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Mount Sinai Group Publishes Allele-Specific PCR Assay for Novel CYP2C19 Variants

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Having published research last year identifying a novel haplotype that may alter the interpretation of CYP2C19 genotyping, clinical researchers from the Icahn School of Medicine at Mount Sinai in New York have now published a paper describing an allele-specific PCR assay to detect and discriminate the various alleles associated with the haplotype.

The researchers believe that their assay, which specifically detects and discriminates the CYP2C19*4A, *4B, and *17 alleles, could provide valuable information on the ability of carriers to metabolize certain drugs, particularly the anti-platelet drug clopidogrel, not currently addressed by available CYP2C19 genotyping tests.

As such, the group plans to seek approval from the New York State Department of Health to use the assay at Mount Sinai Hospital as a reflex test after identifying certain alleles during targeted, pre-emptive CYP2C19 genotyping to aid in therapeutic administration, Stuart Scott, an assistant professor of genetics and genomic sciences at the medical school and corresponding author on the recent publications, told PCR Insider this week.

The importance of assessing variant CYP2C19 alleles to gauge drug response and predict adverse events, particularly with regards to clopidogrel response, has been on the rise, as multiple CYP2C19 genotyping assays have been cleared by the US Food and Drug Administration — most recently Spartan Biosciences' Spartan RX CYP2C19 assay (PCR Insider 8/29/2013); and several medical institutions, such as the University of Ottawa Heart Institute and Vanderbilt University Medical Center, have implemented point-of-care or pre-emptive CYP2C19 genotyping to guide antiplatelet therapy (PGx Reporter 8/11/2010 and 10/6/2010).

Currently, all FDA-cleared CYP2C19 genotyping assays include *17 among other alleles, but not the *4 allele. However, last year, as part of a study examining the frequencies of certain CYP2C19 alleles in various ethnic populations, the Mount Sinai group identified a novel allele called CYP2C19*4B – work that was published in The Pharmacogenomics Journal.

"A couple of years ago we started doing [CYP2C19] population genotyping … to look for novel alleles and establish allele frequencies in different populations," Scott said. "We came across the *4 allele, and it was often found with *17 – the genotype results always came back that both alleles were present, but we didn't know the phase. And we rarely saw a *4 allele by itself. And we would also sometimes see a *17 homozygote in addition to a *4 allele in the same individual. And that really indicated that the two alleles were in cis, or on the same haplotype."

To confirm this theory the group conducted allele-specific PCR cloning and sequencing, and indeed found that the alleles are on the same haplotype sometimes, but not all the time.

"This was something that we found more commonly in the Ashkenazi Jewish population, as well," Scott said. "And it was interesting to us and others, too, because *17 … is the gain-of-function promoter allele, and *4 is a loss-of-function allele. So there was kind of opposing phenotypic forces at play."

Essentially, as the researchers explained in greater detail in a paper published last week in the Journal of Molecular Diagnostics, the *17 allele is common in most populations and many commercial genotyping assays test for it; however, the increased response conferred by this promoter variant is abolished when accompanied by the downstream ATG translation initiation mutation that together define *4B. Furthermore, this translation initiation mutation occurs independent of the *17 variant, an allele that is now designated *4A.

Given that current genotyping and sequencing assays are unable to distinguish the phase of these variants, Stuart and colleagues developed a rapid allele-specific PCR method to detect and differentiate the related *4A, *4B, and *17 alleles for improved pharmacogenetic metabolizer prediction.

"Lots of [assays] test for *17, [and] … the FDA has approved it for a couple of [genotyping] tests already," Scott said. "But they haven't included *4. That's important because [clinicians] can genotype *17 and on occasion call that [patient], depending on what's on that other allele, an ultra-rapid metabolizer [of clopidogrel]. But if you don't look at *4, you don't know if it's there or not."

What's more, some of those *17 carriers – although not a high percentage – end up being *4B carriers, meaning they have a loss-of-function allele and thus would be intermediate or poor metabolizers. As such, the researchers believe that their assay could be used as a reflex test to increase accuracy when CYP2C19 genotyping reveals a *4 or *17 mutation.

The researchers developed their assay based on allele-specific PCR, also known as the amplification-refractory mutation system, a well-established PCR technique for haplotype determination.

"I spend a lot of my time in a molecular diagnostic lab," said Scott, who is also assistant director of the Cytogenetics and Cytogenomics Laboratory and the Molecular Genetics Laboratory at Icahn School of Medicine. "And this seemed like it was an easy thing to do using an established molecular biology technique like allele-specific PCR. That was only possible, though, because the two alleles are relatively close to one another, about 800 or so nucleotides apart. That allowed us to design an assay using multiple different primer combinations to be able to detect it quickly … if and when we detect a *17 or *4 using another commercial genotyping assay."

The researchers currently use electrophoretic detection to analyze the amplification products from their assay, and Scott said that the entire protocol can be completed in about two hours. Scott conceded that this turnaround time may not be compatible with the recent advent of rapid CYP2C19 genotyping tests like Spartan's, which aims to provide results in less than an hour.

"But it depends on the testing strategy," Scott said. "Rapid point-of-care testing is certainly one option. Another option that some larger academic medical centers are moving toward is kind of a pre-emptive genotyping strategy, and that requires a fairly large commitment from the institution to pull it off."

For example, Mount Sinai has initiated pre-emptive genotyping of its biobank, Scott said. "So the genotype goes into the electronic health records, and then only when a prescription is made for these individuals will that genotype be looked at," he said. "If there is an intersection between an at-risk genotype and a prescription for a medication that would interact with that … the physician [is notified] that this person is a poor metabolizer."

That kind of pre-emptive genotyping strategy, Scott noted, does not require such a rapid turnaround time.

Nevertheless, the group is hoping to refine its assay to increase the turnaround time with the idea that it could possibly also provide valuable supplementary information to rapid CYP2C19 genotyping at the point of care, as those tests are developed.

In the meantime, Scott and colleagues are gearing up to submit the assay to the NYS DOH in order to gain clearance to begin using it as part of Mount Sinai's pre-emptive genotyping program. In addition, Mount Sinai has applied for a patent covering the assay and is looking to license it to commercial CYP2C19 test developers, Scott said.

The FDA-approved label for clopidogrel includes a black box warning generally informing doctors and patients that alternative treatments or treatment strategies should be considered for CYP2C19 poor metabolizers. In the pharmacogenomics section of the label, the agency discusses the impact of specific CYP2C19 alleles and includes *4 among less-frequent alleles associated with reduced drug metabolism. The label doesn't mention the *17 allele.

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