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Originally published In Press as doi:10.1194/jlr.M400192-JLR200 on September 1, 2004

Papers In Press, published online ahead of print November 1, 2004
J. Lipid Res., doi:10.1194/jlr.M400192-JLR200
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Journal of Lipid Research, Vol. 45, 2096-2105, November 2004
Copyright © 2004 by American Society for Biochemistry and Molecular Biology

Influence of the APOA5 locus on plasma triglyceride, lipoprotein subclasses, and CVD risk in the Framingham Heart Study

Chao-Qiang Lai1,*, Serkalem Demissie{dagger}, L. Adrienne Cupples{dagger}, Yueping Zhu*, Xian Adiconis*, Laurence D. Parnell*, Dolores Corella*,§ and Jose M. Ordovas*

* Nutrition and Genomics Laboratory, Jean Mayer–United States Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston, MA
{dagger} School of Public Health and School of Medicine, Boston University, Boston, MA
§ Genetic and Molecular Epidemiology Unit, University of Valencia, Valencia, Spain

The online version of this article (available at http://www.jlr.org) contains an additional table. Back

Published, JLR Papers in Press, September 1, 2004. DOI 10.1194/jlr.M400192-JLR200

1 To whom correspondence should be addressed. e-mail: chao.lai{at}tufts.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Several polymorphisms in the APOA5 gene have been associated with increased plasma triglyceride (TG) concentrations. However, associations between APOA5 and lipoprotein subclasses, remnant-like particles (RLPs), and cardiovascular disease (CVD) risk have been less explored. We investigated associations of five APOA5 single-nucleotide polymorphisms (SNPs; –1131T>C, –3A>G, 56C>G IVS3+ 476G>A, and 1259T>C) with lipoprotein subfractions and CVD risk in 1,129 men and 1,262 women participating in the Framingham Heart Study. Except for the 56C>G SNP, the other SNPs were in significant linkage disequilibria, resulting in three haplotypes (11111, 22122, and 11211) representing 98% of the population. SNP analyses revealed that the –1131T>C and 56C>G SNPs were significantly associated with higher plasma TG concentrations in both men and women. For RLP and lipoprotein subclasses, we observed gender-specific association for the –1131T>C and 56C>G SNPs. Female carriers of the –1131C allele had higher RLP concentrations, whereas in males, significant associations for RLPs were observed for the 56G allele. Moreover, haplotype analyses confirmed these findings and revealed that the 22122 and 11211 haplotypes exhibited different associations with HDL cholesterol concentrations.

In women, the –1131C allele was associated with a higher hazard ratio for CVD (1.85; 95% confidence interval, 1.03–3.34; P = 0.04), in agreement with the association of this SNP with higher RLPs.

Supplementary key words apolipoprotein A-V • triglycerides • cardiovascular disease risk • haplotype • remnant-like particles


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Increased plasma triglyceride (TG), low density lipoprotein cholesterol (LDL-C), and reduced high density lipoprotein cholesterol (HDL-C) concentrations are widely accepted as cardiovascular disease (CVD) risk factors (1–3). Their plasma concentrations are regulated by a combination of genetic and nongenetic factors. Whereas much emphasis has been placed on the identification of factors modifying the cholesterol fraction of lipoproteins, less weight has been placed on elucidating modifiers of TG concentrations. LPL has been traditionally considered as the major enzyme involved in plasma TG regulation (4). However, most of the genetic variability for plasma TG concentrations in the general population still remains unexplained. Therefore, other loci need to be identified to account for the bulk of the genetic variability corresponding to this phenotype. Recently, apolipoprotein A-V (apoA-V) has emerged as a significant player in plasma TG metabolism, as shown in several experimental animal models (5–8) and in human primary cell cultures (9). The evidence from mouse models consistently supports the role of apoA-V as an activator of LPL, thus increasing lipolysis and VLDL clearance (7, 8). More recent evidence from a double-knockout mouse model supports the notion that the apoA-V (APOA5) and apoC-III (APOC3) genes independently influence plasma TG concentrations but in an opposing manner (10). However, less agreement exists regarding the potential effect of apoA-V in VLDL synthesis (7, 8). Another line of evidence for the relevance of this apolipoprotein on the regulation of TG concentrations comes from studying its common genetic variants. Several single-nucleotide polymorphisms (SNPs) within this locus (–1131T>C, –3A>G, 56C>G, IVS3+476G>A, 1259T>C, etc.) have been identified in humans (5, 11, 12), and their minor alleles have been reported to be significantly associated with increased plasma TG levels in several ethnic groups (5, 11–16).

