New technology reveals hidden mitochondrial DNA mutations

Human blastocyst like a synthetic embryo called blastoid

A human blastocyst-like synthetic embryo called the blastoid, showing the presence of an enveloping layer of extraembryonic cells, a blastocoel-like cavity, epiblast cells (green, which produce the future embryo) and hypoblast cells (red, which produce the future embryo) . iMiGSeq was used to sequence mtDNA in a single blastoid to model the dynamics of mtDNA mutations during human embryogenesis. Photo credits: © 2023 KAUST; Mo Li

A high-throughput single-cell, single-mitochondrial genome sequencing technology called iMiGseq has provided new insights into mitochondrial mutations[{” attribute=””>DNA (mtDNA) and offers a platform for assessing mtDNA editing strategies and genetic diagnosis of embryos prior to their implantation.

The development of a new high-throughput single-cell single-mitochondrial genome sequencing technology, called iMiGseq, has enabled researchers to uncover previously hidden mutations in mitochondrial DNA (mtDNA) that cause maternally inherited diseases. By allowing for complete sequencing of individual mtDNA in single cells, the iMiGseq method has provided a platform for assessing mtDNA editing strategies, genetic diagnosis of embryos prior to implantation, and understanding the links between mtDNA mutations and complex diseases. The technology has also revealed complex patterns of pathogenic mtDNA mutations, including single nucleotide variants and large structural variants, that were undetectable with conventional next-generation sequencing. Additionally, iMiGseq has shown the potential risks of unintended off-target mutations in a mitochondrial genome editing method called mitoTALEN, highlighting the need for more sensitive methods to assess the safety of editing strategies.

An international team of researchers, led by KAUST stem cell biologist Mo Li, has now quantitatively depicted the genetic maps of mtDNA in single human oocytes (immature eggs) and blastoids (stem cell-based synthetic embryos).[1] It uncovered molecular features of rare mtDNA mutations that cause maternally inherited diseases.

Mitochondria, the “power plants” of the cells, play a crucial role in cellular communication and metabolism. Human mtDNA is a circular genome containing 37 genes encoding 13 proteins and a D-loop noncoding region. Heteroplasmic mutations inherited from oocytes can cause congenital diseases such as maternally inherited Leigh syndrome and are associated with late-onset complex diseases.

“Next-generation sequencing has been used to sequence mtDNA and implicated heteroplasmic mutations as significant contributors to metabolic diseases. However, understanding of mtDNA mutations remains limited due to the limitations of traditional sequencing technologies,” says lead author Chongwei Bi.

“Our new iMiGseq method is significant because it enables complete sequencing of individual mtDNA in single cells, enabling unbiased, high-throughput base-resolution analysis of full-length mtDNA,” says Bi. iMiGseq solves several key questions in the field.

Using third-generation nanopore sequencing technology, researchers characterized mtDNA heteroplasmy in single cells and described the genetic characteristics of mtDNA in single oocytes. They studied mtDNA in induced pluripotent stem cells derived from patients with Leigh syndrome or neuropathy, ataxia or retinitis pigmentosa (NARP). This has revealed complex patterns of pathogenic mtDNA mutations, including single nucleotide variants and large structural variants. “We were able to detect rare mutations with frequencies far below the traditional detection threshold of one percent,” says Mo Li.

In another experiment using the new technology, iMiGseq uncovered the potential risks of an unexpected sharp increase in the frequency of off-target mutations, known as heteroplasmy, in a mitochondrial genome editing method called mitoTALEN – a genome editing tool that cuts a specific sequence in mitochondrial DNA. It is used to cut a mutation that causes mitochondrial encephalomyopathy and stroke-like episode syndrome in patient-derived induced pluripotent stem cells.

“This underscores the benefits of full-length mtDNA haplotype analysis for understanding mitochondrial DNA heteroplasmy alteration; other distant genetic mtDNA variants can be inadvertently affected by editing a genetically linked disease-causing mutation, and there is a need for highly sensitive methods to assess the safety of editing strategies,” says Li.

Researchers also used iMiGseq to analyze single human oocytes from healthy donors and single human blastoids, synthetic embryos derived from stem cells, to identify rare mutations undetectable with conventional next-generation sequencing. These low-level heteroplasmic mutations, which may be inherited through the female germline, are associated with mitochondrial diseases and cancer.[2]

The iMiGseq method provides a novel means to accurately map the complete haplotypes of individual mtDNAs in single cells and provides an ideal platform for elucidating the etiology of mitochondrial mutant diseases, evaluating the safety of different mtDNA editing strategies, and deciphering the connections between mtDNA and Mutations, aging and the development of complex diseases.

References:

  1. “Quantitative haplotype-resolved analysis of mitochondrial DNA heteroplasmy in human single oocytes, blastoids and pluripotent stem cells” by Chongwei Bi, Lin Wang, Yong Fan, Baolei Yuan, Samhan Alsolami, Yingzi Zhang, Pu-Yao Zhang, Yanyi Huang, Yang Yu , Juan Carlos Izpisua Belmonte and Mo Li, April 4, 2023, Nucleic Acid Research.
    DOI: 10.1093/nar/gkad209
  2. “Single-cell individual full-length mtDNA sequencing by iMiGseq reveals unexpected heteroplasmy shifts in mtDNA editing” by Chongwei Bi, Lin Wang, Yong Fan, Baolei Yuan, Gerardo Ramos-Mandujano, Yingzi Zhang, Samhan Alsolami, Xuan Zhou, Jincheng Wang , Yanjiao Shao, Pradeep Reddy, Pu-Yao Zhang, Yanyi Huang, Yang Yu, Juan Carlos Izpisua Belmonte and Mo Li, March 31, 2023, Nucleic Acid Research.
    DOI: 10.1093/nar/gkad208


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