APOA5 and Triglycerides Management

A newest member of the apolipoprotein gene family, APOA5 was not discovered until 2011 (Pennacchio et al, 2001; van der Vliet et al, 2001) due to the extremely low concentration of its gene product, the apolipoprotein Apo A-V (1,000 to 10,000-fold lower comparing to other apolipoproteins) in circulating blood (O’Brien et al, 2005).  Apolipoproteins are components of lipoproteins, which are the transportation vehicles of fat and cholesterol in human blood circulation.  Although low in concentration, Apo A-V plays important roles in the homeostasis of blood triglycerides, the major storage fat in human tissue.  Mice expressing a human APOA5 gene showed a decrease in plasma triglyceride concentrations to one-third of those in control mice; conversely, knockout mice lacking APOA5 had four times as much plasma triglycerides as controls. In humans, SNPs across the APOA5 locus were found to be significantly associated with plasma triglyceride levels.

APOA5 polymorphism

There are 106 SNPs identified on the APOA5 gene to date.  Some of the SNPs belong to the same haplotype.  A haplotype is a set of SNPs that are statistically associated, theoretically inherited together in the same DNA fragment or block. It is assumed that the presence of any SNPs automatically indicates the presence of all the rest in the same haplotype.  Therefore, information about haplotypes is very useful to investigate the influence of polymorphism of genes on diseases. The three most common APOA5 haplotypes are defined by five SNPs.  Haplotype APOA5*1 is the wild type haplotype defined by the common alleles at all sites.  Haplotype APOA5*2 is defined by the rare alleles at four sites: SNP -1131T>C (rs662799), SNP -3A>G (rs651821), SNP 715G>T (rs2072560, previously referred to as IVS3+476G>T), and 1891T>C (rs2266788, previously referred to as c1259T>C). Haplotype APOA5*3 (rs3135506) is distinguished from APOA5*1 by the rare allele of the SNP 56C>G, which results in the switch of amino acid serine to tryptophan at residue 19 of the Apo A-V protein (Fig. 4, Pennacchio et al, 2002).  

Figure 4. (A) APOA5 genomic structure and polymorphism location. The gene is transcribed from left to right as indicated by the large horizontal arrow. Exons are depicted by boxes with protein-encoding regions shaded black. The position and identity of SNPs identified in APOA5 are shown below the schematic. SNPs found within the open reading frame show the predicted amino acid substitution in parentheses (synonymous changes are underlined). SNPs previously identified are indicated as SNPs 1–4 in parentheses. (B) Common APOA5 haplotypes and their relative frequencies in 419 Caucasian samples (Adopted from Pennacchio et al, 2002).

In humans, many studies in several different ethnic populations have shown significant association between two minor APOA5 haplotypes, APOA5*2 and APOA5*3, and elevated plasma triglyceride levels. It is estimated that 53% of Hispanics, 35% of African-Americans, and 24% of Caucasians carry at least one of these two minor haplotypes (Table 1 and Talmud et al, 2005).  Another emerging SNP c553G>T, only reported in Asian population (frequency 3.7%-10.5%), is associated with highly elevated TG concentration and often result in severe hypertriglyceridemia in homologous minor allele carriers.  The rest of known APOA5 SNPs have not shown any functional association with known biological process.

APOA5 and triglycerides level

In literature, the phenotypes of APOA5*2 is often represented by the tag SNP -1131T>C.  A tag SNP is the representative SNP of a haplotype.  APOA5*2 is strongly associated with a higher triglyceride concentration (16% per C allele) and increased risk of coronary heart disease (odds ratio 1.18 and hazard ratio of 1.10 per C allele).  It is also modestly associated with lower HDL cholesterol (mean difference per C allele 3.5%), lower apolipoprotein AI (1.3%), and higher apolipoprotein B (3.2%) concentrations in plasma.  Apolipoprotein AI is a component of HDL lipoprotein.  Apolipoprotein B is a component of chylomicron, VLDL and LDL lipoproteins.
The APOA5*2 carriers often have higher VLDL concentration and smaller HDL particle size, factors that could mediate the effects of triglyceride. In contrast, this allele is not significantly associated with several non-lipid risk factors or LDL cholesterol (Figure 5, Triglyceride Coronary Disease Genetics Consortium and Emerging Risk Factors Collaboration, 2010).

