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JHU Team Awarded $800K to Identify mtDNA Copy Number Markers for Coronary Heart Disease

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NEW YORK (GenomeWeb) – A team of genetic researchers at Johns Hopkins University will use a new, $800,000 National Institutes of Health grant to investigate the link between mitochondrial DNA copy number and coronary heart disease.

The researchers believe that by monitoring mtDNA copy number in blood, they will be able to identify people at greater risk for developing CHD who could benefit from preventative efforts.

Dan Arking, an associate professor in genetic medicine at Johns Hopkins School of Medicine in Baltimore, is the principal investigator on the study, which is slated to run through 2020. He said in an interview that the idea for the effort, that an age-related decline in mtDNA copy number could suggest a higher risk for CHD, evolved out of the theory that links aging and disease risk.

"There is a longstanding hypothesis in aging that there's an energy deficiency as you get older," said Arking. "If your reserves are low, and you have some kind of injury or sickness, you don't have the ability to recover from that, and that leads to frailty and increased mortality."

Arking and colleagues a few years ago became interested in the idea that a depletion in mtDNA — the so-called powerhouse of the cell — could be linked with this process. They called mtDNA copy number in more than 16,000 participants from two prospective cohorts, the Atherosclerosis Risk in Communities (ARIC) study and the Cardiovascular Health Study, that had been genotyped using Affymetrix SNP arrays.

"We figured out how to extract mitochondrial copy number from this existing data and then we were able to show a strong correlation between a decrease in mitochondrial copy number and increased risk of overall mortality," said Arking.

As outlined in the grant's abstract, a lower level of mtDNA copy number was associated with an increased incidence of CHD, independent of traditional CHD risk factors. The findings have been described in two papers: one that appeared in the Journal of the American Society of Nephrology last August, and one that was published in the Journal of Molecular Medicine in 2015.

With the new NIH funding, he and his team would like to validate the finding in two additional cohorts, the Multiethnic Study of Atherosclerosis and the Rotterdam Study. Combining the data from all four cohorts, they hope to "comprehensively evaluate the role of mtDNA copy number in CHD," and to determine whether changes in mtDNA copy number can be associated with atherosclerosis or traditional CHD risk factors.

As part of this process, Arking and colleagues will use DNA from ARIC's baseline samples as well as DNA obtained during follow-up visits to assess how longitudinal changes in mtDNA copy number can impact one's potential CHD risk.

This piece of the study could inform future preventive efforts, Arking noted.  

"When you go to your doctor once a year to measure your cholesterol, potentially you would measure your mitochondrial DNA copy number as well," said Arking. "But whether it's useful to check it every year, [or] every five years, is something we are working on as part of this grant," he said.

Arking and colleagues are conducting a genome-wide association study as part of the current project. They are looking at common and rare variants in all of the aforementioned studies, plus additional samples from the Cohorts for Heart and Aging Research in Genomic Epidemiology Consortium, in a cohort totaling nearly 100,000 samples. The genotyping data was produced using a variety of Affymetrix and Illumina arrays. Arking said he is confident that the team will be able to identify loci that can be used to determine whether a decrease in mtDNA copy number is causative for CHD.

"The GWAS is about establishing causality," said Arking. "The idea is that if I identify genetic variants that modify mitochondrial DNA copy number, I can then test those variants for whether they also modify risk for cardiovascular disease," he said. "If those same variants modify risk for cardiovascular disease, then it's considered causal."

The study should also enable Arking and colleagues to learn if the manipulation of mtDNA copy number might have clinical relevance or health benefits. "That's the critical piece, to understand the clinical utility," said Arking. "It could be a biomarker and have clinical utility to find people at risk for heart attack, or it could be clinical utility as a modifiable risk factor."

Following the GWAS, investigators aim to narrow down the regions of the genome in question, and then identify specific genes, ultimately determining gene function.

He said that the JHU team will accomplish this largely by knocking out or expressing the genes in tissue culture to look at the impact on mtDNA copy number using a variety of approaches, including RNA-seq, run on Illumina instruments at Arking's lab, as well as Agilent Seahorse FX assays to measure mtDNA. Their efforts also will involve CRISPR/Cas9 gene editing to produce mutations and deletions of interest for follow-up investigation. Using these functional assays, the researchers will look at how knocking down these target genes will impact expression of other genes, and they will try to build networks from that information.

Working with bioinformatics teams within JHU, the scientists aim to identify shared patterns of dysregulation, interactions between regulatory genes, and pathways responsible for specific physiological changes. Understanding the regulatory network associated with mtDNA copy number, they hope to better comprehend the mechanisms that both regulate mtDNA copy number and create its downstream phenotypic consequences.

"We want to understand at the molecular level what's going on, and what are the pieces involved in regulating mitochondrial copy number, and what is the potential connection, and if it is correlated," said Arking. "We want to know why is it decreasing with risk for cardiovascular disease, or if it is causal, what are [potentially] the druggable targets," he said.

The envisioned test to measure mtDNA copy number in patients though may be "relatively trivial," Arking noted, perhaps a quantitative PCR assay that costs less than $2 per sample.

"It's relatively straightforward to do qPCR. There are other technologies that work as well," he said. Such a test could be administered regularly to determine which patients are at the greatest risk of developing CHD and require preventative care.

"When you go to your doctor once a year to measure your cholesterol, potentially you would measure your mitochondrial DNA copy number as well," said Arking. "Adding this as a risk factor could help to determine who should start a statin and when they should start taking it."

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