Association of NPC1L1 and HMGCR Gene Polymorphisms with Major Adverse Cardiac and Cerebrovascular Events in Patients with Three-Vessel Disease

Abstract

Three-vessel disease (TVD) is a severe coronary heart disease (CHD) with poor prognosis. Niemann-Pick C1-like 1 (NPC1L1) is a transporter protein for exogenous cholesterol absorption, and 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) is a rate-limiting enzyme for cholesterol synthesis. We aimed to investigate the association between NPC1L1 and HMGCR gene polymorphisms and major adverse cardiac and cerebrovascular events (MACCE) in patients with TVD. A total of 342 TVD patients were consecutively enrolled and followed up for 1-year MACCE (a composite of all-cause death, myocardial infarction, revascularization, readmission, and stroke) as TVD event group, and 344 patients without CHD were control group. Four single-nucleotide polymorphisms (SNPs), rs11763759, rs4720470, rs2072183, and rs2073547, on NPC1L1 gene and four SNPs, rs12916, rs2303151, rs2303152, and rs4629571, on HMGCR gene were genotyped. Multivariate logistic regression analysis showed that rs4720470 of NPC1L1 was associated with higher risk of TVD with MACCE in codominant model (odds ratio [OR]: 1.315; 95% confidence intervals [CI]: 1.007–1.716, p = 0.044), and that rs2303151 of HMGCR was associated with higher in recessive (OR: 3.383; 95% CI: 1.040–10.998, p = 0.043) and codominant (OR: 1.458; 95% CI: 1.038–2.047, p = 0.030) model, respectively. Patients with both variant rs4720470 in codominant model and variant rs2303151 in recessive model related to a higher risk (OR: 6.772, CI: 1.338–34.280; p = 0.021). We reported for the first time that the rs4720470 on NPC1L1 gene and rs2303151 on HMGCR gene were associated with risk of 1-year MACCE in TVD.

INTRODUCTION

Three-vessel disease (TVD) is a severe type of coronary heart disease (CHD), accounting for up to ∼30% of obstructive CHD.¹ Patients with TVD often have diffuse and severe lesions and a high incidence of cardiovascular adverse events. Many studies consistently show that increased low-density lipoprotein cholesterol (LDL-C) is the key factor affecting coronary atherosclerosis.^2,3

The source of cellular cholesterol mainly depends on two regulatory mechanisms: endogenous cholesterol synthesis and exogenous cholesterol uptake by LDL receptor (LDL-R). The former is catalyzed by 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), the target of statins, which is a rate-limiting enzyme of cholesterol synthesis⁴; and the latter is dependent on Niemann-Pick C1-like-1 (NPC1L1), the target of ezetimibe, which participates in the absorption of exogenous food cholesterol.⁵ Previous studies, including a 2 × 2 factorial Mendelian randomization study,⁶ support that NPC1L1 and HMGCR gene polymorphism may affect LDL-C levels and, further, CHD risk.^7,8

TVD is considered to be susceptible to genetic risk factors.^7,8 However, the evidence on gene polymorphism related to progression of CHD in patients with TVD is relatively limited. Although in recent years, genome-wide association studies (GWAS) have discovered a series of cholesterol-related genes,^9,10 only about 10–20% of the total variances in LDL-C could be attributed to the common single-nucleotide polymorphisms (SNPs) identified by GWAS.¹¹

There is a lack of research on the correlation between NPC1L1 and HMGCR gene polymorphisms and major adverse cardiac and cerebrovascular events (MACCE) in patients with TVD. In this study, we detected SNPs of NPC1L1 and HMGCR gene in patients with TVD with 1-year MACCE and controls and aimed to evaluate the relationship between gene polymorphisms of NPC1L1 and HMGCR and MACCE risk in patients with TVD, to determine possible genetic effects on TVD progression.

METHODS

Study Design and Participants

A total of 8,943 consecutive patients diagnosed as TVD by coronary angiography were prospectively enrolled from April 2004 to February 2011 in Fuwai Hospital (Beijing, China). TVD was defined as angiographic stenosis of ≥50% in all three main coronary arteries, including the left anterior descending, circumflex, and right coronary arteries, with or without the left main artery involved. There were no specific exclusion criteria. The methodology has been described previously.^12
–14 General information, baseline data, past history, and laboratory tests of all patients were collected on admission. All patients were recommended to receive standard drug therapy. This was an observational study and there was no provision for treatment intervention. According to the practice guidelines, operator judgments, and their preferences, the patients received percutaneous coronary intervention, coronary artery bypass grafting, or drug therapy alone. Data were collected by follow-up pattern, including telephone interview, letter, and clinic visit. The last follow-up was finished in 2016. All events defined by study protocol were carefully verified by an independent group of clinical physicians.

