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Published on: May 21, 2020
Single-Mitochondrion Sequencing Uncovers Distinct Mutational Patterns and Heteroplasmy Landscape in Mouse Astrocytes
Parnika S Kadam1, Zijian Yang2, Youtao Lu3
1Department of Pharmacology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Background:
Mitochondrial (mt) heteroplasmy can cause adverse biological consequences when deleterious mtDNA mutations accumulate disrupting 'normal' mt-driven processes and cellular functions. To investigate the heteroplasmy of such mtDNA changes we developed a moderate throughput mt isolation procedure to quantify the mt single-nucleotide variant (SNV) landscape in individual mouse neurons and astrocytes In this study we amplified mt-genomes from 1,645 single mitochondria (mts) isolated from mouse single astrocytes and neurons to 1. determine the distribution and proportion of mt-SNVs as well as mutation pattern in specific target regions across the mt-genome, 2. assess differences in mtDNA SNVs between neurons and astrocytes, and 3. Study cosegregation of variants in the mouse mtDNA.
Results:
1. The data show that specific sites of the mt-genome are permissive to SNV presentation while others appear to be under stringent purifying selection. Nested hierarchical analysis at the levels of mitochondrion, cell, and mouse reveals distinct patterns of inter- and intra-cellular variation for mt-SNVs at different sites. 2. Further, differences in the SNV incidence were observed between mouse neurons and astrocytes for two mt-SNV 9027:G>A and 9419:C>T showing variation in the mutational propensity between these cell types. Purifying selection was observed in neurons as shown by the Ka/Ks statistic, suggesting that neurons are under stronger evolutionary constraint as compared to astrocytes. 3. Intriguingly, these data show strong linkage between the SNV sites at nucleotide positions 9027 and 9461.
Conclusion:
This study suggests that segregation as well as clonal expansion of mt-SNVs is specific to individual genomic loci, which is important foundational data in understanding of heteroplasmy and disease thresholds for mutation of pathogenic variants.
Insights
Mitochondrial DNA mutations (mt-SNVs) vary across mouse cell types, with neurons showing stronger evolutionary constraints than astrocytes. This research provides foundational data for understanding heteroplasmy and disease thresholds.
Area of Science:
- Genomics
- Cell Biology
- Mitochondrial Biology
Background:
- Mitochondrial heteroplasmy arises from accumulating mtDNA mutations, disrupting cellular functions.
- Investigating mitochondrial single-nucleotide variant (mt-SNV) landscapes is crucial for understanding disease.
- A moderate-throughput method was developed to quantify mt-SNVs in individual mouse cells.
Purpose of the Study:
- To determine the distribution, proportion, and mutation patterns of mt-SNVs in the mitochondrial genome.
- To compare mtDNA SNV incidence between mouse neurons and astrocytes.
- To investigate the cosegregation of variants within mouse mtDNA.
Main Methods:
- Isolation of 1,645 single mitochondria from individual mouse neurons and astrocytes.
- Amplification of mt-genomes from isolated mitochondria.
- Analysis of mt-SNV landscape, mutation patterns, and variant cosegregation.
Main Results:
- Specific mt-genome sites are permissive to SNVs, while others are under purifying selection.
- Neurons and astrocytes exhibit different mt-SNV incidence, with specific variants (9027:G>A, 9419:C>T) showing cell-type variation.
- Neurons display stronger evolutionary constraint (Ka/Ks statistic) compared to astrocytes, with significant linkage between SNV sites (9027 and 9461).
Conclusions:
- mt-SNV segregation and clonal expansion are locus-specific.
- This study provides foundational data for understanding heteroplasmy and disease thresholds related to pathogenic variants.
