Clinical OMICS

MAY-JUN 2017

Healthcare magazine for research scientists, labs, pathologists, hospitals, cancer centers, physicians and biopharma companies providing news articles, expert interviews and videos about molecular diagnostics in precision medicine

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34 Clinical OMICs May/June 2017 www.clinicalomics.com In the Lab Splicing Error Detectives Using a Newly Developed Assay, Researchers ID Disease- Causing Mutations in the Exome That Alter Gene Splicing By Diana Kwon, Contributing Editor I n recent years, DNA sequencing has become faster, easier, and cheaper. However, pinpointing disease-causing vari- ants in an individual's genome remains a challenge. "Our validation technologies—our means of analyzing vari- ance—is actually very low throughput," said William Fair- brother, Ph.D., a biology professor at Brown University. "So we were looking to develop technology that would match the throughput of the genotyping technologies." To help identify disease-causing variants in the genome, Dr. Fairbrother and his colleagues created MaPSy (Mas- sively Parallel Splicing Assay), a tool that works by produc- ing artificial "minigenes" that either contain disease-causing mutations or the corresponding "normal" versions of the sequence. These synthesized genes are then tested both in vivo in living cells and in vitro by examining the effects of splicing on synthesized RNA. " The main strength of the MaPSy approach is that they couple both in vivo and in vitro massively parallel reporter assays to identify which part of the spliceosome is disrupted by each tested mutation," said Alex Rosenberg, Ph.D., a postdoctoral researcher in bioinformatics at Washington University who was not involved in the work. In a study published April in Nature Genetics , the team used MaPSy to examine 4,964 diseases-causing exonic mutations from the Human Gene Mutation Database, a col- lection of genetic variants that lead to inherited diseases. The analysis revealed that around 10 percent (around 500 alleles) altered splicing both in vivo and in vitro. "That's by pretty stringent criteria—we demand that there's at least 1.5-fold difference in the representation of an allele to call it a splicing mutation," Dr. Fairbrother noted. "It [also] has to happen in two different systems. So we might be under- counting that number." Some exons, the researchers found, had a higher propor- tion of splicing-associated mutations than others. "Some [exons] were robust and everything worked great, but oth- ers were sort of borderline—so if there was no mutation, everything was fine and you'd splice normally, but many mutations would disrupt splicing," Dr. Fairbrother said. In addition, he added, there were certain features of those exons made them easier to disrupt than others. "It was [gen- erally] things that made the exon a worse substrate for splic- ing," he said. They also found that splicing mutations were more likely to appear in haploinsufficient genes, where los- ing on functional copy leads to disease. "In addition to identifying many disease-causing muta- lvcandy / Getty Images

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