Cardiovascular diseases (CVDs) are the leading cause of mortality and morbidity worldwide. In 2012, an estimated 17.5 million people died of CVDs, representing about 31.0% of all global deaths [1]. Atherosclerosis, which refers to the progressive hardening of arteries and narrowing of lumen due to fibro-fatty changes in the vessel wall, is the major pathophysiological process underlying CVDs [2]. The risk factors for atherosclerosis and CVDs are many, such as altered serum lipid levels (also called dyslipidemia), smoking, alcohol, obesity, male gender, advancing age, ethnicity and genetic predisposition, diabetes and hypertension [1-3]. The
Recently, the G-75A and C+83T polymorphisms were described in the native population of Assam, where significant differences were noted in their distribution as compared to populations of neighboring regions [21]. Assam is the most populated state in northeast India, an ethnically distinct region that is often regarded as the ethnological transition zone between the Indian subcontinent and east/southeast Asia. Decreased level of HDL-C is a major risk factor for CVD [1-3] and a common form of dyslipidemia in Assam [22,23]. Moreover,
On this basis, we decided to conduct a pilot study to explore if G-75A and C+83T SNPs of
Individuals undergoing health check-ups at the Guwahati Medical College and Hospital, Assam, India, were screened clinically and with the help of available investigation reports. Information on past medical history, drug history, dietary habits (vegetarian or non-vegetarian), and current smoking and alcohol use status was also obtained. We screened 649 individuals, from which 200 subjects (100 cases and 100 controls) of either sex, who were native to Assam, were finally selected for a case-control study on the basis of the following eligibility criteria.
Subjects enrolled in the case group had decreased HDL-C dyslipidemia (defined as HDL-C levels <40.0 mg/dL) [30]. Individuals with confounding conditions known to decrease the levels of HDL-C, such as diabetes mellitus/hyperglycemia, liver diseases, thyroid disorders and other endocrinal disorders, acute infections and inflammatory conditions, using medications affecting serum lipids (such as β-blockers, statins, oral contraceptive pills, steroids, hormone replacement therapies,
In the control group, healthy subjects with normal values of HDL-C (≥40.0 mg/dL) were included. The other serum lipid fractions were also within the normal range (
The study was conducted as per the guidelines of the Declaration of Helsinki. It was approved by the Institutional Ethics Committee, Guwahati Medical College and Hospital, Assam, India. All subjects voluntarily provided written informed consent to participate prior to enrollment in the study. Some of the participants were selected from our previous studies [21,22].
The physical measurements performed in the subjects were: WC, BMI and BP. Waist circumference and BMI were used as indices of central and general obesity, respectively. They were measured according to World Health Organization guidelines [31]. Systolic and diastolic BP was measured as per Joint National Committee (JNC VII) guidelines [32].
Measurements of TC, TG and HDL-C in fasting blood samples were done photometrically by homogenous enzymatic methods using dry chemistry reagent slides (Ortho-Clinical Diagnostics Inc., Rochester, NY, USA) in VITROS 5600 integrated system autoanalyzer (Ortho-Clinical Diagnostics Inc.). For quality control, these assays were validated using third-party control materials (Christian Medical College, Vellore, India and Bio-Rad Laboratories, Hercules, CA, USA). The LDL-C and VLDL-C were estimated indirectly using Friedewald’s formula [33]. Atherogenic indices were determined as follows: CRI-I = TC/HDL-C, CRI-II = LDL-C/HDL-C, AIP = log10 (TG/HDL-C), AC = (TC – HDL-C)/HDL-C and non-HDL-C = TC – HDL-C [34-36].
Genomic DNA extraction (by a rapid salting-out method) and genotyping for the G-75A and C+83T loci (by a PCR-RFLP method) were done from peripheral blood samples using the protocol described previously [21]. In brief, a 435 bp region at the 5’-end of the
We performed a quality-check by repeating the PCR-RFLP technique randomly in 20.0% of the samples ensuring that no differences were noted in the genotypes. Additional confirmation by direct sequencing (at SciGenom Pvt. Ltd., Cochin, Kerala, India) was done in 5.0% of the samples. All the genotypes detected for the two loci in the study were represented.
Descriptive statistics were performed in Excel spreadsheets (Microsoft Office 2003, Redmont, WA, USA). For continuous data, means with standard deviation (SD) were expressed. The categorical data were summarized as counts and percentages. The allelic and genotype frequencies for the two SNPs were calculated using POPGENE 2.0 software (Molecular Biology and Biotechnology Centre, University of Alberta, Edmonton, AB, Canada). Conformity to Hardy Weinberg equilibrium (HWE) was assessed by the goodness-of-fit χ2 and G-square tests. Diplotype frequencies were also determined.
The statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS Inc., Chicago, IL, USA) version 11.5 software. The continuous data were verified for normality by the Kolmogorov-Smirnov test. The comparisons of allelic, genotype and diplotype frequencies between the case and the control groups were performed by the χ2 test (with Yate’s continuity correction, if required). The association of G-75A and C+83T poly-morphisms with decreased HDL-C was tested under different genetic models (dominant, recessive, additive and allelic). The crude association was first determined by calculating the unadjusted odds ratios (OR) with 95% confidence intervals (CI). Further, adjusted OR with 95% CI was calculated by multiple logistic regressions after adjusting for covariates such as gender, age, smoking, alcohol use, WC, BMI, TC and TG. The associations of the SNPs with other CVD risk factors (
The baseline characteristics of the study subjects are presented in Table 1. The individuals in the case and the control groups were comparable (
Baseline characteristics of the study subjects. (Values are expressed as mean ± SD or HDL-C: high density lipoprotein cholesterol; LDL-C: low density lipoprotein cholesterol; VLDL-C; very low density lipoprotein cholesterol; non-HDL-C: non-high density lipoprotein cholesterol.
Study Subjects (
Variables
Case Group (
Control Group (
Age (years)
43.12 ± 11.64
42.95 ± 11.60
0.92
Sex
males
66 (66.0%)
61 (61.0%)
0.56
females
34 (34.0%)
39 (39.0%)
Smokers
yes
35 (35.0%)
25 (25.0%)
0.16
no
65 (65.0%)
75 (75.0%)
Alcohol use
yes
20 (20.0%)
32 (32.0%)
0.08
no
80 (80.0%)
68 (68.0%)
Body mass index (kg/m2)
24.79 ± 3.08
22.07 ± 3.18
<0.05
Waist circumference (cm)
87.14 ± 5.89
80.72 ± 6.06
<0.05
Systolic blood pressure (mmHg)
133.58 ± 16.44
117.86 ± 8.53
<0.05
Diastolic blood pressure (mmHg)
84.32 ± 8.84
77.20 ± 5.92
<0.05
HDL-C (mg/dL)
30.87 ± 5.19
54.61 ± 12.17
<0.05
Total cholesterol (mg/dL)
175.54 ± 35.98
160.79 ± 25.70
<0.05
Triglycerides (mg/dL)
145.95 ± 29.39
95.35 ± 23.55
<0.05
LDL-C (mg/dL)
116.41 ± 28.19
87.37 ± 22.96
<0.05
VLDL-C (mg/dL)
29.12 ± 7.62
19.03 ± 5.90
<0.05
Non-HDL-C (mg/dL)
144.67 ± 32.47
106.18 ± 24.83
<0.05
Castelli’s risk index I
5.71 ± 0.87
3.04 ± 0.65
<0.05
Castelli’s risk index II
3.78 ± 0.79
1.68 ± 0.56
<0.05
Atherogenic index of plasma
0.66 ± 0.13
0.26 ± 0.14
<0.05
Atherogenic coefficient
4.71 ± 0.87
2.04 ± 0.65
<0.05
The genotype, allelic and diplotype frequencies for G-75A and C+83T polymorphic loci are summarized in Table 2. Both the polymorphisms were in Hardy-Weinberg equilibrium. For the G-75A site, the GG genotype was most common, followed by GA and AA. The frequencies of GG, GA and AA genotypes were comparable between the case and the control groups (χ2 = 1.692,
The distribution of G-75A and C+83T polymorphisms in the case and control groups. [Values are expressed as SNPs: single nucleotide polymorphisms;
Genotype Frequency
Allelic Frequency
SNPs
Group
GG
GA
AA
G
A
G-75A
Case
62 (62.0%)
35 (35.0%)
3 (3.0%)
159 (79.5%)
41 (20.5%)
Control
60 (60.0%)
33 (33.0%)
7 (7.0%)
153 (76.5%)
47 (23.5%)
χ2 = 1.692 (
χ2 = 0.364 (
CC
CT
TT
C
T
C+83T
Case
89 (89.0%)
11 (11.0%)
–
189 (94.5%)
11 (5.5%)
Control
87 (87.0%)
13 (13.0%)
–
187 (93.5%)
13 (6.5%)
χ2 = 0.047 (
χ2 = 0.044 (
GG/CC
GA/CC
AA/CC
GG/CT
GA/CT
Combined
Case
54 (54.0%)
32 (32.0%)
3 (3.0%)
8 (8.0%)
3 (3.0%)
Control
50 (50.0%)
30 (30.0%)
7 (7.0%)
10 (10.0%)
3 (3.0%)
χ2 = 2.041 (
Neither the genotypic variants nor the allelic variants of the G-75A or the C+83T polymorphism detected in the study were associated with HDL-C status (Table 3).
