APOC3 gene has been mapped on the long arm of chromosome11, closely linked to the APOA4 and APOA1 genes [7]. A transversion from C to G in the 3' untranslated region (3'UTR) of exon 4 in the APOC3 gene results in an SstI polymorphism. Several studies have suggested a close association between rare S2 allele of SstI polymorphism and elevated TG levels [8-24] and apoCIII levels [22-25]. However, few other studies have shown contradictory results [26-31].

The genotypic and allelic distribution of APOC3 polymorphism in the study population is shown in Table ​Table2.2. No significant deviation from Hardy-Weinberg equilibrium was observed in the study population. The frequency of the S2 allele in the study population was 0.313 (Table ​(Table2).2). The 95% CI of the allele frequency is also presented in Table ​Table22.

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 ​Table33 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 ​Table3.3. 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 ​Table33 (Chi square = 14.019, df = 2, p = 0.001). S1S1 genotype was more frequent in the NTG group as compared to the HTG group (53.3% vs. 26.5%). Conversely, S2S2 genotype was almost five times more prevalent in the HTG group as compared to the NTG group (23.5% vs. 4.8%). However, not much difference was observed in the S1S2 frequency between the two groups (HTG: 50% vs. NTG: 41.9%). In totality, 73.5% of HTG group (25/34) were S2 carriers out of which 32% individuals (i.e. 8/25) were S2 homozygotes. Comparatively, NTG group had 46.7%(49/105) S2 carriers, of which only 10.2%(5/49) were S2 homozygotes. Consequently, S2 allele was almost two times more frequent in the HTG group compared to the NTG group (p = 0.001) as shown in Table ​Table33.

Logistic analysis revealed a significant association of APOC3 S2 allele with hypertriglyceridemia (Table ​(Table3).3). The crude Odds ratio (OR) for S1S2 genotype (in comparison to S1S1 genotype) was found to be 2.4 (95%CI: 0.98–5.90), which was towards significance (p= 0.056) and did not change much after adjusting for age (OR= 2.43, 95% CI = 0.99–6.01, p= 0.054). The crude OR for S2S2 to develop HTG (in comparison to S1S1) was found to be 9.95 (95% CI: 2.66–37.29, p = 0.0006), which was highly significant and remained significant even after adjusting for age (OR = 9.90, 95%CI = 2.64–37.12, p = 0.0007). In totality, S2 carriers had a crude OR of 3.17 to develop HTG (95%CI = 1.35–7.45, p = 0.008) and 3.22 (95% CI = 1.37–7.60, p= 0.007) after age adjustment.

The intergenotypic variations in lipid profile in the HTG, NTG and total subjects are shown in Table ​Table4.4. 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 ​(Table44).

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. The genotypic distribution was in good agreement with Hardy-Weinberg equilibrium. Our study revealed a higher frequency of rare S2 allele (0.313) than observed for most of the Caucasians (0.00–0.11) [8-13,18,21,26] but within the same range as reported for different non Caucasian populations (0.15–0.39) [14-17,20,28-31]. Earlier Paul et al reported the distribution of APOC3 SstI polymorphism in a much smaller sample of immigrant Asian Indians at U.K. (S2 = 0.19, N = 24) [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 [8-13,18,21-24], 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].

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]. 100–500 ng of DNA was amplified in a thermocycler (PTC-100, MJ Research Inc., USA) using 1 unit of Taq DNA Polymerase (Life Technologies Inc., USA) in a 25 ul reaction mixture containing 10 picomole forward primer: 5'-CAT GGT TGC CTA CAG GAG TTC-3'and reverse primer: 3'-TGA CCT TCC GCA CAA AGC TGT-5' (MWG Biotech GmbH, Germany)[8]. The PCR mixture also contained 10% DMSO and 5 mM dNTPs (Life Technologies Inc., USA). DNA was initially denatured for 5 minutes at 95°C, annealed at 58°C for 3 minutes and at 72°C for 5 minutes. The cycling conditions were set to heat the samples at 95°C for 50 seconds, at 58°C for 45 seconds and at 72°C for 1 minute. The cycle was repeated 40 times followed by final extension at 72°C for 10 minutes.

Nine microlitre of the PCR product was digested at 37°C overnight with 10 units of SacI restriction enzyme (New England Biolabs Inc., USA) in the presence of 1 ul of 10X buffer provided with the restriction enzyme. The digested PCR product was resolved on a 3% agarose gel using 1XTBE buffer (89 mM Tris Borate, 2 mM EDTA, pH8.3) at 80 V for at least 1 hour and visualized by ethidium bromide staining on a U. V. transilluminator. The presence of the SstI site yields two fragments of 225 bp and 371 bp, while in the absence of the site one fragment of 596 bp is observed. The wild type allele lacking the restriction site is called as S1 and the allele containing the SstI restriction site is designated as S2 allele.