Effect of ABCA1-R219K Polymorphism in Serum Lipid Parameters in Patients under Statin Therapy Visiting Tertiary Cardiac Center of Nepal

Introduction

Dyslipidemia is the principle metabolic comorbidities which is associated with excessive body fat like increased level of triglycerides (TG) or low-density lipoprotein-cholesterol (LDL-C), or decreased high-density lipoprotein-cholesterol (HDL-C) levels.^[1] In general abnormality of at least one component of serum lipids, that is, high TC, high LDL-C, low HDL-C and high TG represents dyslipidemia.^[2] It is one of the major contributors to the development of various diseases like atherosclerosis, cardiovascular disease (CVD), and stroke.^[3] Therefore, the timely diagnosis, proper treatment and management of dyslipidemia is crucial.^[4] Treatment mostly includes the use of statin with LDL-C as a major target.^[5] Along with drug, lifestyle changes like increasing physical activity, changing the diet plan, and reducing total calorie intake are intended to reduce morbidity and mortality that are associated with dyslipidemia.^[6] Among the widely used drug, statin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) inhibitor is known to be the most effective class of drugs for the treatment for lipid disorders.^[7] Statins are considered to be the first line of therapeutic drugs for the treatment of hypercholesterolemia.^[8] They are effective on lowering LDL-C and even lowering TG level and increasing HDL-C, thus can reduce risk of heart attack by 25 to 30%.^[9,10] The most commonly used class of statins is lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, and rosuvastatin. Among several statins, atorvastatin is one of the well-established members of statin which has efficacy and safety across its dosage range.^[11] In Nepal, among various statins preferred for treatment of dyslipidemia, atorvastatin is the most popular and the most commonly prescribed one even though different types of statins ought to have similar efficacy.^[12]

Statins are the wonder drugs for lowering lipid but still there is wide interpatient variability in measurable response to these HMG-CoA reductase inhibitors. Individual patient genotype is found to have role in controlling responses to drug and the individual genetic polymorphism can influence response to statins.^[13] Polymorphism in ABCA1 gene is associated with various lipid transport defects and wide-ranging HDL deficiency.^[14] Various single nucleotide polymorphisms (SNPs) of ABCA1 are found to be associated with plasma lipid and susceptibility to coronary artery disease (CAD).^[15] Human ABCA1 gene located on chromosome 9q31-q31^[16] encodes transmembrane peptide, ABCA1, which is responsible for mediating transport of cholesterol, phospholipids, vitamins, and other lipophilic molecules across cell membranes. The extracellular domain of ABCA1 is responsible for binding to apoA-I.^[17,18] Lipid-free apoA-I bound to extracellular domain is loaded with cellular phospholipids and cholesterol, which efflux excess cellular cholesterol from peripheral cell to form nascent HDL. In this way, ABCA1 has important role in the process of reverse transport of cholesterol from peripheral tissue to the liver through HDL. So, ABCA1 is considered to be preventive factor against atherosclerosis and CVDs.^[14,19–21] Among various polymorphisms in ABCA1 gene, one of the most common missense polymorphism in its coding region is rs2230806, also known as Arg219Lys (R219K). The rs223080 (G) allele encodes the amino acid arginine (R) and (A) allele encodes lysine (K).^[22]

R219K polymorphism is a SNP that occurs as a result of G to A transversion at nucleotide position 1051 in exon 7 that results in the substitutions of a lysine for arginine at amino acid residue 219 of ABCA1 protein.^[23,24] And the codon 219 is located at long extracellular loop of ABCA1 and the substitution of an arginine by lysine alters the conformation of the extracellular domain of ABCA1 protein. This leads to increase in the activity of ABCA1 protein that can then increase the efficiency of intracellular lipid transport. It is more likely that R219K variant acts as a functional mutation to regulate HDL-C level. R219K polymorphism is found to alter the development and severity of CAD through alteration of lipid profile.^[16] The R219K has been identified to be associated with protection against CAD risk and severity and this protective effect is supposed to be related to an increased HDL-C and/or reduced TG levels.^[17] One probable mechanism how R219K polymorphism can reduce risk of coronary heart disease (CHD) is that R219K polymorphism can induce the activity of ABCA1 protein that helps in cholesterol efflux from peripheral cells back to liver and this process can occur without being dependent on plasma HDL-C levels. Several studies have shown that the R219K polymorphism is associated with the variation in lipid parameter of individuals.^[18,23] However, the prevalence of genetic polymorphism remarkably differs among ethnic groups^[24] and to the best of our knowledge, not a single study has been done in this gene among Nepalese population till date. Therefore, this study was designed with the aim to examine the effects of the R219Kpolymorphism of ABCA1 on serum lipid levels and responses to atorvastatin therapy in Nepalese patients with dyslipidemia visiting Manmohan Cardiothoracic Vascular and Transplant Center (MCVTC), Kathmandu, Nepal.