Figure 5. APOA5 −1131T>C genotypes and circulating lipid concentration. Size of data markers is proportional to the inverse of the variance of the weighted mean difference (the reference group is represented by a square with an arbitrary fixed size) and the vertical lines represent 95% CIs. To enable comparison of associations across lipids and apolipoproteins, associations are presented as percentage differences (Adopted from Triglyceride Coronary Disease Genetics Consortium and Emerging Risk Factors Collaboration, 2010. Lancet 375: 1634–1639).

Relatively rare in Asian population, the APOA5*3 haplotype (SNP 56C>G, SNP c56C>G, or S19W in literature) is found in 12% of Caucasians, 14% of African-Americans and 28% of Hispanics.  It is associated with increased plasma triglyceride levels in both men and women in each ethnic group studied.  Besides the elevated triglyceride concentrations, this haplotype is also associated with increased VLDL concentration and lower HDL cholesterol (Table 2, Lai et al, 2004).

Table 2. Plasma lipoprotein components affected by the SNP 56C>G polymorphism in the Framingham Heart Study (derived from Lai et al, 2004).

Plasma Lipid Variables	C/C	C/G + G/G
TG (mg/dl)	98.5±1.01	114.4±1.03
HDL-C (mg/dl)	50.3±0.3	48.3±0.7
VLDL-large (mg/dl)	5.09±0.03	6.67±0.09
VLDL-intermediate (mg/dl)	54.41±0.02	67.33±0.05
VLDL-small (mg/dl)	19.58±0.03	21.32±0.07
Chylomicron (mg/dl)	1.18±0.02	1.42±0.07

The APOA5 SNP c553G>T is remarkable in two aspects: it is present only in 4.5% to 10.5% Asian population; and it is associated with severe hypertriglyceridemia in homozygote carriers.  This SNP results in a change of amino acid residual from glycine to cysteine at position 185 of the protein product.  In one study, the frequency of the T allele is 4-fold higher in the high-TG group (15.1% when TG > 150 mg/ml) than in the low-TG group (3.7% when TG < 150 mg/ml), corresponding to a 4.45 times higher risk of hypertriglyceridemia.  In the high-TG group, the effect of the T allele shows gene dosage dependent increase of TG level (Fig. 6). Notably, all the TT homozygotes had severe hypertriglyceridemia, with a mean TG of 2,292 mg/dl (Kao et al, 2003, Tang et al, 2006, Pullinger et al, 2008).

Figure. 6. Association of the APOA5 c.553G>T (protein 185Gly185Cys) single nucleotide polymorphism with plasma levels of triglycerides in the Chinese-American population. The mean values are shown next to the boxes (Adapted from Pullinger et al, 2008).

Disease Association

Elevated plasma triglyceride (TG) levels (hypertriglyceridemia), one of the characteristics of the metabolic syndrome (MS), have been identified as an independent risk factor of coronary heart disease (CHD). APOA5 variants that are associated with elevated TG also associate with increased risk for CHD (Fig.7). This is especially true in Asian populations in which minor allele frequencies are notably higher.  In a Chinese study of 483 CHD patients and 502 control non-CHD subjects, the minor allele 56C>G was only observed in CHD patients (4.7%) and the minor allele -1131T>C was significantly higher in CHD patients (39.1%, comparing to 29.9 in controls). In another Chinese study involving APOA5 SNP c.553G>T in 486 patients with CHD and 501 controls, individuals who carried T allele (TT + GT genotype) had an increased risk of CHD (OR = 1.753) compared to GG genotype. These results suggest that APOA5 polymorphism represented by three haplotypes, APOA5*2 (tag SNP -1131T>C), APOA5*3 (56C>G), and SNP c.553G>T are risk factors for CHD susceptibility among Chinese (Liu et al, 2006, Yuan et al, 2011).

Figure 7. Association of APOA5 −1131T>C genotypes and equivalent differences in circulating triglyceride concentration with risk of coronary heart disease (Adopted from Triglyceride Coronary Disease Genetics Consortium and Emerging Risk Factors Collaboration, 2010).

