Genetic Polymorphisms, LDL-C and sdLDL-C Help Optimize Coronary Heart Disease (CHD) Therapy

Background: Low-density lipoprotein cholesterol (LDL-C) and small, dense LDL-C (sdLDL-C) are important risk indicator of coronary heart disease (CHD), but their application in therapy monitoring of CHD is still far from being elucidated. Following the concept of precision medicine, we investigated whether the scientic medication based on medication-sensitive genes can reverse the LDL-C and sdLDL-C status in human bloodstream, so as to reveal the possibility of them as a monitoring indicator of CHD ecacy. Methods: A prospective study of CHD cohort containing 208 Chinese CHD patients (158 males and 50 females) and 20 healthy people (14 males and 6 females) was recruited. LDL-C and its subfractions were detected before and after treatment. Polymorphism of medication-sensitive genes, including SLCO1B1 (rs4149056, 521T>C), CYP2C19*2 (rs4244285, c.681G>A), and CYP2C19*3 (rs4986893, c.636G>A) were detected for medication guidance. Results: Nearly half of Chinese CHD patients (47.60%, 99/208) had genetic polymorphisms with homozygous or heterozygous mutations within these three genes. LDL-1 and LDL-2, subfractions of LDL-C, had a 100% positive rate in CHD patients and healthy people. However, sdLDL-C components of LDL-5 to LDL-7 were only enrichment in CHD patients. Moreover, the mean amount of sdLDL-C subfractions in CHD patients was signicantly higher than that in healthy people. Among 180 patients with treatment remission, 81.67% (n=147) of CHD patients had decreased LDL-C, while 61.67% (n=111) of patients had decreased sdLDL-C. Conclusion: sdLDL-C has better accuracy on than suitable detection would optimize the treatment strategy for CHD patients.


Introduction
Coronary heart disease (CHD) is an important public health disease that causes death and disability [1].
Based on the data from the National Health and Nutrition Examination Survey (NHANES) 2011 to 2014, an estimated 16.5 million Americans ≥20 years of age had CHD, and approximately 720,000 new coronary events occurred in America in 2018 [1]. In China, more than 10 million people have been affected by this disease [2]. Low-density lipoprotein cholesterol (LDL-C) plays a pivotal role in the progression of CHD, as veri ed by genetic, pathology, observational, and intervention studies [3][4][5][6]. In 36,375 participants and a low 10-year risk cohort with long-term follow-up, LDL-C and non-HDL-C ≥160 mg/dL were independently associated with a 50% to 80% increased relative risk of cardiovascular disease (CVD) mortality [7]. The risk of CHD is accelerated by 10 to 20 years in men and 20 to 30 years in women with LDL-C levels ≥190 mg/dL [8], and the Adult Treatment Panel (ATP III) cholesterol guideline from 2001 [9] and the 2013 American College of Cardiology (ACC)\American Heart Association (AHA) guideline [10] recommend intensive treatment of patients with primary elevation of LDL-C [8,11]. CHD treatment may include reducing the level of LDL-C to prevent recurrent cardiovascular events (CEs) [5], and LDL-C has now largely replaced total cholesterol as a risk marker and the primary treatment target for hyperlipidemia [12].
LDL-C is a heterogeneous class of particles, and accumulating evidence suggests that different LDL subfractions vary in their risk pro les [13,14]. Lipoprotein pro les consisting of primarily LDL-1 and LDL-2 subfractions have been designated as Pattern A, while pro les with predominantly small and dense   subfractions (LDL-3 to LDL-7), known as small, dense LDLs (sdLDLs), have been designated as Pattern B [15]. Patients with the same LDL-C levels may have different cardiovascular risks, due to the difference on sdLDL-C subfractions [16][17][18]. In 808 participants from the Chinese Multiprovincial Cohort Study, sdLDL-C was independently associated with the progression of carotid atherosclerosis [19]. The study showed that sdLDL-C was the most effective predictor of residual risk of future CEs in those using statins and in highrisk coronary artery disease patients with diabetes or hypertriglyceridemia [20].
Although there are many reports about the excellent predictive performance of LDL-C and sdLDL-C for cardiovascular disease, the clinical application research especially in drug curative evaluation is still lacking; therefore, we wanted to assess patient status by LDL-C or sdLDL-C concentration change before and after using clopidogrel or statins to obtain more clinical application value, especially in Chinese CHD patients. A total of 208 CHD patients from the Quanzhou First Hospital A liated to Fujian Medical University (Fujian, China) were enrolled. CHD patients were assessed for LDL-C and sdLDL-C before and after treatment with statins or clopidogrel until the disease was alleviated.
To improve the treatment effect, genetic testing before medication administration was recommended to patients [21][22][23]. Simvastatin is the most common prescription medication for cholesterol reduction, but a single nucleotide polymorphism (SNP), rs4149056 T>C, in SLCO1B1 increases systemic exposure to simvastatin and the risk of muscle toxicity [21]. Individuals carrying the CYP2C19*2 allele have impaired pharmacodynamic responses to clopidogrel [22]. CYP2C19*2 and CYP2C19*3 can be genetically screened, and appropriate dose adjustments can be made on the basis of a patient's CYP2C19 genotype [23]. Therefore, we intended to perform pretreatment screening of SLCO1B1 (rs4149056, 521T>C), CYP2C19*2 (rs4244285, c.681G>A), and CYP2C19*3 (rs4986893, c.636G>A) with an Agena Bioscience MassARRAY system, which is an advanced detection system based on MALDI-TOF MS technology, with superfunction to detect dozens of gene loci in one sample [24]. Finally, the polymorphisms of these three genes were analyzed to help implement more effective treatment, and LDL-C and sdLDL-C data before and after treatment were analyzed. Overall, the clinical values of multigene testing, LDL-C or sdLDL-C were evaluated to acquire the best therapeutic effect for CHD patients. The ow chart is shown in Figure 1A.

