Association of Blood Lipids With Common DNA Sequence Variants at 19 Genetic Loci in the Multiethnic United States National Health and Nutrition Examination Survey IIICLINICAL PERSPECTIVE
Background— Using the genome-wide association approach in individuals of European ancestry, we and others recently identified single-nucleotide polymorphisms (SNPs) at 19 loci as associated with blood lipids; 8 of these loci were novel. Whether these same SNPs associate with lipids in a broader range of ethnicities is unknown.
Methods and Results— We genotyped index SNPs at 19 loci in the Third United States National Health and Nutrition Examination Survey (n=7159), a population-based probability sample of the United States comprised primarily of non-Hispanic blacks, Mexican Americans, and non-Hispanic whites. We constructed ethnic-specific residual blood lipid levels after adjusting for age and gender. Ethnic-specific linear regression was used to test the association of genotype with blood lipids. To summarize the statistical evidence across 3 racial groups, we conducted a fixed-effects variance-weighted meta-analysis. After exclusions, there were 1627 non-Hispanic blacks, 1659 Mexican Americans, and 2230 non-Hispanic whites. At 5 loci (1p13 near CELSR2/PSRC1/SORT1, HMGCR, CETP, LPL, and APOA5), the index SNP was associated with low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, or triglycerides in all 3 ethnic groups. At the remaining loci, there was mixed evidence by ethnic group. In meta-analysis, we found that, at 14 of the 19 loci, SNPs exceeded a nominal P<0.05.
Conclusions— At 5 loci including the recently discovered region on 1p13 near CELSR2/PSRC1/SORT1, the same SNP discovered in whites associates with blood lipids in non-Hispanic blacks and Mexican Americans. For the remaining loci, fine mapping and resequencing will be required to definitively evaluate the relevance of each locus in individuals of African and Hispanic ancestries.
Received October 16, 2008; accepted February 13, 2009.
Genome-wide association (GWA) studies in subjects of European ancestry have successfully identified genetic loci associated with plasma level of low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglycerides. We and others recently identified single-nucleotide polymorphisms (SNPs) at 19 loci as associated with blood lipids; 8 of these loci were novel.1–6 However, the relevance of these variants in the diverse US population is unclear.
Clinical Perspective on p 238
When investigating these loci in diverse ethnic populations such as black and Hispanic groups, 3 potential outcomes are possible. First, association of the same polymorphism at the same locus may be confirmed. Second, a different polymorphism at the same locus may be associated in differing ethnicities. Finally, loci identified in cohorts of European ancestry may not replicate in individuals of non-European ancestry, suggesting that unique loci in the genome are responsible for blood lipid traits in groups of differing ancestries. Here, we test the first of these possibilities-the hypothesis that the index SNP at each of 19 loci validated in whites will also be associated with blood lipid traits in individuals of black and Hispanic ancestry. We address this hypothesis in the United States Third National Health and Nutrition Examination Survey (NHANES III), a multiethnic representative sample of noninstitutionalized persons in the United States.
The NHANES III cohort and DNA sample collection have been previously described.7 Briefly, NHANES III is a representative, cross-sectional sample of the noninstitutionalized US population surveyed between 1988 and 1994 by the National Center for Health Statistics at the Center for Disease Control and Prevention. Minority groups (non-Hispanic blacks and Mexican Americans), children, and the elderly were oversampled. The survey consisted of a household interview, including a detailed dietary questionnaire. In addition, a physical examination was performed, and blood samples were collected. For the genetic component of NHANES III, DNA was isolated from cell lines created from human blood samples from participants of NHANES III phase 2 (1991 to 1994). The total number of participants in NHANES III phase 2 was 16 530. DNA was available from 7157 of these individuals aged 12 and older.7 The present study was approved by the Centers for Disease Control/National Center for Health Statistics institutional review board and the institutional review board at the Massachusetts Institutes of Technology, and all subjects provided written informed consent.
HDL-C and triglycerides were measured using standard enzymatic methods. LDL-C was calculated using the Friedewald formula with assignment of a missing value for participants with triglycerides exceeding 400 mg/dL. Fasting blood samples were available in 41% of individuals (n=2916), and the remainder were nonfasting. We included both fasting and nonfasting samples in our analyses to increase statistical power.
