We analysed the CYP2C19 genotype screening results of 1406 patients genotyped at the genetic diagnosis centre of the First Hospital of Jilin University from October 2016 to December 2019, and the frequency ofCYP2C19LOF variant allele carriers was 58.04%. Furthermore, the allelic frequencies of CYP2C19 variants display significant interethnic differences7. In Asian countries, CYP2C19LOF variant allele carriers are observed at a relatively high frequency14,15, and the frequencies of the CYP2C19*2 and *3 alleles in Chinese Han populations are significantly higher than those in other racial groups7. A previous study reported that the allele frequency of CYP2C19LOF variant allele carriers is 62.38% in China16.The results of our study are similar to the frequencies of the variant alleles CYP2C19*2 and *3 reported in previous studies.
The rate of MACCEs in the conservative treatment group was 14.49%, and that in the cerebral artery stent groupwas 9.14% (Table S1). A 403-patient study showed that MACCEs were observed in 8.19% of patients after treatment with CAS17, which is in line with our study. Considering the limited number of patients, the rate of MACCEs in this studymay not reflect the overall situation. This could also be driven by the patients who underwent conservative treatment, and carotid artery stents were almost all moderate-severe cerebral artery stenosis disease patients. Because conservative treatment patients whose own condition is not good enough to undergo surgery, they cannot tolerate carotid intervention operations, such as age factors, fragility of blood vessels and cardiac insufficiency, only orally. In this way, the rate of MACCEs in the conservative treatment group was higher than that in the cerebral artery stent group.
As a commonly prescribed antiplatelet, clopidogrel is usually used to prevent secondary ischaemia in patients treated by endovascular techniques.Composite MACCE outcomes, such as death, stroke, and stent thrombosis, limit the long-term success rate of interventions through the recurrence of symptoms. Although intervention has been associated with minimal complications, previous studies have reported that CYP2C19 loss-of-function polymorphisms may be a significant risk factor for in-stent restenosis18. Clopidogrel plus aspirin is used to reduce the risk of recurrent stroke. As one of the most prominent genetic polymorphisms, CYP2C19 polymorphisms can be used to explain a poor response to clopidogrel, and CYP2C19*2 is the strongest predictor of high residual platelet reactivity19.However, few studies have reported the significance of CYP2C19polymorphismsin affecting the incidence of MACCEs aftercarotid artery stenosis, especially carotid artery stents.
It has been reported that patients with CYP2C19 LOF variant alleles have a 3.58 times higher risk for death and stroke than patients with the CYP2C19 wild-type genotype20. A recent systematic review also demonstrated that CYP2C19 loss-of-function alleles were associated with clinical outcomes for ischaemic stroke21.In addition, a previous study reported that CYP2C19*2 was an independent risk factor for the primary outcomes of clopidogrel treatment in patients with acute ischaemic stroke22. In our study, the incidence of MACCEs in CYP2C19 LOF allele noncarriers (n = 343) was 7.14% (9/126), and in carriers, it was 14.69% (26/177). Patients with CYP2C19 LOF variant alleles had a 2.05 times higher risk of MACCEs than patients with the CYP2C19 wild-type genotype. Table 2 shows that there was no significant difference in the frequency of CYP2C19 LOF alleles in the conservative treatment group regardless of whether the patients experienced MACCEs. The frequency of CYP2C19 LOF alleles of the patients who experienced MACCEs was significantly higher than that of the non-MACCE patients. In the log-rank tests and multivariable Cox analysis, we found that CYP2C19 LOF allele variants were associated with an increased risk of MACCEs in patients who underwent cerebral artery stents. In this way, we infer that CYP2C19 LOF allele carriers are related to MACCE incidence in the cerebral artery stent group. These results could be driven by the fact that within conservative treatment patients, the patient's condition is so poor that theCYP2C19 gene does not work.
