The rs2070895 (-250G/A) Single Nucleotide Polymorphism in Hepatic Lipase (HL) Gene and the Risk of Coronary Artery Disease in North Indian Population: A Case-Control Study

Coronary Artery Disease (CAD) is a condition in which atherosclerotic plaque builds up within the wall of the coronary arteries leading to narrowing and the clinical manifestations of acute coronary syndrome [1]. It is one of the most common causes of mortality and morbidity in both developed and developing countries. It is also predicted to be the most common cause of death globally, including India, by 2020 [2,3]. The occurrence, morbidity and mortality from CAD among Asian Indians have been reported to be elevated among Europeans, Americans and other Asians, irrespective of whether they live in India or abroad [4]. The CAD rates in large Indian cities are reported as high or higher than that of Indians living overseas [5–7].

The occurrence of CAD has gradually increased in India during the latter half of the last century, predominantly among the urban population [8]. The predictable risk factors, namely hypertension, Diabetes Mellitus (DM), hypertriglyceridaemia, low levels of high-density lipoprotein cholesterol (HDL-C), central obesity, high low-density lipoprotein) cholesterol (LDL-C), low levels of antioxidants (vitamin A, E, beta-carotene), escalating affluence, rapid modernization associated with sedentary but stressful lifestyle in summation are suggested as additional risk factors for CAD [9].

Hepatic Lipase (HL) is an enzyme synthesized and secreted into the Disse space where it binds to the surface of sinusoidal endothelial cells and the external surface of microvilli of parenchymal cells [10]. It catalyzes the hydrolysis of triglycerides and phospholipids from plasma lipoproteins, contributing to the remodelling of Very Low- Density Lipoprotein (VLDL) remnants, LDL and HDL [11,12]. Independently of its lipolytic function, HL also plays a role in the hepatic uptake of remnants, HDL and LDL particles. Low HL activity has been related to high HDL concentration and more buoyant, less atherogenic LDL particles, but also to hypertriglyceridemia and the accumulation of remnant lipoproteins [13].

The human hepatic lipase (LIPC) gene encodes HL, an enzyme involved in lipoprotein metabolism and regulation [14]. Therefore, variants in LIPC gene may influence plasma lipoprotein levels. It is found to be on chromosome 15q21 and consist of nine exons and eight introns, covers over up 30 kb of DNA and encodes a protein with 449 amino acids [15,16]. The four common SNPs in the promoter region, consist of four extremely associated polymorphism in the 5’-Flanking region of the LIPC gene (-250 G/A,-514C/T,-710T/C and -763A/G) with respect to the transcription star site, which are in complete linkage disequilibrium, have been identified [17,18]. A substitution in the promoter region of the LIPC gene (-250G/A) has been reported to be related to modifications of plasma lipid levels [19–26] and the risk of CAD [27,28]. The association between hepatic lipase and CAD has been controversial [29]. The inverse relationship between HL activity and plasma HDL-C [30], a well known protective factor against CAD and the positive association of HL with small dense LDL-C [31], a possible risk factor of CAD, have pointed towards the pro-atherogenic role of HL [32]. However, reports that patients with the HL deficiency developed premature CAD [33].

The A allele of G-250A SNP are associated with lower HL activity and higher HDL levels in healthy subjects [29,34–38]. The distribution and clinical significance of G-250A polymorphisms have been extensively investigated among the Europeans, Asians and Americans, while they are still widely unknown among Indians [39–47]. Therefore, in this study, we plan to investigate the HL promoter -250G/A gene polymorphisms and risk of CAD in North Indian population.

This was a population based case control study conducted in the Department of Physiology and Cardiology at King George’s Medical University, Uttar Pradesh, Lucknow, India. Total 477 subjects (n=233 cases and n=244 control subjects) between the age group of 40 to 70 years were enrolled in the study on the basis of well defined inclusion and exclusion criteria. The sample size was statistically calculated with 80% of power [48]. The study group was recruited from the Intensive Care Unit (ICU) of Department of Cardiology, King George’s Medical University, Uttar Pradesh, Lucknow, a tertiary care hospital in India, between March 2011 and September 2014.