SNP Selection and Genotyping
We selected 21 SNPs from 19 loci that exceeded genome-wide statistical significance (P<5×10−8) in 2 recently published GWA studies for LDL-C, HDL-C, and triglycerides.2,3 Eight of these loci had been newly identified. SNPs at the new loci included the following: rs3794991 (near CILP2/PBX4, LDL-C and triglycerides); rs646776 (near CELSR2/PSRC1/SORT1, LDL-C); rs4846914 (in GALNT2, HDL-C and triglycerides); rs10774708 (in MMAB/MVK, HDL-C); rs17145738 (near BCL7B/TBL2/MLXIPL, triglycerides); rs17321515 (near TRIB1, triglycerides); rs1748195 (ANGPTL3/DOCK7/ATG4C, triglycerides); and rs1260326 (GCKR, triglycerides). SNPs at the 11 additional loci included the following: rs6511720 and rs1529729 (LDLR, LDL-C); rs11591147 (PCSK9, LDL-C); rs12654264 (HMGCR, LDL-C); rs4420638 (APOE-C1-C4-C2 cluster, LDL-C); rs693 (APOB, LDL-C); rs2156552 (LIPG, HDL-C); rs1800588 (LIPC, HDL-C); rs1800775 (CETP, HDL-C); rs328 (LPL, HDL-C, triglycerides); rs3890182 (ABCA1, HDL-C); and rs3135506 and rs662799 (APOA5, HDL-C and triglycerides).8–16 rs6511720 and rs1529729 have a low degree of linkage disequilibrium within the LDLR gene in populations of European (r2=0.085) and African (r2=0.167) ancestry. rs3135506 and rs662799 tag 2 known haplotypes within the APOA5 gene.17
Genotyping was performed on the Sequenom platform, which uses matrix-assisted laser-desorption ionization time-of-flight mass spectroscopy as described previously.18 Using 356 samples in duplicate, we found the genotyping error to be 0.34%. SNP rs693 in the APOB gene was excluded from analysis due to a low genotyping call rate (88%). SNP rs10774708 near the MMAB/MVK genes was excluded from analysis due to a low genotyping call rate (70%). In sum, 19 SNPs from 17 loci were successfully genotyped and the genotyping success rate for each of these 19 SNPs exceeded 92%.
All statistical analyses were performed at the National Center for Health Statistics at the Center for Disease Control and Prevention and conducted using SAS software version 9.0 (Cary, NC). Participants in NHANES III who were missing phenotype data, those younger than 18 years, and those on lipid-lowering therapy were excluded from analysis. LDL-C, HDL-C, and triglyceride values were log transformed.
Ethnic-specific residual lipid levels were generated after adjusting for age and gender. Linear regression stratified by self-reported race/ethnicity was used to test the association of residual values with genotype, assuming an additive model of inheritance. To summarize the statistical evidence across the 3 race/ethnic groups, we conducted a fixed-effects variance-weighted meta-analysis. We computed a weighted average of the β-coefficient estimates and SEs (from the linear regression models earlier) for each genotype, using the inverse of the variance in each group as weights.
We denote significance to be an association P≤0.05 for the same allele at the same SNP for the same trait as reported in the original discovery GWA studies.1–3 The authors had full access to the data and take responsibility for its integrity. All authors have read and agree to the manuscript as written.
Participant characteristics are summarized in Table 1. After exclusions, there were 1692 non-Hispanic blacks, 1715 Mexican Americans, and 2295 non-Hispanic whites with genotype and phenotype data available for analysis.
Associations of genotypes with LDL-C concentrations are detailed in Table 2. The association between rs646776 (at 1p13 near CELSR2/PSRC1/SORT1) and LDL-C was highly significant in all 3 ethnic groups (P=0.0005 in non-Hispanic blacks, P=1.8×10−6 in Mexican Americans, and P=0.002 in non-Hispanic whites). The effects of rs646776 on LDL-C are summarized in the Figure 1. In each ethnic group, individuals with at least 1 copy of the minor G allele had lower LDL-C. Each copy of the minor allele lowered LDL-C by ≈5 mg/dL in non-Hispanic blacks, ≈11 mg/dL in Mexican Americans, and ≈4 mg/dL in non-Hispanic whites.
