The Prevalence of CYP2C19 Polymorphism in Patients with Symptomatic Intracranial Atherosclerosis Open Access

Ischemic stroke is a common disease and the leading cause of disability and death. Large vessel atherosclerosis accounts for most of the ischemic stroke cases. Among patients with atherosclerosis, intracranial artery stenosis is common among Asian population [1]. In our previous study, intracranial atherosclerosis is the cause of ischemic stroke in 51% of the patients who had large vessel atherosclerosis, categorized by Trial of ORG 10172 in Acute Stroke Treatment (TOAST) classification [2].

Symptomatic intracranial stenosis can lead to significant cerebrovascular events such as transient ischemic stroke (TIA) or ischemic stroke. In managing this condition, dual antiplatelet therapy (DAPT), which typically involves the administration of aspirin and clopidogrel, has been a subject of extensive research and discussion. The efficacy of DAPT in preventing recurrent strokes in patients with symptomatic intracranial stenosis has been supported by various studies [3, 4]. For instance, the SAMMPRIS trial highlighted the potential benefits of aggressive medical therapy, including DAPT, for patients with high-grade intracranial atherosclerosis. Therefore, the recent American Stroke Association guideline also suggests combination of aspirin and clopidogrel for 3 months in patients with symptomatic intracranial atherosclerosis [5].

Clopidogrel is a medication used to prevent blood clots in patients with certain cardiovascular conditions, and its effectiveness can be influenced by genetic variations. The CYP2C19 enzyme, encoded by the CYP2C19 gene, is crucial for converting clopidogrel into its active form. Variations in the CYP2C19 gene can lead to different metabolic rates which can affect the drug’s efficacy and safety. The Clinical Pharmacogenetics Implementation Consortium (CPIC) provides guidelines for clopidogrel therapy based on CYP2C19 genotype, suggesting alternative treatments for those identified as poor metabolizers to ensure optimal therapeutic outcomes [6]. Poor metabolizers due to having little or no CYP2C19 enzyme activity have been reported in 2–14% of the population, depending on the ethnicity [7].

Previous study in Thai population demonstrated a 9–13% prevalence of poor metabolizer [8, 9]. However, patients with intracranial atherosclerosis are a special group for whom clopidogrel is required as the standard treatment. This study aims to find the types of genetic variation and more importantly, the prevalence of poor metabolizers among patients with intracranial atherosclerosis.

We performed a prospective study among ischemic stroke patients aged >18 years who presented with acute ischemic stroke at the King Chulalongkorn Memorial Hospital and were found to have symptomatic intracranial atherosclerosis during October 2014 and January 2016. Patients with ethnics other than Thai were excluded. The exclusion criteria include non-atherosclerotic intracranial stenosis and patients who required anticoagulant for treatment, due to associated cardiac conditions.

Significant intracranial atherosclerosis (>50% stenosis) was diagnosed and confirmed by 2 of the following methods: transcranial Doppler ultrasound (TCD), computed tomography angiography (CTA), or magnetic resonance angiography (MRA). TCD: more than 50% stenosis of M1 MCA and A1 ACA are diagnosed by mean flow velocity more than 80 cm/s of basilar artery and intracranial vertebral artery are diagnosed by mean flow velocity more than 60, and 50 cm/s, respectively. CTA and MRA: the North American Symptomatic Carotid Endarterectomy Trial (NASCET) criteria were used for stenosis calculations: $Dn-Ds/Dn×100$⁠, where Dn is normal diameter and D_s is stenosed diameter.

All participants’ blood samples were collected for detection of CYP2C19 polymorphisms (CYP2C19*2, CYP2C19*3, and CYP2C19*17) using real-time polymerase chain reaction (rt-PCR) technique. The binomial test was performed for comparative analysis of allelic frequencies for CYP2C19 polymorphism. The CYP2C19 genotypes are classified into 4 predicted phenotypes which based on genotype including normal metabolizer (CYP2C19*1/*1), intermediate metabolizer (CYP2C19*1/*2, CYP2C19*1/*3, CYP2C19*2/*17, CYP2C19*3/*17), poor metabolizer (CYP2C19*2/*2, CYP2C19*2/*3, CYP2C19*3/*3), and rapid metabolizer(CYP2C19*1/*17, CYP2C19*17/*17) [6, 10].

Continuous variables were tested for a normal distribution. If the data were in normal distribution, the data would be reported as mean and standard deviation and analyzed with an independent t test. If the data were not in a normal distribution, the data would be reported as a median and interquartile range and analyzed with the Mann-Whitney U test. Categorical variables were reported as frequency in percentage (%) analyzed with Fisher’s exact test or chi-square test. Differences were considered statistically significant at p < 0.05. We performed data collection by Microsoft Excel in Microsoft Office Home and Student 2016 version. Statistical analysis was performed by using SPSS statistics version 23.0 for Windows.

During the study period, there were 1,028 patients with ischemic stroke. One hundred patients met the inclusion criteria and were enrolled into the study. The demographics and clinical characteristics are presented in Table 1. The mean age of patients was 66 years old and 54% of them were male. Hypertension was the most common risk factor and was found in 79% of the cases.

The frequency of alleles CYP2C19*1, *2, *3, and *17 was 70.5%, 26%, 2.5%, and 1%, respectively, as shown in Table 2. Normal metabolizer (*1/*1 homozygous genotype) was observed in 53 patients. Poor metabolizer was found in 10 patients. This consisted of 7 patients (7%) with CYP2C19*2/*2 genotype and 3 patients (3%) with CYP2C19*2/*3 genotype. Intermediate metabolizer was observed in 35% of the cases. The genotype and metabolize activities are shown in Table 3.

