Single neuron may carry over 1,000 mutations

"We found that the genes that the brain uses most of all are the genes that are most fragile and most likely to be mutated," says Christopher Walsh, an HHMI investigator at Boston Children's Hospital who led the research. Walsh, Peter Park, a computational biologist at Harvard Medical School, and their colleagues reported their findings in the October 2, 2015, issue of the journal Science.

It's not yet clear how these naturally occurring mutations impact the function of a normal brain, or to what extent they contribute to disease. But by tracing the distribution of mutations among cells, Walsh and his colleagues are already learning new information about how the human brain develops. "The genome of a single neuron is like an archeological record of that cell," Walsh says. "We can read its lineage in the pattern of shared mutations. We now know that if we examined enough cells in enough brains, we could deconstruct the whole pattern of development of the human brain."

Cells of many shapes, sizes, and function are intimately intertwined inside the brain, and scientists have wondered for centuries how that diversity is generated. Scientists are further interested in genome variability between neurons due to evidence from Walsh's lab and others that mutations that affect only a small fraction of cells in the brain can cause serious neurological disease. Until recently, however, scientists who wanted to explore that diversity were stymied by the scant amount of DNA inside neurons: Although researchers could isolate the genetic material from an individual neuron, there was simply not enough DNA to sequence, so cell-to-cell comparisons were impossible.

Walsh's team undertook its current study thanks to technology that has become available in the last few years for amplifying the full genomes of individual cells. With plenty of DNA now available, the scientists could fully sequence an individual neuron's genome and scour it for places where that cell's genetic code differed from that of other cells.

The scientists isolated and sequenced the genomes of 36 neurons from healthy brains donated by three adults after their deaths. For comparison, the scientists also sequenced DNA that they isolated from cells in each individual's heart. That effort yielded mountains of data, and Walsh's group teamed up with Park and Semin Lee, a postdoctoral fellow in Park's group, to make sense of it all.

What they found was that every neuron's genome was unique. Each had more than 1,000 point mutations (mutations that alter a single letter of the genetic code), and only a few mutations appeared in more than one cell. What's more, the nature of the variation was not quite what the scientists had expected.

"We expected these mutations to look like cancer mutations," Walsh says, explaining that cancer mutations tend to arise when DNA is imperfectly copied in preparation for cell division, "but in fact they have a unique signature all their own. The mutations that occur in the brain mostly seem to occur when the cells are expressing their genes."

Neurons don't divide, and most of the time their DNA is tightly bundled and protected from damage. When a cell needs to turn on a gene, it opens up the DNA, exposing the gene so that it can be copied into RNA, the first step in protein production. Based on the types and locations of the mutations they found in the neurons, the scientists concluded that most DNA damage had occurred during this unwinding and copying process.

While most of the mutations in the neurons were unique, a small percentage did turn up in more than one cell. That signaled that those mutations had originated when future brain cells were still dividing, a process that is complete before birth. Those early mutations were passed on as cells divided and migrated, and the scientists were able to use them to reconstruct a partial history of the brain's development.

"We knew that cells that shared a certain mutation were related, so we could look at how different cells in the adult were related to each other during development," explains Mollie Woodworth, a postdoctoral researcher in Walsh's lab. Their mapping revealed that closely relatedly cells could wind up quite distant from one another in the adult brain. A single patch of brain tissue might contain cells from five different lineages that diverged before the developing brain had even separated from other tissues in the fetus. "We could identify mutations that happened really early, before the brain existed, and we found that cells that had those mutations were nestled next to cells that had totally different mutations," Woodworth says. In fact, the scientists found, a particular neuron might be more closely related to a cell in the heart than to a neighboring neuron.

The scientists say intermingling cells with different developmental origins might protect the brain from the effects of early-arising, potentially harmful mutations. Although most of the mutations the scientists catalogued were harmless, they did encounter mutations that disrupted genes that, when impaired throughout the brain, can cause disease. "By having very mixed populations, cells that are next to each other and responsible for a similar task are not very closely related to each other, so they're not likely to share the same deleterious mutation," says Michael Lodato, who is also a postdoctoral researcher in Walsh's lab. That could reduce the risk of a particular mutation interfering with a localized brain function, he explains.

Still, the scientists say, this abundance of mutations could influence the function of a normal brain. "To what extent do these clonal mutations normally shape the development of the brain, in a negative way or a positive way?" says Walsh. "To what extent do we have a patch of brain that doesn't work quite right, but not so much that we would call it a disease? That's a big open question."