Comprehensive evaluation of apolipoprotein H gene (APOH) variation identifies novel associations with measures of lipid metabolism in GENOA

Apolipoprotein H (apoH, also named β-2 glycoprotein I) is found on several classes of lipoproteins, and is involved in the activation of lipoprotein lipase in lipid metabolism. We have comprehensively investigated the association of variation in the apoH gene (APOH) with lipid traits in hepatic cholesterol transport, dietary cholesterol transport (DCT), and reverse cholesterol transport (RCT). Our study population consisted of families from the Genetic Epidemiology Network of Arteriopathy multicenter study that include African Americans, Mexican Americans, and European Americans. We individually tested 36 single-nucleotide polymorphisms (SNPs) that span the APOH locus, including nonsynonymous variants that result in known apoH charge isoforms. In addition, we constructed haplotypes from SNPs in the 5′ promoter region that comprise cis-acting regulatory elements, as well as haplotypes for multiple amino acid substitutions. We found point-wise significant associations of APOH variants with various lipid measures in the three racial groups. The strongest associations were found for DCT traits (triglyceride and apoE levels) in Mexican Americans with a nonsynonymous variant (SNP 14917, Cys306Gly) that may alter apoH protein folding in a region involved in phospholipid binding. In conclusion, family-based analyses of APOH variants have identified associations with measures of lipid metabolism in three American racial groups. Apolipoprotein H (apoH, also named β-2 glycoprotein I) is found on several classes of lipoproteins, and is involved in the activation of lipoprotein lipase in lipid metabolism. We have comprehensively investigated the association of variation in the apoH gene (APOH) with lipid traits in hepatic cholesterol transport, dietary cholesterol transport (DCT), and reverse cholesterol transport (RCT). Our study population consisted of families from the Genetic Epidemiology Network of Arteriopathy multicenter study that include African Americans, Mexican Americans, and European Americans. We individually tested 36 single-nucleotide polymorphisms (SNPs) that span the APOH locus, including nonsynonymous variants that result in known apoH charge isoforms. In addition, we constructed haplotypes from SNPs in the 5′ promoter region that comprise cis-acting regulatory elements, as well as haplotypes for multiple amino acid substitutions. We found point-wise significant associations of APOH variants with various lipid measures in the three racial groups. The strongest associations were found for DCT traits (triglyceride and apoE levels) in Mexican Americans with a nonsynonymous variant (SNP 14917, Cys306Gly) that may alter apoH protein folding in a region involved in phospholipid binding. In conclusion, family-based analyses of APOH variants have identified associations with measures of lipid metabolism in three American racial groups. Apolipoprotein H (apoH, also named β-2 glycoprotein I) is synthesized mainly in the liver, but the specific functions of apoH remain unknown. ApoH was first thought to be involved mainly in lipid metabolism, because much of the circulating protein is bound to lipoproteins (1Polz E. Kostner G.M. The binding of beta 2-glycoprotein-I to human serum lipoproteins: distribution among density fractions.FEBS Lett. 1979; 102: 183-186Crossref PubMed Scopus (191) Google Scholar, 2Polz E. Kostner G.M. Holasek A. Studies on the protein composition of human serum very low density lipoproteins: demonstration of the beta 2-glycoprotein-I.Hoppe Seylers Z. Physiol. Chem. 1979; 360: 1061-1067Crossref PubMed Scopus (20) Google Scholar), and apoH activates lipoprotein lipase (LPL) in triglyceride (TG) metabolism (3Nakaya Y. Schaefer E.J. Brewer Jr., H.B. Activation of human post heparin lipoprotein lipase by apolipoprotein H (beta 2-glycoprotein I).Biochem. Biophys. Res. Commun. 