Association of the Lipoprotein Lipase S447X and Neuropeptide Y Leu7Pro Genetic Polymorphisms with the Lipid Profiles of Individuals with and without Evidence of Coronary Artery Disease

Background: Coronary artery disease (CAD) is a multifactorial disease whose etiogenesis involves a number of environmental, genetic and lifestyle-related factors. Genetic polymorphisms are noteworthy among these factors because they alter gene expression and, thus, the functions of the respective products. Methods: A case-control study was conducted in the Cardiology Institute, with 79 subjects classified as cases with CAD, and 96 subjects as controls without CAD or other disease. In this study, we evaluated the association between the single-nucleotide polymorphisms (SNPs) of S447X and Leu7Pro of the lipoprotein lipase (LPL) and neuropeptide Y ( NPY ) genes, respectively. Results: No differences were found in the frequencies of LPL SNP between the cases and controls. However, the LPL 447X allele carriers exhibited a near-significant difference in the triglycerides ( p=0.086 ) and higher mean in the HDL-c ( p=0.018 ). NPY polymorphisms proved to be infrequent in this study population, and no significant difference was observed between the groups. Conclusions: Our findings provide further support of the genetic polymorphisms effect on the lipid metabolism control. So, further studies are needed to assess the functional effect of this and other polymorphisms, on LPL and in the NPY activity, and their impact on CAD risk.


INTRODUCTION
Coronary artery disease (CAD) stands out among cardiovascular diseases as the leading cause of death worldwide, it's responsible for approximately 17.3 million deaths per year, and 7 million because CAD. These figures are predicted to be even higher in the future, primarily in developing countries. In Brazil, cardiovascular diseases, especially CAD, are the leading cause of natural deaths, accounting for 286 deaths per 100,000 inhabitants [1].
Many genetic risk markers have been associated with cardiovascular diseases, such as HLA-DRB1 or HLA-DQB1 polymorphism that are related to an increased frequency of autoantibodies (AAs) against angiotensin AT1 receptors (AT1-AAs) found in patients with essential hypertension, [2] others provides some genetic and environmental influences in the cardiometabolic disorders [3] Indeed, mutations may promote changes in gene products in key pathways involving lipid metabolism, eating behaviors, the response to the inflammatory process or blood coagulation [4].
The genes encoding the lipoprotein lipase (LPL) enzyme and the neuropeptide Y (NPY) neurotransmitter stand out among candidate genes for CAD susceptibility, because they are involved in circulatory homeostasis [5]. LPL plays a key role in triglyceride (TG) metabolism, as it is able to hydrolyze the TG contained in lipoproteins, including chylomicrons and very low-density lipoprotein (VLDL), when adhered to the endothelial surface of blood capillaries [6].
NPY has been shown to be a key neurotransmitter that is able to regulate satiety, and have been associated with high serum lipid levels and cardiovascular disease [7] Findings reported by Kaipio et al. [8], showed that NPY is associated with appetite control and mutations in this gene affect food intake and obesity, risk factors for CAD.
The present study assessed whether polymorphisms in the lipoprotein lipase (S447X) and neuropeptide Y (Leu7Pro) genes affect the onset of CAD and the lipid profiles of cases and controls.

Subjects
A case-control study was conducted with 175 participants; 79 (45.14%) classified as cases with CAD, and 96 (54.86%) as controls without CAD or other disease. Were included in the case group, CAD carriers with documented disease or an episode of myocardial infarction and the presence of electrocardiographic characteristic alterations. Each group was paired according to gender and age group (5-year intervals). Individuals without CAD history were evaluated by clinical and cardiac exams and only those who had exhibited normal resting and stress test electrocardiograms were enrolled in control group. Carriers of rheumatic, hepatic, renal, endocrine and neurological diseases were excluded. The study was approved by the Research Ethics Committee of Santa Cruz State University (Protocol 324/09). All study participants were volunteers and willingly signed an Informed Consent Form.

