Using deep sequencing of candidate genes, researchers have identified mosaic mutations – mutations that exist in only some of the body's cells, because they arose as a result of a copying error during cell division rather than being present in the sperm or egg – in about a third of patients with malformations of the cerebral cortex.

The extensive sequencing was able to identify mutations that are so rare as to be missed in whole-exome and Sanger sequencing. In fact, almost two-thirds of the mutations that the team identified did not show up when they tested their samples with Sanger sequencing.

Despite the fact that technological advances have made DNA sequencing vastly quicker and cheaper to perform than even a few years ago, there remains a tradeoff between depth and breadth of sequencing that in practice means that a mutation that is present in only some cells may be missed because it is classified as an error, rather than a rare but real genetic variant within a patient – somewhat like rare variants can be missed in genomewide association studies.

In their work, which they published in the Aug. 20, 2014, issue of the New England Journal of Medicine, the team looked at a panel of candidate genes in 158 patients with structural abnormalities in the cerebral cortex in depth. The team used next-generation sequencing, reading each piece of DNA at least 200 times. In Sanger sequencing, each piece of DNA is read an average of 30 times.

By covering those blood samples in great depth, "we were able to separate noise from the truth" and identify causal mutations in about 30 percent of patients, first author Saumya Jamuar told BioWorld Today. Jamuar was at Boston Children's Hospital when the studies were performed, and is now at KK Women's and Children's Hospital in Singapore.

Strikingly, "we could have a phenotype with as few as 10 percent of cells being affected in the blood." Mutations are classified as errors in the bioinformatics methods used for Sanger sequencing if they are present in fewer than 15 to 20 percent of cells.

Twenty of the patients had undergone previous genetic testing, via Sanger sequencing or whole exome sequencing, that had not turned up any mutations related to their disorders, and senior author Christopher Walsh said in a prepared statement that the work shows that detecting somatic mutations requires more sensitive methods than searching for germline mutations, which are present in sperm or egg cells (or both) at conception and, hence, appear in every cell of the body.

"This tells us just how poorly other methods perform in detecting somatic mutations," he said. "You're not going to find these things unless you go looking for them – unless you have a clinical test that is set up to detect them in a sensitive way."

Whether the same proportion of cells is affected in the blood and the brain has not been measured. But Jamuar and his team contended that the proportion would be roughly the same, which means that serious neurodevelopmental disorders can result from mutations that are present in only a relatively small fraction of cells. Jamuar and his team's results, too, are not comprehensive. Since they sequenced candidate genes rather than the whole exome, mutations in any gene that was not in the panel would not be picked up.

Even within the genes the team looked at, "what we could be missing is very low-level mosaics, and mosaics present only in the tissue, not in the blood," he said. Such mutations could occur during development after the lineages for blood and brain cells have diverged.

But the approach opens up "definite diagnostic opportunities," he said. And if a mutation is within a cellular pathway that also contains drug targets, identifying the genetic cause of a malformation might also open up therapeutic possibilities.

The diagnostic possibilities of deep sequencing could go beyond cortical malformations, or indeed, any visible malformations at all.

A recent study looking at genetic contributions to psychiatric disorders also found mosaic mutations in three of 10 patients, matching the roughly 30 percent contribution to cortical malformations that Jamuar and his team showed in their paper. And earlier work out of the lab of senior author Walsh had shown that somatic mutations can play a role in epilepsy.

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