Study finds mitochondrial mutations single cells

New knowledge about mitochondrial DNA (mtDNA) mutations has been obtained through the use of a high-throughput single-cell, single-mitochondrial genome sequencing technique known as iMiGseq. This technology also provides a platform for evaluating mtDNA editing techniques and genetically diagnosing embryos before implantation.

The genetic maps of mtDNA in individual human oocytes (immature eggs) and blastoids (synthetic embryos derived from stem cells) have recently been quantitatively described by an international team of researchers lead by KAUST stem cell biologist Mo Li[1]. Research has uncovered the molecular characteristics of uncommon disorders caused by maternally inherited mtDNA abnormalities.

Mitochondria, the "powerhouses" of cells, play a crucial role in cellular communication and metabolism. Human mtDNA is a circular genome containing 37 genes, encoding 13 proteins and a noncoding D-loop region. Heteroplasmic mutations, inherited from egg cells, can cause congenital diseases, like 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 disease. Yet the understanding of mtDNA mutations remains limited due to the constraints of traditional sequencing technologies," said lead author Chongwei Bi.

"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," said Bi. iMiGseq resolves several key questions in the field.

Using third-generation nanopore sequencing technology, the researchers have characterized mtDNA heteroplasmy in single cells and described the genetic features of mtDNA in single oocytes. They have examined 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," said Mo Li.

In another experiment using the new technology, iMiGseq revealed the potential risks of unexpected large increases 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 episodes syndrome in patient-derived induced pluripotent stem cells.

"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," said li.