Association of decreased high density lipoprotein cholesterol with G-75A and C+83T polymorphisms under different genetic models. [Values are expressed as Odds ratio was adjusted for gender, age, smoking, alcohol use, WC, BMI, TC and TG. In every model, for each OR value, the corresponding 95% CI extended across 1. None of the OR values were statistically significant ( SNPs: single nucleotide polymorphisms; OR: odds ratio; 95% CI: 95% confidence interval. Association between C+83T SNP and decreased HDL-C was not assessed by separate genetic models as no TT homozygotes were detected.
SNPs
Model
Genotype/Allele
Case
Control
Crude OR (95% CI)
Adjusted OR
G-75A
Additive: GG
GG
62 (62.0%)
60 (60.0%)
reference
reference
Dominant: GG
GG
62 (62.0%)
60 (60.0%)
reference
reference
Recessive: (GG + GA)
GG + GA
97 (97.0%)
93 (93.0%)
reference
reference
Allelic: G
G
159 (79.5%)
153 (76.5%)
reference
reference
C+83 T
CC
CC
89 (89.0%)
87 (87.0%)
reference
reference
Allelic: C
C
189 (94.5%)
187 (93.5%)
reference
reference
The G-75A SNP was associated with LDL-C levels (Table 4). Values of LDL-C were significantly (
Influence of G-75A polymorphism on cardiovascular risk factors. [Values are expressed as adjusted mean±standard error (SE). Adjusted means compared between genotypes by analysis of covariance (ANCOVA). Statistically significant ( Means adjusted taking age, gender, smoking, alcohol use, TC, TG, WC and BMI as covariates. Means adjusted taking age, gender, smoking, alcohol use, WC and BMI as covariates. Means adjusted taking age, gender, smoking, alcohol use, WC and BMI as covariates. Means adjusted taking age, gender, smoking, alcohol use, WC and BMI as covariates. Means adjusted taking age, gender, smoking, alcohol use, WC and BMI as covariates. Means adjusted taking age, gender, smoking, alcohol use, WC and BMI as covariates. Means adjusted taking age, gender, smoking, alcohol use, WC and BMI as covariates. Means adjusted taking age, gender, smoking, alcohol use, WC and BMI as covariates. Means adjusted taking age, gender, smoking, alcohol use, WC and BMI as covariates. Means adjusted taking age, gender, smoking, alcohol use, WC and BMI as covariates. Means adjusted taking age, gender, smoking, alcohol use, TC, HDL-C and TG as covariates. Means adjusted taking age, gender, smoking, alcohol use, TC, HDL-C and TG as covariates. Means adjusted taking age, gender, smoking, alcohol use, TC, HDL-C, TG, BMI and WC as covariates. Means adjusted taking age, gender, smoking, alcohol use, TC, HDL-C, TG, BMI and WC as covariates. HDL-C: high density lipoprotein cholesterol; TC: total cholesterol; TG: triglycerides; LDL-C: low density lipoprotein cholesterol; VLDL-C; very low density lipoprotein cholesterol; non-HDL-C: non-high density lipoprotein cholesterol; CRI-I: Castelli’s risk index I; CRI-II: Castelli’s risk index II; AIP: atherogenic index of plasma; AC: atherogenic coefficient ; BMI: body mass index; WC: waist circumference; Systolic BP: systolic blood pressure; Diastolic BP: diastolic blood pressure.