APOA5 polymorphism has been associated with the risk of myocardial infarction (MI).  In an Italian nationwide case-control study involving 1864 early-onset MI cases,  of 20 single nucleotide polymorphisms (SNPs) in the candidate genes that are associated  CHD risk factors, APOA5 SNP -1131C stands out as the only SNP that strongly affects the risk for early-onset MI, even after adjusting for triglycerides. This raises the possibility that APOA5-1131T>C may affect the risk of early MI over and above effects mediated by triglycerides (De Caterina et al, 2011).

APOA5 polymorphism is also associated with carotid intimal medial thickness (IMT), a surrogate measure of atherosclerosis burden.  It was found in the Framingham Heart Study, that the haplotype APOA5*3 (SNP 56C>G) was associated with higher carotid IMT compared with the wild-type, whereas the haplotype APOA5*2 (−1131T>C) associated with higher carotid IMT only in obese subjects (Elosua et al, 2006).

In general, high level of triglycerides in blood, as known as hypertriglyceridemia, can lead to inflammation.  In clinic diagnostics, the blood level of triglycerides between 200 mg/dl to 450 mg/dl is considered moderate hypertriglyceridemia; more than 450mg/dl is considered severe hypertriglyceridemia and often leads to acute pancreatitis (Durrington, 2007).

Dietary response

The associations of APOA5 SNPs  -1131T>C  and SNP 56C>G with intakes of carbohydrate, protein, total fat, saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and total polyunsaturated fatty acids (PUFA) were calculated for each individual have been reported for different populations.  In general, APOA5 polymorphism does not respond to the intakes of carbohydrate or protein while shows a strong correlation with the quantity and quality of fat intake.

As mentioned in the previous section, a significant and consistent interaction between the APOA5 -1131T>C polymorphism and ω-6 PUFA intake on the concentration of plasma TGs, RLP-TGs (remnant-like particle triglycerides), and RLP-C (remnant-like particle cholesterol) as well as on VLDL and LDL sizes was observed. When PUFA consumption was 6% or more of total energy intake, carriers of the -1131T>C exhibited significantly higher concentrations of fasting TGs and remnant lipoproteins (RLP-TGs and RLP-C) than the major allele carriers. Moreover, carriers of the -1131T>C displayed larger VLDL and smaller LDL molecules, which have been reported to increase CVD risk. This gene– diet interaction showed a clear dose-dependent effect. However, the interaction observed for SNP -1131T>C was not shared by SNP 56C>G. In addition, the ω-6 PUFA effect on APOA5 -1131T>C was not observed when ω-3 PUFA was replaced (Lai et al, 2006).

In other studies, SNP -1131T>C carriers tend to have a decreased BMI as a function of increased total fat intake (Fig. 1) and this feature is not shared by the SNP 56C>G (Corella et al, 2007).  A recent study on APOA5 gene polymorphism (-1131T > C) and dietary fat response in 1465 overweight and obese individuals attending outpatient obesity clinics show that in the -1131T > C carriers, despite the concentrations of TG-rich lipoproteins were elevated, higher fat intakes were not associated with higher BMI, in contrast to the -1131T major allele carriers who had a positive association between fat intake and obesity. It is therefore confirmed the hypotheses that the minor C-allele may protect those consuming a high-fat diet from obesity (Sánchez-Moreno et al. 2011).  Consistent with these results, an earlier intervention study on the effects of short-term fat restriction on lipid traits and body mass index (BMI) of -1131T>C carriers and non-carriers in a group of 606 hyperlipidemic and overweight men shows that the reduction of BMI was significantly higher in -1131T>C carriers than in the major allele carriers (Aberle et al, 2005).

