Splice-Break finds key DNA deletions

A new approach to studying deletions in the mitochondria could inform disease study

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IRVINE, Calif.—Research originating at the University of California, Irvine (UCI) demonstrates the efficacy of a reliable, high-throughput technique to analyze mitochondrial DNA (mtDNA) and identify significant deletions that lead to disease. Building on an existing RNA-seq alignment algorithm, the team developed a process to be able to use it to detect and quantify mitochondrial deletions. Preliminary testing has indicated that significant deletion burdens are present in brain samples from patients with major depressive disorder, which may eventually lead to new psychiatric treatment possibilities.
Deletions in the 16.6 kb mitochondrial genome have been implicated in numerous disorders that often display muscular and/or neurological symptoms due to the high-energy demands of these tissues. Dr. Marquis Vawter in the Department of Psychiatry and Human Behavior at UCI has been exploring changes in mitochondrial DNA for several years. His team has worked to evaluate single nucleotide variants, rare point mutations and one of the known deletions in mitochondrial DNA, specifically looking in brain tissue of subjects with mental illnesses. Vawter challenged Dr. Brooke E. Hjelm, then the Della Martin Fellow in Psychiatric Genetics in his lab, to develop a computational approach to find large mitochondrial DNA deletions in the next-generation sequencing data.
Hjelm is a talented molecular and cellular biologist with extensive experience in genomics and bioinformatics, but she is not a computer programmer. Drawing inspiration from ways that science has successfully repurposed drugs to treat different diseases, she looked at exploiting a readily available RNA-seq algorithm called MapSplice. The resulting pipeline, called Splice-Break, allowed the team to scan the mitochondrial genome for deletion breakpoints.
“The way these RNA-Seq algorithms typically work is they try to map an RNA molecule that we sequenced back to the human genome (DNA) sequence, and determine how many molecules are there and what the molecules look like in terms of overall structure,” says Hjelm. “Because the RNA molecules that go on to make proteins are ‘spliced,’ there are large gaps in between where an RNA sequence will match the DNA sequence, so RNA-Seq algorithms are unique and different from DNA-Seq algorithms because they need to be able to handle these gaps in the sequence and identify where they occur. To me, this was a perfect strategy to exploit for detecting mitochondrial deletions, because these molecules likewise have large gaps due to the deleted (or missing) piece of DNA.”
The UCI team published a paper, “Splice-Break: exploiting an RNA-seq splice junction algorithm to discover mitochondrial DNA deletion breakpoints and analyses of psychiatric disorders,” which appeared recently in the journal Nucleic Acids Research. According to the paper, Splice-Break can be used to study large datasets of individual mtDNA deletions rates as well as cumulative deletion metrics with respect to tissue, brain region, age and disease. They discovered 4,489 mtDNA deletions, what they refer to as “a stunning catalog,” given that only about 800 deletions had previously been detected in mitochondrial DNA. Interestingly, their findings only include those with breakpoints between positions 357–15925 (NC_012920.1) of the mitochondrial genome, as they filtered out the majority of the control region in their analysis.
As the authors note in their conclusions in the paper, “This study demonstrates the efficacy and reliability of the Splice-Break pipeline to detect and quantify mtDNA deletions. Future studies may include analysis of mtDNA deletion breakpoints in the D-loop and extended control region, which includes a 3′ breakpoint ‘hotspot’ at position 16071, perhaps using different primers (with different binding positions) for the LR PCR. Indeed, any analysis of mtDNA deletions that utilizes LR PCR will be limited to only discover molecules that can be successfully amplified with the primers chosen.”
While there are no current therapies available to repair mitochondrial deletions, the research has significant potential to aid in the search for effective therapies down the road. According to Hjelm, Splice-Break could be used as a high-throughput drug-screening pipeline to potentially quantify deletions across many drug tests simultaneously and decrease the time it would take to find an effective therapy. In addition, the catalog of almost 5,000 unique mitochondrial deletions may be used to understand more about why mitochondrial deletions are forming and if they are more or less likely to get worse over time, possibly leading to additional strategies aimed at preventing deletion formation/ accumulation in human tissues. And while many mitochondrial disorders are considered rare, the initial pipeline also identified mitochondrial deletion as a factor in many symptoms of aging.
Hjelm, who is now an assistant professor of clinical translational genomics in the Department of Translational Genomics at the University of Southern California, is excited for the next steps in this research. By examining different subsets of patients, researchers may be able to determine which mitochondrial diseases are caused by deletions, and which may be caused by environmental factors or genetic mutations. Splice-Break can pave the way for truly personalized medicine, helping to determine which subsets of patients respond to which therapies, and facilitating more predictable outcomes. It can also play a significant role in the development of precisely fine-tuned clinical trials aimed at even small groups of afflicted people.
“I am working to expand this work into neurodegenerative diseases, specifically Parkinson’s and Alzheimer’s disease, as well as cancer. I am also working on a deeper characterization of the aging effects we observed, as I believe it will have important implications for many diseases that are age-related or have an adult onset, and may lead to collaborative studies outside my neurological disease research,” Hjelm says. “Dr. Vawter and I are also continuing to work together on evaluating the roles these deletions play in psychiatric disorders, and we are starting to look in other diagnoses like bipolar disorder, as well as other brain regions like the thalamus for their effect on depression. Ultimately, we need to perform larger screening efforts on more subjects and brain regions to truly understand how big of a role this plays in mental illness.”

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