The study complied with the Declaration of Helsinki. The ethics committee of Fuwai Hospital approved the research protocol. Written informed consent was obtained from all participants.

Definitions and Groups

The definition of MACCE included all-cause death, myocardial infarction, revascularization, readmission, and stroke in the study. The diagnostic criteria for myocardial infarction were in accordance with the third global standard definition.¹⁵ Stroke included ischemic stroke and hemorrhagic stroke. Revascularization was defined as ischemia-driven revascularization. Readmission was defined as rehospitalization due to ischemia exacerbation. Five hundred twelve patients with 1-year MACCE in follow-up were included, and we excluded 170 participants due to unavailable blood samples or failed deoxyribonucleic acid (DNA) quality test. Finally, 342 patients were included as TVD event group for analysis (Fig. 1). With a nearly 1:1 ratio, 344 patients was enrolled as control group according to the following criteria: age ≥40 years, no coronary artery stenosis <50% observed on multidetector computed tomography cardiac scanning or coronary artery angiography, no CHD history, no manifestation of myocardial ischemia or infarction on electrocardiogram, and no regional wall motion abnormalities, wall thinning, ventricular enlargement, or lowered ejection fraction on echocardiography.

Figure 1.

Patient flow chat. Flowchart shows a total of 342 TVD patients and 344 patients of control group were enrolled and 4 SNPs of NPC1L1 and 4 SNPs of HMGCR were genotyped in this study. *Student's t-test, Pearson χ ²–test, and logistic regression analysis adjusted for age and sex. DNA, deoxyribonucleic acid; HMGCR, 3-hydroxy-3-methylglutaryl-coenzyme A reductase; MACCE, major adverse cardiac and cerebrovascular events; NPC1L1, Niemann-Pick C1-like 1; SNPs, single-nucleotide polymorphisms; TVD, three-vessel disease.

Determination of SNPs

Fasting blood samples were drawn from all patients within 24 h of admission and genomic DNA was extracted from leukocytes through the standard salting-out method.¹⁶ The samples were stored in a refrigerator at −80°C. By consulting the previous literature and HapMap project (http://hapmap.ncbi.nlm.nih.gov) of the Chinese in Beijing with a minor allele frequency ≥0.05, the following SNPs were selected, including four NPCIL1 sites (rs11763759, rs4720470, rs2072183, nd rs2073547) and four HMGCR sites (rs12916, rs2303151, rs2303152, and rs4629571). The SNP genotyping was performed using an improved multiplex ligation detection reaction technique.¹⁷

Statistical Analysis

Continuous variables with a normal distribution were expressed as mean ± standard deviation, and non-normally distributed continuous variables as median with quartiles. Categorical variables were expressed as number (%). Student's t-tests were used to compare continuous variables, while χ ²-tests were applied to compare categorical variables between the two groups. SNP association analysis was applied in three allele models, including dominant, recessive, and codominant model. χ ²-tests were used to compare frequency of individual with minor allele in each SNP between the two groups. Multivariate logistic regression analysis was conducted to evaluate the correlation between NPC1L1 and HMGCR gene polymorphisms and MACCE in patients with TVD. The results were reported as odds ratios (ORs) with 95% confidence intervals (95% CIs). Statistical significance was defined as two-sided p < 0.05. All analyses were performed using SPSS software version 23.0 (IBM Corp., Armonk, NY).

RESULTS

Baseline Data

A total of 686 persons with baseline data available were finally included (Fig. 1), among which there were 342 patients with TVD (mean age, 62.8 ± 10.2 years) and 344 control subjects (mean age, 50.5 ± 11.5 years). Compared with control group, TVD event group had more males and higher systolic blood pressure, accompanied with more comorbidities (hypertension, diabetes mellitus, hyperlipidemia, and peripheral vessel disease), and had more smoking history. White blood cell count and serum creatinine elevated, hemoglobin, high-density lipoprotein cholesterol level, and LDL-C level declined, and fasting plasma glucose and high-sensitivity C-reactive protein elevated in TVD event group (all p < 0.05) (Table 1).

