In this study, we evaluated statistical associations between blood lipid levels and candidate genes involved in a number of biological pathways, such as nutrient metabolism, immune response and inflammation, oxidative stress, and homeostasis. To our knowledge, there is only one other study (i.e., Keebler et al.) [40
] published that describes genetic associations with blood lipid levels using a nationally representative sample of the U.S. population. This study also used data from the NHANES III survey, but associations were examined at 19 genome-wide validated loci on fasting and nonfasting samples. Those data were not available for our use while the present study was being conducted. We examined a different set of polymorphisms which had been identified previously through candidate gene association studies. In our analyses, we used only fasting samples in accordance with guidelines from The National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III, ATPIII) [34
Our findings suggest, before and after adjustment for numerous demographic and behavioral characteristics in one or more race/ethnic groups, that blood lipid levels differ by an increasing number of minor alleles of polymorphisms in APOE, ITGB3, NOS3, and PON1. Our results also show that the A-G haplotype of ADRB2 was associated with elevated HDL-C among Mexican Americans. However, these results from crude and adjusted models are unstable (wide confidence intervals) and would need more data collected to support the association. We found that the group of rare haplotypes (frequency <1%) within MTHFR and TNF were associated with several blood lipids across race/ethnic groups; but, we are unable to identify which rare haplotype(s) contribute to these findings. Consequently, we cannot interpret these associations.
In analyses of individual variants and of haplotypes, we found strong statistical associations between genetic variation in APOE
and LDL-C and TC levels in non-Hispanic blacks and Mexican Americans. APOE
, one of the most studied genes in risk assessment of cardiovascular disease, plays a key role in the metabolism of cholesterol and triglycerides by binding to receptors on the liver and helping to mediate the clearance of chylomicrons and very low-density lipoproteins from the bloodstream [41
]. Allelic variation in APOE
has been associated consistently with plasma concentrations of total cholesterol and LDL cholesterol [42
], and with protein levels of APOB (the major protein of LDL, VLDL, and chylomicrons).
Our findings suggested an association of the NOS3
rs1799983 variant and T-T haplotype with LDL-C in Mexican Americans. NOS3 serves as a key enzyme of the endogenous nitrovasodilator system, which is essential for the regulation of vascular function and blood pressure, through the production of nitric oxide. The Glu298Asp variant (rs1799983) has been significantly associated with higher plasma LDL cholesterol, LDL particle size, and lower plasma HDL cholesterol; but no significant associations were found with the T-786C variant [48
]. Numerous studies have also reported a positive association with the Glu298Asp variant and haplotypes containing this variant with higher triglycerides and LDL cholesterol in Venezuelans [49
] and Greeks [50
We found higher HDL-C among Mexican American carriers of the PON1 rs854560 (Leu55Met) variant and A-A and A-G haplotypes. Conversely, we found higher LDL-C in non-Hispanic white carriers of the A-G haplotype. PON1
is an HDL-associated esterase that hydrolyzes products of lipid peroxidation and prevents the oxidation of HDL and LDL. In fact, the antioxidant activity and anti-atherogenic effect of HDL is thought to be largely because of the paraoxonase located on the HDL particle. Variants in PON1
previously have been associated with serum HDL and LDL cholesterol levels [51
], and with increased risk for stroke [53
]. There have been multiple studies and meta-analyses evaluating the association of PON1
variants with blood lipids in several populations or community-based samples, but with inconsistent results [51
Our results suggest a strong association of ITGB3
with triglycerides in non-Hispanic blacks. ITGB3 is a membrane receptor for fibrinogen and von Willebrand factor that has an important role in platelet aggregation. The Pro33 allele (rs5918) has been associated with coronary thrombosis [62
] and stroke [64
]. A previous study examined associations between 15 single nucleotide polymorphisms across ITGB3
and cardiovascular disease-related traits in the Hutterites (e.g., plasma levels of HDL and LDL cholesterol and triglycerides) and suggested that ITGB3
has sex-specific associations with plasma lipoprotein(a) [66
Although we did not assess racial/ethnic difference in the genetic effects, we observed that two associations, both involving the rs7412 variant in APOE
, were significant in two racial/ethnic groups. No variants were significant across all three racial/ethnic groups after the FDR adjustment. Limited power and statistical chance may explain, at least, in part, the lack of consistent findings across the three race/ethnicities. Alternatively, these differences may be caused by varying linkage disequilibrium patterns at causal loci across different race/ethnic populations or by gene-environment interactions that have not been identified or measured. As a result, it might not be unusual to find varying risks for a disease or trait at a given genomic locus across population subgroups. In agreement, a recent study examined 12 newly discovered genetic variants known to predict lipid levels in Europeans and also evaluated local ancestry at validated genes that influence lipid levels [67
]. This study found genetic differences between the determinants of lipid phenotypes across different African and European populations. Such findings might suggest that many of the truly causal variants in different race/ethnic groups have yet to be discovered, as most genetic epidemiology studies have been performed in populations of European descent.