Clopidogrel is thienopyridine inhibitor of P2Y12 ADP platelet receptor. Hepatic metabolism is essential to generate clopidogrel’s active metabolite. There are 2 sequential oxidative steps in this pathway. Cytochrome P450 (CYP) system is involved in oxidative drug metabolism. The first metabolic step, which leads to 2-oxo-clopidogrel, is catalyzed by CYP1A2, CYP2B6, and CYP2C19. The second step, which converts to the active metabolite, is involved in CYP2B6, CYP2C9, CYP2C19, and CYP3A4 [11].

As CYP2C19*1 represents the wild-type allele, genetic polymorphisms of CYP2C19 have been identified. The CYP2C19*2 and *3 are common alleles associated with no functional metabolism of clopidogrel. CYP2C19*17 is another variant which was found to increase enzyme activity. Patients with two loss-of-function alleles (either *2 or *3) are considered as poor metabolizer status whereas those with only one loss-of-function allele are the intermediate metabolizer [6].

Report of poor metabolizer phenotype in Caucasian and African American population occurs between 2 and 4% while the prevalence of poor metabolizer among Chinese, Japanese, and Koreans is higher [7]. The frequency of poor metabolizer in Asian population is about 14%. In Sen et al. [12] study, CYP2C19 polymorphism in ischemic stroke patients in Turkey found intermediate metabolizer (*1/*2) 13/51 (25%) and poor metabolizer (*2/*2) 2/51 (4%). None of patients in Turkey had *3 allele [12]. Sukasem et al. [8] reported CYP2C19 polymorphism in Thai population. Intermediate metabolizer and poor metabolizer phenotype were 42 and 13 percent, respectively. Thai population had both *2 and *3 alleles: allele frequency as 27 and 6 percent, respectively [8]. Our study of CYP2C19 polymorphism in symptomatic intracranial atherosclerosis was comparable to previous Thai studies.

Report of the effect of CYP2C19 phenotype on the pharmacokinetics and pharmacodynamics of clopidogrel showed that patients with poor metabolizer genotype exhibited significant lower plasma concentration and antiplatelet effect after 300-mg loading dose and 75-mg daily from day 2 to 7 [13]. According to the US FDA recommendation, patients who can poorly metabolize clopidogrel are recommended to use other antiplatelets or alternative dosing strategies. More importantly, patients who are the intermediate metabolizers have increased risks for serious cardiovascular outcomes, including stent thrombosis. Therefore, some recommendations suggest switching from clopidogrel to an alternative antiplatelet of patients who are intermediate metabolizer [14]. Another study comparing the efficacy of clopidogrel plus aspirin to ticagrelor plus aspirin on secondary stroke prevention among CYP2C19 loss-of-function allele carriers with high-risk TIA or minor ischemic stroke (CHANCE-2 study) showed that the incidence of recurrent stroke was significant lower in the ticagrelor group than in the clopidogrel group [15].

A meta-analysis of 25 studies enrolling patients with acute ischemic stroke and transient ischemic attack found that carriers of CYP2C19 loss-of-function alleles are at greater risk of stroke and composite vascular events than noncarriers [16]. However, a small study in Caucasian with intracranial stenosis showed conflicting results.

This study demonstrates that among Thai patients with symptomatic intracranial atherosclerosis where combination of clopidogrel and aspirin is indicated, 29% were carriers for the loss-of-function allele (CYP2C19*2 or *3 polymorphism). This group of patients may not benefit from clopidogrel therapy as much as those without loss-of-function mutation. Although genotype-directed therapy may help identify patients who are likely to benefit most from clopidogrel or alternative antiplatelet therapy, genotype testing is costly and not widely available, especially in the developing countries. For clinicians, individualized clinical judgment is very important and the test is to justify in patients who had recurrent stroke despite ongoing clopidogrel treatment.

Almost one-half of Thai patients with symptomatic intracranial stenosis patients had at least one of loss-of-function alleles. Combination of aspirin and clopidogrel in this group of patients might not be effective to prevent further stroke or any vascular events. Additional CYP2C19 loss-of-function alleles test in patients who have indication to take clopidogrel, will assign appropriate therapy to patients who need.

This study protocol was reviewed and approved by Chulalongkorn Institutional Review Board of Faculty of Medicine, Chulalongkorn University, Approval No. IRB 974/2557 and the principles for this research were based on the Declaration of Helsinki. Written informed consent was obtained from participants provided that the participants were fully conscious. A proxy was given access to detailed information in case the participants were at risk of coercion. Consequently, a proxy provided the written informed consent.

The authors have no conflicts of interest to declare.

This study received a grant from eived a grant from The Neurological Society of Thailand, Thailand. The Neurological Society of Thailand, Thailand.

Nijasri C. Suwanwela, the corresponding author, had full access to all the data in the study, and takes responsibility for the integrity of the data and the accuracy of the data analysis. Concept and design, acquisition, analysis, or interpretation of data: Songchai Kittipanprayoon, Aurauma Chutinet, and Pajaree Chariyavilaskul. Drafting of the manuscript and statistical analysis: Songchai Kittipanprayoon and Pongpat Vorasayan. Critical revision of the manuscript for important intellectual content: Pongpat Vorasayan, Aurauma Chutinet, and Nijasri C. Suwanwela. Administrative, technical, or material support: Pajaree Chariyavilaskul. Supervision: Nijasri C. Suwanwela.

The data that support the findings of this study are not publicly available due to their containing information that could compromise the privacy of research participants but are available from the corresponding author (N.C.S.) upon reasonable request.