Ma et al. BMC Medical Genetics 2010, 11:55 http://www.biomedcentral.com/1471-2350/11/55 Open Access RESEARCH ARTICLE BioMed Central �� 2010 Ma et al licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons At- tribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Research article Genome-wide association analysis of total cholesterol and high-density lipoprotein cholesterol levels using the Framingham Heart Study data Li Ma1, Jing Yang1, H Birali Runesha2, Toshiko Tanaka3,4, Luigi Ferrucci4, Stefania Bandinelli5 and Yang Da*1 Abstract Background: Cholesterol concentrations in blood are related to cardiovascular diseases. Recent genome-wide association studies (GWAS) of cholesterol levels identified a number of single-locus effects on total cholesterol (TC) and high-density lipoprotein cholesterol (HDL-C) levels. Here, we report single-locus and epistasis SNP effects on TC and HDL-C using the Framingham Heart Study (FHS) data. Results: Single-locus effects and pairwise epistasis effects of 432,096 SNP markers were tested for their significance on log-transformed TC and HDL-C levels. Twenty nine additive SNP effects reached single-locus genome-wide significance (p 7.2 �� 10-8) and no dominance effect reached genome-wide significance. Two new gene regions were detected, the RAB3GAP1-R3HDM1-LCT-MCM6 region of chr02 for TC identified by six new SNPs, and the OSBPL8- ZDHHC17 region (chr12) for HDL-C identified by one new SNP. The remaining 22 single-locus SNP effects confirmed previously reported genes or gene regions. For TC, three SNPs identified two gene regions that were tightly linked with previously reported genes associated with TC, including rs599839 that was 10 bases downstream PSRC1 and 3.498 kb downstream CELSR2, rs4970834 in CELSR2, and rs4245791 in ABCG8 that slightly overlapped with ABCG5. For HDL-C, LPL was confirmed by 12 SNPs 8-45 kb downstream, CETP by two SNPs 0.5-11 kb upstream, and the LIPG-ACAA2 region by five SNPs inside this region. Two epistasis effects on TC and thirteen epistasis effects on HDL-C reached the significance of "suggestive linkage". The most significant epistasis effect (p = 5.72 �� 10-13) was close to reaching "significant linkage" and was a dominance �� dominance effect of HDL-C between LMBRD1 (chr06) and the LRIG3 region (chr12), and this pair of gene regions had six other D �� D effects with "suggestive linkage". Conclusions: Genome-wide association analysis of the FHS data detected two new gene regions with genome-wide significance, detected epistatic SNP effects on TC and HDL-C with the significance of suggestive linkage in seven pairs of gene regions, and confirmed some previously reported gene regions associated with TC and HDL-C. Background Total cholesterol (TC) is related to coronary diseases and high-density lipoprotein (HDL-C) cholesterol is anti- atherogenic. Genome-wide association studies (GWAS) and human genetic studies have identified a number of genes and gene regions affecting cholesterol phenotypes including TC and HDL-C [1-11]. A meta-analysis of HDL-C levels that include the FHS data has previously been published [2]. An early report on FHS [12] analyzed TC and HDL-C but used 100 k SNPs and a sample size that was much smaller than the current FHS sample size. Epistasis analysis of TC and HDL-C was unavailable. Here, we apply a quantitative genetics approach to detect additive or dominance single-locus effects and epistasis effects on log-transformed TC and HDL-C using 432,096 SNP markers and over 6000 individuals in FHS. The epistasis effects we tested included additive �� additive (A �� A), additive �� dominance (A �� D) or dominance �� addi- tive (D �� A), and dominance �� dominance (D �� D) effects, * Correspondence: yda@umn.edu 1 Department of Animal Science, University of Minnesota, USA Full list of author information is available at the end of the article