Controls were recruited from teaching/non-teaching staff of the institute as well as from other OPDs of our institute. Subjects having a previous history of medication, endocrinological disorder, pregnancy, chronic diseases, infection, gynaecological problem, were excluded. The diagnosis of CAD was defined as 50% stenosis in any major coronary artery, as revealed by coronary angiography [49]. On the basis of angiography results, patients were classified into two groups: with and without CAD. The study was approved by the ethical committee of King George’s Medical University, Lucknow. Written informed consent for the participation in the study was obtained prior to enrollment from all the subjects. “We certify that all applicable institutional and governmental regulations concerning the ethical use of human volunteers were followed during this research”. This study was conducted under the principles of the Declaration of Helsinki [50].

All subjects were evaluated, for Waist to Hip Ratio (WHR), waist circumference (WC); was measured at the narrowest point superior to the hip and was divided by the circumference of the hip measured at its greatest gluteal protuberance, height (Ht), weight (Wt), Blood Pressure (BP), and Pulse Rate (PR) and Body Mass Index (BMI); calculated as weight (in kilograms) divided by height (in meters) squared [51].

A 5 ml of venous blood samples were collected from all subjects and controls after 12 hours fasting. Blood samples were centrifuged at 5000 rpm for 15 min and plasma/serum was separated and stored at -20°C until being assayed further. Estimation of plasma glucose was done by GOD-POD method (Randox Laboratories Ltd., Antrim, UK). Lipid profile concentrations (TG: triglyceride, TC: total cholesterol and HDL: high density lipoprotein) were done by enzymatic method (Randox Laboratories Ltd., Antrim, UK). Low density lipoprotein (LDL) and very low density lipoprotein (VLDL) were calculated by Friedewald equation [52].

The genomic DNA was extracted from peripheral blood lymphocytes by salting out method [53]. The genotyping was performed using Polymerase Chain Reaction–Restriction Fragment Length Polymorphism (PCR-RFLP) technique. To improve the genotyping quality and validation, all variants and heterozygous samples were re-genotyped and the results were noted only for those samples which were reproducible and with no discrepancy. The transition polymorphism G to A of (LIPC-250) polymorphic site was amplified by the polymerase chain reaction (PCR). The amplification was performed using 5’-CCTACCCCGACCTTTGGCAG-3’ and 5’-GGGGTCCAGGCTTTCTTGG-3’ (Zambon A et al., 1998) as forward and reverse primer pair, respectively. The reaction was carried out in a final volume of 25 μl containing 3 mmol/l MgCl₂, 0.5 mmol of each dNTP (Bangalore GeNie), 0.2 μmol of each primer, and 2.5 U of taq DNA polymerase (Bangalore GeNie). DNA was amplified with cycling conditions of 95°C for 4 min, 95°C for 45 sec, 60°C for 45 sec, 72°C for 45 sec and for 30 cycles with final extension of 7 min at 72°C. The 560bp amplicon was digested with Dral restriction enzyme (Fermantas) to identify the G and A allele. The digested PCR product was analysed by gel electrophoresis using 2% agarose gel. G/A heterozygote mutant genotype shows three bands of 560 bp, 449 bp and 111 bp and G/ G homozygote wild genotype shows one band of 560 bp [Table/Fig-1].

[Table/Fig-1]:

Restriction Fragment Length Polymorphism Analysis of LIPC-250G/A (rs2070895) gene: L: 100 bp DNA molecular weight marker, Lane1,2,4 and 5 G/G Homozygous wild, Lane 3 G/A Heterozygous mutant.

Statistical analysis was carried out using the INSTAT 3.0 (Graph Pad Software, San Diego, CA). Quantitative variables are presented as the mean ± standard deviation. Comparisons of continuous data between two independent groups were done by Student’s unpaired t-test. Minitab version 15.0 was used for univariate to assess the association with each variable in turn and then with adjustment for other significant associations in coronary artery disease. All statistical tests were two-tailed, and p < 0.05 was chosen as the level of significance. The 2 test was used to perform the analysis of the association of the polymorphism with CAD and categorical features. Allelic and genotypic frequencies were estimated by genotype count. Hardy–Weinberg equilibrium assumptions were assessed by comparing the observed and expected numbers of genotypes. The association between genotypes and clinical characteristics was expressed as Odds Ratio (OR) with 95% confidence interval (95% CI).