Medicine response

People carrying different APOA5 variants respond differentially to fenofibrate, an agonist of the peroxisome proliferative activated receptor alpha protein (PPARa) that has been used as a serum TG reduction medicine. In one study, the association between tag SNPs (-1131T>C and 56C>G) at APOA5 and TG and HDL-C response to fenofibrate in 393 men and 398 women was examined. After 3-weeks of fenofibrate treatment, APOA5 56C>G carriers displayed more significant decrease in TG (35.8%), and increase in HDL-C (11.8%) levels in the fasting state when compared with non-carriers (a TG reduction 27.9 and HDL-C decrease 6.9%); in contrast, subjects with the APOA5-1131T>C genotypes showed no differential response to fenofibrate intervention when compared with non-carriers (Lai et al. 2007). More specifically, 56C>G and -1131T>C carriers have a higher baseline of TG and lower base line of HDL-C than non-carriers.  After fenofibrate intervention, the TG and HDL-C in 56C>G carriers change to the same levels as the non-carriers while in -1131T>C carriers, the TG level remains higher and the HDL-C level remains lower than the non-carriers (Fig. 8).

Figure 8. Changes in TG and HDL-C concentrations (mg/dL) in responses to fenofibrate according to APOA5 genotypes (Adopted from Lai et al, 2007).

In another study, the association between APOA5 SNPs (-1131T>C, 56C>G and 457G>A) and efficacy of three months of low doses statin treatment in 188 adult Caucasians was examined. No significant response to statin was found among 56C>G and 457G>A and non-carriers.  However, the -1131T>C carriers were less responsive to statin (Δ LDL-C – 29.9%) treatment in comparison to the non-carriers (Δ LDL-C –36.3%) (Hubáček et al. 2009).

Exercise response

Currently the interaction between APOA5 polymorphism and exercise is not well understood.  Reports on carefully controlled studies are lacking. In one report, the effect of 9 weeks of combined energy intake and aerobic exercise program in 98 overweight and obese non-diabetic females (BMI over 27.5 kg/m2) were examined. No significant association between BMI decrease and APOA5 variants was found, but the SNP -1131T carriers have significantly higher body weight both before and after intervention. Furthermore, plasma TG levels decreased (from 1.42±0.62 mmol/l to 1.28±0.48 mmol/l) in the major allele carriers but increased (1.15±0.47 mmol/l to 1.41±0.80 mmol/l) in SNP 56C>G carriers.  Similarly, in carriers of at least one minor APOA5 allele, plasma LDL-cholesterol levels did not decreased as they did in the major allele carriers (Suchánek et al. 2008).

Molecular mechanisms

The molecular mechanisms of Apo A-V in controlling blood triglyceride level are not fully understood.  Several hypotheses have been proposed based on works on transgenic mouse models: (1) Apo A-V increases LPL mediated lipolysis of TG-rich VLDL, leading to a faster clearance and non hepatic tissue uptake of triglycerides, (2) Apo A-V increases the hepatic uptake of lipoprotein core remnants, caused by an enhanced affinity for the LDL receptor, and (3) Apo A-V decreases the hepatic production of VLDL-TG.

In vitro experiments show that Apo A-V enhances the TG lipolysis in a dose dependent and Apo C-II dependent manner.  In vivo experiments show that intravenously injected [3H]TG-rich emulsion particles have shortened half-life in the plasma of APOA5 over expressing mice than in the control (3 min. vs. 10 min).  In the injected mice, the accumulation of 3H activity into LPL-expressing tissues is also higher in the plasma of APOA5 over expressing mice than in the control.  Consistent with these observations, in APOA5 null mice the TG rich VLDL particles are 30% larger, intravenously injected 14C-palmitate containing chylomicrons and chylomicron remnant are cleared at a 50% lower rate comparing to the control mice. These data suggest that Apo A-V enhance the LPL lipolysis activity (Fruchart-Najib et al, 2004; Grosskopf et al, 2004; Schaap et al, 2004).  It is speculated that Apo A-V facilitates the access of LPL to the TG molecules in the core of the lipoprotein particle due to the high hydrophobic feature of this protein. It is also possible that Apo A-V interacts directly with LPL and increases the efficiency of TG hydrolysis by enhancing enzymatic activity through other mechanism such as stabilization of the LPL dimer.  Hypothesis of Apo A-V changing the Apo E: Apo C-II ration in lipoproteins are also proposed.