Two independent APOA5 (–1131T>C and 56C>G) variants represented by two haplotypes have been statistically significantly associated with hypertriglyceridemia (11, 12). The frequencies of the minor alleles vary significantly among ethnic groups, with frequencies ranging from 0.1% [in Chinese (16)] to 15% [in Hispanics (11)] for the 56C>G variant and from 6% [in Caucasians (5, 12)] to 34% [in Japanese (13)] for the –1131T>C variant. In addition to the TG-increasing characteristics of APOA5 variants, they have been shown to be associated with reduced HDL-C and increased LDL-C concentrations in some Asian populations (13, 14, 16); however, the specific associations of these TG-increasing alleles on lipoprotein subclasses, particle size, and remnant-like particles (RLPs) and CVD risk have been less explored.

Given the correlation between fasting and postprandial TG-rich particles, it is conceivable that APOA5 variants may affect postprandial triglyceride-rich lipoproteins (TRLs) and lipoprotein remnant concentrations. The mechanism linking postprandial TRLs and lipoprotein remnants to the development of atherosclerosis was proposed more than 20 years ago (17). Since then, several studies have demonstrated the association between postprandial remnants and the occurrence of CVD (18). Lipoprotein remnants of both intestinal and hepatic origin have been found to be atherogenic on the basis of experiments in cell culture (19), animal models (20), and humans (18, 21, 22). However, it has been technically difficult and labor-intensive to separate, isolate, and standardize the measurements of these lipoprotein subfractions. In this regard, an assay was developed (23–25) that facilitates the immunoseparation of RLPs and the measurement of their cholesterol (RLP-C) and TG (RLP-TG) contents. Fasting plasma RLP-C and RLP-TG levels have been tightly correlated with the accumulation of TRLs during the postprandial state (26–29). RLP-C has been shown to be an independent and significant CVD risk factor (27, 29–31) as well as a risk factor for sudden death in the absence of coronary atherosclerosis (32). Indeed, APOA5 variants have been shown to increase the risk for familial combined hyperlipidemia in whites (33–35) and to affect postprandial lipemia (36, 37). Therefore, the aim of our study was to investigate the association between common APOA5 gene variants and well-characterized phenotypes related to TG metabolism (RLP-TG, RLP-C, lipoprotein subclass concentrations, and particle size), using both SNP and haplotype approaches in the Framingham Heart Study, to provide further insight about the contribution of the APOA5 locus to plasma TG metabolism and, thus, to the risk of CVD in humans.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects and study design
The design and methods of the Framingham Offspring Study (FOS) have been presented elsewhere (38). Starting in 1971, 5,124 predominantly white subjects were enrolled. Blood samples for DNA were collected between 1987 and 1991. Lipids, genotypes, coronary heart disease risk factors, and dietary intake were determined for participants who attended the fifth examination visit, conducted between 1992 and 1995 (n = 3,515). The Institutional Review Board for Human Research at Boston University and the Human Investigation Research Committee at Tufts University/New England Medical Center approved the protocol. All participants provided written informed consent, underwent standardized clinical examination, and provided fasting blood samples. Subjects taking lipid-lowering medications (n = 166) were excluded from this analysis. The present study describes the results of genotype (SNP)-phenotype association analyses performed in 1,129 men and 1,262 women (aged 28–79 years) who had APOA5 genotype data and plasma lipid determinations. Among them, 863 men and 877 women were unrelated and were included in the haplotype analyses based on the specific requirements of the analytical tools applied. The sample studied did not differ from the total population for the variables of interest. In addition, to estimate the risk of CVD associated with the APOA5 variations and to increase the statistical power, we performed survival data analyses at exam 6 (~22.5 years of follow-up). This analysis included 1,214 men and 1,364 women who were free of CVD at exam 1 and had genotype data and CVD information. At exam 6, 163 men and 91 women were diagnosed as having CVD according to the criteria and protocols established in the Framingham Heart Study. At each clinic visit, a CVD history was obtained routinely and hospitalization records were collected for subjects with suspected interim CVD events. For subjects who did not attend a clinic examination, a health history update was obtained by telephone and records from interim hospitalizations were obtained and reviewed. A diagnosis of myocardial infarction was established by a panel of three physicians when at least two of the following three criteria were simultaneously present: symptoms consistent with myocardial infarction; diagnostic electrocardiographic changes of myocardial infarction; and diagnostic increase of biomarkers.