Materials And Methods
Patients and sample collection Subjects were enrolled from the Quanzhou First Hospital A liated to Fujian Medical University (Fujian, China) from Jan 2018 to May 2020. There were a total of 228 subjects, including 208 CHD patients (158 males and 50 females) with a mean age of 64.48 and 20 healthy people (14 males and 6 females) with a mean age of 45.05 (Table 1). Peripheral blood samples (0.5 mL each) were extracted from subjects and centrifuged at 800 × g for 10 minutes to obtain supernatant plasma samples (0.2 mL each) for LDL-C detection and peripheral blood cell sediment for MassARRAY SNP detection. Under the guidance of the results of SNP, all CHD patients were given precise medication (mainly statins) according to the dosage recommended in Technical Guidelines for Gene Detection of Drug Metabolic Enzymes and Drug Targets (2015, China).

Statistical analysis
Statistical analysis was performed using GraphPad Prism 5 statistical software (GraphPad Software, Inc., San Diego, CA, USA). P<0.05 indicated a statistically signi cant difference.

Results
Multitudinous gene polymorphisms associated with drug e cacy in Chinese CHD patients.
After testing, 47.60% (99/208) of patients had genetic polymorphisms of homozygous or heterozygous mutations; in speci c, SLCO1B1 (521T>C) TT, TC, and CC were identi ed in 80.29%, 19.23%, and 0.48% of patients, respectively; CYP2C19*2 (681G>A) GG, GA, and AA were identi ed in 60.10%, 34.13%, and 5.77% of patients, respectively, while CYP2C19*3 (636G>A) GG, GA, and AA were identi ed in 92.31%, 7.69%, and 0.00% of patients, respectively ( Figure 1B). The results showed that these three genes had multitudinous polymorphisms in Chinese CHD patients and further a rmed the signi cance of polymorphism detection before statin or clopidogrel therapy. CHD patients had higher expression and more types of sdLDL-C subfractions.
For subfractions of LDL-C and sdLDL-C that were related to CVD [16][17][18], the precise subfraction expression levels of LDL-C and sdLDL-C in CHD patients and healthy people were detected using a Quantimetrix Lipoprint system. After analysis, the typical lipoprotein subfraction images showed that LDL-1 and LDL-2, known as Pattern A, collectively existed in the plasma of healthy people and CHD patients (Figure 2A), while CHD patients had extra LDL-3 to LDL-7, known as Pattern B or sdLDL-C ( Figure  2B). To ensure robust results, samples from 228 subjects were analyzed, including 208 CHD patients and 20 healthy people ( Table 1).
The clinical effect of LDL-C and sdLDL-C was then studied on CHD patient monitoring. A total of 208 CHD patients (158 males and 50 females) were enrolled, and their disease was alleviated after treatment. In total, 169 patients (81.25%) and 126 patients (60.58%) exhibited LDL-C and sdLDL-C decreases ( Figure  3A). All 208 CHD patients were evaluated for LDL-C and sdLDL-C before treatment; after treatment and disease remission, 180 CHD patients (134 males and 46 females) underwent detection once, and the percentage of patients with an LDL-C decrease was 81.67% (n=147), while the percentage of patients with an sdLDL-C decrease was 61.67% (n=111) ( Figure 3A). Twenty patients (17 males and 3 females) underwent posttreatment detection twice; the percentages with LDL-C and sdLDL-C decreases were 80.00% (n=16) and 55.00% (n=11), respectively ( Figure 3A).
To observe the dynamic changes of LDL-C and sdLDL-C in the process of disease remission, 8 CHD patients (7 males and 1 female) who underwent detection four times (one time before treatment and three times after treatment) were analyzed ( Figure 3B). As shown in from Figure 3A, the percentages of patients with LDL-C and sdLDL-C decreases were 75.00% (n=6) and 50.00% (n=4), respectively. However, the expression changes of LDL-C and sdLDL-C in the same patient were not exactly the same. For instance, the expression levels of LDL-C and sdLDL-C in patient 6 were both signi cantly decreased and remained relatively stable, with low expression in follow-up monitoring, while patient 5 had low expression sdLDL-C after therapy but had higher expression of LDL-C, though anesis of the CHD occurred after treatment ( Figure 3C). Overall, the expression of LDL-C was more consistent with the disease course of the patient than sdLDL-C.
LDL-C had a better monitoring effect than other clinical biomarkers.
To better understand the monitoring utility of LDL-C and sdLDL-C in CHD patients, they were compared with other clinical biomarkers, such as total cholesterol, total triglycerides, plasma HDL-C, plasma LDL-C, Apo A1, and Apo B. Forty-seven CHD patients were chosen from the 208 patients, and all of these biomarkers were detected before and after treatment. When CHD anesis occurred, the expression levels of total cholesterol, total triglycerides, plasma LDL-C, Apo A1, Apo B, LDL-C and sdLDL-C were decreased, and plasma HDL-C was possibly increased.
Among the 47 CHD patients (39 males and 8 females), the percentages of those with decreased total cholesterol, total triglycerides, plasma LDL-C, Apo A1, Apo B, LDL-C, and sdLDL-C and increased plasma HDL-C were 82.98%, 40.43%, 80.85%, 53.19%, 65.96%, 76.60%, 65.96%, and 25.53%, respectively ( Figure   3D). Although 82.98% of the CHD patients had decreased total cholesterol, total cholesterol is composed of LDL-C and HDL-C and thus was not clinically signi cant ( Figure 3D). LDL-C was a better biomarker for CHD treatment monitoring than sdLDL-C and other clinical biomarkers.
Conclusion. sdLDL-C was more suitable for CHD screening than LDL-C, while LDL-C was more suitable for CHD monitoring than sdLDL-C. Combined medication-related gene polymorphism, LDL-C and sdLDL-C detection would better optimize the treatment strategy for Chinese CHD patients.

