Apolipoprotein C3 SstI polymorphism and triglyceride levels in Asian Indians

Background A close association between Sst I polymorphism in the 3' untranslated region of the apolipoproteinC3 (APOC3) gene and levels of plasma triglycerides (TG) had been reported by different investigators. Hypertriglyceridemia(HTG) is a known risk factor for coronary artery disease (CAD) in the context of Asian Indians. We conducted a study on the relationship between APOC3 SstI polymorphism (S1S1, S1S2 and S2S2 genotypes) and plasma TG levels in a group of 139 male healthy volunteers from Northern India. Methods DNA samples were analyzed by polymerase chain reaction (PCR) followed by SstI digestion. Digested PCR products were run on 3% agarose gel and visualized by ethidium bromide staining. Results Rare S2 allele was highly prevalent in our study population (0.313) as compared to the Caucasians (0.00–0.11). The genotypic distribution was in agreement with Hardy-Weinberg equilibrium. S2 allele was almost two times more prevalent in the HTG group (N = 34) as compared to NTG group (N = 105) (p = 0.001). Multiple logistic regression revealed S1S2 individuals had age-adjusted odds ratio of 2.43 (95%CI = 0.99–6.01, p = 0.054) and S2S2 had 9.9 (95%CI = 2.66–37.29, p = 0.0006) for developing HTG in comparison to S1S1 genotype. Conclusions Our study shows a significant association between rare S2 allele and HTG in Asian Indians.


Background
Apolipoprotein CIII (apoCIII protein; APOC3 gene) is a 79 amino acids long glycoprotein that is synthesized predominantly in the liver and to a lesser degree in the intes-tine [1]. It is present on very low density lipoproteins (VLDLs) and chylomicron remnants; and to some extent on high density lipoproteins (HDLs) [1]. Although the precise function of apoCIII is not clearly understood, sev-eral lines of evidence suggest its involvement in the regulation of triglyceride (TG) levels. In vitro, apoCIII inhibits lipoprotein lipase (LPL), a rate-limiting enzyme for TG hydrolysis, resulting in the delayed catabolism of TG-rich particles [2]. Furthermore, it also decreases apoE-mediated remnant removal by displacement of apoE from the VLDL particles in vivo [3,4]. Additional copies of human APOC3 gene in transgenic mice were associated with hypertriglyceridemia (HTG) [5], whereas the absence of the gene in knock out mice leads to reduced TG [6].
We have investigated the association of APOC3 SstI polymorphism with TG levels in a group of healthy volunteers from Northern India; considering the high prevalence of CAD in Asian Indians, HTG as one of the underlining risk factors in the progression of coronary atherosclerosis [32] and no information available on Asian Indians in this context.

Results
The characteristics of the NTG and HTG groups are shown in Table 1. There was no significant difference in the mean age between the two groups (p = 0.748). TC (p = 0.0001) and TG levels (p < 0.0001) were significantly higher in the HTG group as compared to the NTG group. There was no significant difference in LDL, HDL and LDL/HDL ratio.
The genotypic and allelic distribution of APOC3 polymorphism in the study population is shown in Table 2. No observed in the study population. The frequency of the S2 allele in the study population was 0.313 ( Table 2). The 95% CI of the allele frequency is also presented in Table 2. Values expressed in mmol/L  To determine the association of APOC3 SstI polymorphism with TG levels, the study population was divided into NTG and HTG groups. Out of 139 individuals, 34 subjects (24.46%) were in HTG group i.e. TG levels beyond 1.921 mmol/L. Table 3 summarize the distribution of various genotypes and alleles of APOC3 polymorphism between the NTG and HTG groups. The 95% CI of the allele frequencies is also presented in Table 3. No significant difference was observed between the expected and observed genotype frequencies of the two groups (NTG: Chi square = 0.9852, df = 1, p = 0.3209 and HTG: Chi square = 0.000, df = 1 and p = 0.9960; in Hardy-Weinberg equilibrium). There was a significant difference in the genotypic distribution between the NTG and HTG groups as shown in  Table 3.
The intergenotypic variations in lipid profile in the HTG, NTG and total subjects are shown in Table 4. TG was significantly different among various genotypes in the HTG (p = 0.015, log TG: p = 0.015), the total subjects (p < 0.0001) and insignificant in the NTG group (p = 0.114, logTG: p = 0.137). In particular, the S2S2 individuals were associated with highest concentration of TG followed by S1S2 and then by S1S1 in HTG group & total study population. No significant differences were observed in TC, LDL, HDL and LDL/HDL ratio in any of the study group (Table 4).