Phenotypic Variables
Case Group (n = 100)
Control Group (n = 100)
Study Subjects (n = 200)
HDL-C
30.7±0.5
31.3±0.6
29.5±2.3
55.4±1.5
54.8±2.0
47.0±4.6
42.7±1.1
42.9±1.5
42.2±3.9
TC
179.0±4.7
172.9±6.3
134.9±21.9
156.5±3.3
166.1±4.5
172.9±10.1
168.2±2.9
169.2±3.9
161.3±10.5
TG
145.5±4.9
148.1±6.7
129.6±23.2
93.8±3.7
94.9±4.9
110.3±11.2
120.4±3.5
121.1±4.8
121.5±12.7
LDL-C
120.7±3.8
113.3±5.1
87.2±17.8
102.4±2.7
101.6±3.6
97.9±9.6
VLDL-C
29.0±0.9
29.6±1.3
25.8±4.7
18.7±0.7
18.9±0.9
21.9±2.2
24.2±2.5
24.2±0.9
24.0±0.7
Non-HDL-C
147.9±4.2
141.9±5.7
109.5±19.8
125.5±3.1
126.1±4.1
120.2±10.9
CRI-I
5.8±0.1
5.6±0.2
5.4±0.5
4.4±0.1
4.3±0.2
4.1±0.5
CRI-II
3.9±0.1
3.6±0.1
3.4±0.5
2.8±0.1
2.7±0.1
2.5±0.4
AIP
0.66±0.02
0.67±0.02
0.69±0.08
0.24±0.02
0.26±0.02
0.36±0.05
0.46±0.02
0.46±0.03
0.48±0.07
AC
4.8±0.1
4.6±0.2
4.4±0.5
3.4±0.1
3.3±0.2
3.1±0.5
BMI
24.5±0.4
25.1±0.5
26.3±1.9
21.9±0.4
22.3±0.6
21.9±1.3
23.3±0.3
23.7±0.4
23.1±1.1
WC
86.7±0.7
88.3±0.9
83.7±3.4
80.6±0.8
80.8±1.1
81.2±2.4
83.7±0.6
84.6±0.8
81.7±2.2
Systolic BP
135.4±2.1
130.7±2.8
129.1±9.7
118.1±1.0
117.2±1.4
118.7±3.1
127.0±1.2
124.0±1.6
121.5±4.5
Diastolic BP
85.1±1.1
83.7±1.6
77.2±5.4
77.1±0.7
77.1±0.9
78.3±2.2
120.4±3.5
80.3±0.9
78.4±2.4
Influence of C+83T polymorphism on cardiovascular risk factors. [Values are expressed as adjusted mean±SE. Adjusted means compared between genotypes by analysis of covariance (ANCOVA). Statistically significant ( Means adjusted taking age, gender, smoking, alcohol use, TC, TG, WC and BMI as covariates. Means adjusted taking age, gender, smoking, alcohol use, WC and BMI as covariates. Means adjusted taking age, gender, smoking, alcohol use, WC and BMI as covariates. Means adjusted taking age, gender, smoking, alcohol use, WC and BMI as covariates. Means adjusted taking age, gender, smoking, alcohol use, WC and BMI as covariates. Means adjusted taking age, gender, smoking, alcohol use, WC and BMI as covariates. Means adjusted taking age, gender, smoking, alcohol use, WC and BMI as covariates. Means adjusted taking age, gender, smoking, alcohol use, WC and BMI as covariates. Means adjusted taking age, gender, smoking, alcohol use, WC and BMI as covariates. Means adjusted taking age, gender, smoking, alcohol use, WC and BMI as covariates. Means adjusted taking age, gender, smoking, alcohol use, TC, HDL-C and TG as covariates. Means adjusted taking age, gender, smoking, alcohol use, TC, HDL-C and TG as covariates. Means adjusted taking age, gender, smoking, alcohol use, TC, HDL-C, TG, BMI and WC as covariates. Means adjusted taking age, gender, smoking, alcohol use, TC, HDL-C, TG, BMI and WC as covariates. HDL-C: high density lipoprotein cholesterol; TC: total cholesterol; TG: triglycerides; LDL-C: low density lipoprotein cholesterol; VLDL-C; very low density lipoprotein cholesterol; non-HDL-C: non-high density lipoprotein cholesterol; CRI-I: Castelli’s risk index I; CRI-II: Castelli’s risk index II; AIP: atherogenic index of plasma; AC: atherogenic coefficient; BMI: body mass index; WC: waist circumference; Systolic BP: systolic blood pressure; Diastolic BP: diastolic blood pressure.