Materials and Method

DNA Extraction and Genotyping of the R219K Polymorphism of the ABCA1 Gene

Genomic DNA was first extracted from whole blood sample by a manual method using the protocol by Iranpur et al.^[25] Isolated DNA was then amplified by polymerase chain reaction (PCR) technique using cleaver scientific-thermal and gradient thermal cycler. In PCR, a 309 bp fragment of ABCA1 gene was amplified using specific primers. The forward primer sequence: 5ʹ-AAAGACTTCAAGGACCCAGCTT-3ʹ and reverse primer sequence: 5ʹ-CCTCACATTCCGAAA-GCATTA-3ʹ was selected for amplification based on previously described PCR assay for R219K genotyping.^[26] The amplified gene was then cleaved by restriction endonuclease EcoNI, producing fragments of 309 bp for R allele and fragments of 184bp and 125bp for K allele (►Fig. 1). The fragments obtained after restriction digestion were analyzed in the 3% agarose gel electrophoresis and the result was visualized under ultraviolet transilluminator. Wild-type alleles RR showed a single band of 309 bp (no digestion). The KK homozygotes variant genotype showed two bands of 184 bp and 125 bp fragments when digested with EcoN1. However, for heterozygotes R219K polymorphic alleles (R/K), three bands of 309bp, 184bp, and 125 bp were observed.

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Fig. 1

Restriction fragment length polymorphism fragments of different samples. Lane 1 shows the DNA ladder; Lane 2, 4, 5, and 7 are undigested polymerase chain reaction product representing wild-type homozygote RR genotype; lane 8 represents homozygote KK and lane 3 and 6 represent heterozygotes RK.

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Results

Discussion

Dyslipidemia is a condition with derangement of lipid and/or lipoproteins level including increased total cholesterol level or TGs level or LDL cholesterol level or decreased level of HDL cholesterol level. It is considered as a major risk factor for CVDs.^[27] Therefore, the prevention and sensible management of dyslipidemia can greatly alter the CVDs risk and related morbidity and mortality.^[28] Dyslipidemia is treated with numbers of medication, the most common being statins.^[29] The action of the statin in both lipid modifying and reducing risk of CVDs is found to be variable in various patient population receiving similar drug as same dose. The heterogeneity in lowering TC, LDL-C, TG, and increasing HDL-C was found in various patients' population even at the highest statin doses available. The cause behind this may be various polymorphisms in genes that are responsible for modulating the action of statins. Variability in gene responsible for statin absorption, distribution, and/or excretion may also affect several drug-related phenotypes.^[30] Various SNPs are associated with drug metabolism and their actions like SNPs in ABCA1 gene may play role in the different responses to the lipid lowering therapy.^[17] One of the frequent R219K SNP of ABCA1 genes is found to be associated with decreased severity of atherosclerosis, decreased TG levels, and increased HDL-C levels. Consequently, R219K seems to be associated with decreased risk and severity of CAD.^[18] But the results obtained from different studies are not replicable.^[17] Also, similar studies are not performed in Nepalese population yet.

The result of this study revealed that there was significant association of R219K polymorphism with higher degree of percentage reduction in value of TG/HDL ratio, and TC/HDL ratio in atorvastatin users. However, no association of R219K polymorphism was seen in reduction of serum TC, LDL-C, TG, and VLDL levels and increase of HDL-C levels. The effect of R219K seen in TG, VLDL, TG/HDL, and HDL in this study is also reported earlier.^{[17,18,31,32]} Contrast to this, in a study by Kolovou et al,^[26] no association was observed between R219K gene polymorphism and demographic and lipid parameters. In addition to our study, Akao et al^[33] also demonstrated nonsignificant relationship between the presence and absence of R219K variant and statin-induced LDL-C response or changes in other lipid profiles including TG or HDL-C levels.