In addition to 1p13, HMGCR rs12654264 was associated with LDL-C in non-Hispanic blacks (P=0.006), Mexican Americans (P=0.01), and non-Hispanic whites (P=0.05). As in the original discovery studies, the minor T allele was associated with higher LDL-C in each of the ethnic groups from NHANES III.2,18
SNPs at 3 loci (TRIB1, LDLR, and CILP2/PBX4) were significantly associated with LDL-C in at least 1 nonwhite ethnic group. TRIB1 rs17321515 and LDLR rs1529729 were significantly associated with LDL-C (P=0.0004 and P=0.02, respectively) in Mexican Americans. CILP2/PBX4 rs3794991 and LDLR rs6511720 were associated with LDL-C in non-Hispanic blacks (P=0.04 and P=8.6×10−5, respectively).
At PCSK9, rs11591147 (or R46L) failed to reach statistical significance in both non-Hispanic blacks or Mexican Americans but was highly significant in non-Hispanic whites (P=0.0002).
The results for HDL-C are summarized in Table 3. SNPs at CETP and LPL were strongly associated with HDL-C in all 3 ethnic groups. Each increase in minor C allele copy number in CETP polymorphism rs1800775 resulted in a 0.05 SD unit decrease in HDL-C across all ethnicities (P=5.0×10−20). LPL rs328 was associated with an increase of 0.04 SD unit in HDL-C per allele (P=5×10−8).
At 1 locus, GALNT2, the same SNP discovered in whites was significantly associated with HDL-C in at least 1 nonwhite ethnic group. SNP rs4846914 at GALNT2 was associated with HDL-C in Mexican Americans (P=0.02) but not in non-Hispanic blacks. SNPs at the remaining loci failed to replicate in either nonwhite ethnic groups.
The results for triglycerides are summarized in Table 4. At APOA5, rs3135506 was associated with triglyceride concentrations across all ethnicities. In each ethnicity, increase in minor allele copy number resulted in a ≈0.12 SD increase in triglyceride level (P=4.3×10−11).
SNPs at 3 loci (MLXIPL, TRIB1, and CILP2/PBX4) were significantly associated with triglycerides in at least 1 nonwhite ethnic group. SNPs rs17321515 near MLXIPL and rs17145738 near TRIB1 replicated in Mexican Americans (P=0.01 and 7.1×10−5, respectively). In non-Hispanic blacks, we observed an association between rs3734991 near CILP2/PBX4 and triglycerides (P=0.01). LPL rs328 was strongly associated with triglycerides in non-Hispanic whites (P=0.0002). SNPs at the remaining 4 loci failed to replicate in either nonwhite ethnic group.
Because nonfasting status may affect triglyceride levels, we conducted secondary analyses with the phenotype of race-specific triglyceride residuals adjusted for age, gender, and fasting status and the results were not materially different (data not shown).
In a multiethnic sample representative of the US population, we tested DNA sequence variants at 19 loci that had been discovered in individuals of European descent. We addressed the hypothesis that these same variants would relate to blood lipid traits in other ethnic groups. At 5 loci (CELSR2/PSRC1/SORT1, HMGCR, CETP, LPL, and APOA5), we found that the same variant identified in whites was nominally associated in both non-Hispanic blacks and Mexican Americans. At other loci, there was mixed evidence for SNPs by ethnic group.
For SNPs at 5 loci discovered in whites, the same polymorphism was confirmed across all 3 NHANES III ethnic groups. This suggests that these loci impact blood lipid traits across multiple ethnic groups. These results set the stage for fine mapping and resequencing of these gene regions in all 3 ethnic groups.
For the remaining loci, variable replication among ethnicities likely occurred for 1 or more of the following reasons. First, individual genetic variants contributing to complex traits have very modest effect sizes. Therefore, each individual ethnic group may have been underpowered to detect an association. This problem could be addressed by genotyping the polymorphisms in additional individuals of African or Hispanic ancestry. Second, the locus discovered in whites may be relevant in other ethnicities, but the specific polymorphisms associated with lipids may differ across ethnicities. Older populations such as those of African descent have a more complex linkage disequilibrium (LD) structure.19 Therefore, if the causal variant in blacks is poorly correlated with the index SNP discovered from whites, no association will be detected. In this case, testing a comprehensive set of variants that tag the entire locus (ie, fine mapping the region in other ethnicities) would be required to detect the relevant polymorphisms. Finally, genetic variants in different ethnicities may lie in regions of the genome distinct from those observed in whites. New variant discovery through additional GWA studies or resequencing efforts in other ethnicities would be necessary to locate these variants. Such efforts are currently underway.
