Multimodal single-cell analysis of nonrandom heteroplasmy distribution in human retinal mitochondrial disease
Nathaniel K. Mullin, Andrew P. Voigt, Miles J. Flamme-Wiese, Xiuying Liu, Megan J. Riker, Katayoun Varzavand, Edwin M. Stone, Budd A. Tucker, Robert F. Mullins
Nathaniel K. Mullin, Andrew P. Voigt, Miles J. Flamme-Wiese, Xiuying Liu, Megan J. Riker, Katayoun Varzavand, Edwin M. Stone, Budd A. Tucker, Robert F. Mullins
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Research Article Genetics Ophthalmology

Multimodal single-cell analysis of nonrandom heteroplasmy distribution in human retinal mitochondrial disease

Abstract

Variants within the high copy number mitochondrial genome (mtDNA) can disrupt organelle function and lead to severe multisystem disease. The wide range of manifestations observed in patients with mitochondrial disease results from varying fractions of abnormal mtDNA molecules in different cells and tissues, a phenomenon termed heteroplasmy. However, the landscape of heteroplasmy across cell types within tissues and its influence on phenotype expression in affected patients remains largely unexplored. Here, we identify nonrandom distribution of a pathogenic mtDNA variant across a complex tissue using single-cell RNA-Seq, mitochondrial single-cell ATAC sequencing, and multimodal single-cell sequencing. We profiled the transcriptome, chromatin accessibility state, and heteroplasmy in cells from the eyes of a patient with mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) and from healthy control donors. Utilizing the retina as a model for complex multilineage tissues, we found that the proportion of the pathogenic m.3243A>G allele was neither evenly nor randomly distributed across diverse cell types. All neuroectoderm-derived neural cells exhibited a high percentage of the mutant variant. However, a subset of mesoderm-derived lineage, namely the vasculature of the choroid, was near homoplasmic for the WT allele. Gene expression and chromatin accessibility profiles of cell types with high and low proportions of m.3243A>G implicate mTOR signaling in the cellular response to heteroplasmy. We further found by multimodal single-cell sequencing of retinal pigment epithelial cells that a high proportion of the pathogenic mtDNA variant was associated with transcriptionally and morphologically abnormal cells. Together, these findings show the nonrandom nature of mitochondrial variant partitioning in human mitochondrial disease and underscore its implications for mitochondrial disease pathogenesis and treatment.

Authors

Nathaniel K. Mullin, Andrew P. Voigt, Miles J. Flamme-Wiese, Xiuying Liu, Megan J. Riker, Katayoun Varzavand, Edwin M. Stone, Budd A. Tucker, Robert F. Mullins

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Figure 2

Nonrandom distribution of m.3243G in the retina and choroid measured by mt-scATAC–Seq.

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Nonrandom distribution of m.3243G in the retina and choroid measured by ...
(A) Schematic of the neural retina, RPE, and choroid indicating the cell classes and regions captured by scRNA-Seq, mt-scATAC–Seq, and LCM-dPCR. (B) Two-dimensional UMAP embeddings of cells recovered from the MELAS neural retina. Individual cells are colored based on proportion of m.3243A>G mutant allele (red indicating 100% mutant allele and blue indicating 100% WT allele). (C) Two-dimensional UMAP embeddings of cells recovered from the MELAS RPE and choroid colored based on proportion of m.3243A>G mutant allele. (D) Violin plot demonstrating the proportion of m.3243A>G mutant allele in individual cells isolated from the neural retina sample as measured by mt-scATAC–Seq. Cells are grouped based on cell type cluster identity. (E) Violin plot demonstrating the proportion of m.3243A>G mutant allele in individual cells isolated from the RPE/choroid sample as measured by mt-scATAC–Seq. Cells included in D and E have at least 10× coverage of the m.3243 locus.