Anthropometric Data
Weight and height were measured with the subjects standing barefoot and wearing light clothing, using a Plenna ® portable scale with 100-g accuracy and a 150-kg capacity and a portable stadiometer with a 0.1 cm accuracy. Circumference measurements were performed at the waist, hip and abdomen using a measuring tape made of non-expandable synthetic material, graduated in millimeters.

Biochemical Assessment
Blood samples were collected in vacuum tubes by puncture of the median cubital vein and were used for assessing TG, total cholesterol and high-density lipoprotein cholesterol (HDL-C), lowdensity lipoprotein cholesterol (LDL-C) and VLDL cholesterol, according to protocols described by the manufacturers of the applied diagnostic kits (Liquiform: Labtest Diagnóstica S.A. Lagoa Santa-MG, Brazil).

DNA Extraction and Amplification
Genomic DNA extraction was performed in the Laboratory of Immunopathology and Genetics of the Federal University of Recôncavo da Bahia according to the manufacturer's recommendations (KiagenFlexigene 250 ® ). The extracted DNA was spectrophotometrically quantified (Smart SpecTM Plus, BioRad) by reading the absorbance at 260 nm.

Restriction Fragment Length Polymorphism (PCR-RFLP) Analysis
Following amplification, 20 µl of LPL DNA was digested using 1.25 U of MnLI at 37ºC for 18 h, and the resulting fragments were separated via electrophoresis in a 2% agarose gel (Pronadisa), which was stained using GELRED dye (Bitium

Statistical Analysis
The data were entered into EpiData 3.0, and analyses were performed in STATA 9.0. The description of cases and controls was conducted according to the presence of lipoprotein lipase and neuropeptide Y polymorphisms, gender, age and socio-economic status, beyond the lipid profile. Continuous variables were compared using Student's t test and categorical variables using the chi-squared test. A p-value of less than 0.05 was considered statistically significant. The frequencies of all of the identified alleles, genotypes and phenotypes were represented as percentages.
The genotype frequencies observed for LPL and NPY SNPs were tested for Hardy-Weinberg equilibrium (HWE) between cases and controls.

