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High throughput single cell analysis of mitochondrial heteroplasmy in mitochondrial diseases.

ABSTRACT: Mitochondrial heteroplasmy, which fundamentally means intracellular heterogeneity of mitochondrial DNA (mtDNA), has been measured in a group of cells, regardless of intercellular heterogeneity. Ordinal methods for mitochondrial heteroplasmy cannot discriminate between an intercellular homogenic population composed of cells with similar intracellular heterogeneity for mtDNA and an intercellular heterogenic population composed of cells with different rates of mutated mtDNA. A high-throughput method to determine mitochondrial heteroplasmy in a single cell was developed by using droplet digital PCR with TaqMan polymerase in this study. This technique revealed that there are three different cell populations of cultured fibroblasts derived from patients with mitochondrial disease carrying a mutation in the mtDNA; cells with homoplasmy of either mutated or healthy mtDNA; and cells mixed with mutated and healthy mtDNA. The presence of intercellular heterogeneity, even in uniformed cultured fibroblasts, suggests that heterogeneity should exist among different kinds of cells. The diagnosis of intercellular heterogeneity with respect to mitochondrial heteroplasmy by this methodology could provide novel insight into developing a treatment strategy for mitochondrial diseases.

SUBMITTER: Maeda R

PROVIDER: S-EPMC7331593 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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High throughput single cell analysis of mitochondrial heteroplasmy in mitochondrial diseases.

Maeda Ryotaro R Kami Daisuke D Maeda Hideki H Shikuma Akira A Gojo Satoshi S

Scientific reports 20200702 1

Mitochondrial heteroplasmy, which fundamentally means intracellular heterogeneity of mitochondrial DNA (mtDNA), has been measured in a group of cells, regardless of intercellular heterogeneity. Ordinal methods for mitochondrial heteroplasmy cannot discriminate between an intercellular homogenic population composed of cells with similar intracellular heterogeneity for mtDNA and an intercellular heterogenic population composed of cells with different rates of mutated mtDNA. A high-throughput metho ...[more]

PMID: 32616755

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Project description:BackgroundMitochondrial dysfunction is linked to numerous pathological states, in particular related to metabolism, brain health and ageing. Nuclear encoded gene polymorphisms implicated in mitochondrial functions can be analyzed in the context of classical genome wide association studies. By contrast, mitochondrial DNA (mtDNA) variants are more challenging to identify and analyze for several reasons. First, contrary to the diploid nuclear genome, each cell carries several hundred copies of the circular mitochondrial genome. Mutations can therefore be present in only a subset of the mtDNA molecules, resulting in a heterogeneous pool of mtDNA, a situation referred to as heteroplasmy. Consequently, detection and quantification of variants requires extremely accurate tools, especially when this proportion is small. Additionally, the mitochondrial genome has pseudogenized into numerous copies within the nuclear genome over the course of evolution. These nuclear pseudogenes, named NUMTs, must be distinguished from genuine mtDNA sequences and excluded from the analysis.ResultsHere we describe a novel method, named MitoRS, in which the entire mitochondrial genome is amplified in a single reaction using rolling circle amplification. This approach is easier to setup and of higher throughput when compared to classical PCR amplification. Sequencing libraries are generated at high throughput exploiting a tagmentation-based method. Fine-tuned parameters are finally applied in the analysis to allow detection of variants even of low frequency heteroplasmy. The method was thoroughly benchmarked in a set of experiments designed to demonstrate its robustness, accuracy and sensitivity. The MitoRS method requires 5 ng total DNA as starting material. More than 96 samples can be processed in less than a day of laboratory work and sequenced in a single lane of an Illumina HiSeq flow cell. The lower limit for accurate quantification of single nucleotide variants has been measured at 1% frequency.ConclusionsThe MitoRS method enables the robust, accurate, and sensitive analysis of a large number of samples. Because it is cost effective and simple to setup, we anticipate this method will promote the analysis of mtDNA variants in large cohorts, and may help assessing the impact of mtDNA heteroplasmy on metabolic health, brain function, cancer progression, or ageing.

Dataset Information

High throughput single cell analysis of mitochondrial heteroplasmy in mitochondrial diseases.

Publications

High throughput single cell analysis of mitochondrial heteroplasmy in mitochondrial diseases.

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