GIFT Exome Analysis Unearths Two New Genes Related to Platelet Function

March 24, 2012

March 24, 2012 (Chicago, Illinois) — The first whole exome analysis of the genome of almost 150 white patients taking part in the GRAVITAS trial has revealed two new genes that appear to be critical to platelet function.

"CYP2C19*2 only explains a small portion of the overall variability in the clopidogrel response," Dr Matthew Price (Scripps Clinical and Translational Science Institute, La Jolla, CA), who presented the new findings, told heartwire . "So the question is, can we find other markers that further explain the variability? And therefore have a comprehensive genetic panel, so we can make better decisions before the procedure about which antiplatelet therapy to use?"

Price presented the results of the Genotype Information and Functional Testing (GIFT) exome study--the first to use whole exome sequencing to identify novel genetic determinants of clopidogrel response variability--during a featured interventional session here at the American College of Cardiology 2012 Scientific Sessions this morning.

The first new gene identified, the ATP2B2 variant, "is a very provocative and interesting molecule that plays a critical role in maintaining intracellular calcium homeostasis," said Price, and is associated with higher on-treatment platelet reactivity. The second, TIAM2, codes for a protein involved in activation of secondary messengers involved in platelet aggregation and is associated with low platelet reactivity.

The bigger picture is the power of exploratory pharmacogenomics for the assessment of the response to drugs.

"This is only the beginning, it's preliminary. There is much more work to do, more validation," he says, but adds, "It would be easy now to make a test of the two genes identified.

"But the bigger picture is the power of exploratory pharmacogenomics for the assessment of the response to drugs," Price stresses. "Our findings demonstrate the feasibility and potential of exploratory pharmacogenomics using exome sequencing to pinpoint unidentified mechanisms of drug response. What it allows you to do is to really understand both the variability in response and also adverse effects, like bleeding and so forth, without needing to know mechanisms."

What Is Exome Sequencing?

Price explained that exome sequencing is the sequencing of the entire protein-coding regions of the human genome and as such is more likely to detect mutations with a greater impact on disease. "It identifies both single nucleotide polymorphisms [SNPs] and insertion/deletions [indels] and, unlike genomewide association studies [GWAS], can identify actual, causative variants associated with disease. GWAS looks at linking the response with certain zip-codes in the genome, whereas exome sequencing looks at the base pairs and the relationship of the actual mutation with the response. It's a different approach, and they are complementary."

Exome sequencing looks at the base pairs and the relationship of the actual mutation with the response.

Price and colleagues used genetic samples from the GRAVITAS study, which was designed to see if double-dose clopidogrel (150 mg) would be of any benefit in patients with high on-treatment platelet reactivity after PCI; it wasn't. On-treatment platelet reactivity in GRAVITAS was assessed using the VerifyNow P2Y12 test at baseline and at 30 days. Price and colleagues selected 147 self-identified white subjects for exome analysis.

"You get the base pairs for about 1.5% of the human genome, you get the exons in more than 21 000 genes. This is a lot of data. It's not just sequencing, it’s the counting--we are talking about a load of information and a lot of processing time. The real challenge now with sequencing is not getting the base pairs but actually the analysis, because of the amount of data one needs to look at. And you need to have good quality checks because there can be a lot of junk, a lot of misreads; this takes a lot of time. It takes supercomputers, and it is a fairly expensive and very rigorous analysis."

Findings Preliminary But Exome Sequencing Becoming More Feasible

In their analysis, there were three distinct loci associated with on-treatment platelet reactivity at baseline (12–24 hours post-PCI, after standard periprocedural clopidogrel regimen): the ATP2B2 gene, which they think is present in around 27% of the general white population; TIAM2, found in about 13% of the population; and the CYP2C18 and CYP2C19 alleles.

The limiting factor is not sequencing, it's the computational biology. But we are getting there.

At 30 days after PCI, they saw only the CYP2C18 and CYP2C19 cluster "and nothing else." This singular influence of CYP2C18/9 locus at 30 days of maintenance clopidogrel after PCI means it's "unlikely that other protein-coding variants have a large effect on response variability at this time point," Price noted.

He added that while the findings have to be considered "preliminary, identification of two genes critical to platelet function among the 21 000 sequenced genes lends credibility to the validity of the result."

The next step is to validate these results in more than 1000 patients, which is ongoing, he says. He adds that exome sequencing is becoming more feasible: "The most expensive part is the reagents, but we were down to a little more than a $1000 per genome (per person), and every month it's getting cheaper and cheaper. The limiting factor is not sequencing, it's parsing through the data, the computational biology. But we are making progress, we are getting there."

GIFT was supported through a grant from Bristol-Myers Squibb/Sanofi, and exome analysis was made possible by a grant from Molecular Response and in-kind support from Agilent Technologies. Price reports consulting honoraria from Bristol-Myers Squibb/Sanofi, Accumetrics, DSI/Lilly, Merck, Janssen, AstraZeneca, the Medicines Company, and Medicure; speaker honoraria from DSI/Lilly and AstraZeneca; research support from Bristol-Meyers Squibb/Sanofi, Quest Diagnostics, Accumetrics, and Molecular Response; and equity interest in Iverson Genetics.

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