RESULTS AND DISCUSSION
In order to correlate the LPL and Leu7Pro gene polymorphism with the development of CAD, were evaluated 175 individuals, 79 (45.14%) classified as cases with CAD, and 96 (54.86%) as controls without CAD or other disease. The mean age of case group was 64.8±8.8 years, including 54 men and 25 women. The mean age of the control group was 63.0±9.2 years, including 62 men and 34 women. No statistic difference in age was found between the groups (Table 1).
Similarly, no significant difference was found between the ethnic groups, consisting of 11 men and twelve white women patients and eight men and fourteen white women controls; four men and twelve black women patients and six men and eighteen black women controls; ten men and twenty admixed women patients and thirty men and twenty admixed women controls ( Table 1).
The S447X polymorphism is one of the most frequent among the various proteins involved in the intravascular lipid metabolism, showing frequencies ranging from 17 to 22% in Caucasian populations [9]. Similar values were found in Brazil when assessing carriers and noncarriers of this polymorphism [5]. In our study we found 16% of carriers for the LPL S447X polymorphism in groups, case and control.
More specifically, according to the work of Pena et al. 2010 [10] and the self-declaration of the participants with regard to ethnicity, 14% white, 13% black, 17% admixed and one native people carry the LPL heterozygous genotype in the control group. And in the case group, 18% white, 11% black, 18% admixed carries the LPL heterozygous genotype, showing no significant differences between than (p=0.807). We also found in the control group, a black man and a admixed woman homozygous mutant (G/G) ( Table 2).
Others studies with different populations have shown different frequencies, as in Sudan, where the allele frequency LPL heterozygous genotype was 7.1% [11]. Similarly, the LPL heterozygous genotype variant in the Venezuela population was 9.4%, and the homozygous mutant was not identified [12] Minor frequencies of LPL 447X allele were 11.1% and 6.2% among subjects without CAD compared with CAD subjects in the Turkey population [13]. Statistically significant differences were observed between the cases and controls in relation to total cholesterol and LDL-c concentrations in the blood plasma, with the concentrations being lower in the cases than in the controls (p<0.001; Table 1). These results were probably because the cases used hypocholesterolemic medication, such as statins. On the other hand, the abdominal circumference measurements were significantly larger in the cases than controls (p=0.034; Table 1), evidenced by the higher BMI in case group, demonstrating greater amount of overweight or obese individuals, although there was no statistical difference between groups ( Table 1). As can be seen, there is a prevalence of overweight and obesity in the Northeast Brazilian population, in this age group, confirmed in other study carrying out in the same country region [14].
The distribution of the lipid and anthropometric profiles was assessed among the carries and no-carries LPL heterozygous genotype variant individuals ( Table  2). Higher mean concentrations of TG and total cholesterol and lower mean HDL-c concentrations occurred in the non-carrier group, although there was a lack of statistical significance between the groups.
When we comparative the lipid profiles just in the case group, LPL heterozygous genotype carriers variant showed lower mean TG levels compared with non-carriers. In addition, a statistically significant difference was observed in the mean HDL-c concentrations between carriers and noncarriers polymorphism, with higher levels being found among carriers (p=0.018) ( Table 3).
In another study with an Iranian population, the LPL S447X (rs328-GG-GC) genotype was significantly related to a higher concentration of TG, compared to the CC wild-type [15] Likewise, triglycerides were significantly lower in African American women with the -93GG compared to the -93TT LPL genotype [16].   In addition to the direct correlation of LPL polymorphism with lipid levels in the blood and consequently a higher risk for CAD, these mutations have been implicated in other diseases. A meta-analysis compared with 447SS homogeneous carriers, those with 447X variant had a lower risk of hypertension. Similarly, compared with 447S allele carriers, those with 447X allele carriers also had a lower risk of hypertension [17]. The genotype CC of S447X was associated with high systolic blood pressure, high diastolic blood pressure, and with increase of total cholesterol in Mexican families [18]. It was shown that, diabetes mellitus led to loss of the protective lipid profile in patients with CC genotype in Egyptians. In addition, rs328 is associated with a favorable lipid profile in diabetic Egyptian patients [19]. Genetic variants of LPP, including rs328 also showed significant correlations with incident ischemic stroke in Chinese diabetic population [20].
The results of the present study suggest a trend of the rs328 has a protective effect against CAD by increasing the LPL enzymatic activity. These findings highlight lower mean lipid levels among carriers of rs328 compared with non-carriers. Our findings corroborate with what were reported by other authors, [13] although just few studies had been published associating the LPL polymorphism with CAD. Research on the association of the rs328 with lipid concentrations and the risk of CAD indicate significant differences, with lower levels of TG and higher HDL-c levels found in carriers, suggesting that this polymorphism provides protection against CAD and is associated with a significant reduction in disease severity [13,21]. High HDL-c levels and lower TG concentrations help to prevent early CAD, [22] and reducing the risk of developing cardiovascular disorders related to lipid metabolism [23].