Plasma lipid, lipoprotein, and apolipoprotein subfraction measurements
Fasting venous blood samples were collected and plasma was separated from blood cells by centrifugation and immediately used for the measurement of lipids. Plasma lipids, lipoproteins, and apolipoproteins were measured as previously described (39). Lipoprotein subclass distributions were determined by proton NMR spectroscopy as previously described (40, 41). Each profile displayed the concentrations of six VLDL, one intermediate density lipoprotein (IDL), three LDL, and five HDL subclasses and the weighted-average particle size of VLDL, LDL, and HDL. The lipoprotein subclass categories used were the following: large VLDL and remnants (80–220 nm), intermediate VLDL (35–80 nm), small VLDL (27–35 nm), large LDL (21.3–27.0 nm), intermediate LDL (19.8–21.2 nm), small LDL (18.3–19.7 nm), large HDL (8.8–13.0 nm), intermediate HDL (7.8–8.8 nm), and small HDL (7.3–7.7 nm). Levels of VLDL subclasses are expressed in units of TG (mg/dl), and those of LDL and HDL subclasses are expressed in units of cholesterol (mg/dl).

RLP analyses
RLP-C and RLP-TG concentrations were measured in plasma aliquots that were stored at –80°C. RLP isolation was based on the removal of apoA-I-containing particles and most apoB-containing particles using a well-validated immunoseparation technique (42). In brief, RLPs were separated by mixing 5 µl of plasma with 300 µl of immunoseparation gel consisting of monoclonal antibodies to apoB-100 and apoA-I. After 2 h of incubation at room temperature, cholesterol and TGs in the unbound fraction were measured by sensitive cholesterol and TG assays. Normal ranges and disease associations for these measurements have been reported previously in this population (26, 27, 43).

SNP genotyping
DNA was isolated from blood samples using DNA blood Midi kits (Qiagen, Hilden, Germany) according to the vendor's recommended protocol. Five previously reported SNPs (–1131T>C, –3A>G, 56C>G IVS3+476G>A, and 1259T>C) at APOA5 (11, 12) were analyzed in this population. Our nomenclature is in agreement with that suggested by Human Genome Variation Society (44). Genotyping was done using the ABI Prism SNapShot multiplex system (Applied Biosystems, Foster City, CA). The primers and probes used for genotyping were described previously (16). Standard laboratory practices were used to ensure the accuracy of genotype data. Internal controls and repetitive experiments were used.

Haplotype analyses
In addition to the association analysis using SNPs, we carried out haplotype analyses. First, subjects were assigned all possible haplotypes, consistent with their genotypes, and then their inferred haplotype probabilities were computed via expectation-maximization algorithm (45) [snphap (http://www-gene.cimr.cam.ac.uk/clayton/software/)].

Statistical analysis
Allele frequencies were estimated by direct counting using unrelated subjects only. The pair-wise linkage disequilibria (LDs) between SNPs at the APOA5 locus were estimated as correlation coefficient r (46) with the HelixTree program using unrelated subjects in the FOS only.

Statistical analyses were carried out using the SAS Windows version 8.2. Plasma TG, chylomicron, VLDL, RLP-TG, and RLP-C concentrations were natural log-transformed to achieve approximate normal distributions before analysis. Chi-square tests were used for comparisons of binary variables across groups, and ANOVA was used for means of continuous variables. Analysis of covariance (ANCOVA) was used to determine the association between genotypes and dependent variables, adjusting for age, body mass index (BMI), smoking, diabetic status, alcohol use, and ß blocker use as well as menopausal status and estrogen use for women. To adjust for correlated observations, because of familial relations, we used the generalized estimating equations approach as implemented in the GENMOD procedure in SAS.

ANCOVA was used to evaluate the haplotype-phenotype association. In the ANCOVA model, inferred haplotypes were used as predictors and the potential confounding factors listed above were used as covariates. The strategy used here is similar to that implemented in HAPLO.STAT (http://www.mayo.edu/hsr/people/schaid.html). Haplotype association analysis was carried out using unrelated subjects only.

A two-tailed P value of <0.05 was considered as statistical significance.

