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Blood-Based Biomarker of Neural Cell Death Could Lead to Molecular Multiple Sclerosis Test


NEW YORK (GenomeWeb) ­– The first non-invasive test for the most common form of multiple sclerosis could be on the horizon thanks to recent work by scientists at Winthrop-University Hospital, who have zeroed in on a blood-derived biomarker of the disease.

Specifically, the researchers used methylation-specific qPCR of circulating cell-free DNA to show in mouse and human models that the demethylated form of the myelin oligodendrocyte glycoprotein gene is a suitable biomarker for oligodendrocyte death, a hallmark of relapsing-remitting MS, work that they published in late June in EBioMedicine.

The investigators are now looking to test their biomarker in larger patient cohorts, and are searching for additional markers of relapsing-remitting MS and other, rarer forms of the disease to develop a biomarker panel that could help guide pharmaceutical companies testing new MS therapies or eventually be used as a clinical diagnostic, corresponding author Eitan Akirav told GenomeWeb.

"The pharma companies really deal with a black box with these drugs," Akirav said. "The only way they can measure efficacy is by really subjective phenotypic measurements of movement and cognition, [or to] dump these huge amounts of money into MRIs. For us it's exciting because it would allow rapid testing of … new treatment options."

Multiple sclerosis, a degenerative disease of the central nervous system, is notoriously difficult to diagnose quickly and accurately. Relapsing-remitting MS is the most common form of the disease, comprising some 80 percent of all MS cases. When it is suspected in a patient after a first episode, physicians typically need to wait for a second episode to occur to make a diagnosis — "a bit counter-productive since you're waiting for the disease to get worse," Akirav said.

Furthermore, "it is really expensive and not very comfortable for patients to undergo the current diagnostic methods: MRIs, which are costly and time-consuming, and spinal taps, which are invasive," he added.

Considering these issues, Akirav and colleagues, with research funding from the National Multiple Sclerosis Society and the Conrad N. Hilton Foundation, sought to identify a biomarker for the disease based on the differential methylation of cell-free DNA derived from dying oligodendrocytes (ODCs), the myelin-producing cells of the CNS that are primarily affected by the disease.

Although other researchers had previously demonstrated proof of principle for this approach using two other genes, MBP and WM1, the Winthrop researchers instead focused on the MOG gene, which codes for a CNS-specific protein expressed solely by ODCs in the myelin sheath.

To measure the methylation of this gene in both mouse and human samples, the researchers used a so-called "nested PCR" process, which first involves non-methylation-specific PCR to increase template availability, followed by quantitative real-time PCR using methylation-specific primers designed by the team. All reactions were run using Roche LightCycler 480 SYBR Green I master mix on a Bio-Rad CFX96 Real-Time System.

The team was able to use its methylation-specific PCR method to detect cell-free, circulating demethylated MOG in the blood of mice that had been injected with a plasmid insert of the biomarker, as well as in mice in which ODC injury had been chemically induced.

Then, the Winthrop researchers examined blood samples from 40 patients who had been diagnosed with relapsing-remitting MS (20 with active disease, and 20 with inactive disease), as well as 20 control subjects. They found that demethylated MOG levels were 3.6-fold higher in patients with active disease than those with inactive disease, showing statistical significance.

Akirav noted that the group now needs to test the biomarker in a larger patient cohort, something that it has begun under an approximately $500,000 grant from the Conrad N. Hilton Foundation. Under Phase II of this grant, which is slated to begin in January, the team plans to recruit MS patients both locally and in collaboration with other medical centers around the country.

In addition, the team will begin vetting biomarkers for primary progressive and secondary progressive MS, the rarer and less well-understood forms of the disease.

"We are increasing the repertoire of genes, with the idea that we will have several genes, maybe a dozen, half a dozen, or maybe two dozen that will give us a risk index," Akirav said. In addition, "the genes that we're going to look for will cover several different cell types in the CNS," Akirav said. "This particular paper is on oligodendrocytes, which are the cells that produce myelin. We have now expanded our repertoire to different subsets of neurons and so on, to get a more complete picture."

For all of these potential tests, the detection technology is crucial, as circulating cell-free DNA is found at extremely low levels in the blood, making the nested PCR approach with pre-amplification a requirement. However, in order to further increase sensitivity and potentially multiplexing capabilities, the researchers are testing droplet-based digital PCR from RainDance Technologies to measure absolute copy number of the differentially methylated genes.

"I would say that eventually [the test] is going to be some sort of a droplet-based, chip-based type of kit," Akirav said.

He noted that next-generation sequencing would also be a feasible approach, but that technology is still too expensive for a non-profit organization like Winthrop-University Hospital to use, although the price is rapidly dropping.

"Being a basic research lab operating on philanthropic support, we do have a limited budget, and even the [droplet-based] PCR, which we are using right now for other studies, is not the cheapest, but it's still about $30 an assay," Akirav said. "Once you go to NGS, the cost starts climbing quite a bit. Probably, if we were a biotech with more resources at our disposal, this is something we'd be looking at."

But even a digital PCR test would be a much cheaper way to diagnose patients than, for instance, MRI, which can cost thousands of dollars per patient, he noted.

Winthrop-University Hospital has filed for a patent on behalf of the researchers covering the differentially methylated MOG biomarker, and Akirav said he would be happy to have a biotech firm license the IP and pump more resources into test development. Akirav and Winthrop have some experience in this regard, having previously licensed a PCR assay for identifying beta cell death in type 1 diabetes patients to biotech firm Islet Sciences

Further down the road, Akirav and colleagues plan to pursue a similar approach in patients with other neurodegenerative diseases such as Parkinson's and Alzheimer's.

"We are now testing it in Parkinson's disease in dopamine-producing neurons," Akirav said. "We've identified some signatures in these cells, and that would be really great. If you can diagnose this disease before the tremors start, it's a completely different ballgame with these patients."