Table 1.

Comparison of clinical characteristics between three-vessel disease and controls

Variables	Control Group (n = 344)	TVD Event Group (n = 342)	p^a
Age, years	50.5 ± 11.5	62.8 ± 10.2	<0.001
Sex, male	198 (57.6)	270 (78.9)	<0.001
HR, bpm	70.2 ± 15.7	71.3 ± 12.2	0.303
SBP, mmHg	124.7 ± 26.2	129.4 ± 20.5	0.008
BMI, kg/m²	25.3 ± 3.1	25.7 ± 3.2	0.147
Hypertension, %	44 (12.8)	241 (70.5)	<0.001
DM, %	14 (4.1)	134 (39.2)	<0.001
Hyperlipidemia, %	25 (7.3)	209 (61.1)	<0.001
PVD, %	0 (0.0)	31 (9.1)	<0.001
Smoking history, %	1 (0.3)	56 (16.4)	<0.001
WBC count, × 10⁹/L	6.25 ± 1.73	7.21 ± 2.08	<0.001
Serum creatinine, μM	72.6 ± 18.4	84.5 ± 22.0	<0.001
Hemoglobin, g/L	144.9 ± 12.3	136.7 ± 14.9	<0.001
TC, mM	4.68 ± 1.15	4.61 ± 1.07	0.389
TG, mM	1.72 ± 1.19	1.77 ± 0.94	0.533
HDL-C, mM	1.15 ± 0.32	1.05 ± 0.24	<0.001
LDL-C, mM	2.87 ± 0.88	2.56 ± 0.81	<0.001
FPG, mM	5.61 ± 1.23	6.31 ± 2.32	<0.001
Hs-CRP, mg/L	2.23 ± 1.57	3.77 ± 3.62	<0.001

Student's t-test or Pearson χ ²-test.

Data are expressed as mean ± standard deviation or percentage (%).

BMI, body mass index; DM, diabetes mellitus; FPG, fasting plasma glucose; HDL-C, high-density lipoprotein cholesterol; HR, heart rate; Hs-CRP, hypersensitive C-reactive protein; LDL-C, low-density lipoprotein cholesterol; PVD, peripheral vessel disease; SBP, systolic blood pressure; TC, total cholesterol; TG, triglycerides; TVD, three-vessel disease; WBC, white blood cell.

The Frequency of Genetic Polymorphism

The genotype distribution of polymorphism in both the TVD event group and control group was conformed to Hardy–Weinberg equilibrium (both p > 0.05). In dominant allele model, frequency of individual with minor allele of rs12916 on HMGCR gene was higher in TVD event group (189 cases, 56.25%) compared with control group (142 cases, 41.64%; p < 0.001) with statistical significance (Table 2).

Table 2.

