Abstract

Introduction: Statins are frequently prescribed for patients with hyperlipidemia to prevent cardiovascular disease, however, there is an inter-individual variability in their efficacy due to many factors, including genetic polymorphism. This study aimed to determine the association between single nucleotide polymorphism (SNP) in the gene cluster SORT1-CELSR2-PSRC1 (rs646776) and lipid profiles in a subset of statin users in Malaysia.

Methods: In total, 122 statin-treated patients were recruited in this cross-sectional study. Genomic DNA from whole blood samples (3 mL) was extracted and genotyped using amplification-refractory mutation system-polymerase chain reaction (ARMS-PCR) for the rs646776 polymorphism. The association between the SNP and statin-related lipid profile changes was evaluated using a dominant genetic model (AA vs. GA + GG genotypes).

Results: The minor allele frequency of the rs646776 was 0.08 and the allele frequency of each genotype was in the Hardy–Weinberg equilibrium (P = 0.6149). Variant allele carriers of rs646776 showed higher (P < 0.05) high-density lipoprotein cholesterol (HDL-C) levels after statin treatment in females but not in males. Conversely, AA genotypes were linked to a significant decrease in total cholesterol and low-density lipoprotein cholesterol (P < 0.01).

Conclusion: Our study provides the first frequency data for PSRC1/CELSR2/SORT1 rs646776 in the Southeast Asian region and further confirms the SNP association with improved HDL-C levels, especially in females using statins. The findings warrant further replication studies to validate the SNP association among different ethnicities in Asia.

Introduction

Statin, a cholesterol-lowering drug, is commonly used to prevent cardiovascular disease because it is highly effective at lowering cholesterol levels. However, statin efficacy has an inter-individual variability on lipid profiles which can be partially caused by genetic variation. As indicated by a genome-wide association study (GWAS) on Europeans and South Asians, the SORT1/CELSR2/PSRC1 rs646776 polymorphism has a strong association with plasma lipoproteins, hence the increased risk of coronary artery disease (CAD)1. In brief, rs646776 is a single nucleotide polymorphism (SNP) on chromosome 1 (i.e., 1p13.3) which resides in the intergenic region of three distinct genes: CELSR2, PSRC1, and SORT12. Minor allele carriers of the SNP are associated with decreased cardiovascular disease risk since it has been shown to lower circulating cholesterol levels, particularly low-density lipoprotein cholesterol (LDL-C), among statin users3. Similarly, another GWAS found that rs646776 was associated with significantly larger LDL-C reductions of up to 0.47%4.

Evidence from animal and in vitro studies showed that SORT1, out of the three candidates in the gene cluster, was the main regulator of lipoprotein metabolism, especially of LDL-C. However, there was no unidirectional evidence for the effect of SORT1 expression on plasma lipid profiles5. In fact, a large meta-analysis from a GWAS on Europeans has associated SORT1 rs646776, particularly owing to its direct involvement in lipid metabolism, with an enhanced statin-related LDL-C response from interacting with apoB at the Golgi apparatus to stimulate LDL-C uptake6. Therefore, we speculate that individuals who inherit these genetic variants have improved lipid profiles, most likely in terms of reduced LDL-C or increased high-density lipoprotein cholesterol (HDL-C) levels, and hence a lower risk of CAD.

Although statins are well-known for their ability to reduce cardiovascular morbidity, an individual's response to a given drug can be influenced by several factors. Besides genetics, the demographic profile of the subject (i.e., age, body mass index, gender) and clinical factors determine the inter-individual variability of statin efficacy7. This study aimed to determine the association between the SORT1/CELSR2/PSRC1 rs646776 polymorphism and statin-related lipid profile changes in a subset of the Malaysian population. Stratification according to the subject’s gender allowed us to evaluate the gene–gender effects on statin-affected lipid profiles. To our knowledge, this study provides the first frequency data for the SNP of Southeast Asian origin.