Estimation of variance attributable to APOA5 variants
The variance associated with APOA5 SNPs or haplotypes was estimated by subtracting the total correlation coefficient (r 2) based on the GLM model without the APOA5 genotype from that of the same GLM model with the APOA5 genotype in the model, adjusting for age, BMI, smoking, diabetic status, alcohol use, and ß blocker use as well as menopausal status and estrogen use for women. The estimation was calculated among unrelated subjects only and without making an assumption of additive or dominant effect of the APOA5 variants.

APOA5 variants associated with CVD risk
To investigate the CVD risk associated with APOA5 variants, survival analysis with the Cox regression model was performed for men and women, separately, and for both genders combined, after adjusting for covariates (age, BMI, smoking, diabetic status, alcohol use, and ß blocker use as well as menopause for women).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Lipid profiles and clinical characteristics of men and women in the Framingham Study
Table 1 displays means and standard deviations for the study subjects (1,129 men and 1,262 women) for the relevant biochemical data, NMR-based lipoprotein measures, and clinical characteristics grouped by gender.


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  		TABLE 1. Plasma lipids, RLPs, lipoprotein subclasses, and clinical characteristics of the Framingham Heart Study participants according to gender

 
Strong LDs within APOA5 regions in the Framingham population
Allele frequencies of five SNPs at the APOA5 locus were investigated in 1,740 unrelated subjects (863 men and 877 women) participating in the FOS. Their minor allele frequencies (Table 2) were in the range of 0.059 (56C>G) to 0.072 (1259T>C). We estimated the pair-wise LDs (see supplementary table) between these five SNPs as correlation coefficient r (46). With the exception of the 56C>G SNP, the other four SNPs were almost in complete LD, with r ranging from 0.873 to 1. However, SNP 56C>G is independent of the other four SNPs.


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  		TABLE 2. Minor allele frequencies and SEM (in parentheses) of five SNPs at APOA5 in unrelated subjects of the Framingham Heart Study

 
Two independent APOA5 variants are strongly associated with lipid and RLP concentrations
To determine if APOA5 variants were associated with lipid and RLP concentrations, statistical analysis was carried out with all unrelated and related subjects (1,129 men and 1,262 women) after adjusting for familial relationships. In this white population, the frequencies of the minor alleles were relatively low; therefore, we combined heterozygotes (i.e., 12) and homozygotes (i.e., 22) of the minor allele (2) to increase the statistical power. We first tested for associations between individual SNPs of all five common polymorphisms at the APOA5 locus and plasma lipid levels as dependent variables. We examined genotype-phenotype associations for men and women separately as well as combined. Because the four SNPs (–1131T>C, –3A>G, IVS3+476G>A, and 1259T>C) were in very strong LD and showed similar patterns of association with dependent variables, only the results of one representative SNP (–1131T>C) are given in Table 3. The patterns of association for the independent 56C>G SNP are described in Table 4.


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  		TABLE 3. Plasma lipids, RLPs, and lipoprotein subclasses according to the –1131T>C polymorphism in the Framingham Heart Study

 

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  		TABLE 4. Plasma lipids, RLPs, and lipoprotein subclasses according to the 56C>G polymorphism in the Framingham Heart Study

 
Table 3 displays significant associations between the –1131T>C SNP and plasma total cholesterol and TG concentrations for men (P = 0.003 and P = 0.001), women (P = 0.024 and P = 0.005), and both genders combined (P < 0.001 and P < 0.001). In addition, we observed significant associations with LDL-C concentrations in men (P = 0.046) and both genders combined (P = 0.020) but not in women alone (P = 0.244). Consistent with LDL-C concentration, we observed that both male and female carriers of the –1131C allele have significantly higher apoB concentrations (P = 0.019 and P = 0.012, respectively). Most interestingly, these results reveal significant associations between the –1131T>C SNP and concentrations of RLP (RLP-C and RLP-TG) and intermediate VLDL (VLDLINT) for women (P = 0.025, 0.018, and 0.032 for RPL-C, RPL-TG, and VLDLINT, respectively) and both genders combined (P = 0.003, P = 0.029, and P < 0.001, respectively). For men, the association was marginally significant (P = 0.057) with RLP-C, highly significant (P < 0.001) with VLDLINT, and not significant (P = 0.314) but in the same trend with RPL-TG. These significant associations were even stronger for the other three SNPs (–3A>G, IVS3+ 476G>A, and 1259T>C; data not shown), which were in almost complete LD with –1131T>C. Furthermore, the minor allele carriers of –1131T>C had smaller LDL size (P = 0.045) compared with CC homozygotes when both genders were combined. Similar trends, particularly in men, were observed for the other three SNPs (–3A>G, IVS3+ 476G>A, and 1259T>C; data not shown). The –1131T> C SNP showed significant association with apoA-I concentration when both genders were combined (P = 0.044). However, this SNP displayed no significant associations with HDL-C, chylomicron, IDL, large and small VLDL concentrations, and HDL size. Overall, the covariates included in our statistical model accounted for 11.7% and 27.1% of the TG variance in men and women, respectively, whereas the –1131T>C SNP accounted for 0.78% and 0.51% of the variance in men and women, respectively. Likewise, for LDL size the covariates in the model accounted for 3.3% and 9.1% of the variance in this trait in men and women, whereas this SNP accounted for 0.4% and 0.2% of the variance in men and women, respectively.