Frequency of genetic polymorphism in case–control patients

Polymorphisms	Allele Model	Control Group	TVD Event Group	p^a
rs11763759	DO TT/CC+CT	300 (87.21%)/44 (12.79%)	300 (87.72%)/42 (12.28%)	0.840
	RE CC/CT+TT	1 (0.29%)/343 (99.71%)	2 (0.58%)/340 (99.42%)	0.559
	CO CC/CT/TT	1 (0.29%)/43 (12.50%)/300 (87.21%)	2 (0.58%)/40 (11.70%)/300 (87.72%)	0.804
rs4720470	DO CC/TT+CT	185 (54.25%)/156 (45.75%)	172 (50.44%)/169 (44.56%)	0.319
	RE TT/CT+CC	30 (8.80%)/311 (91.2%)	46 (13.49%)/295 (86.51%)	0.052
	CO TT/CT/CC	30 (8.80%)/126 (36.95%)/185 (54.25%)	46 (13.49%)/123 (36.07%)/172 (50.44%)	0.144
rs2072183	DO GG/CC+GC	122 (35.57%)/221 (64.43%)	133 (38.89%)/209 (61.11%)	0.808
	RE CC/GC+GG	58 (16.91%)/285 (83.09%)	58 (16.96%)/284 (83.04%)	0.986
	CO CC/GC/GG	58 (16.91%)/163 (47.52%)/122 (35.57%)	58 (16.96%)/151 (44.15%)/133 (38.89%)	0.628
rs2073547	DO AA/GG+GA	124 (36.80%)/213 (63.20%)	134 (39.64%)/204 (60.36%)	0.446
	RE GG/GA+AA	61 (18.10%)/276 (81.90%)	56 (16.87%)/282 (83.13%)	0.599
	CO GG/GA/AA	61 (18.10%)/152 (45.10%)/124 (36.80%)	56 (16.87%)/148 (43.79%)/134 (39.64%)	0.721
rs12916	DO TT/CC+CT	142 (41.64%)/199 (58.36%)	189 (56.25%)/147 (43.75%)	<0.001
	RE CC/CT+TT	77 (22.58%)/264 (77.42%)	84 (25%)/252 (75%)	0.460
	CO CC/CT/TT	77 (22.58%)/122 (35.78%)/142 (41.64%)	84 (25%)/105 (31.25%)/147 (43.75%)	0.443
rs2303151	DO CC/TT+CT	235 (68.31%)/109 (31.69%)	220 (64.3%)/122 (35.7%)	0.269
	RE TT/CT+CC	6 (1.74%)/338 (98.26%)	11 (3.2%)/331 (96.8%)	0.215
	CO TT/CT/CC	6 (1.74%)/103 (29.94%)/235 (68.31%)	11 (3.2%)/111 (32.5%)/220 (64.3%)	0.323
rs2303152	DO GG/AA+GA	270 (78.49%)/74 (21.51%)	281 (82.16%)/61 (17.84%)	0.226
	RE AA/GA+GG	3 (0.87%)/341 (99.13%)	4 (1.17%)/338 (98.83%)	0.698
	CO AA/GA/GG	3 (0.87%)/71 (20.64%)/270 (78.49%)	4 (1.17%)/57 (16.67%)/281 (82.16%)	0.389
rs4629571	DO AA/GG+GA	270 (78.49%)/74 (21.51%)	282 (82.46%)/60 (17.54%)	0.190
	RE GG/GA+AA	3 (0.87%)/341 (99.13%)	4 (1.17%)/338 (98.83%)	0.698
	CO GG/GA/AA	3 (0.87%/71 (20.64%)/270 (78.49%)	4 (1.17%)/56 (16.37%)/282 (82.46%)	0.338

Pearson χ ²-test.

CO, codominant model; DO, dominant model; RE, recessive model.

Logistic Regression Analysis

SNPs of NPC1L1

According to univariate logistic regression analysis, no statistical correlation was found between the polymorphisms of NPC1L1 gene and MACCE in TVD patients in various models (all p > 0.05). After multivariate logistic regression analysis adjusted for age and sex was performed, in the codominant model, the risk of TVD with 1-year MACCE in the TVD event group with rs4720470 on NPC1L1 gene was 1.315 times higher than that in the control group (OR: 1.315; 95% CI: 1.007–1.716, p = 0.044) (Table 3).

Table 3.

Univariate and multivariate adjusted logistic regression analysis of NPC1L1 and HMGCR gene single-nucleotide polymorphisms