Plasma levels of NPY become elevated due to the increased sympathetic activation present in stress-related cardiac conditions. Also, NPY and Y receptor polymorphisms have been identified that may predispose individuals to increased risk of hypertension and cardiac complications [24] The Leu7Pro polymorphism originally has been reported to be associated with high levels of plasma lipids, which are associated, in turn, with an increased risk for cardiovascular disease [25,26,27], and more recently with myocardial infarction, ischemic stroke and accelerated progression of atherosclerosis, [28,29] and hypertension [30].
The allele frequency of the NPY rs16139 was investigated in 6,626 subjects from Europe, North-America, South-America, Asia and the Middle-East, and shows significant differences between some populations with different genetic backgrounds, ranging from 0.5 to 10% [31,32]. The NPY rs16139 was observed in all populations of European descent and the Israeli population, with a mean frequency of about 4%. The highest frequencies of this polymorphism have been observed in Nordic countries, providing a geographic pattern of its distribution with a decreasing gradient from north to south.
[31] On the other hand, the frequency of rs16139 of NPY was 5.9% in the Iranian population [33].
A previous Brazilian study showed a 0.03 minor allele frequency of rs16139 allele frequency [34]. The occurrence of rs16139 of NPY proved to be infrequent too in our study population. Only 6 of the 175 (3.4%) individuals showed a heterozygous genotype (A/C) and homozygous polymorphisms were not found; which indicate that it is expected based on known minor allele frequency (Table 4).
Nevertheless, a significantly higher frequency of the Leu7Pro genotype it was observed in Iranian CAD patients, where the Pro7 had significantly higher values for weight, BMI, hip circumference and prevalence of diabetes mellitus [33]. Furthermore, there was a significant difference in Pro7 frequency between diabetics versus nondiabetics, dyslipidemic versus nondyslipidemic, and obese versus nonobese Iranian CAD patients [28]. A logistic regression analysis indicated that the NPY polymorphism rs16147 was nominally associated with an increased risk of CAD. In stratified analyses, the C allele was significantly associated with a reduced risk of CAD in Iranian males and subjects who were <50 years of age [35]. On the other hand, the frequency of the variant G allele of the NPY gene was significantly higher in CAD patients without metabolic syndrome. Compared to the AA genotype of the NPY gene, where Iranian individuals carrying the GG genotype had a reduced risk of metabolic syndrome [35].
In a multicentric study, the functional NPY variant rs16147, C allele was associated with a greater reduction in waist circumference. In addition, the authors found statistically significant genotypedietary fat interaction on the change in total abdominal adipose tissue, visceral adipose tissue, and subcutaneous adipose tissue at 24 months, where the rs16147 T allele appeared to associate with more adverse change in the abdominal fat deposition in the high-fat diet group than in the low-fat diet group [36]. A study with Caucasian women found the CT-genotype of the SNP rs16147 to be significantly associated with lower waist-to-hip ratio, compared to homozygote gene carriers. However, no association between rs16147, waist-to-hip ratio and serum leptin levels was found in Caucasian men in the same study [37].
The NPY polymorphism distribution in our study population was analyzed in relation to carriers (Leu7Pro) and non-carriers of NPY Leu7Leu, and no significant difference (p=0.151) were observed between the groups (Table 4), although higher concentrations of total cholesterol and LDL-c were found among carriers than noncarriers individuals. Additionally, lower levels of TG and VLDL and a lower BMI and hip circumference were observed in carriers versus non-carriers of the NPY Leu7Pro polymorphism in the analysis of the lipid and anthropometric profile in this study.
Both rs16147 and NPY alleles (rs16131) were associated with the risk of obesity, and the latter was also associated with insulin resistance, triacylglycerol's leptin, and HDL-c in Spanish children, confirming the association of these NPY alleles with obesity, and its possible impact on the early onset of metabolic syndrome features, mainly triacylglycerol [38]. Another study, conducted with children, adolescence, and young adults in a German community, homozygote carriers of the NPY alleles exhibited significantly lower BMI scores when compared with individuals carrying the T allele. In addition, a significant genotype by age interaction emerged, indicating that the genotype effect increased during the course of development [39]. Two SNPs (rs17149106 (G>T) and NPY alleles (T>C), with minor allele frequencies of 4%, were associated with elevated risks of obesity. TTCC carriers had an increased risk of obesity compared with those carrying the common haplotype GCTT. Carriers of the NPY alleles had greater BMI than non-carriers. The NPY alleles were associated with weight gain since adolescence/early adulthood, but were not associated with abdominal adiposity as measured by waist circumference and waist to hip ratio in that study [40].