Table 4 displays associations between the 56C>G SNP and plasma lipids. Similar to the –1131T>C SNP, the 56C>G SNP revealed significant associations with TG concentrations for men (P = 0.002), women (P = 0.006), and both genders combined (P < 0.001) and with RLP-C and RLP-TG concentrations only in men (P = 0.036 and P = 0.020) and both genders combined (P = 0.027 and P = 0.004). In addition, this variant was also significantly associated with intermediate VLDL concentrations in men (P = 0.012) and women (P = 0.005) and with large VLDL concentrations in men (P = 0.002) only. The 56C>G variant displayed no significant associations with chylomicron, IDL, and large and small VLDL concentrations. Unlike the –1131T>C SNP, the 56C>G SNP was significantly associated with lower HDL-C concentrations in women (P = 0.019) but not in men (P = 0.144). Finally, unlike the –1131T>C SNP, the 56C>G SNP was not significantly associated with LDL-C, total cholesterol, apoA-I, and apoB concentrations. The genotype-based variance attributed to this SNP for TG concentrations was similar to that explained by the –1131T>C SNP (0.78% and 0.45% for men and women, respectively). A much greater contribution was observed for RLP-TG variance in men, amounting to 1.2%, whereas the model using all of the covariates explained only 8.6% of the variance.

APOA5 haplotypes and plasma lipid and lipoprotein subclass concentrations
To understand the combined effect of all five APOA5 SNPs, we constructed haplotypes with the five SNPs using the expectation-maximization algorithm (45). Only 1,740 unrelated subjects (863 men and 877 women) were used for the haplotype analyses. We identified three major haplotypes: 11111, 22122, and 11211 (the five SNPs were ordered from 5' to 3') with frequencies greater than 0.01. The most common haplotype, 11111, represented 86.3% of all haplotypes; the other two haplotypes, 22122 and 11211, accounted for 6.2% and 5.7%, respectively. In total, these three haplotypes accounted for 98.2% of all haplotypes in this population.

We next investigated the overall association between common haplotypes and plasma lipid variables. We first conducted the analyses separately by sex and adjusting for age, BMI, smoking, diabetic status, alcohol use, and ß blocker use as well as menopause for females, and then by combining both genders (data not shown). The effect associated with haplotypes for which the associations were highly significant is shown in Fig. 1. Our analysis shows that both TG-increasing haplotypes (22122 and 11211) have strong and significant effects associated with increased TG and intermediate VLDL levels in men and both genders combined, but to a lesser extent in women. Interestingly, we found both TG-increasing haplotypes to be associated with reduction in LDL size predominantly in men. This effect was not observed in women.



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  		Fig. 1. Mean effects and standard errors (bars) for each haplotype on lipid, lipoprotein subclasses, and remnant-like particles (RLPs). Single-nucleotide polymorphisms were arranged in the haplotype descriptions from 5' to 3' as follows: –1131T>C, –3A>G, 56C>G, IVS3+476G>A, and 1259T>C. Haplotype effects were log-transformed and normalized to the effect of the 11111 haplotype (reference haplotype); only the effects for haplotypes 11211 and 22122 are presented. Haplotype effects were estimated by analysis of covariance with SAS, a strategy similar to that used in HAPLO.STAT (http://www.mayo.edu/hsr/people/schaid.html), and adjusted for age, BMI, smoking, diabetic status, alcohol use, and ß blocker use as well as menopausal status and estrogen use for women. Open, shaded, and closed bars represent the effects for men, women, and both genders combined, respectively. Asterisks indicate a significant effect at P < 0.05. HDL-C, high density lipoprotein cholesterol concentration; LDL-SIZE, LDL diameter; RLP-TG, RLP triglyceride; TG, triglyceride; VLDLINT, intermediate low density lipoprotein.