In addition, the baseline characteristics showed that in the conservative treatment group, the glucose of the patients who experienced MACCEs was significantly higher. In addition, in the cerebral artery stent group, the frequency of CYP2C19 LOF alleles and the ALT of the patients who experienced MACCEs was significantly higher, and the total protein was significantly lower. To identify the influencing factors of the clinical endpoint after cerebral artery stenosis, we first performed log-rank tests. We found that CYP2C19 LOF allele variants, high ALT levels, low total protein levels and high blood glucose levels were associated with an increased risk of MACCEs in patients who underwent cerebral artery stents. However, only a high blood glucose level is associated with an increased risk of MACCEs in patients who underwent conservative treatment.These findings suggested that glycaemic control was key to MACCEprevention in all carotid artery stenosis patients.In addition, we generated Kaplan-Meier curves to assess the association of CYP2C19 LOF alleles and the incidence of MACCEs stratified by patient status. We found that the CYP2C19 LOF allele carriers were identified as a predictor of the incidence of MACCEs at 6 months after carotid artery stenting only when the patients had high levels of glucose (glucose > 6.5 mmol/L).
A Danish cohortof nearly 60 000 patients with myocardial infarction demonstrated that the clinical efficacyof clopidogrel in diabetes mellitus (DM) was impaired23. It has also been reported that DM significantly increases the risk of stroke recurrence and poor outcome in the small-artery occlusion subtype22. In our study, the rate of MACCEs in CYP2C19 LOF allele carrierswas higher than that in noncarriers in patientswith high blood glucose levels who underwentcarotid artery stents, while in patients with normal blood glucose levels, it was not. This could be ascribed to high blood glucose levels decreasing the active metabolite of clopidogrel24, especially impairing the clopidogrel clinical efficacy of CYP2C19LOF allele carriers with genetically poor clopidogrel metabolism. The mechanism of this has been reported: the active metabolite of clopidogrel could increase plateletreactivity by upregulating platelet P-selectin25 and proteinkinase C26 via the glycation of platelet surface proteinsand osmotic effects of glucose27. These findings remind usthat it is necessary to performCYP2C19genotype tests in patients who underwent cerebral artery stents with dysglycaemia.
Next, we used the Cox proportional hazards regression model to assess the association between CYP2C19 LOF allele carriers and patient status with MACCE risk.As shown in Table 3, the univariable analysis and multivariable Cox analysis revealed that glucose > 6.5 mmol/L was significantly associated with MACCEs at 6 months in the conservative treatment group. Nevertheless, the univariable analysis revealed that CYP2C19 genotypes, ALT > 35 U/L, total protein < 65 g/L and glucose > 6.5 mmol/L were significantly associated with MACCEs at 6 months in the cerebral artery stent group (Table 4). We found that CYP2C19 genotypes, total protein < 65 g/L and glucose > 6.5 mmol/L remained significant in the multivariable Cox analysis. Collectively, these results indicated that CYP2C19 LOF allele variants, total protein levels anddysglycaemiasynergically impact the risk of MACCEs.On the one hand, malnutrition has an important prognostic value in patients who undergo cerebral artery stents28,and total protein < 65 g/L may reflect themalnourished status of the patient. On the other hand, it could be due to poor kidney function, as the protein is lost in the urinary system, leading to a decrease in total protein. treatment resistance for clopidogrel. It has been reported that chronic kidney disease is associated with impaired drugabsorption and transport and platelet abnormalities, which are thought to decrease the response to clopidogrel11,29. In addition, CYP2C19 LOF wasreported to be associated with an increased risk of adverse cerebrovascularoutcomes in patients in the lowest quintile of eGFR.In this way, low total protein levels may reflect poor kidney function, and the response to clopidogrel is decreased.
In this way, we conclude thatCYP2C19 LOF allele variants, total protein levels and dysglycaemia synergically impact the risk of MACCEs in patients who underwent cerebral artery stents. In addition, it is necessary to perform CYP2C19 genotype tests in patients who underwent cerebral artery stents with dysglycaemiaand low total protein levels.
There are several limitations in our study. Considering that this is a single-centre study, further multicentre and large sample studies are needed to expand upon our findings. Glucoselevels were assessed only at baseline, and a dynamic evaluationof glucose levels could be more informative. In addition, glucose levels can only reflect the current status, and glycated albumin and haemoglobin A1c can more accuratelyreflect the actual status of glycaemic control. Moreover, we did not assess the status of insulin resistance, which could lead to abnormal plateletP2Y12 receptor signalling in diabetic patients.