Ma et al. BMC Medical Genetics 2010, 11:55 http://www.biomedcentral.com/1471-2350/11/55 Page 2 of 11 with genetic interpretations of allele �� allele, allele �� gen- otype or genotype �� allele, and genotype �� genotype interactions. The single-locus analysis was intended to detect new targets or confirm existing targets using a method of analysis different from those used in previous reports based on an extended Kempthorne model that allows Hardy-Weinberger disequilibrium and linkage dis- equilibrium [13] for GWAS analysis of the FHS data while the epistasis analysis of TC and HDL-C was the first such attempt using the FHS data and the 500 k SNP panel. Results The single-locus tests detected nine SNPs with additive (or allelic) effects on TC and twenty SNPs with additive effects on HDL-C that reached genome-wide significance (Tables 1-2). No dominance effect reached genome-wide significance. Among the twenty nine SNP effects, twenty were new effects that were not reported in previous stud- ies and nine were previously reported to be associated with various cholesterol phenotypes [1-12]. Seven SNPs identified two new gene regions while the remaining twenty two SNPs confirmed previously reported gene regions. Two epistasis effects on TC and thirteen epista- sis effects on HDL-C representing seven pairs of gene regions reached the significance of "suggestive linkage". Single-locus effects For TC, nine SNPs with additive (or allelic) effects reached genome-wide significance with p 7.2 �� 10-8 (Table 1). Six SNPs inside or near four genes identified a new chr02 region containing RAB3GAP1, R3HDM1, LCT and MCM6 to be associated with TC (Figure 1A). Of the six SNPs in the RAB3GAP1-R3HDM1-LCT-MCM6 region, five SNPs were inside genes and one SNP was 4.2 kb upstream MCM6. The most significant SNP in this region was rs2322660 in intron 12 of LCT (Table 1). The RAB3GAP1-R3HDM1-LCT-MCM6 region contained two other genes (ZRANB3 and UBXD2) that did not have sig- nificant SNPs. Eleven other SNPs spanning a 1.23 Mb region (Figure 1A) that includes RAB3GAP1-R3HDM1- LCT-MCM6 had p-values between 1.27 �� 10-5 and 7.13 �� 10-7, including one SNP upstream ACMSD, one SNP in ACMSD, two SNPs in YSK4, one SNP in R3HDM1, one SNP in UBXD2 (also named UBXN4 according to NCBI Table 1: Single-locus SNP effects for TC with genome control (GC) adjusted P 7.2 �� 10-8. Effect Type & P value SNP Chr Position Gene Region Reported SNP effect MAF Genotype Additive Effect Size rs4970834 1 109814880 CELSR2a (intron 28) Non-HDL-C [5-7] 0.18 1.10E-08 1.75E-09 0.146 �� 0.023 TC [8] rs599839 1 109822166 10 bases downstream LDL-C [6,7,9,10] 0.22 8.72E-14 2.46E-14 0.174 �� 0.021 PSRC1a Non-HDL-C [5] rs4245791 2 44074431 ABCG8 (intron 3) 0.32 1.82E-07 3.33E-08 -0.127 �� 0.021 rs6730157 2 135907088 RAB3GAP1 (intron 17) LDL-C: P = 0.018 [2]b 0.45 8.51E-08 2.16E-08 0.113 �� 0.019 rs12465802 2 136381348 R3HDM1 (intron 7) LDL-C: P = 0.022 [2]b 0.44 2.63E-08 7.98E-09 0.117 �� 0.019 rs4954280 2 136420690 R3HDM1 (intron18) LDL-C: P = 0.007 [2]b 0.33 1.49E-07 5.87E-08 0.114 �� 0.02 rs2322660 2 136557319 LCT (intron 12) LDL-C: P = 0.055 [2]b 0.35 2.42E-08 7.08E-09 -0.120 �� 0.019 TC: P = 0.003-0.005 [17] LDL-C: P = 0.002-0.0005 [17] rs309180 2 136614255 MCM6 (intron 11) LDL-C: P = 0.057 [2]b 0.36 2.43E-08 8.39E-09 -0.119 �� 0.019 rs632632 2 136638216 4.2 kb upstream MCM6 LDL-C: P = 0.216 [2]b 0.36 2.50E-08 1.03E-08 -0.118 �� 0.019 a This gene was reported to be associated with TC [1]. b Available at http://www.sph.umich.edu/csg/abecasis/public/lipids2008/