Apo A-V enhancement to the hepatic uptake of TG-rich lipoprotein remnants is supported by the delayed removal of normal remnants in the APOA5 knock-out mice.
It is postulated that in the absence of Apo A-V, the composition of TG-rich lipoprotein is changed in a manner that affects its ability to bind an appropriate receptor. Indeed, studies of LDL binding with cultured cells revealed that VLDLs from APOA5 knock-out mice were poor competitors for binding to the LDL receptor as compared with VLDL from normal mice. The weak binding could be caused by the 30% larger VLDL size, by the altered Apo E to C apolipoprotein ratios or by the direct effect of APO A-V absence (Grosskopf et al, 2004).

Based on structural analysis, it has been proposed that, at the intracellular level, Apo A-V may affect hepatic VLDL production (Weinberg et al, 2003).  However, experimental support provided by transgenic mouse model systems is controversial.  Some data support this hypothesis (Schaap et al, 2004) while others dispute (Grosskopf et al, 2004). It is speculated that the inconsistence between different experimental systems might be the result of other genetic factors (Rensen et al, 2005).

How the haplotype APOA5*2 contribute to the elevated triglycerides remains speculative.  As mentioned previously, the haplotype APOA5*2 is defined by four SNPs: SNP -1131T>C in the promoter, SNP -3A>G in the Kozak sequence, SNP 715G>T in intron 3, and 1891T>C in the 3’-UTR region of APOA5 gene.  Therefore, none of the SNPs in this haplotype is in the coding region of APOA5 gene.  It could be the result of altered transcriptional regulation leading to a decreases APOA5 gene expression or impaired Kozak sequence leading to translation dysfunction, or both.  Although functional assays using reporter system to test the impact of each of these SNPs individually did not yield any difference between the minor alleles and the major one (Talmud et al, 2005), it is nevertheless observed that homozygous for the major allele had significantly higher plasma Apo A-V levels than those homozygous for the minor allele -1131T>C (Ishihara et al, 2005).   Alternatively, the impact of haplotype APOA5*2 could be due to strong linkage with functional variants in the nearby APOC3 gene. Haplotype analysis of the APOA5/A4/C3 gene cluster that SNP -1131T>C is in strong linkage disequilibrium with the -482C>T SNP of APOC3 gene, which disrupts the normal insulin responsiveness of Apo C-III (Talmud et al, 2005; Ahituv et al, 2007).

The first 23 N-terminal residues of Apo A-V is a membrane translocation signal peptide.  The S19W mutation in the APOA5*3 haplotype seems impaired the proper function of the signal peptide.  Molecular modeling of the 19W variant predicted reduced translocation across the endoplasmic reticulum.  In vitro studies confirmed a two-fold reduction in protein secretion in comparison to the common 19S allele at this position (Talmud et al, 2005). In addition, introducing the APOA5*3-defining allele (19W) into transgenic mouse resulted in three fold lower Apo A-V plasma levels.  All these results point to a reduced protein translocation in this haplotype (Ahituv et al, 2007).

Little is known regarding the regulation of Apo A-V levels, but PPARa (peroxisome proliferator-activated receptor-a) and the farnesoid X receptor have been implicated in the transcriptional regulation of the APOA5 gene.   A PPARa response element was identified in the promoter region of APOA5. The PPARa agonist fenofibrate is known to induce APOA5 expression in human primary hepatocytes (Vu-Dac et al, 2003; Prieur et al, 2003).  It is hypothesized that expression through PPARa-RXR dimer binding, and this increased expression could compensate for the structural, functional defect of the 19W APOA5. In other words, the effect of 19W APOA5 is normalized on fenofibrate intervention and 56G carriers respond as effectively as non-carriers in clearance of plasma TG and in response to a fat load (Lai et al, 2007).

Conclusions

APOA5 gene polymorphism plays important role in blood triglycerides level.  The three haplotypes represented by the tag SNP-1131T>C, SNP 56C>G and SNP c553G>T are distributed in human population in high frequency and predispose risks for cardiovascular diseases.  Specific diet regimens emphasizing an optimized quantity and quality of fat, especially the ratio between ω-6 to ω-3 fatty acids, have been designed for each of them to optimize the effects.  Personalized triglycerides management plans are waiting for you to explore at GB.

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