Discussion
Statins are currently effective in the treatment of CHD and lowering blood lipids [26], and clopidogrel has been recommended to treat CHD in current clinical practice guidelines [27]. Before use, SLCO1B1 (rs4149056, 521T>C), CYP2C19*2 (rs4244285, c.681G>A), and CYP2C19*3 (rs4986893, c.636G>A) were detected to ensure the best treatment was administered [21][22][23]. In this study, the SNPs of these genes were detected by a Agena Bioscience MassARRAY system, which had the advantages of detecting dozens of gene loci in one sample [24], requiring fewer samples, having a lower cost, and obtaining results in a shorter time than the Sanger sequencing method [28]. The results showed that there were three genotypes associated with SLCO1B1 (521TT, 521TC, 521CC), CYP2C19*2 (681GG, 681GA, 681AA) and two genotypes associated with CYP2C19*3 (636GG, 636GA) in these 208 CHD patients ( Figure 1B). A total of 47.60% patients had homozygous or heterozygous mutations ( Figure 2C), and polymorphisms detection before statin or clopidogrel therapy could lead to better treatment strategies for Chinese CHD patients.
Numerous studies have shown that LDL-C and sdLDL-C were related to CEs [18,20]. LDL-C subfractions were detected prior to treatment in CHD patients and healthy people, and we identi ed the presence of LDL-5 C, LDL-6 C, and LDL-7 C in CHD patients, but not in healthy people ( Figure 2C and Figure 2D, Table  1). The mean amounts of sdLDL-C subfractions were calculated; CHD patients also had higher expression of sdLDL-C subfractions than healthy people ( Figure 2D). These results suggested that sdLDL-C (LDL-3 C to LDL-7 C) was a high-risk factor for CHD; thus, sdLDL-C detection had greater clinical signi cance in CHD screening. Our results were consistent with previous studies [16][17][18].

Conclusions
In the meantime, the monitoring effect of LDL-C and sdLDL-C was a rmed; after treatment and remission, CHD patients with LDL-C and sdLDL-C decreases attained 81.25% and 60.58% ( Figure 3A). Although the change curves of LDL-C and sdLDL-C expression were not necessarily consistent in one patient ( Figure 3C), in general, the coincidence rate of LDL-C in the disease state was greater than that of sdLDL-C. Other clinical biomarkers, total cholesterol, total triglycerides, plasma HDL-C, plasma LDLC, Apo A1, and Apo B versus LDL-C and sdLDL-C, and ultimately LDL-C were also better biomarkers for CHD treatment monitoring ( Figure 3D).