Identification of unique and shared mitochondrial DNA mutations in neurodegeneration and cancer by single-cell mitochondrial DNA structural variation sequencing (MitoSV-seq)

Abstract

Background: Point mutations and structural variations (SVs) in mitochondrial DNA (mtDNA) contribute to many neurodegenerative diseases. Technical limitations and heteroplasmy, however, have impeded their identification, preventing these changes from being examined in neurons in healthy and disease states. Methods: We have developed a high-resolution technique-Mitochondrial DNA Structural Variation Sequencing (MitoSV-seq)-that identifies all types of mtDNA SVs and single-nucleotide variations (SNVs) in single neurons and novel variations that have been undetectable with conventional techniques. Findings: Using MitoSV-seq, we discovered SVs/SNVs in dopaminergic neurons in the Ifnar1 À/À murine model of Parkinson disease. Further, MitoSV-seq was found to have broad applicability, delivering high-quality, full-length mtDNA sequences in a species-independent manner from human PBMCs, haematological cancers, and tumour cell lines, regardless of heteroplasmy. We characterised several common SVs in haematological cancers (AML and MDS) that were linked to the same mtDNA region, MT-ND5, using only 10 cells, indicating the power of MitoSV-seq in determining single-cancer-cell ontologies. Notably, the MT-ND5 hotspot, shared between all examined cancers and Ifnar1 À/À dopaminergic neurons, suggests that its mutations have clinical value as disease biomarkers. Interpretation: MitoSV-seq identifies disease-relevant mtDNA mutations in single cells with high resolution, rendering it a potential drug screening platform in neurodegenerative diseases and cancers.

Unsupervised reference-free inference reveals unrecognized regulated transcriptomic complexity in human single cells

“…Mitochondrial genes also had high diversity, highest in an anchor with compactors mapping to MT-ND5 , with 24 compactors in donor 1 lung, the greatest number of compactors generated in a single donor-tissue (Table 3) by any anchor with compactors mapping to an annotated gene. MT-ND5 is a component of the transmembrane electron transport chain in mitochondria with previously reported recurrent mutations with clinical significance (Jaberi et al 2020; Wang et al 2022).…”

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

“…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.…”

“…More recently, various next-generation sequencing (NGS) methods have revolutionized the study of heteroplasmy because they could detect and quantify very rare heteroplasmic variants using a great variety of sophisticated experimental techniques and bioinformatic tools [ 54 , 55 , 56 , 57 , 58 ]. The NGS methods revealed the extent of heteroplasmy in tissues and organisms, gave insight on its causes, and identified its role in mitochondrial diseases and aging [ 6 , 30 , 32 ]. Most of the modern methodological strategies include the generation of mtDNA-enriched libraries either with capture- or with targeted PCR-based methods, followed by NGS.…”

Isolation of highly purified genomic material from mitochondria of muscle tissue cells

“…Harmful mutations in the mitochondrial genome can be compensated for by an increased copy number of the mitochondrial genome 1 .…”

“…Next, we focus on a high level of variation in mtDNA heteroplasmy, which is unexplained by age. Harmful mutations in the mitochondrial genome can be compensated for by an increased copy number of the mitochondrial genome 1…”