Gene	SNPs	Allele Model	Univariant Logistics Regression		Multivariant Logistics Regression
Gene	SNPs	Allele Model	Crude OR (95% CI)	p	Adjusted OR (95% CI)	p^a
NPC1L1	rs11763759 T > C	DO TT/CC+CT	0.955 (0.607–1.500)	0.840	0.843 (0.488–1.459)	0.543
		RE CC/CT+TT	2.018 (0.182–22.355)	0.567	7.286 (0.246–216.134)	0.251
		CO CC/CT/TT	0.982 (0.640–1.509)	0.935	0.902 (0.533–1.525)	0.699
	rs4720470 C > T	DO CC/TT+CT	1.165 (0.863–1.574)	0.319	1.399 (0.973–2.012)	0.070
		RE TT/CT+CC	1.616 (0.994–2.630)	0.053	1.564 (0.876–2.793)	0.131
		CO TT/CT/CC	1.201 (0.963–1.499)	0.104	1.315 (1.007–1.716)	0.044
	rs2072183 G > C	DO GG/CC+GC	0.846 (0.620–1.154)	0.291	0.738 (0.508–1.073)	0.111
		RE CC/GC+GG	1.004 (0.673–1.496)	0.986	1.094 (0.685–1.746)	0.707
		CO CC/GC/GG	0.937 (0.758–1.158)	0.937	1.015 (0.790–1.303)	0.908
	rs2073547 A>G	DO AA/GG+GA	0.886 (0.650–1.209)	0.446	0.986 (0.679–1.432)	0.940
		RE GG/GA+AA	0.899 (0.603–1.339)	0.599	1.005 (0.632–1.600)	0.983
		CO GG/GA/AA	0.918 (0.743–1.134)	0.426	0.995 (0.776–1.277)	0.970
HMGCR	rs12916 T > C	DO TT/CC+CT	0.917 (0.677–1.244)	0.579	0.852 (0.591–1.227)	0.389
		RE CC/CT+TT	1.143 (0.802–1.629)	0.460	1.101 (0.721–1.683)	0.655
		CO CC/CT/TT	1.005 (0.831–1.215)	0.959	0.966 (0.770–1.212)	0.766
	rs2303151 C > T	DO CC/TT+CT	1.196 (0.871–1.642)	0.270	1.406 (0.961–2.057)	0.079
		RE TT/CT+CC	1.872 (0.684–5.121)	0.222	3.383 (1.040–10.998)	0.043
		CO TT/CT/CC	1.216 (0.915–1.614)	0.178	1.458 (1.038–2.047)	0.030
	rs2303152 G > A	DO GG/AA+GA	0.792 (0.543–1.156)	0.227	0.814 (0.516–1.284)	0.375
		RE AA/GA+GG	1.345 (0.299–6.056)	0.699	0.571 (0.099–3.282)	0.530
		CO AA/GA/GG	0.832 (0.587–1.181)	0.304	0.813 (0.534–1.237)	0.334
	rs4629571 A>G	DOAA/GG+GA	0.776 (0.531–1.134)	0.191	0.783 (0.495–1.237)	0.294
		RE GG/GA+AA	1.345 (0.299–6.056)	0.699	0.571 (0.099–3.282)	0.530
		CO GG/GA/AA	0.818 (0.576–1.162)	0.263	0.786 (0.516–1.198)	0.263

Logistic regression analysis adjusted for age and sex.

CI, confidence interval; HMGCR, 3-hydroxy-3-methylglutaryl-coenzyme A reductase; NPC1L1, Niemann-Pick C1-like 1; OR, odds ratio; SNPs, single-nucleotide polymorphisms.

SNPs of HMGCR

According to univariate logistic regression analysis, no statistical correlation was found between the polymorphisms of HMGCR gene and MACCE in TVD patients in various models (all p > 0.05). After multivariate logistic regression analysis adjusted for age and sex was performed, in the recessive model, the risk of TVD with 1-year MACCE in the TVD event group with rs2303151 on the HMGCR gene was 3.383 times (OR: 3.383; 95% CI: 1.040–10.998, p = 0.043) higher than that in the control group, and meanwhile, in codominant model, it was 1.458 times higher (OR: 1.458; 95% CI: 1.038–2.047, p = 0.030) (Table 3).

Combined Analysis of SNPs

After multivariate logistic regression analysis adjusted for age and sex was performed, the risk of TVD with 1-year MACCE in patients with both variant rs4720470 of NPC1L1 in codominant model and variant rs2303151 of HMGCR in recessive model was 6.772 times higher (OR: 6.772; CI: 1.338–34.280, p = 0.021) compared with no variant controls. No statistical significance was found in patients with variant rs4720470 of NPC1L1 in codominant model and variant rs2303151 of HMGCR in codominant model (OR: 1.611; CI: 0.987–2.628, p = 0.056) (Table 4).

Table 4.

Combined analysis of gene model

Combination of Gene Model	Univariant Logistics Regression		Multivariant Logistics Regression
Combination of Gene Model	Crude OR (95% CI)	p	Adjusted OR (95% CI)	p^a
rs4720470 CO and rs2303151 RE	1.403 (0.935–2.106)	0.102	1.611 (0.987–2.628)	0.056
rs4720470 CO and rs2303151 CO	2.707 (0.712–10.291)	0.144	6.772 (1.338–34.280)	0.021

Logistic regression analysis adjusted for age and sex.

