Skip to main content
Premium Trial:

Request an Annual Quote

Regulatory Alterations Overrepresented in Single-Cell Sequence Data From Individual Neurons

NEW YORK – The relatively low-frequency small insertions and deletions (indels) that accumulate over time in human neurons appear to be particularly common in regulatory element sequences, according to a new single-cell DNA sequencing (scDNA-seq) study by an international team that included investigators in the US, Belgium, South Korea, and the Netherlands.

"Our data suggest that indels in gene-regulatory elements have a considerable effect on genome integrity in human neurons," co-senior and co-corresponding authors Peter Park, a biomedical informatics researcher at Harvard Medical School, and Christopher Walsh, a genetics and genomics, orphan disease, and medical researcher at Boston Children's Hospital, and their colleagues wrote in Nature Genetics on Monday.

After comparing single-cell genome sequences generated with two amplification methods — either multiple displacement amplification (MDA) or primary template-directed amplification (PTA) — the team relied on PTA, scDNA-seq, and a "single-cell analysis 2" (SCAN2) somatic genotyping approach to profile somatic mutations in individual prefrontal cortex (PFC) neurons isolated from frozen postmortem human brain tissues in a National Institutes of Health Neurobiobank housed through the University of Maryland's Brain and Tissue Bank.

"Our study establishes a methodology for somatic mutation detection from scDNA-seq of PTA-amplified whole genomes," the authors wrote, noting that "our approach can analyze genomes with low mutation burden and [cases] where somatic mutations may not be shared by multiple cells."

With this approach, the team generated new genome sequence data for 52 individual PFC neurons from a dozen neurotypical individuals, sequenced to an average depth of 30-fold to 60-fold coverage. Together with published single-cell sequence data for 15 more neurons from another five neurotypical individuals, the sequences helped to refine the relationship between individuals' age and somatic mutation accumulation in PFC neurons from infancy to old age.

Single nucleotide variants (SNVs) accumulated more slowly than small insertions and deletions (indels), the researchers explained. They estimated the annual uptick of new non-clonal somatic mutations at around 16 SNVs, while indels increased in each neuron at an average rate of three per year.

Even so, the single-cell sequence data pointed to a preponderance of indels in regulatory regions of the neurons profiled, prompting the team to examine the gene expression consequences of such regulatory changes and to propose ties between neuronal genome integrity and somatic indel accumulation in gene regulatory regions of the genome.

"Our catalog confirms a previously discovered age-related SNV signature (with a slightly revised rate of accumulation) and reveals an enrichment of somatic mutations — particularly indels — in transcribed genes and brain-specific regulatory elements," the researchers reported, adding that "increased enrichment level of indels in brain-specific regulatory regions suggests that somatic indels may interfere with neuronal regulatory programs."

More generally, the authors suggested that the PTA-amplified scDNA-seq strategy "will enable a wide range of studies, including somatic mutation analysis of neurons from individuals with neurodegenerative diseases, further characterization of mutations caused by exposures to mutagenic compounds and measuring the efficiency and accuracy of CRISPR … editing at the single-cell level."