 
The associations between haplotypes and HDL-C concentration and RLPs (RLP-C and RLP-TG) indicate interesting differences between haplotypes. Although both the 11211 and 22122 haplotypes had similar effects on TG and intermediate VLDL levels, they differed in their associations with HDL-C concentrations. Haplotype 11211 was significantly associated with lower HDL-C levels, especially in women, whereas the 22122 haplotype had little effect on HDL-C concentrations, indicating that carriers of the 11211 haplotype have significantly lower HDL-C levels compared with those who carry the 22122 haplotype; both haplotypes were consistently associated with higher TG levels. Moreover, this HDL-C effect was independent of TG, as the APOA5 locus remained significantly associated with HDL-C levels after adjusting for TG levels. Furthermore, the 11211 haplotype had a stronger influence on increased levels of RLPs (RLP-C and RLP-TG) than the 22122 haplotype in men, but this difference was less obvious in women.

Compared with the variation explained by each of the SNPs, the haplotype resulted in a dramatic increase in the variance explained by the APOA5 locus, much greater than that expected from the slight decrease in the number of cases included in the haplotype analyses. For males, haplotype explained 2.9, 1.2, 1.9, 3.6, and 1.6% of the variance for TG, HDL-C, RLP-TG, VLDLINT, and LDL size, respectively. For women, haplotype explained a lesser amount of the variance, 1.2, 1.1, 1.1, 1.1, and 0.2% for TG, HDL-C, RLP-TG, VLDLINT, and LDL size, respectively.

APOA5 variants associated with CVD risk
To investigate the CVD risk associated with APOA5 variants, survival analysis with the Cox regression model was performed for men and women, separately, and both genders combined, after adjusting for covariates (age, BMI, smoking, diabetic status, alcohol use, and ß blocker use as well as menopausal status and estrogen use for women). Hazard ratios for CVD risk associated with the minor allele of each SNP are given in Table 5. The highest risk for CVD was observed in the female carriers of the –1131T>C minor allele, which was associated with an almost 2-fold increased likelihood of the risk (1.85; 95% confidence interval, 1.23–3.34; P = 0.040). Further adjustment for total cholesterol, HDL, and TG concentrations did not substantially modify the observed risk (hazard ratio = 1.90; 95% confidence interval, 1.26–3.26; P = 0.034). The other SNPs in strong LD with the –1131T>C SNP showed similar trends but did not reach statistical significance in women. In men, despite following a similar trend, none of the SNPs reached a statistically significant increase in CVD risk.


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  		TABLE 5. HR for cardiovascular disease associated with the minor allele of each SNP at APOA5

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our results demonstrate that common variants at the APOA5 locus are significantly associated with increased plasma TG concentrations in the Framingham Heart Study, consistent with our previous report in Asians (16) and the findings of others in several other populations (5, 11–15, 33–36). More importantly, the availability of lipoprotein subfractions and RLP data in the Framingham population provides us with a better understanding of the potential association of APOA5 variants with CVD risk. Our results reveal several important findings related to these APOA5 variants. First, we have demonstrated that APOA5 haplotypes were significantly associated with fasting RLP concentrations (i.e., RLP-C and RLP-TG) and LDL size. In addition, our results reveal that APOA5 haplotypes are significantly associated with VLDL and HDL-C concentrations. Second, we estimated the effect of each haplotype on these lipid-related variables. Third, we have demonstrated that the two APOA5 haplotypes exhibit different effects on HDL-C and RLP concentrations. Fourth, we have shown that variability at this locus is associated with increased risk for CVD in women in the Framingham Heart Study. Overall, our results demonstrate that APOA5 is a major locus involved in both TG metabolism and CVD risk; the latter is likely manifested by undetermined mechanisms independent of the traditional lipid risk factors. The fact that we carried out a large number of statistical comparisons raises the issue of whether to adjust for multiple testing. In this case, we did not adjust for multiple testing based on the arguments proposed by several investigators (35, 47). Namely, we studied highly correlated phenotypes and used marker alleles that were interdependent. Moreover, type I errors are random (47); therefore, patterns in results, such as those reported here, which also confirm previous reports, should be given more weight than isolated results with a single low P value. In addition, it is known that the correction for multiple comparisons largely increases the likelihood of type II errors, so that truly important differences are considered nonsignificant.

