Non-random cellular distribution of the mitochondrial 3243A>G variant in the human retina and choroid and its impact on disease phenotype.

Abstract

Purpose : The dynamics of mitochondrial genome partitioning in complex tissues such as the retina have implications for how mitochondrial variants cause disease. In this study, we sought to understand how the most common pathogenic mitochondrial variant (m.3243A>G) is distributed across ocular cell types in eyes from patients with mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), and how this distribution relates to gene expression in ocular cells. We hypothesized that in the eyes of MELAS patients with severe retinal atrophy, the photoreceptors and retinal pigment epithelium (RPE) would harbor high levels of m.3243G.

Methods : We profiled the proportion of the m.3243A>G variant in single cells from the retina, RPE, and choroid of a MELAS patient, as well as from a healthy control donor. Chromatin accessibility and heteroplasmy were assessed using the mitochondrial single-cell assay for transposable chromatin by sequencing (mt-scATACseq), which captures both nuclear and mitochondrial DNA reads. The transcriptomes of single cells from the same samples were profiled in parallel using single-cell RNA sequencing (scRNAseq). Data were analyzed using Seurat and Signac in R.

Results : We find that the proportion of the pathogenic m.3243G allele is neither evenly nor randomly distributed among ocular cell types. While all classes of neural retinal cells and the RPE exhibited a high degree of heteroplasmy, endothelial and lymphocyte populations of the choroid are near homoplasmic for the wildtype m.3243A allele. Single cell gene expression analysis reveals that the presence of the m.3243G allele in neural retinal cells deranges expression of pathways involved in energy metabolism and the response to oxidative stress. However, many of the same pathways in choroidal cell classes homoplasmic for the wildtype allele are also dysregulated.

Conclusions : This work illuminates the non-random segregation of a pathogenic mitochondrial variant in the retina and choroid and demonstrates that the m.3243A>G variant causes non-cell autonomous tissue dysfunction in the complex and metabolically linked light sensing tissue of the eye. Expanding our understanding of the mechanisms that underlie the non-random partitioning of m.3243A>G will enhance management of this and potentially other mitochondrial diseases.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

 

Overview of experimental approach.