The allele frequency (R allele = 0.665 and K allele = 0.335) and genotype frequency (RR = 42.0%, RK = 48.9%, and KK= 9.1%) of R219K polymorphism found in this study is almost similar to allele frequency (R allele = 0.617 and K allele = 0.383) and genotype frequency (RR = 38.3%, RK = 46.7%, and KK= 16.1%) reported by Li et al. in Chinese population.^[34] The genotype frequency (RR = 50.5%, RK = 42.8%, and KK= 6.7%) published by Kolovou et al,^[26] in Greek population also showed similar frequency with our finding, major similarity being in the KK genotype (6.7%).

Our study also reports that mean percentage reduction in TG/HDL-C ratio, TC/HDL ratio, and TG and VLDL was significantly higher in individuals having KK genotypes than those having RR genotypes (p < 0.05). Consistent with our results, Genvigir et al^[17] demonstrated that the carriers of 219 RK/KK genotypes had lower TG/HDL-C ratio in comparison to that of 219 RR genotype and 219 RK/KK carrier had lower basal serum TG and VLDL than those with 219 RR carrier. Wu et al^[23] also demonstrated that subjects carrying K allele had significantly decreased TC/HDL-C ratio when compared with RR genotype. Therefore, our study could provide the supporting fact that rare allele of R219K SNP showed association with decrease in TG/HDL-C and TC/HDL-C ratio. The study done by Lutucuta et al^[18] and Clee et al^[31] also found that R219K variant is associated with decrease in TG level. Further, our study also demonstrated that the mean percentage increase in HDL-C was higher in KK genotype than in RR genotype (p = 0.05). This favors the fact that homozygous K genotype is associated with higher rate of rise in HDL-C as compared with other in statin users. Similar to our study, the study conducted in Chinese population also demonstrated that the action of statin on HDL-C levels is modified by R219K polymorphism of ABCA1 gene and they also found that the mean percentage rise in HDL-C was higher in patients who were homozygous for KK than that in patients who were homozygous for RR. This supports the finding of our study that HDL-C increasing effect of statin can be affected by R219K polymorphism.^[34]

Our study is also in accordance with meta-analysis of Mokuno et al^[35] which analyzed that the HDL-C concentration was higher in both men and women with RK or KK genotype than those with RR genotype. Moreover, the study conducted by Maruyama et al^[36] supports the evidence that serum HDL-C level of homozygotes of K219 allele is higher than those of carriers of homozygous R219 allele. All these findings suggest that 219KK allele is an antiatherogenic allele that might contribute in cholesterol efflux activity. Moreover, the degree of percentage reduction in TG/HDL-C ratio and TC/HDL ratio was significantly higher in individuals with RK genotypes than in individual with RR genotype (p < 0.05). This indicates that carrier of K allele of R219K polymorphism either of heterozygous or homozygous types is associated with greater reduction in TC/HDL-C ratio and TG/HDL-C in comparison to nonpolymorphic RR genotype. Thus, it can be concluded that the lipid rising or lipid reduction response of atorvastatin could be affected by R219K polymorphism of ABCA1 gene mainly by K allele of R219K.

However, this study could not provide the evidence for mechanism by which R219K polymorphism affects the statin effect in improving lipid parameters. Also, best to our knowledge there are no studies that have assessed the mechanism by which R219K ABCA1 genetic variant affects responses to statin treatment in terms of lipid modification.

Our observations could provide a possible genetic explanation for this finding, but our study was conducted in a sample of modest size and also this study does not determine the effects of other polymorphism of ABCA1 gene that are associated with lipid level and lipid lowering action of statin. This study also could not provide the possible explanation for observations. Thus, further clinical studies need to be conducted in large sample size including effect of other genetic variants to extend our observation to wide range of population.

Conclusion

This is the first study conducted in Nepal to demonstrate the frequency of ABCA1 R219K polymorphism and its effect in the lipid parameters and the lipid lowering action of statins. Our study concluded that among the statin users, the KK genotype of R219K polymorphism is associated with greater degree of reduction in TG, VLDL, TG/HDL ratio and TC/HDL ratio. It was also demonstrated that carrier of K allele relative to carrier of R allele had significantly greater reduction in value of TG, VLDL, TG/HDL ratio and TC/HDL ratio and higher level of increase in level of HDL-C. This result might indicate the K allele to be protective against subclinical cardiovascular disease in Nepalese population. In conclusion, R219K polymorphism of ABCA1 gene might be a novel genetic determinant of statin treatment response in patients with dyslipidemia.