DISCUSSION

We performed genetic polymorphism association study on two key genes of cholesterol metabolism for their relevance to MACCE in patients with TVD. We reported for the first time in the setting of CHD that the risk of TVD with 1-year MACCE in TVD event group with variant rs4720470 on NPC1L1 gene increased significantly in codominant model, which was 1.315 times higher than that in the control group. The SNP of rs2303151 was first reported in our study and the results implied that compared with control group, the risk of TVD with 1-year MACCE in TVD event group with variant rs2303151 on HMGCR gene significantly increased, which was 3.383 times higher in recessive model and 1.458 times higher in codominant model. The risk of TVD with 1-year MACCE in patients with both variant rs4720470 of NPC1L1 in codominant model and variant rs2303151 of HMGCR in recessive model was 6.772 times higher compared with no variant controls.

NPC1L1 is a key protein for cholesterol absorption in intestine. Exogenous cholesterol is ingested through food and absorbed through the intestine, which may lead to an increase in plasma cholesterol level. The gene polymorphism of NPC1L1 can affect the absorption efficiency of cholesterol, and then affect the plasma cholesterol level. Meanwhile, NPC1L1 is the target of ezetimibe, a lipid-lowering drug. rs4720470 of NPC1L1 is an intronic variant, located on human chromosome 7. Rudkowska et al. ¹⁸ analyzed effects of SNPs of NPC1L1 gene to individual differences on responsiveness to plant sterols, including rs4720470 and three other SNPs (rs2072183, rs10264715, and rs217434) and results explained that an uncommon NPC1L1 polymorphism (T mutant heterozygote rs4720470) and other infrequent polymorphisms of cholesterol pathway may underline nonresponsiveness. Interestingly, our study found that in codominant model, rs4720470 of NPC1L1 was associated with MACCE in patients with TVD. Previous SNP studies on NPC1L1 have indicated that loss of function variant of NPC1L1 could reduce LDL-C level and CHD risk,^19,20 meanwhile, other studies have shown variation of NPC1L1 could also increase CHD event risk,^8,21 all of which consistently suggest that NPC1L1 was closely related to the occurrence, progression, and event risk of CHD. Our study reported for the first time that variant rs4720470 of NPC1L1 was related to MACCE in patients with TVD in codominant model, which further supported the opinion that the variation of NPC1L1 is closely related to the risk of CHD events. We speculated that the variant rs4720470 may lead to the activation of NPC1L1 gene function, which may increase the risk of progression of atherosclerosis and cardiovascular events in TVD patients. In addition, racial and ethnic differences usually can be seen in gene polymorphisms. Further and larger studies to investigate whether the same genetic variation exists in different populations are warrant in the future.

HMGCR is an important rate-limiting enzyme in cholesterol synthesis and its activity directly affects the speed of cholesterol synthesis and the level of cholesterol in plasma. When the activity of HMGCR increases, the synthesis of cholesterol in liver increases. Meanwhile, HMGCR is the target of statins. rs2303151 of HMGCR is located on human chromosome 5, belonging to intron variation. Our study showed rs2303151 of HMGCR gene was related to increased risk of TVD with MACCE in both codominant and recessive modes. Especially in the recessive mode, the risk of TVD with MACCE in patients was significantly increased, which was 3.383 times higher than that in the control group, suggesting a strong correlation between the SNP of rs2303151 and the risk of TVD with MACCE. In review of previous SNP studies on HMGCR gene, we did not retrieve any report on rs2303151. Therefore, our results for the first time reported the importance of variant rs2303151 in the field of CHD. More importantly, the variant rs2303151 can lead to the risk of serious coronary lesion events, which is of great harm. The SNP of rs2303151 is worthy of further study.

It is believed that patients carrying multiple susceptible SNPs possess increased risk of diseases. We combined the two gene polymorphisms that remain significant after multivariate logistic regression analysis and discovered that when variant rs4720470 of NPC1L1 in codominant model and variant rs2303151 of HMGCR in recessive model coexist, the risk of TVD with MACCE in these patients was higher compared with controls, which suggested that we may find ultra-high-risk patients in clinic by joint detection of multiple genes. These ultra-high-risk patients should be given early detection and intervention, so as to prevent from the occurrence and development of serious CHD and the risk of events. In the future, adopting genetic risk score combined with clinical risk factors and/or plasma risk markers may further improve the detection rates of these ultra-high-risk patients, which is conducive to accurate guide of individualized diagnosis and treatment.^22,23

Recently, although GWAS have confirmed that genetic factors play an important role in CHD, the association between a substantial part of variant SNPs and the occurrence and prognosis of the disease is still unclear.^{9,10,24
–26} Our study reported that the polymorphisms of rs4720470 of NPC1L1 and rs2303151 of HMGCR gene were associated with the risk of MACCE in patients with TVD. The findings of these novel relationships might help us to predict high risk of events in patients with TVD, and seek for new therapeutic targets, which is significant for the individualized treatment and assessment of prognosis.