1980; 95: 1168-1172Crossref PubMed Scopus (113) Google Scholar). However, apoH is also thought to be involved in coagulative and atherosclerotic pathways through the immunological response (4Schousboe I. Binding of beta 2-glycoprotein I to platelets: effect of adenylate cyclase activity.Thromb. Res. 1980; 19: 225-237Abstract Full Text PDF PubMed Scopus (97) Google Scholar, 5Schousboe I. Beta 2-glycoprotein I: a plasma inhibitor of the contact activation of the intrinsic blood coagulation pathway.Blood. 1985; 66: 1086-1091Crossref PubMed Google Scholar, 6Nimpf J. Bevers E.M. Bomans P.H. Till U. Wurm H. Kostner G.M. Zwaal R.F. Prothrombinase activity of human platelets is inhibited by beta 2-glycoprotein-I.Biochim. Biophys. Acta. 1986; 884: 142-149Crossref PubMed Scopus (311) Google Scholar). Recent studies have shown that apoH binds to phospholipid particles, forming a major epitope for auto-antibodies involved in antiphospholipid syndrome (7McNeil H.P. Simpson R.J. Chesterman C.N. Krilis S.A. Anti-phospholipid antibodies are directed against a complex antigen that includes a lipid-binding inhibitor of coagulation: beta 2-glycoprotein I (apolipoprotein H).Proc. Natl. Acad. Sci. USA. 1990; 87: 4120-4124Crossref PubMed Scopus (1628) Google Scholar, 8Jones J.V. James H. Tan M.H. Mansour M. Antiphospholipid antibodies require beta 2-glycoprotein I (apolipoprotein H) as cofactor.J. Rheumatol. 1992; 19: 1397-1402PubMed Google Scholar, 9Sanghera D.K. Wagenknecht D.R. McIntyre J.A. Kamboh M.I. Identification of structural mutations in the fifth domain of apolipoprotein H (beta 2-glycoprotein I) which affect phospholipid binding.Hum. Mol. Genet. 1997; 6: 311-316Crossref PubMed Scopus (81) Google Scholar). The gene for apoH (APOH) on chromosome 17q is 18 kb in length and contains 8 exons (10Mehdi H. Nunn M. Steel D.M. Whitehead A.S. Perez M. Walker L. Peeples M.E. Nucleotide sequence and expression of the human gene encoding apolipoprotein H (beta 2-glycoprotein I).Gene. 1991; 108: 293-298Crossref PubMed Scopus (72) Google Scholar). Genetic variations in APOH result in five charge isoforms, including H*1, H*2, H*3, H*3B, and H*3W (9Sanghera D.K. Wagenknecht D.R. McIntyre J.A. Kamboh M.I. Identification of structural mutations in the fifth domain of apolipoprotein H (beta 2-glycoprotein I) which affect phospholipid binding.Hum. Mol. Genet. 1997; 6: 311-316Crossref PubMed Scopus (81) Google Scholar, 11Kamboh M.I. Ferrell R.E. Sepehrnia B. Genetic studies of human apolipoproteins. IV. Structural heterogeneity of apolipoprotein H (beta 2-glycoprotein I).Am. J. Hum. Genet. 1988; 42: 452-457PubMed Google Scholar, 12Sanghera D.K. Kristensen T. Hamman R.F. Kamboh M.I. Molecular basis of the apolipoprotein H (beta 2-glycoprotein I) protein polymorphism.Hum. Genet. 1997; 100: 57-62Crossref PubMed Scopus (48) Google Scholar). H*2 is the most common isoform, with a relative allele frequency >85% in all studied populations, whereas frequencies for the other isoforms range only as high as 6% (9Sanghera D.K. Wagenknecht D.R. McIntyre J.A. Kamboh M.I. Identification of structural mutations in the fifth domain of apolipoprotein H (beta 2-glycoprotein I) which affect phospholipid binding.Hum. Mol. Genet. 