Introduction
Clopidogrel is an antiplatelet drug widely used in patients with acute
coronary syndrome (ACS), including those with non-ST-segment elevation
ACS (unstable angina UA or non-Q wave myocardial infarction) and
ST-segment elevation myocardial infarction
(NSTEMI)(1-3). Specifically,
non-ST-segment elevation ACS includes patients with stent implantation
after percutaneous coronary intervention, patients with peripheral
arterial disease, and patients with recent myocardial infarction or
recent ischemic stroke(4-6). Clopidogrel,
a prodrug, has no pharmacological
activity(7,
8), but active metabolites are mainly
produced by the cation of the CYP2C19
enzyme(9-11). The active metabolites
produced irreversibly bind to the P2Y12 receptor on the surface of
platelets, inhibiting their aggregation and interfering with
ADP-mediated platelet activation for an overall antiplatelet
effect(12,
13). Genetic variation of the CYP2C19
gene leads to individual differences in CYP2C19 enzyme activity,
resulting in four phenotypes: ultrafast metabolizer (UM), fast
metabolizer (EM), intermediate metabolizer (IM) and slow metabolizer
(PM)(14). CYP2C19 UM patients treated
with conventional doses of clopidogrel generate increased levels of
active metabolites, with increased platelet inhibition, enhanced
antiplatelet function, and increased risk of
bleeding(15). Treatment of CYP2C19 PM
patients with conventional doses of clopidogrel results in decreased
inhibition of platelets, decreased antiplatelet function, and increased
risk of thrombosis(16). CYP2C19 * 2
(rs4244285, c.681G > A) and CYP2C19 * 3 (rs4986893, c.636G
> A) are two major alleles in the Chinese
population(17) with frequencies of 23.1%
-35% and 2% -7%, respectively. The CYP2C19 * 17 (rs12248560, c.-806C
> T)-encoded CYP2C19 enzyme displays increased activity,
and the frequency of occurrence in the Chinese population is
approximately 0.5%-4%(18). In general,
methods that detect and distinguish CYP2C19 genotypes should be reliable
and rapid, especially when the purpose is for clinical medication
guidance, which is related to safety and treatment costs.
Several methods have been developed to detect CYP2C19 gene
polymorphisms. However, these methods are almost all performed by highly
skilled technicians in well-equipped referral hospitals, and the process
is complex and time-consuming. This approach may exclude those who need
the medication, which is not helpful for rapid guidance clinically.
Sanger sequencing (referred to as polymerase chain reaction-Sanger
sequencing) of PCR-amplified fragments from patient samples is the
current ’gold standard’ method for detecting CYP2C19
genotypes(19,
20). It facilitates accurate detection of
all nucleotide mutations, including new mutations not previously
reported. Nonetheless, Sanger sequencing instrumentation is often
unaffordable for most local hospitals. In addition, the sequencing
process is complex and time-consuming, requiring very careful operation
to avoid contamination by PCR amplification. This is also true for other
sequencing-based analyses, such as pyrosequencing and next-generation
sequencing, as well as analyses based on high-end instruments, such as
DNA microchips and mass spectrometry(21).
Several simpler and more cost-effective methods have been developed to
detect CYP2C19
genotypes(22). Real-time fluorescent
quantitative polymerase chain reaction is a good platform for clinical
diagnosis because its closed detection format makes it easy to use and
fast and reduces the contamination of amplification
products(23,
24), and
a real-time amplification
polymerase chain reaction (PCR) method to detect CYP2C19 genotype
polymorphisms has been established(25,
26). However, due to the limited coverage
of multiple detection of mutation sites, it is far from practical.
In this study, we developed a detection strategy based on PCR
amplification combined with melting curve analysis that uses a
combination of adjacent probes and TaqMan probes for amplification. This
strategy can detect 9 genotypes of CYP2C19 in a single tube. Here, we
describe a new rapid PCR combined with melting curve analysis method for
rapid (within 1 h) detection of CYP2C19 * 2/3/17 sites in one tube. We
named this method Rapid PCR Melting Curve Method (RPCR-MC). We
systematically evaluated its analytical performance, including mutation
detection accuracy, analytical sensitivity, specificity and detection,
in clinical samples. We tested the RPCR-MC method by analyzing samples
from 93 patients with a high risk of thrombosis after percutaneous
coronary intervention(PCI) and compared the results with those of Sanger
sequencing.