In previously published literature, several of the loci have been studied in black and/or Hispanic Americans including LDLR, PCSK9, CETP, LPL, and APOA5. Our study supports and builds on these previous reports. Although other variants have been reported in multiethnic groups,20 we are the first to describe associations of the LDLR intronic SNPs rs6511720 and rs1529729 with LDL-C in a sample including blacks and Mexican Americans. We confirm that rs11591147 (R46L) in the PCSK9 gene is common and decreases LDL-C in whites (minor allele frequency of 3%) but is rare in blacks (minor allele frequency of 0.4%).9,21 As in our study, associations between polymorphisms in the CETP and LPL genes and HDL-C have been consistently reported in cohorts including black and/or Hispanic subjects.22–24 Finally, we confirm the robust association between APOA5 and triglycerides in all 3 ethnic groups.25,26 By reproducing previously reported findings, we demonstrate that our patient sample is representative and that our methods are valid.
Our study has several strengths and limitations. To our knowledge, this is the first study to assess loci recently discovered through GWA studies in a multiethnic cohort. Next, compared with previous studies assessing few variants in multiethnic cohorts, our study queried a larger set of lipid loci. Finally, because the NHANES III cohort oversampled minority groups in the US population, our sample includes a large number of blacks and Mexican Americans. That being said, due to the modest contribution of each polymorphism to trait variation, low statistical power is a key limitation. Lipids in NHANES III were measured in both fasting and nonfasting samples and our use of both may have further reduced statistical power, particularly for lipid variables sensitive to fasting status such as triglycerides. In addition, in NHANES III, ethnic groups are designated by self-reported ethnicity and this assessment is not always accurate. For a more precise assessment of ethnicity using genetic data, ancestry-informative markers are necessary. Last, we did not evaluate a comprehensive set of “tag” SNPs across each of the 19 loci. Such an effort would be required to definitively study each locus in blacks and Mexican Americans.
In conclusion, we evaluated 21 SNPs in 19 loci from recent GWA studies for blood lipids in a US population-based multiethnic sample. We found that at 5 loci, the exact same SNP identified in whites was also associated with lipids in both blacks and Mexican Americans. At 1p13 near CELSR2/PSRC1/SORT1, the strongest new locus identified in GWA studies of LDL-C, the same index SNP was associated with LDL-C in all 3 ethnic groups, suggesting that this locus is of broad relevance. Our findings set the stage for comprehensive fine mapping and sequencing efforts across these loci in multiethnic groups.
Sources of Funding
This work was supported by the Charles A. King Trust, Bank of America (to M.K.), the Doris Duke Charitable Foundation Clinical Scientist Development Award, a charitable gift from the Fannie E. Rippel Foundation, the Donovan Family Foundation, a career development award from the United States National Institutes of Health, and the Department of Medicine and Cardiovascular Research Center at Massachusetts General Hospital (to S.K.).
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A principal goal of genetic association studies has been to augment current disease prediction algorithms by identifying genetic variants associated with common diseases. Genome-wide association studies along with candidate gene studies have identified many variants associated with plasma lipid traits. However, most genome-wide association studies published to date and most candidate gene studies have been conducted exclusively in samples of European ancestry. Therefore, it is unclear whether these variants are relevant in a broader range of ethnic groups such as blacks and Hispanic Americans. In the current study, we tested polymorphisms from candidate gene loci and loci identified through genome-wide association in the Third United States National Health and Nutritional Examination Survey, a population-based probability sample consisting of non-Hispanic black, Mexican American, and non-Hispanic white participants. Our study takes the first step toward addressing whether the same genetic variants identified in populations of European ancestry will be associated with blood lipids and, therefore, possibly predictive in other ethnic groups. Although further work is needed, our findings suggest that for many loci, the same variant identified in whites will also be relevant in other ethnic groups.
Article handled by Guest Editor Donna K. Arnett, PhD.