High concentrations of remnant lipoproteins are considered to be risk factors for CVD (17, 18). The measurements of RLP-C and RLP-TG have been shown to serve as surrogates for postprandial lipoprotein remnants and to be associated with CVD (27, 29–31) and sudden death in the absence of coronary atherosclerosis (32). In our population, RPL has been previously demonstrated to be an independent risk factor for CVD in women (27). In the current study, we demonstrated that APOA5 individual SNPs and haplotypes have significant associations with RLP-C and RLP-TG concentrations. The increase in RLP and intermediate VLDL concentrations suggests that the clearance of postprandial remnants may be delayed in carriers of the haplotype 22122 and 11211, suggesting an increased risk for CVD. This is in good agreement with experimental data from one of our feeding studies (37) and from population studies that have shown an association between the –1131T>C and S19W (56C>G) rare alleles and hypertriglyceridemia (48); however, in the latter study, none of the variants influenced responsiveness to the oral fat tolerance test after correcting for baseline TG. Conversely, in another study, no association was observed between these SNPs and the postprandial lipid response (36).

Haplotype frequency estimation reveals three common haplotypes (11111, 22122, and 11211) that account for 98% of all haplotypes at the APOA5 locus in this population sample. The 22122 and 11211 haplotypes are two independent haplotypes, with the former representing the four SNPs that are strong LD, and the latter representing the 56C>G SNP. The estimated percentage of carriers with haplotypes 22122 and 11211 are 11.4% and 10.9%, respectively. The total percentage carrier of either one or both haplotypes is ~21%, thus posing significant potential risk in this population. These results are in agreement with previous studies in white populations, although the frequencies of these two haplotypes in our population are slightly lower than those reported previously (11, 12). However, the frequency of the 11211 haplotype represented by the G allele of SNP 56C>G in the Framingham population is much higher than that observed in our study (16) in Chinese, Malays, and Indians, with frequencies of <1, 1.4, and 3.2%, respectively. In addition, the amount of variance attributable to the APOA5 locus in this population is much less than that estimated in Singaporean populations (16), in which the APOA5 locus explained 6.9% of the TG variance in Malays, 5.2% in Asian Indians, and 2.7% in Chinese. The most likely reason for these differences between studies is that the frequencies of the high-TG-associated variants, particularly the –1131T>C (SNP or haplotypes), are much lower in whites (~0.07) than in Chinese (0.29), Malays (0.30), and Asian Indians (0.23) (16).

In our study, both haplotypes 22122 and 11211 were significantly associated with increases in TRL and had similar frequencies (6.2% and 5.7%). However, these haplotypes exhibit different associations with HDL-C concentration. Haplotype 22122 has little influence on HDL-C concentrations, whereas haplotype 11211 displays significant associations with low HDL-C levels. Interestingly, the association of the 11211 haplotype with HDL-C was independent of its association with increased TG levels. In addition, this haplotype has a stronger effect on the increased levels of RLP than the 22122 haplotype in men. Therefore, these results suggest that each of these two haplotypes may be associated with different mechanisms enhancing atherogenic risk.