1997; 6: 311-316Crossref PubMed Scopus (81) Google Scholar, 11Kamboh M.I. Ferrell R.E. Sepehrnia B. Genetic studies of human apolipoproteins. IV. Structural heterogeneity of apolipoprotein H (beta 2-glycoprotein I).Am. J. Hum. Genet. 1988; 42: 452-457PubMed Google Scholar, 12Sanghera D.K. Kristensen T. Hamman R.F. Kamboh M.I. Molecular basis of the apolipoprotein H (beta 2-glycoprotein I) protein polymorphism.Hum. Genet. 1997; 100: 57-62Crossref PubMed Scopus (48) Google Scholar). A recent effort to resequence APOH in 23 European Americans and 24 African Americans discovered 150 single-nucleotide polymorphisms (SNPs) and characterized linkage disequilibrium (LD) patterns for APOH variants (13Chen Q. Kamboh M.I. Complete DNA sequence variation in the apolipoprotein H (beta-glycoprotein I) gene and identification of informative SNPs.Ann. Hum. Genet. 2006; 70: 1-11Crossref PubMed Scopus (15) Google Scholar).Previous studies have associated apoH charge isoforms and other amino acid substitutions with various lipid traits (14Sepehrnia B. Kamboh M.I. Adams-Campbell L.L. Bunker C.H. Nwankwo M. Majumder P.P. Ferrell R.E. Genetic studies of human apolipoproteins. VIII. Role of the apolipoprotein H polymorphism in relation to serum lipoprotein concentrations.Hum. Genet. 1989; 82: 118-122Crossref PubMed Scopus (29) Google Scholar, 15Kaprio J. Ferrell R.E. Kottke B.A. Kamboh M.I. Sing C.F. Effects of polymorphisms in apolipoproteins E, A-IV, and H on quantitative traits related to risk for cardiovascular disease.Arterioscler. Thromb. 1991; 11: 1330-1348Crossref PubMed Scopus (118) Google Scholar, 16Cassader M. Ruiu G. Gambino R. Guzzon F. Pagano A. Veglia F. Pagni R. Pagano G. Influence of apolipoprotein H polymorphism on levels of triglycerides.Atherosclerosis. 1994; 110: 45-51Abstract Full Text PDF PubMed Scopus (35) Google Scholar, 17Takada D. Ezura Y. Ono S. Iino Y. Katayama Y. Xin Y. Wu L.L. Larringa-Shum S. Stephenson S.H. Hunt S.C. et al.Apolipoprotein H variant modifies plasma triglyceride phenotype in familial hypercholesterolemia: a molecular study in an eight-generation hyperlipidemic family.J. Atheroscler. Thromb. 2003; 10: 79-84Crossref PubMed Scopus (19) Google Scholar). However, population-based associations of the extensive variation in APOH characterized by Chen and Kamboh (13Chen Q. Kamboh M.I. Complete DNA sequence variation in the apolipoprotein H (beta-glycoprotein I) gene and identification of informative SNPs.Ann. Hum. Genet. 2006; 70: 1-11Crossref PubMed Scopus (15) Google Scholar) have not yet been investigated in relation to lipid traits. In this study, we have comprehensively investigated this extensive APOH variation in the family-based Genetic Epidemiology Network of Arteriopathy (GENOA) study, which includes African Americans (AAs), Mexican Americans (MAs), and European Americans (EAs) (18Daniels P.R. Kardia S.L. Hanis C.L. Brown C.A. Hutchinson R. Boerwinkle E. Turner S.T. Genetic Epidemiology Network of Arteriopathy Study. The familial aggregation of hypertension treatment and control in the GENOA study.Am. J. Med. 2004; 116: 676-681Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). We genotyped GENOA subjects for 36 SNPs, including variants that underlie the known apoH charge isoforms. These APOH genotypes were used for family-based statistical analyses to identify associations with cardiovascular disease risk factors involved in hepatic cholesterol transport (HCT), dietary cholesterol transport (DCT), and reverse cholesterol transport (RCT).MATERIALS AND METHODSStudy subjectsThe study subjects participated in the GENOA study, which is composed of AAs from Jackson, MS, MAs from Starr County, TX, and EAs from Rochester MN (18Daniels P.R. Kardia S.L. Hanis C.L. Brown