The demographic and biochemical features of the study and control groups are presented in [Table/Fig-2]. Among the 477 participants, 233 patients had angiographically proven CAD and 244 had normal coronary arteries (without CAD). The CAD cases had significantly higher fasting blood sugar (FBS) (p = <0.0001), BMI (p =<0.0001) systolic blood pressure (p= <0.0001), diastolic blood pressure (p= 0.046), total cholesterol (p = <0.0001), TG (P = 0.0001), LDL-C (p <0.0001), and lower HDL-C (p= < 0.0001) than controls. For the demographic data the Weight (p= <0.0001), Waist Circumference (p = <0.0001) and Waist to Hip Ratio (p = <0.0001) were significantly higher in CAD cases as compare to controls. An elevated level of LDL-C, TG and lower level of HDL-C have been considered to increase the risk of CAD.

The allele frequencies were G (wild type): 88.42% vs. 94.67% and A (mutant type): 11.58% vs. 5.33% in with and without coronary artery disease. The frequencies of LIPC genotypes and alleles were compared between with and without coronary artery disease subjects [Table/Fig-3]. In all genetic models (co-dominant, dominant and recessive) LIPC-250G/A gene had a strong association with coronary artery disease. The genotype distribution of LIPC-250G/A deviates significantly. [Table/Fig-2] shows the distribution of genotype and allele frequencies of the case and control groups for LIPC-250G/A genotypes. In LIPC-250G/A genotype, the frequencies of GG, GA and AA genotype in CAD group was 80.69%, 15.45% and 3.86%, respectively; in the control group, the corresponding frequencies were 90.16%, 9.02% and 0.82%, respectively. Significant difference was found in the genotype (LIPC-250G/A) distribution between the two groups. Further logistic regression analysis indicated that the GA and AA genotypes in SNP -250G/A were significantly associated with CAD in all genetic models (In co-dominant model- GA vs. GG, OR=1.91, 95% CI=1.09-3.37, p=0.03 and AA vs. GG, OR= 5.26, 95% CI= 1.10-24.60, p=0.04; in dominant model- GA+AA vs GG, OR=2.19, p=0.004 and in recessive model- AA vs GG+GA, OR=5.26, p=0.04 whereas, A allele at nucleotide −250G/A in the LIPC gene had an association with increased risk of CAD (OR=2.33, p=<0.0008).

Comparison of anthropometrical and biochemical characteristics of the cases and controls according to the LIPC-250 genotype among non-carriers (GG) and carriers (GA+AA) of LIPC -514C/T promoter gene polymorphism with and without CAD are summarized in [Table/Fig-4]. In our study, we could not find the significant association between LIPC-250 G/A gene polymorphism and anthropometrical as well as biochemical characteristics among cases and controls. Only WHR was significantly (0.0001) different between LIPC genotypes among the cases. There was a significant association between LIPC-250 G/A gene polymorphism and its circulating serum level.

India is predicted to bear the greatest CAD burden, according to the estimates from the Global Burden of Disease Study [54]. A matter of serious concern is that 52% of the CAD deaths in India occurred in people aged below 70 years, while the same was just 22% in developed countries [55,56]. Reports on CAD in Indians from different parts of the world have shown that Asian Indians are at 3-4 times higher risk of CAD than white Americans, 6 times higher than Chinese, and 20 times higher than Japanese [57–60].

In the present study, we are reporting the association of LIPC promoter gene polymorphism, lipoprotein levels and coronary artery disease among the North Indian population. In our study, we observed 233 CAD and 244 without CAD, in which 286 male and 191 female were between age group of 40-70 years. Age is a powerful risk factor for coronary heart disease [61]. The development of atherosclerosis increases noticeably with age up to 65, regardless of sex and ethnic background [62,63]. According to the inherent study, the median age for developing CAD in the South Asian population is 53 years, whereas in western Europe, China & Hong Kong it is 63 years [64].

