Genetic Variation in ABCG1 and Association with Lipid Phenotypes in MESA

1. Genetic Variation in ABCG1 and Association with Lipid Phenotypes in MESA Stephen Rich, Ph.D. Lynn Hedrick, Ph.D. University of Virginia

2. ABCG1 and Atherosclerosis • Members of the ATP-binding cassette transporter family (ABC transporters) regulate cholesterol efflux • Contributes to foam cell formation • Contributes to development of atherosclerotic lesions • ABCA1 and ABCG1 regulate cellular lipid metabolism in macrophages • ABCA1 mediates cholesterol efflux to lipid-free apoA-I • ABCG1 mediates cholesterol efflux to HDL • ABCG1 plays a role in lipid metabolism and atherosclerosis • Expression is regulated by chronic glucose exposure in type 2 diabetes model (db/db mouse) (Mauldin 2006) • Decreased ABCG1 function increases foam cell formation and atherosclerosis risk (Mauldin 2006) • Western diet-fed mice resulting from crossing ABCG1 transgenic mice with LDLr-KO mice have increased atherosclerotic lesion area and increased MCP-1 and TNF-a plasma levels (Basso 2006)

3. ABCG1 in Reverse Cholesterol Transport

4. ABCG1 Molecular Structure 3 4 2 5 6 1 C LL N B A S ABCG1 is expressed in macrophages; it functions in RCT to HDL; dimerizes for function ABC JPM

5. Research Hypotheses • Primary • Variation in ABCG1 contributes to the observed variation in levels of HDL • Variation in ABCG1 influence the distribution of HDL particle size • Secondary • Variation in ABCG1 are not associated with other measures of lipids • Variation in ABCG1 is associated with variation in markers of subclinical atherosclerosis (IMT, CAC), independent of HDL

6. ABCG1 Gene Organization

7. ABCG1 Gene Structure

8. LD Structure of ABCG1 INTRON 1 INTRON 2 INTRON 5 I-8 I-11 I-14

9. Analytic Plan • Outcome Variables: Lipid phenotypes • Total Cholesterol • HDL domain • HDL cholesterol • HDL particles, HDL size • Large HDL, Medium HDL, Small HDL • IDL, VLDL particles, VLDL size, remnant-like particles • LDL domain • LDL cholesterol • LDL particles, LDL size • Large LDL, Small LDL, Very Small LDL • Triglyceride level • Predictor Variables: ABCG1 SNPs (and ABCG1 haplotypes) • Covariates: Age, sex, site of ascertainment, diabetes status, smoking, ethnicity (for pooled analysis)

10. 1/1 1/2 2/2 Association Analysis of SNPs with Quantitative Traits Significance under 2-df model; then test AD, Add, AR models (add model fits here) % Trait

11. ABCG1 in MESA • Selection of SNPs • HapMap data for EUC • Minor allele frequency (MAF) > 0.05, r2 threshold of 0.8 • High Illumina genotyping ‘designability’ score • Repeat for other ethnic groups and develop combined SNP list • For ABCG1, 40 haplotype-tagging SNPs identified and genotyped • MAF ranged from 0.026 (rs8129752) to 0.496 (rs492338) • Average inter-SNP distance of 2.7kb • Significant (P < 0.001) deviations from Hardy-Weinberg Equilibrium • All groups pooled, 9 out of HWE • rs1378577, rs8129752, rs8127716, rs225444, rs225371, rs225393, rs425215, rs3788010, rs1541290 • EUA: none • AFA: none • CHN: none, but 5 SNPs monomorphic • HIS: 2 out of HWE • rs225412, rs235371

12. Association of ABCG1 SNPs with Lipid Phenotypes (all groups) Int 1 Int 2 I5 I8 I11 Total Cholesterol HDL HDL Particles Large HDL Medium HDL Small HDL HDL Size IDL 0.05 < P < 0.01 LDL P < 0.01 LDL Particles Large LDL Small LDL Excludes those 9 SNPs out of HWE (P<0.001) Very Small LDL LDL Size • P-values determined from 2-df test of association • Model adjusts for age, sex, center, diabetes status, pack-yrs smoking VLDL Particles VLDL Size Remnant-like Particles Triglycerides

13. Association of ABCG1 SNPs with Lipid Phenotypes (EUC, AFA, HIS) Int 1 Int 2 I5 I8 I11 Total Cholesterol HDL HDL Particles Large HDL Medium HDL Small HDL HDL Size 0.05 < P < 0.01 IDL P < 0.01 LDL LDL Particles Large LDL Excludes CHN SNPs and SNPs out of HWE (P<0.001) Small LDL Very Small LDL LDL Size • P-values determined from 2-df test of association • Model adjusts for age, sex, center, diabetes status, pack-yrs smoking VLDL Particles VLDL Size Remnant-like Particles Triglycerides

14. Effect of ABCG1 SNP rs6586298 P = 0.008 2579 237 31 P = 0.007 P = 0.06 Model adjusts for age, sex, center, diabetes status, pack-yrs smoking

15. Summary • In all populations, there is evidence of association of SNPs in ABCG1 with phenotypes relating to HDL and LDL, but not IDL, VLDL remnant-like particles, triglycerides, or total cholesterol • There are differences in association between ABCG1 SNPs by ethnic group, with limited association in the CHN population • In the non-CHN populations, • there is strong evidence of association of SNPs in intron 1 and intron 2 with HDL, HDL size and HDL particles • There is evidence of association of SNPs in intron 2 with IDL • There is evidence of association of SNPs in introns 8 and 11 with HDL level and particles • SNPs in intron 11 appear to be highly associated with VLDL particles and VLDL size • LDL phenotypes continue to show weak association with ABCG1 SNPs

16. ABCG1 Gene Structure

17. ABCG1 in Reverse Cholesterol Transport

18. Summary and Conclusions • These results are preliminary • Additional covariates in the models • Use of haplotypes in the analyses • A number of ABCG1 SNPs exhibited significant association with HDL phenotypes • ABCG1 SNP associations with lipid phenotypes are, for the most part, ethnic-specific (not observed in CHN) • Future studies include • Genotyping additional SNPs that occur in exons • Characterizing ABCG1 variants in replicate MESA samples • Extending associated SNP studies to model systems • Cellular • Mouse