Methods Study subjects

After obtaining informed written consent, subjects diagnosed with hyperlipidemia and on statin medication were recruited from the Klinik Rawatan Keluarga at Hospital Universiti Sains Malaysia (HUSM) between May 2018 and October 2020. The recruited subjects included individuals aged between 18 and 75 who had been diagnosed with hyperlipidemia and prescribed statins for at least six weeks. The patients’ medical records were obtained from their clinic files and the hospital’s database. The exclusion criteria are as follows: patients who have been (i) prescribed other lipid-lowering medications as concurrent treatment; (ii) diagnosed with malignancy, liver disease, thyroid disease, kidney disease, or familial hypercholesterolemia; (iii) receiving other medications known to interfere with statin’s efficacy.

SNP genotyping

The DNA from the whole blood (3 mL) was extracted according to the manufacturer’s protocols (GeneAll Biotechnology, Korea). The extracted DNA was then subjected to genotyping using amplification-refractory mutation system-polymerase chain reaction (ARMS-PCR). The temperatures during the ARMS-PCR cycles were as follows: initial denaturation at 95 °C for 5 minutes, 30 denaturation cycles at 94 °C for 30 seconds, annealing at 60.9 °C for 30 seconds, extension at 72 °C for 30 seconds, and an additional extension at 72 °C for 5 minutes. Table 1 lists the primer sets used in this study, which were designed using a website (http://primer1.soton.ac.uk/primer1.html); see Appendix 1 for a supplementary file highlighting the primer sequences within the gene sequences. The products of the ARMS-PCR were visualized on the 2% agarose gel for 60 minutes.

Statistical analysis

The data analysis was carried out using the SPSS software version 26.0 (IBM, United States). The data were reported as frequency (%) and mean ± standard error of the mean (SEM) for categorical and continuous data, respectively. The normality of the continuous data was checked using a test of normality, the Kolmogorov–Smirnov test. For the categorical data, the Chi-square test was used in a contingency table to obtain the odds ratio (OR); the test required at least 80% of the cells to have an expected count greater than 5. Student’s t-test was used to assess differences between two continuous data; in the case of non-parametric data, the Mann–Whitney test was used instead. Repeated measures ANOVA was used to compare means across one or more variables that are based on related subjects; in the case of non-parametric data, the Friedman test was used. When conducting multiple statistical tests, the Bonferroni correction was used to adjust the probability (P) values to avoid a Type I error. The observed genotype frequency was checked for the Hardy–Weinberg equilibrium (HWE) using https://gene-calc.pl/hardy-weinberg-page. Since the homozygous mutant GG genotypes are rare, a genetic dominant model (AA vs. GA + GG genotypes) was applied to assess the SNP association. Multiple binary logistic regression was used to determine which independent factors were associated with the likelihood of achieving the LDL target of P-value of 0.05 or lower was considered statistically significant.

× Figure 1 . The impact of SORT1/CELSR2/PSRC1 rs646776 on the HDL-C levels in (a) females and (b) males at the baseline and after statin treatment. Figure 1 . The impact of SORT1/CELSR2/PSRC1 rs646776 on the HDL-C levels in (a) females and (b) males at the baseline and after statin treatment.

Table 1.

Primers used and the interpretation of the genotypes according to the band sizes in ARMS-PCR

Primer sequences Product size Interpretation of the genotypes FO: 5'-TGGTGAAAAGGACACCTTCC-3' Outer; 500 bp Heterozygous; 500 bp, 420 bp and 129 bp FI (G allele): 5'-TATTTGGGAGCAGTGTCATGGACGTG-3' G allele; 420 bp Homozygous recessive; 500 bp and 420 bp RI (A allele): 5'-GGCTGATAAGCCTGTCCCTCTGACT -3' A allele; 129 bp Wild type; 500 bp and 129 bp RO: 5'-CTTTTCTGCCGTGGATTCTC- 3'

Table 2.

Demographic and clinical features of the patients

Characteristics n = 122 Age (years + SEM) 53.62 ± 6.67 Gender, n (%) Female 71 (58.2) Male 51 (41.8) Race, n (%) Malay 117 (95.9) Chinese 3 (2.5) Indian 1 (0.8) Others 1 (0.8) Genotype of rs646776, n (%) a Present study AA 102 (83.6) GA 20 (16.3) GG 0 Reference population AA 461 (91.4) GA 40 (7.9) GG 3 (0.5) Type and dosage of statin, n (%) Atorvastatin 10 mg/day 33 (27.0) 20 mg/day 32 (26.2) 30 mg /day 4 (3.3) 40 mg /day