders in aged individuals, including neurodegenerative diseases, cancers, and even senescence. Here, we present a procedure to generate mitochondrial DNA-replaced somatic cells with a combination of a temporal reduction in endogenous mitochondrial DNA and coincubation with exogeneous isolated mitochondria. Heteroplasmy in mitochondrial disease patient-derived fibroblasts in which the mutant genotype was dominant over the wild-type genotype was reversed. Mitochondrial disease patient-derived fibroblasts regained respiratory function and showed lifespan extension. Mitochondrial membranous components were utilized as a vehicle to deliver the genetic materials into endogenous mitochondria-like horizontal genetic transfer in prokaryotes. Mitochondrial DNA-replaced cells could be a resource for transplantation to treat maternal inherited mitochondrial diseases.

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Generation of somatic mitochondrial DNA-replaced cells for mitochondrial dysfunction treatment

Kami

et al. 2021

Sci Rep

Self Cite

“…One existing application is genetic mosaicism detection [18,20,28,29]. Other potential applications include monitoring of chimerism in patients after stem cell transplantation [30], analysis of mitochondrial heteroplasmy in mitochondrial diseases [31,32], rare microbe detection for microbiome pro ling [33] and con rmation of metagenomic sequencing results [34].…”

Section: Discussionmentioning

confidence: 99%

Confirming Putative Variants at ≤5% Allele Frequency Using Allele Enrichment and Sanger Sequencing

Yan¹,

Chen

Cheng

et al. 2021

Preprint

Whole exome sequencing (WES) is used to identify mutations in a patient’s tumor DNA that are predictive of tumor behavior, including the likelihood of response or resistance to cancer therapy. WES has a mutation limit of detection (LoD) at variant allele frequencies (VAF) of 5%. Putative mutations called at ≤5% VAF are frequently due to sequencing errors, therefore reporting these subclonal mutations incurs risk of significant false positives. Here we performed ~1000x WES on fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissue biopsy samples from a non-small cell lung cancer patient, and identified 226 putative mutations at between 0.5% and 5% VAF. Each variant was then tested using NuProbe NGSureTM, to confirm the original WES calls. NGSure utilizes Blocker Displacement Amplification to first enrich the allelic fraction of the mutation and then uses Sanger sequencing to determine mutation identity. Results showed that 52% of the 226 (117) putative variants were disconfirmed, among which 2% (5) putative variants were found to be misidentified in WES. In the 66 cancer-related variants, the disconfirmed rate was 82% (54/66). This data demonstrates Blocker Displacement Amplification allelic enrichment coupled with Sanger sequencing can be used to confirm putative mutations ≤5% VAF. By implementing this method, next-generation sequencing can reliably report low-level variants at a high sensitivity, without the cost of high sequencing depth.

“…Modern methods have been developed to detect directly or indirectly heteroplasmy in single cells. Such methods include an initial step of single cell isolation or propagation which is followed by massive parallel sequencing [30][31][32]. These advanced methods identify heteroplasmy at the single cell level but they cannot distinguish yet between levels four and five.…”

Section: The Hierarchical Levels For Studying Heteroplasmymentioning

confidence: 99%

mtDNA Heteroplasmy: Origin, Detection, Significance, and Evolutionary Consequences

Parakatselaki

Ladoukakis

2021

Life

Mitochondrial DNA (mtDNA) is predominately uniparentally transmitted. This results in organisms with a single type of mtDNA (homoplasmy), but two or more mtDNA haplotypes have been observed in low frequency in several species (heteroplasmy). In this review, we aim to highlight several aspects of heteroplasmy regarding its origin and its significance on mtDNA function and evolution, which has been progressively recognized in the last several years. Heteroplasmic organisms commonly occur through somatic mutations during an individual’s lifetime. They also occur due to leakage of paternal mtDNA, which rarely happens during fertilization. Alternatively, heteroplasmy can be potentially inherited maternally if an egg is already heteroplasmic. Recent advances in sequencing techniques have increased the ability to detect and quantify heteroplasmy and have revealed that mitochondrial DNA copies in the nucleus (NUMTs) can imitate true heteroplasmy. Heteroplasmy can have significant evolutionary consequences on the survival of mtDNA from the accumulation of deleterious mutations and for its coevolution with the nuclear genome. Particularly in humans, heteroplasmy plays an important role in the emergence of mitochondrial diseases and determines the success of the mitochondrial replacement therapy, a recent method that has been developed to cure mitochondrial diseases.