Discussion
ApoCIII provides a strong negative charge on the surface of lipoproteins preventing nonspecific interactions with cell surfaces [33] and perhaps with other lipoproteins. This may serve the function of reducing futile cycles in TG transport by preserving the particles for high affinity interactions such as with lipoprotein lipase or specific cell surface receptors e.g., such as those binding to apoE or apoB. Plasma concentrations of apoC-III in human populations correlate well with TG levels [16,34]. In vivo apoCIII modulates the postprandial management of the TG [6] and inhibits the hepatic uptake of VLDL remnants [35]. The genetically determined deficiency of apoCIII in humans has been shown to increase the rate of TG clearance from plasma by 6-to 7-fold [36]. A similar enhancement of TG clearance was observed in mice made apoCIII deficient by gene knockout experiments [6]. Overexpression of apoCI-II produces hypertriglyceridemia in transgenic mouse models via inhibition of clearance of TG-rich particles [4].
It is now clear that normal physiological systems responsible for TG transport are partially determined by the plasma content of apoCIII. Although it is not clear how this protein contributes to the familial hypertriglyceridemic syndromes, recent studies have found that two classes of drugs that are effective in lowering plasma TG in these patients act through suppression of APOC3 gene transcription in rodents [37,38].
The human APOC3 gene expression is controlled by positive and negative elements that are spread through out the APOA1-C3-A4 gene cluster on the long arm of chromosome 11 [39]. Various restriction fragment length polymorphisms in and around the human APOC3 gene have been associated with hypertriglyceridemia in several distinct populations [40]. The present study on SstI polymorphism was carried out on a random sample of 139 individuals inhabiting plains of Northern part of India.  [41]. We attempted at elucidating the association of APOC3 SstI polymorphism with TG levels. Individuals having TG levels up to 1.921 mmol/L were grouped in NTG and more than 1.921 mmol/L were grouped in HTG group. S2S2 individuals had the highest levels of TG followed by S1S2 and S1S1 in HTG and total study population. Significantly higher frequency of S2 allele in the HTG group as compared to the NTG group suggests a strong association of the S2 allele with higher levels of TG. Such an association of S2 allele with higher levels of TG has been reported in studies carried out on Caucasians [8][9][10][11][12][13]18,[21][22][23][24], Chinese [14], Mayans [15], Japanese [16], Koreans [17], South Africans [19] and Arabs [20]. The biochemical basis for the association of S2 allele with hypertriglyceridemia has yet to be established. Dallinga-Thie et al [22,23] and Shoulders et al [24,25] reported an association between levels of apoCIII and S2 allele. The SstI polymorphism is located in the 3' untranslated region of APOC3 gene. Therefore, it is more likely that S2 allele is not etiological but in linkage disequilibrium with other causative mutation hitherto unknown in APOC3 or nearby gene involved in determining the TG levels. It has been suggested that certain haplotypes generated from SstI polymorphism and promoter polymorphism of APOC3 gene may protect or predispose to hypertriglyceridemia [17]. In addition, SstI polymorphism may also influence mRNA stability [17]. Few of the studies carried out on Caucasians [26,27], Taiwanese [28], Japanese [29,30] and Arabs [31] did not find any significant association between SstI polymorphism and HTG. It has been speculated that the linkage disequilibrium between this polymorphic site and the causative mutation is weakened or absent in some populations [24].
Ours is the first study on Asian Indians to report a strong association of APOC3 S2 allele with hypertriglyceridemia in Indians. The logistic analysis revealed that individuals carrying S2 allele were 3.2 times more prone to develop hypertriglyceridemia as compared to S1S1. Thus, S2 allele may serve as a significant risk marker for susceptibility to hypertriglyceridemia. This is an important finding as Asian Indians are highly sensitive to the adverse effects of hypertriglyceridemia [32], the risk of which is likely to increase manifold with growing shift towards affluent lifestyle and sedentary habits in larger fraction of our population.

Conclusion
We found a high prevalence of rare S2 allele of APOC3 gene in HTG individuals. Further, this allele was more frequent in our study population as compared to Caucasians. Since HTG is considered as a risk factor for CAD in Asian Indians, there is an urgent need to evaluate the association of APOC3 SstI polymorphism with the risk of developing coronary artery disease in Asian Indians.

Materials and Methods
One hundred and thirty nine healthy male volunteers from plains of northern part of India (mean age: 52.32 ± 11.02 years) were enrolled in the study. The subjects were scrutinized on the basis of standard questionnaire. They shared fairly common socio-cultural background and comparable dietary habits. All underwent routine biochemical tests (hemoglobin, urea & sugar) and blood pressure measurement. Subjects having angina or any his-tory of myocardial infarction were excluded from the study. The research was undertaken with the approval of ethical committee set by All India Institute of Medical Sciences, New Delhi and its guidelines were observed.
Venous blood was collected from each individual after at least 12 hours of fasting. Lipid profile was monitored using enzymatic kits (Randox laboratories limited, UK).
The study subjects were classified into normotriglyceridemic group (NTG: N = 105, TG < = 1.921 mmol/L) and hypertriglyceridemic group (HTG: N = 34, TG>1.921 mmol/L) based on the normal range of the TG calibrated at AIIMS, which is 0.791-1.921 mmol/L. The calibration was done on data obtained from huge number of serum samples. All the chemicals used in the study were procured from Sigma Chemical Co., USA, if not specified.
DNA was extracted from blood by salting out method [42]. Allelic frequencies were estimated by gene-counting method. The sample-size dependent standard error of alleles was calculated in terms of 95% confidence interval (CI) of the estimates. Chi-square goodness-of-fit was used to verify the agreement of the observed genotype frequencies with those expected ones (Hardy-Weinberg equilibrium) in various study groups. Chi-square test was applied to compare genotypic frequencies between the two groups. Contingency table approach (Fisher's RxC test) was used to determine if there is significant differences in allele frequencies among the group of individuals. The biochemical characteristics of the individuals in various genotypic groups were expressed in terms of mean ± standard deviation (S.D.) and were compared using analysis of variance (Annova). Triglycerides values were also log transformed because the test of homogeneity of variance was found to be significant. Logistic analysis with enter method was performed. S1S1 genotype was taken as the reference and the odds ratio with 95% confidence interval was calculated for S1S2 and S2S2 genotypes individually and taken together. Hypertriglyceridemia was entered as dependent variable with NTG = 0 and HTG = 1.
All the statistical analysis was preformed using SPSS (Statistical Package for Social Sciences) for windows (version 7.5.10, SPSS Inc., Chicago). Statistical significance was set at p < 0.05.