Tweet
A team of international scientists utilized high-throughput single-cell single-mitochondrial genome sequencing technology to quantitatively investigate the genetic landscape of mitochondrial DNA (mtDNA) in single human oocytes and blastoids. This method, called individual Mitochondrial Genome sequencing (iMiGseq), has allowed the researchers to identify rare variants previously undetected in healthy cells and provide new insights into mtDNA genetics.
Investigating mitochondrial genomes is crucial to understanding heteroplasmic mtDNA mutations inherited from oocytes (eggs cells), which are common causes of metabolic diseases and often associated with late-onset diseases. Previous mtDNA studies have relied on short-read technologies, amplicon sequencing, and bulk sequencing methods, which have limited variant discovery and cell-to-cell heterogeneity.
“Next-generation sequencing has been used to sequence mtDNA and implicated heteroplasmic mutations as significant contributors to metabolic disease,” said Chongwei Bi, the study’s lead author. “Yet the understanding of mtDNA mutations remains limited due to the constraints of traditional sequencing technologies.”
“Our new iMiGseq method is significant because it enables complete sequencing of individual mtDNA in single cells, allowing for unbiased, high-throughput base-resolution analysis of full-length mtDNA,” Bi added. Utilizing Oxford Nanopore’s long-read technologies, the team was able to characterize mtDNA heteroplasmy from single cells, as well as determine individual genetic features of mtDNA in single oocytes.
After examining mtDNA in induced pluripotent stem cells from patients with conditions such as Leigh syndrome or neuropathy, ataxia or retinitis pigmentosa (NARP), they discovered many complex pathogenic mutation patterns. “We were able to detect rare mutations with frequencies far below the traditional detection threshold of one percent,” said the study’s leader Mo Li from King Abdullah University of Science and Technology (KAUST).
The team’s additional efforts involving iMiGseq exposed the risks of off-target mutations in the mitochondrial genome editing method, mitoTALEN. This tool is used to edit mtDNA by cutting specific sequences, particularly cutting at mutation sites known to cause mitochondrial encephalomyopathy and stroke-like episodes in induced pluripotent stem cells from patients.
Li explained that, “This highlights the advantages of full-length mtDNA haplotype analysis for understanding mitochondrial DNA heteroplasmy change. Other distant mtDNA genetic variants may be unintentionally affected by the editing of a genetically linked disease-relevant mutation and there is a need for ultrasensitive methods to assess the safety of editing strategies.”
Additionally, the research team applied iMiGseq to investigate single human oocytes from known healthy donors and synthetic embryos derived from stem cells (single human blastoids). This allowed the researchers to identify different types of rare mutations that were previously undetectable using conventional sequencing methods. Their identification shows that these low-level disease-associated heteroplasmic mutations commonly exist in single healthy human oocytes and can be potentially inherited.
The application of iMiGseq has broadened our understanding of mtDNA mutations and offers researchers a chance to obtain insights into mitochondrial-related diseases. The authors believe this method could be further used for preimplantation genetic diagnosis of mitochondrial diseases, particularly in in vitro fertilization settings.
Both studies related to this work are published in Nucleic Acids Research.
Chongwei Bi et al, Quantitative haplotype-resolved analysis of mitochondrial DNA heteroplasmy in Human single oocytes, blastoids, and pluripotent stem cells, Nucleic Acids Research (2023). DOI: 10.1093/nar/gkad209
Chongwei Bi et al, Single-cell individual full-length mtDNA sequencing by iMiGseq uncovers unexpected heteroplasmy shifts in mtDNA editing, Nucleic Acids Research (2023). DOI: 10.1093/nar/gkad208
Investigating mitochondrial genomes is crucial to understanding heteroplasmic mtDNA mutations inherited from oocytes (eggs cells), which are common causes of metabolic diseases and often associated with late-onset diseases. Previous mtDNA studies have relied on short-read technologies, amplicon sequencing, and bulk sequencing methods, which have limited variant discovery and cell-to-cell heterogeneity.
“Next-generation sequencing has been used to sequence mtDNA and implicated heteroplasmic mutations as significant contributors to metabolic disease,” said Chongwei Bi, the study’s lead author. “Yet the understanding of mtDNA mutations remains limited due to the constraints of traditional sequencing technologies.”
“Our new iMiGseq method is significant because it enables complete sequencing of individual mtDNA in single cells, allowing for unbiased, high-throughput base-resolution analysis of full-length mtDNA,” Bi added. Utilizing Oxford Nanopore’s long-read technologies, the team was able to characterize mtDNA heteroplasmy from single cells, as well as determine individual genetic features of mtDNA in single oocytes.
After examining mtDNA in induced pluripotent stem cells from patients with conditions such as Leigh syndrome or neuropathy, ataxia or retinitis pigmentosa (NARP), they discovered many complex pathogenic mutation patterns. “We were able to detect rare mutations with frequencies far below the traditional detection threshold of one percent,” said the study’s leader Mo Li from King Abdullah University of Science and Technology (KAUST).
The team’s additional efforts involving iMiGseq exposed the risks of off-target mutations in the mitochondrial genome editing method, mitoTALEN. This tool is used to edit mtDNA by cutting specific sequences, particularly cutting at mutation sites known to cause mitochondrial encephalomyopathy and stroke-like episodes in induced pluripotent stem cells from patients.
Li explained that, “This highlights the advantages of full-length mtDNA haplotype analysis for understanding mitochondrial DNA heteroplasmy change. Other distant mtDNA genetic variants may be unintentionally affected by the editing of a genetically linked disease-relevant mutation and there is a need for ultrasensitive methods to assess the safety of editing strategies.”
Additionally, the research team applied iMiGseq to investigate single human oocytes from known healthy donors and synthetic embryos derived from stem cells (single human blastoids). This allowed the researchers to identify different types of rare mutations that were previously undetectable using conventional sequencing methods. Their identification shows that these low-level disease-associated heteroplasmic mutations commonly exist in single healthy human oocytes and can be potentially inherited.
The application of iMiGseq has broadened our understanding of mtDNA mutations and offers researchers a chance to obtain insights into mitochondrial-related diseases. The authors believe this method could be further used for preimplantation genetic diagnosis of mitochondrial diseases, particularly in in vitro fertilization settings.
Both studies related to this work are published in Nucleic Acids Research.
Chongwei Bi et al, Quantitative haplotype-resolved analysis of mitochondrial DNA heteroplasmy in Human single oocytes, blastoids, and pluripotent stem cells, Nucleic Acids Research (2023). DOI: 10.1093/nar/gkad209
Chongwei Bi et al, Single-cell individual full-length mtDNA sequencing by iMiGseq uncovers unexpected heteroplasmy shifts in mtDNA editing, Nucleic Acids Research (2023). DOI: 10.1093/nar/gkad208