Although we identified associations of APOE, ITGB3, NOS3, and PON1 with blood lipid levels by examining polymorphisms individually, our results suggest that assessing genetic variation using haplotype methods might be more comprehensive and more informative. We found that although a single genetic variant might have a small (if any) effect in identifying a susceptibility locus for an outcome, the effect might reach statistical significance when combined with other variants within the gene. For example, after adding a single variant (APOE rs7412) to a regression model containing non-genetic risk factors, we were able to explain only slightly more variation in LDL-C (R2 = 0.1448 for non-Hispanic white persons, 0.2065 for non-Hispanic black persons, and 0.1462 for Mexican American persons) compared to the variation explained by non-genetic risk factors alone (R2 = 0.1163, 0.1533, and 0.1230, respectively). However, we observed that a larger proportion of the variation in LDL-C is explained by the model that contains the APOE T-T haplotype compared to the model containing the rs7412 variant alone (R2 = 0.1521, 0.2073, and 0.1636, respectively). Overall, the variance in blood lipid levels explained by the contribution of each individual variant or haplotype is considerably small (<5%; data not shown).
The present study has many notable strengths. First, the study was conducted using a large population-based and nationally representative survey of the United States. The wealth of data in NHANES facilitated the examination of genetic, environmental, and clinical data for each of the three major race/ethnicities in the United States. Moreover, whereas many previous reports were limited to a single population or were based on smaller study populations, we were able to conduct the analyses separately in each race/ethnicity, and were therefore able to account for the differences in allele frequencies, disease prevalence, and linkage disequilibrium patterns between these subpopulations. Finally, the control of hypercholesterolemia is an important clinical and public health objective. Awareness of, and screening for, hypercholesterolemia have become more common in recent years. Accordingly, treatment of the condition has increased since the initiation of the National Cholesterol Education Program in 1985. The use of cholesterol-lowering medications has increased steadily in U.S. adults aged ≥ 20 years, from 8.2% in 1999-2000 to 14.0% in 2005-2006, as measured in NHANES [68
]. Among those diagnosed with hypercholesterolemia, the proportion on treatment increased from 32.4% to 38.9% in the 8-year period from 1999 to 2006 [69
]. Association analyses of genetic variants involved in influencing blood lipid levels may therefore be complicated by a high prevalence of study participants who take lipid-lowering drugs. An advantage of this study in NHANES III is that a small number of participants taking such medication needed to be excluded (n = 75; 3% of fasting samples). Evaluation of such genetic associations in subsequent NHANES surveys will result in a loss of a higher number of participants in the analyses.
In addition to these strengths, we acknowledge several limitations. To help reduce the chance of potential false-positive results from multiple testing, we adjusted p-values to control the false discovery rate [39
]. This method assumes that the set of tests are independent. Yet, we know that many of the test statistics might be correlated because of linkage disequilibrium between genetic variants [70
]. The FDR adjustment, therefore, might result in overly conservative p-values, thus decreasing our ability to identify true associations.
Although we stratified the analysis by race/ethnicity, we cannot eliminate completely the possibility of confounding of our study results by population stratification. We were not able to assess population structure in our analysis and grouped participants by broad categories on the basis of self-reported race and ethnicity. Substantial admixture in the African American and Hispanic populations has been documented [71
]. However, previous research conducted on the U.S. population has found little evidence for population substructure in whites [75
Although the NHANES III data may be more representative of the U.S. population than other non-population-based samples, the statistical power to detect genetic associations was limited in this study. For example, we determined the beta-coefficients that correspond to the genetic variant explaining 1% of the variation in LDL-C. The beta-coefficients ranged from 5.2 to 26.3 depending on the frequency of the minor allele (MAF = 0.01 to 0.5). Using these beta-coefficients and corresponding allele frequencies described above for LDL-C, we found that our power would be 42-82% for non-Hispanic whites, 24-66% for non-Hispanic blacks, and 27-70% for Mexican Americans (Additional file 1
, Table S7).