C.A. Hutchinson R. Boerwinkle E. Turner S.T. Genetic Epidemiology Network of Arteriopathy Study. The familial aggregation of hypertension treatment and control in the GENOA study.Am. J. Med. 2004; 116: 676-681Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). For AAs and EAs, sibships were recruited that contained at least two hypertensive subjects. For MAs, sibships were recruited based on at least two subjects with type II diabetes. GENOA provided demographic and pedigree information, medical history, anthropometric measures, informed consent, and various plasma measures for study subjects. Genomic DNA samples were available for 4,941 individuals, and pedigree information and phenotypic measurements were available for 5,242 individuals. After exclusion of subjects who were missing data, the current study included a total of 4,748 subjects (1,696 AA subjects from 583 families, 1,643 MAs from 415 families, and 1,409 EAs from 498 families). All GENOA protocols were approved by appropriate institutional review boards for the protection of human subjects, and all GENOA subjects provided written informed consent.APOH genotypingWe selected 36 SNPs spanning APOH from the SeattleSNPs Program for Genome Application database (http://pga.gs.washington.edu/data/APOH) that were identified by resequencing of 23 EAs and 24 AAs (13Chen Q. Kamboh M.I. Complete DNA sequence variation in the apolipoprotein H (beta-glycoprotein I) gene and identification of informative SNPs.Ann. Hum. Genet. 2006; 70: 1-11Crossref PubMed Scopus (15) Google Scholar). We included SNPs based on minor allele frequencies (MAFs) >5%, and potential functional properties (nonsynonymous SNPs). Although many of these SNPs were correlated, we chose to genotype multiple SNPs rather than a few tag SNPs that rely on LD relationships that differ among the racial groups. The potentially functional SNPs included nucleotide substitutions that underlie the known apoH protein isoforms (9Sanghera D.K. Wagenknecht D.R. McIntyre J.A. Kamboh M.I. Identification of structural mutations in the fifth domain of apolipoprotein H (beta 2-glycoprotein I) which affect phospholipid binding.Hum. Mol. Genet. 1997; 6: 311-316Crossref PubMed Scopus (81) Google Scholar, 11Kamboh M.I. Ferrell R.E. Sepehrnia B. Genetic studies of human apolipoproteins. IV. Structural heterogeneity of apolipoprotein H (beta 2-glycoprotein I).Am. J. Hum. Genet. 1988; 42: 452-457PubMed Google Scholar, 12Sanghera D.K. Kristensen T. Hamman R.F. Kamboh M.I. Molecular basis of the apolipoprotein H (beta 2-glycoprotein I) protein polymorphism.Hum. Genet. 1997; 100: 57-62Crossref PubMed Scopus (48) Google Scholar). Genotyping of APOH SNPs in GENOA DNA samples used several high-throughput genotyping platforms, including Taqman assays (Applied Biosystems), the LightTyper platform (Roche Applied Science), and the SNPlex platform (Applied Biosystems). In addition, we resequenced exon 5 of APOH to obtain genotypes for several closely spaced SNPs using the Applied Biosystems 3730xl DNA analyzer. The identity and location of the 36 SNPs are provided in the Results section (Table 2), and the primer and probe sequences are available from the authors upon request.TABLE 2APOH SNP positions and minor allele frequencies in African Americans, Mexican Americans, and European AmericansMAFPositionReferenceAlleleFunctional LocationAAMAEA−1283rs8178818C/GPromoter00.0200.002−1218rs8178819G/APromoter0.0120.0250.080−1054rs8178897T/GPromoter0.01100−758rs8178820A/GPromoter0.0510.0900.266−700rs3760291C/APromoter0.0640.0850.257−643hcv268405T/CPromoter0.1230.2340.133−627rs8178898A/CPromoter0.0390.0050−581rs8178899A/CPromoter0.0510.0080.001−32rs8178822C/A5′ UTR0.0580.0420.06971rs8178901C/TSplicing0.0470.00103333aSNPs that determine known apoH protein charge isoforms.rs8178833G/ASer88Asn0.0140.0270.0415247rs8178835T/CIntron 30.4390.4520.2275956rs8178838A/GIntron 40.0810.0500.0726482rs3785617A/GIntron 40.2670.1720.3788627reference 14A/CSplicing0.0800.0490.0708643aSNPs that determine known apoH protein charge isoforms.rs1803122T/CIle122Thr0.0830.0480.0698682aSNPs that determine known apoH protein charge isoforms.rs8178847G/AArg135His0.0840.0500.0708698rs8178925A/GSer140Ser0.010008700aSNPs that determine known apoH protein charge isoforms.reference 14C/AAla141Asp0.019008853hcv265411T/CIntron 50.1190.3600.3938906rs8178926G/AIntron 50.059009926rs2215415T/CIntron 50.1320.3600.39510099rs7212060A/CIntron 50.3470.4860.70313255rs4366742G/AIntron 60.1190.3600.39413324rs8178857C/AIntron 60.0970.0750.11113524rs2873966C/TIntron 60.1870.1250.30614740rs3176975G/TVal247Leu0.4700.5420.77314917rs4791077T/GCys306Gly0.0070.0490.02614969rs1544556C/TIntron 70.0510.0890.26815937rs8178943T/GIntron 70.0970.001015957rs4791079C/AIntron 70.1950.3650.39616219rs4790914C/GIntron 70.1870.3640.39317212aSNPs that determine known apoH protein charge isoforms.rs8178862G/CThr316Ser0.0050.0410.06118368rs8178818G/A3′ Flanking region0.1100.2600.14318774rs8178819T/G3′ Flanking region0.1140.2630.14418801rs8178897A/T3′ Flanking region0.2780.3960.419SNP, single-nucleotide polymorphism; AA, African American; MA, Mexican American; EA, European American; MAF, minor allele frequency; UTR, untranslated region.a SNPs that determine known apoH protein charge isoforms. Open table in a new tab Statistical analysisCorrelations among APOH SNPs were estimated using LDSelect (r2 > 0.8) (19Carlson C.S. Eberle M.A. Rieder M.J. Yi Q. Kruglyak L. Nickerson D.A. Selecting a maximally informative set of single-nucleotide polymorphisms for association analyses using linkage disequilibrium.Am. J. Hum. Genet. 2004; 74: 106-120Abstract Full Text Full Text PDF PubMed Scopus (1325) Google Scholar). For association studies, we grouped plasma lipoprotein and apolipoprotein measures according to their involvement in HCT [total cholesterol (TC), LDL-cholesterol (LDL-C), and apoB], DCT (TG and apoE), and RCT (HDL-C and apoA-I). Plasma levels of TC, HDL-C, TGs, apoA-I, and apoE were ln-transformed to provide normalized trait distributions. All traits were adjusted for sex, age, height, weight, and waist-hip ratio. We also adjusted for diabetes and hypertension status because GENOA ascertainment resulted in enrichment of diabetes in MAs and hypertension in AAs and EAs. To adjust for covariates, we used the Generalized Estimating Equation (GEE) that accounts for pedigree structures. Heritabilities of the lipid and lipoprotein measures were estimated with SOLAR (Sequential Oligogenic Linkage Analysis Routines) (20Almasy L. Blangero J. Multipoint quantitative-trait linkage analysis in general pedigrees.Am. J. Hum. Genet. 1998; 62: 1198-1211Abstract Full Text Full Text PDF PubMed Scopus (2563) Google Scholar, 21Blangero J. Williams J.T. Almasy L. Quantitative trait locus mapping using human pedigrees.Hum. Biol. 2000; 72: 35-62PubMed Google Scholar). F-tests were used to test for differences in variances of quantitative measures among the three GENOA racial groups. Association analysis used the Family Based Association Test (FBAT) algorithm with an additive model for genotypic effects (22Laird N.M. Horvath S. Xu X. Implementing a unified approach to family-based tests of association.Genet. Epidemiol. 