High-Throughput DNA Sequencing at Single-Cell Level Provides Insight into Mitochondrial Disease

Subscribe to the latest updates from Neuroscience Advances in Motion

Thank you for subscribing!

Error: Please enter a valid email address.

Email Address Submit

Mitochondrial diseases are often caused by inherited mutations in mitochondrial DNA (mtDNA). Affected patients' cells contain a mixture of mutant and nonmutant mtDNA, a phenomenon called heteroplasmy. It's generally accepted that the proportion of mutated mtDNA is what determines whether a patient will exhibit disease.

Single-cell analysis is needed in order to better understand the effects of different levels of heteroplasmy. However, until recently, such analyses were limited to one cell type and at most a few dozen cells. Now, Melissa A. Walker, MD, PhD, physician in the Department of Neurology, Vamsi K. Mootha, MD, investigator in the Departments of Molecular Biology and Medicine at Massachusetts General Hospital, and colleagues have used new technology to determine mtDNA heteroplasmy in thousands of cells simultaneously. They explain the clinical implications of their findings in The New England Journal of Medicine.

Study Details

The researchers studied patients with a syndrome called mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS). This syndrome is associated with the most common heteroplasmic pathogenic mtDNA mutation, A3243G. Heteroplasmy of A3243G varies widely, even among siblings and across tissues within the same patient, resulting in a broad spectrum of symptoms and severity.

To begin, the researchers collected 6,000 to 7,200 peripheral blood mononuclear cells (PBMCs) from each of three unrelated men with MELAS, all of whom had stroke-like episodes and other symptoms. PBMCs consist of multiple cell types that originate from a common progenitor pool.

Using a new genomics technology (per research published in Nature Biotechnology), a single-cell assay for transposase-accessible chromatin sequencing, the researchers determined mtDNA heteroplasmy and cell type for the thousands of cells simultaneously.

Results

In each patient and cell type assessed, individual cells spanned a broad range of heteroplasmy. But levels of heteroplasmy tended to be lower within lymphocytes (B cells, T cells and natural killer cells) than within monocytes or dendritic cells. T cells consistently showed the lowest levels, even though the total numbers of T cells were normal.

The results were validated in:

• Two of the same three patients by using different methods to purify the T cells
• Six additional patients who carry the A3243G mutation but have not had stroke-like episodes

Laying the Groundwork

The simplest explanation of these observations is that they reflect purifying selection—selective removal of the deleterious A3243G allele from the T-cell lineage. It's conceivable that a T-cell–specific process in the bone marrow, thymus or peripheral circulation selects against high heteroplasmy.

Currently, there is no FDA-approved therapy for MELAS or other mitochondrial diseases. Understanding the determinants of reduced T-cell heteroplasmy may lead to new therapies that exploit the body's own process to guard against pathogenic mtDNA mutations.

view original journal article Subscription may be required

Learn more about research in the Department of Neurology

Learn more about the Department of Neurology