Since genetic factors are irreversible, CHD treatment has started to focus on relevant gene and shown tremendous prospects. Prior studies have shown that lipid metabolism is an attractive target.^27

–30 It is possible to prevent CHD by permanently reducing lipid levels through gene editing, implying that the risk of atherosclerosis or MACCE may be reduced if one could perform gene editing in patients with the variant alleles on the NPC1L1 and HMGCR genes. Excitingly, HMGCR has made great progress in nonviral gene therapy. Hibbitt et al. found that RNA interference (RNAi)-mediated HMGCR gene knockout enhanced LDL-R gene therapy for familial hypercholesterolaemia.³¹ On this basis, Kerr et al. generated a new combined vector with LDLR and HMGCR gene targets and further found that HMGCR gene knockout could result in long-term reductions in serum lipids, thereby slowing the progression of atherosclerosis.³² Our study found that the risk of MACCE in TVD was much higher when the specific variant SNP of NPC1L1 and HMGCR gene coexisted. Thus, we envision a combined gene therapy approach affecting both the NPC1L1 and HMGCR genes. Furthermore, antisense oligonucleotide (ASO) drugs like Mipomersen³³ have been launched, thanks to the rapid development of ASO technology. Hyperlipidemia therapies based on ASO technology have made great progress and provided ASO drugs targeting angiopoietin-like 3³⁴ and AKCEA-APO(a)-LRx.³⁵ However, as far as we know, there is no ASO directly targeting NPC1L1 and HMGCR gene. Although these therapies are technically feasible, ethical problems may still arise. Despite these, we expect that gene therapy targeting high-risk alleles will be available and could decrease the risk of MACCE in patients with TVD.

In short, this study of CHD-related gene polymorphisms is of substantial significance both for screening high-risk populations and for providing important leads for new gene therapeutic strategies.

Limitations

The study had several limitations. First, all patients were enrolled in a single center, which may limit the universality of our results. Second, despite a great deal of patients being screened in this study, the sample size was still relatively small. Third, our study has enrolled Chinese as study population, and the results of related genes in different races are worth further verification in the future. Fourth, this study reported for the first time the correlation between rs4720470 of NPC1L1 and rs2303151 of HMGCR and patients with TVD events, further research on the biological mechanism of related gene polymorphism is needed in the future.

CONCLUSION

To the best of our knowledge, in the field of CHD, our study reported for the first time that the variant rs4720470 of NPC1L1 gene was associated with MACCE in patients with TVD, and the variant rs2303151 of HMGCR gene significantly increases the risk of TVD with MACCE. Patients with both variant rs4720470 in codominant model and variant rs2303151 in recessive model had the higher risk of TVD with MACCE.

Footnotes

AUTHORS' CONTRIBUTIONS

X.Z., J.L., X.T., L.S., and J.Y. contributed to the conception or design of the work. J.L., J.X., L.X., L.J., J.T., X.F., Y.W., Y.Z., D.W., K.S., B.X., and W.Z. contributed to the acquisition, analysis, or interpretation of data for the work. J.L. and K.H. contributed to statistical analysis. X.Z. and J.L. drafted the article. R.H., R.G., L.S., and J.Y. critically revised the article. All authors gave final approval and agreed to be accountable for all aspects of work, ensuring integrity and accuracy.

ACKNOWLEDGMENTS

We thank all the staff for their data collection, analysis, revision, and monitoring in this study.

AUTHOR DISCLOSURE

All authors have completed the ICMJE uniform disclosure form. The authors have no conflicts of interest to declare.

FUNDING INFORMATION

This work was supported by Beijing Natural Science Foundation (7181008); the CAMS Innovation Fund for Medical Sciences (2016-I2M-1-002); the National Key Research and Development Program of China (Nos. 2016YFC1301300, 2016YFC1301301); and young and middle-aged talents in the XPCC Science and Technology Project (2020CB012).

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