Researchers at Washington University School of Medicine (WUSM) in St. Louis have developed a new technique to cheaply and rapidly create countless sets of DNA fragments that detail all possible genetic variants within a particular stretch of DNA. By studying such DNA fragments, scientists can more easily distinguish between genetic variants linked to disease and those that are innocuous. The new study was published recently in Nature Methods through an article entitled “Massively parallel single-nucleotide mutagenesis using reversibly terminated inosine.”
Roughly 10 million points of genetic variation are scattered across a molecule of DNA, and those variations make us who we are as individuals. However, in some cases, those variants contribute to diseases, and it's a major challenge for scientists to distinguish between harmless variants and those that are potentially hazardous to our health.
“As a pediatric neurologist who does a lot of genetic studies of kids with developmental disabilities, I frequently will scan a patient's whole genome for genetic variants,” explained senior study investigator Christina Gurnett, M.D., Ph.D., associate professor of neurology and of pediatrics at WUSM. “Sometimes I'll find a known variant that causes a particular disease, but more often than not I find genetic variants that no one's ever seen before, and those results are very hard to interpret.”
The new method allows researchers to create sets of DNA variants in a single day for a few hundred dollars—which could eliminate current methods that take up to a week and cost tens of thousands of dollars. Previously, scientists laboriously tested the effect of genetic variants one by one. At a single point in the DNA sequence, they replaced the correct DNA letter—an A, T, C or G—with one of the other three options. Then, they translated that DNA sequence into a protein and evaluated whether the mutated protein behaved differently than the original one.
In recent years, researchers have begun creating sets of hundreds of variants in which each letter in a particular DNA sequence is changed, and then testing the set all at once. Such studies have been limited, however, by the high cost of creating those sets.
The WUSM team realized that they could could harness common laboratory techniques and tools to create sets of DNA variants without the expensive equipment and reagents that drove up the price.
“We demonstrated the construction of a systematic allelic series (SAS) using massively parallel single-nucleotide mutagenesis with reversibly terminated deoxyinosine triphosphates (rtITP),” the authors wrote. “We created a mutational library containing every possible single-nucleotide mutation surrounding the active site of the TEM-1 β-lactamase gene. When combined with high-throughput functional assays, SAS mutational libraries can expedite the functional assessment of genetic variation.”
The scientists copied a DNA sequence using the four standard DNA letters and a nonstandard letter known as inosine. Each copy of the sequence was identical except for one inosine, which was located at a random spot and served as a placeholder. Then, the team replaced the inosine with one of the standard DNA letters, creating a single mutation in each copy of the sequence.
The investigators are looking to apply this technique to genes associated with aortic aneurysms, a weakening and ballooning of the aortic wall that can be fatal. Over the long term, the scientists envision the creation of a catalog listing the effects of every possible variant—a scenario that is now feasible due to the speed and cheapness of the new technique.
Then, when clinicians find a variant that's never been seen before in one of these genes associated with aortic aneurysm, they can go through this catalog and say, 'Yes, this mutation does have a negative effect on that protein, so it's likely harmful',” Dr. Gurnett concluded. “It would help them decide what to tell the patient. This would be one piece of the big interpretation puzzle for genetic mutations.”