In the present study we reported significant difference in the distribution of allelic and genotype frequencies of LIPC gene (–250G/A) polymorphisms in between study and control group. We also found the significant association between polymorphisms and anthropometric parameters. This agreement supported by Pihlajamäki et al. However, Hegel et al., could not found such an association between the genetic variation in the promoter of LIPC gene and plasma lipoproteins in the Canadian population. Possibly it may be due to the differences in study populations, sample size and different ethnic group [65,66].

Ramachandran et al., reported the clustering of various cardiovascular risk factors in Asian Indians. Total serum cholesterol and LDL cholesterol are considered to be important risk factors for CAD in some studies and hyper triglyceridemia with low HDL is reported to be the major risk-factor in other studies [67]. In the present study, total cholesterol, LDL cholesterol and TG were found to be risk factors for CAD. It is well known that lower HDL-C levels could be one of the risk factors for premature CAD in Asian Indians [68]. The findings of our study were in agreement with these reports, as in our study the serum level of total cholesterol, triglyceride LDL and VLDL were found significantly higher in all study group, and high density lipoprotein was significantly lower in study group compared with control subjects. The inherent study showed that hypertension and diabetes were more important risk factors in younger Indian women than men. These studies indicate that abnormalities in lipid metabolism play an important role in development of CAD in young Indians [69].

In human, LIPC gene encodes HL enzyme that is involved in the metabolism and regulation of plasma lipoprotein with well-documented clinical importance of G-250A SNPs in LIPC gene the G-250A polymorphism has been found to be associated with type 2 diabetes, peripheral arterial disease and postprandial lipemic response [20,39,47,70,71]. This involvement has been reported in many populations including Austrians, Finnish, Spanish, and Turkish; however, it is less apparent in Japanese, Iranians, Chinese, Koreans, Americans, and Brazilians [14].

The findings of our study show that the -250G allele was more abundant among North Indian than -250A allele. The homozygous mutant allele (A) was singnificantly higher in the study group (p=0.0008), while homozygous wild allele (G) was higher in the control group(p=0.04). We also found that GA+AA genotype was significantly higher in study group (p=0.004). Here, we have evaluated that homozygous mutant were found at increased risk of CAD. Zambon et al., reported the allele frequency of the LIPC-250A polymorphism is 47% in a small group of Japanese Americans [34]. Another study De Andrade et al., was reported 32% allelic frequency of LIPC-250A polymorphism in Brazilian population. The findings of our study were agreement with these reports. These results indicate that the allelic variation of the LIPC -250G/A may have an ethnic specificity [27]. The distribution of this SNP is similar to that reported in Austrian, Spanish, Finnish, Turkish Brazilians and Americans, however, the -250 G/Allele is relatively less frequent in Japanese and Koreans [14]. This inconsistency may be due to the differences in genetic makeup of population, ethnic specificity and life style in our study population.

Epidemiological studies have suggested that both low HDL-C10 and the presence of small, dense LDL are associated with increased risk of CAD [31,72]. These lipid abnormalities often coexist in the same subject as part of a multifaceted phenotype referred to as an atherogenic lipoprotein profile [73]. In the current study, we showed that the FPG, TC, TG levels are significantly higher in GA + AA genotype and HDL-C level significantly higher in GG genotype of –250G/A polymorphism. This observation is in agreement with previous studies demonstrating an important role of HL activity as a major player in determining LDL size and density, and it provides further evidence for genetic regulation of LDL subclass distribution. A number of other factors have also been shown to affect HL activity in association with changes in LDL size and density, HDL2-C levels, and CAD risk [74]. In a study reported by Jimenez et al, there was no significant associations between the –250G/A polymorphism and plasma HDL-C levels [20].

The variations in the lipid profile levels, may due to environmental factors, life style and physical activity. A study on larger sample size is needed to explore the biological pathways to underlying coronary artery disease and identify the functional variants.

On the basis of our findings, we concluded that higher frequency of heterozygous mutant genotype (G/A) and mutant allele A of LIPC-250G/A promoter gene polymorphism was significantly associated with the risk of CAD among the North Indians. The alteration of lipid levels was also a significant risk factor for CAD

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