Phenotypic Variables
Case Group (
Control Group (
Study Subjects (
Lipid Profile
CC
CT
CC
CT
CC
CT
HDL-C
30.8 ± 0.4
31.1 ± 1.1
54.7 ± 1.2
53.7 ± 3.1
43.1 ± 0.9
39.9 ± 2.5
TC
176.5 ± 3.9
167.8 ± 11.2
162.6 ± 2.7
148.4 ± 7.1
169.7 ± 2.4
157.2 ± 6.5
TG
96.6 ± 3.0
87.1 ± 7.9
LDL-C
116.9 ± 3.2
112.4 ± 9.1
88.7 ± 2.5
78.6 ± 6.4
102.9 ± 2.2
94.8 ± 5.9
VLDL-C
19.3 ± 0.6
17.3 ± 1.6
Non-HDL-C
145.6 ± 3.5
137.5 ± 10.1
107.7 ± 2.7
95.9 ± 6.9
126.7 ± 2.5
116.2 ± 6.8
CRI-I
5.7 ± 0.1
5.6 ± 0.3
3.1 ± 0.1
2.9 ± 0.2
4.4 ± 0.1
4.2 ± 0.3
CRI-II
3.8 ± 0.1
3.8 ± 0.2
1.7 ± 0.1
1.5 ± 0.2
2.7 ± 0.1
2.7 ± 0.2
AIP
0.27 ± 0.01
0.23 ± 0.04
AC
4.7 ± 0.1
4.6 ± 0.3
2.1 ± 0.1
1.8 ± 0.2
3.4 ± 0.1
3.2 ± 0.2
BMI
24.9 ± 0.3
23.5 ± 0.9
22.1 ± 0.3
22.1 ± 0.9
23.5 ± 0.3
22.8 ± 0.7
WC
87.3 ± 0.6
85.5 ± 1.7
80.6 ± 0.6
81.4 ± 1.7
84.0 ± 0.5
83.3 ± 1.4
Systolic BP
133.6 ± 1.7
133.7 ± 4.9
117.7 ± 0.8
119.1 ± 2.2
125.6 ± 1.0
126.3 ± 28
Diastolic BP
83.8 ± 0.9
88.3 ± 2.7
77.0 ± 0.6
77.9 ± 1.5
80.4 ± 0.6
82.9 ± 1.6
The G-75A polymorphism was associated with several atherogenic indices (Table 4). The AA homozygotes had significantly (
The G-75A and C+83T loci were not associated with obesity and BP in the subjects under the present study. Values of WC, BMI and systolic and diastolic BP were comparable (
The CVD risk factors did not differ significantly across the five diplotypes obtained in the current study (data not shown).
The G-75A locus produced medium-to-large sized effects (0.25 < Cohen’s
We explored the associations of G-75A and C+83T SNPs in the study population with cardiovascular risk factors. Although the G-75A and C+83T polymorphic sites were reported to be associated with HDL-C levels in some studies [9,15-17], we observed no such association in our study. These polymorphisms did not confer greater risk of developing decreased HDL-C dyslipidemia in the current population. Lack of association between these SNPs and low HDL-C levels was also reported by other investigators [7,12,38]. We verified our findings in different genetic models (additive, dominant, recessive and allelic) and no relationship was found. It is possible that changes in HDL-C values across the genotypic variants of G-75A and C+83T loci reported in some of the earlier studies were actually modulated by confounding factors. This is substantiated by the fact that the effects of G-75A and C+83T SNPs on phenotypic characteristics have been found to be influenced by gender differences [13,39], smoking [39] and alcohol use [40]. Such biases have not been controlled for in many of the previous studies [9,16,17]. We took care to ensure that the effects of these SNPs were assessed after controlling for potential confounders in the data analysis. Moreover, many conditions that may spuriously alter lipid levels in the cases were excluded in the design stage itself.
Among the other serum lipids, we found that the minor A allele in the G-75A locus was associated with adverse LDL-C levels. The AA and GA genotypes had high LDL-C values. This was consistent with the findings of previous investigators [4,38]. However, few studies have found the A allele to produce beneficial effects on LDL-C [41]. These discordant results may be due to linkage disequilibrium between the G-75A SNP and some other regulatory elements in close proximity that actually influence the levels of LDL-C. For example, strong linkage was observed between the A allele (of the G-75A site) and X2 allele (of the
On the other hand, we found that the C>T transition at C+83T locus favorably affected TG and VLDL-C concentrations. Our results were in agreement to those by Ma
The association of the two SNPs with several atherogenic indices was a novel and significant finding of the current study. Genetic factors affecting atherogenic indices have been investigated infrequently. We found that the minor A allele at the G-75A site was associated with adverse values of CRI-I, CRI-II, non-HDL-C and AC, whereas, the minor T allele at the C+83T site was associated with favorable values of AIP. These pro-atherogenic effects of the G-75A polymorphism and the anti-atherogenic effects of the C+83T polymorphism on the atherogenic indices may be important modulators of CVD risks that are often described in relation to these two SNPs. Recent data have shown that atherogenic indices (
There are contradictory reports about the effects of G-75A and C+83T SNPs on indices of obesity and BP. These SNPs were not associated with WC, BMI, systolic and diastolic BP in the current population. In conformity to the present findings, no association of the G-75A locus was detected with obesity and BP by Ma
A limitation of our study was its design with respect to the secondary objectives. As our primary objective was to investigate the association of the polymorphisms with the risk of developing decreased HDL-C, we therefore had to recruit the subjects as case and control groups on the basis of HDL-C status. Thus, we could not use contingency tables and risk estimates (
The results from our pilot study suggested that the G-75A and C+83T polymorphisms of