ApoA5 is expressed in liver and is found in plasma associated with large HDL particles (6). Pennacchio et al. (5) reported that the human APOA5 (hAPOA5) transgenic mouse has significantly decreased TG and VLDL concentrations, whereas the APOA5 knockout exhibited the opposite phenotype compared with wild-type mice (5). If we assume that the APOA5 variants are associated with a loss of function, the transgenic data are consistent with the finding in humans showing that the minor alleles at the APOA5 locus were significantly associated with increases in plasma TG and VLDL concentrations. The specific mechanism by which these genetic variants may affect the function of the apoA-V protein remains to be elucidated. It is enticing to suggest that the –1131T>C and –3A>G variants contained in haplotype 22122 could alter the expression of APOA5, leading to the increase in TG and VLDL concentrations. In this regard, the transcription start site and the promoter of the human apoA-V gene have been characterized (49) and two response elements have been identified and localized. Thus, bile acids and the farnesoid X-activated receptor induced the APOA5 gene promoter activity through an element localized at positions –103/–84. In addition, the peroxisome proliferator-activated receptor {alpha} also specifically enhanced APOA5 promoter activity, and its response element was localized 271 bp upstream of the transcription start site. Therefore, no potential transcriptional binding site has been identified at or near the –1131T>C polymorphism. Likewise, the –3A>G SNP is 3 bp upstream of the predicted start codon of APOA5, and the base change at this position could potentially reduce the rate of apoA-V translation. However, no functional studies have been reported in support of these hypotheses. Finally, the haplotype 22122 may be in strong LD with a functional mutation in the APOA1/C3/A4 cluster. In fact, the –1131T>C SNP has been shown to be in strong LD with the APOC3 –482T>C SNP (12), which was proposed to abolish the insulin-dependent downregulation of APOC3 gene transcription (50). In fact, one study presented preliminary evidence for allele-specific differences in APOC3 mRNA expression in vivo and suggested that such differences may contribute to the observed associations with hypertriglyceridemia (51). However, other in vitro experiments discount this site as being a crucial player in the regulation of expression of the APOC3 gene (52). Therefore, we cannot dismiss the possibility that this haplotype could be in strong LD with other unknown functional mutations within the APOA1/C3/A4 cluster.

The haplotype 11211 defined by the minor allele of the 56C>G SNP results in a nonsynonymous change at position 16 of apoA-V from serine (Ser) to tryptophan (Trp), which may potentially change the structure and function of apoA-V. We investigated this possibility using bioinformatics tools. Analysis of both versions of apoA-V (Ser-16 and Trp-16) by three different secondary structure prediction algorithms [JPRED (53), PHD (54), and PSIPRED (55)] produced a remarkable consensus. In all cases, the presence of Trp increased the likelihood that residues from positions 14–16 form an {alpha} helix, whereas the presence of Ser favors a break in the helix or a turn (56). This agrees with widespread observations that Ser breaks the {alpha} helix through its ability to hydrogen-bond to the carbonyl group of residue n-3. Signal peptide prediction indicates with high confidence that cleavage occurs between residues Ala-20 and Arg-21 [SignalP 3.0 (57)]. Therefore, we propose that the 56C>G allele causes a change in the secondary structure of apoA-V, with a concomitant change in tertiary structure, by lengthening the initial {alpha} helix segment by increasing the tendency of residues 14–16 to adopt an {alpha} helix conformation. Thus, the structure alteration could potentially lead to the malfunction of apoA-V in lipid metabolism, likely via altered efficiency of either insertion of the nascent polypeptide chain into the endoplasmic reticulum lumen or cleavage of the signal peptide, or by altered lipid affinity.

We found that the female carriers of the –1131T>C minor allele have an almost 2-fold increased risk for CVD. Moreover, the increased CVD risk in female carriers remains even after adjusting for total cholesterol, HDL-C, and TG concentrations. The APOA5 gene is distal to the APOA1/C3/A4 cluster, the four genes being tightly linked within a region spanning ~60 kb on the long arm of chromosome 11 (58–60), and it has been clearly demonstrated that genetic variants within this cluster are in strong linkage LD with each other. This study supports the presence of strong LD between four SNPs within the APOA5 locus. Reports from other white populations (5, 11, 12) also suggest strong LD within this cluster. Therefore, the influence of the APOA5 locus on plasma lipid levels and CVD could be confounded by the neighboring loci. Thus, further studies should focus on the construction of haplotypes with variants from other loci within this cluster and determine the overall haplotype association with lipid measurements and other atherogenic risk predictors. Furthermore, possible interaction between diet and genotypes will be explored in future studies.


   ACKNOWLEDGMENTS
 
This study was supported by grants from the American Heart Association (0335432T), the U.S. Department of Agriculture/Foreign Agricultural Service/International Cooperation and Development Research and Scientific Exchanges Division/Scientific Cooperation Research Program, and National Institutes of Health/National Heart, Lung, and Blood Institute Grant HL-54776, by contracts 53-K06-5-10 and 58-1950-9-001 from the U.S. Department of Agriculture Research Service, and by Grant PR2004-0054 (to D.C.) from the Ministerio de Educación y Cultura, Spain.

Manuscript received May 25, 2004 and in revised form August 10, 2004. and in re-revised form August 19, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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