Study reveals molecular signature of rare mtDNA mutation

A high-throughput single-cell single mitochondrial genome sequencing technology known as iMiGseq provides new insights into mitochondrial DNA (mtDNA) mutations and provides a platform for evaluating mtDNA editing strategies and genetic diagnostics in preimplantation embryos. Offers.

An international research team led by KAUST stem cell biologist Mo Li quantitatively delineated the genetic map of mtDNA in single human oocytes (immature eggs) and blasts (stem cell-based synthetic embryos). This has revealed the molecular signature of rare mtDNA mutations that cause maternally inherited disorders.

Mitochondria, the “power plants” of cells, play a key role in cell communication and metabolism. Human mtDNA is a circular genome containing 37 genes encoding 13 proteins and a non-coding D-loop region. Heteroplasmic mutations inherited from the egg cell can cause congenital disorders 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 important causes of metabolic disease. However, our understanding of mtDNA mutations is still limited due to the limitations of conventional sequencing technology. ”

Chongwei Bi, first author

“Our new iMiGseq method is important because it enables complete sequencing of individual mtDNA within a single cell, enabling unbiased, high-throughput base-resolution analysis of full-length mtDNA,” said Bi. says. iMiGseq solves some important problems in this area.

The researchers used third-generation nanopore sequencing technology to characterize mtDNA heteroplasmy in single cells and describe the genetic signature of mtDNA in single oocytes.they looked at mtDNA induced pluripotent stem cells Derived from patients with Leigh syndrome or neuropathy, ataxia or retinitis pigmentosa (NARP). This revealed a complex pattern of pathogenic mtDNA mutations, including single-nucleotide variants and large structural variants. “He was able to detect rare mutations at frequencies well below 1%, the traditional detection threshold,” said Mo Li.

In another experiment using the new technology, iMiGseq demonstrated an unexpected frequency of off-target mutations known as heteroplasmy in a mitochondrial genome-editing method called mitoTALEN, a genome-editing tool that cuts specific sequences in mitochondria. revealed a potential risk of significantly increased DNA. Used to sever mutations that cause mitochondrial encephalomyopathy and stroke-like episode syndrome in patient-derived induced pluripotent stem cells.

“This highlights the advantage of full-length mtDNA haplotype analysis for understanding alterations in mitochondrial DNA heteroplasmy. It can be unintentionally affected and should be evaluated in an ultra-sensitive way: the safety of any editing strategy,” says Li.

The researchers also used iMiGseq to analyze a single human oocyte from a healthy donor and a single human blast, a synthetic embryo made from stem cells, which conventional next-generation sequencing would not detect. We have identified rare mutations that are not possible. These low-level heteroplasmic mutations can be inherited through the female germline and are associated with mitochondrial diseases and cancer.

The iMiGseq method provides a new means to accurately delineate the complete haplotype of individual mtDNA within single cells, explain the pathogenesis of mitochondrial mutation-associated diseases, assess the safety of different mtDNA editing strategies, and improve mtDNA It provides an ideal platform for elucidating associations between mutations. , aging and the development of complex diseases.