2000; 19: 36-42Crossref PubMed Scopus (744) Google Scholar). Haplotypes were statistically estimated using the Expecation Maximization algorithm as part of FBAT, and association tests were performed by haplotype-based association tests. In some cases, GEE was used to confirm the FBAT results. Corrections for multiple testing were based on false discovery rates (FDRs) (23Benjamini Y. Drai D. Elmer G. Kafkafi N. Golani I. Controlling the false discovery rate in behavior genetics research.Behav. Brain Res. 2001; 125: 279-284Crossref PubMed Scopus (2645) Google Scholar). FDR-adjusted P values were calculated separately for each trait and racial group. Briefly, FBAT P values were ranked, and Q values for each SNP were calculated as qi = kpi/i, where i denotes rank and k gives the total number of tests (k = 34 for AAs, 27 for MAs, and 28 for EAs). The FDR-adjusted P value for SNPi is the minimum q value between qi and qk. We performed conditional linkage analysis using two approaches, including incorporation of associated SNPs as covariates for linkage analysis, and by adjusting apolipoprotein measures for SNP genotypes by regression (GEE) for linkage analysis with residual values.RESULTSTable 1shows the number of GENOA subjects and families, diabetes and hypertension status, plasma levels of lipoproteins and apolipoproteins, and anthropometric measures by racial group. Higher numbers of diabetics were found in MAs owing to ascertainment by diabetes status. Similarly, higher numbers of hypertensives were found in AAs and EAs owing to ascertainment by hypertensive status. All traits were significantly different among the three racial groups (P < 0.05). We calculated highly significant heritability estimates for the lipoprotein and apolipoprotein measures (all significant at P < 0.0001), indicating a strong genetic determinant of these traits in GENOA subjects. Heritabilities ranged from 0.42 (TG) to 0.71 (HDL-C) in AAs, 0.39 (LDL-C) to 0.54 (HDL-C) in MAs, and 0.26 (TC) to 0.64 (HDL-C) in EAs. We genotyped GENOA subjects for 36 SNPs spanning APOH based on MAFs >0.05 and potential functionality (13Chen Q. Kamboh M.I. Complete DNA sequence variation in the apolipoprotein H (beta-glycoprotein I) gene and identification of informative SNPs.Ann. Hum. Genet. 2006; 70: 1-11Crossref PubMed Scopus (15) Google Scholar). Table 2shows a list of the 36 SNPs, as well as their gene locations and MAFs in AAs, MAs, and EAs. Among the potentially functional SNPs, we genotyped 8 SNPs that result in amino acid substitutions, including those that underlie known apoH charge isoforms (9Sanghera D.K. Wagenknecht D.R. McIntyre J.A. Kamboh M.I. Identification of structural mutations in the fifth domain of apolipoprotein H (beta 2-glycoprotein I) which affect phospholipid binding.Hum. Mol. Genet. 1997; 6: 311-316Crossref PubMed Scopus (81) Google Scholar, 11Kamboh M.I. Ferrell R.E. Sepehrnia B. Genetic studies of human apolipoproteins. IV. Structural heterogeneity of apolipoprotein H (beta 2-glycoprotein I).Am. J. Hum. Genet. 1988; 42: 452-457PubMed Google Scholar, 12Sanghera D.K. Kristensen T. Hamman R.F. Kamboh M.I. Molecular basis of the apolipoprotein H (beta 2-glycoprotein I) protein polymorphism.Hum. Genet. 1997; 100: 57-62Crossref PubMed Scopus (48) Google Scholar).TABLE 1General characteristics of GENOA subjectsGENOAEuropean AmericansAfrican AmericansMexican AmericansF-TestSubjects1,4091,6961,643Families498583415Diabetics1613541002Hypertensives1,0671,268815Age55.92 ± 10.8957.94 ± 10.2655.72 ± 12.08<0.0001Height (cm)168.73 ± 9.25168.71 ± 8.81162.94 ± 9.21<0.0001Weight (kg)86.67 ± 19.9387.77 ± 18.6981.88 ± 17.30<0.0001BMI (kg/m2)30.36 ± 6.3030.90 ± 6.5930.83 ± 6.080.042W/H0.913 ± 0.090.910 ± 0.070.974 ± 0.08<0.0001TC210.49 ± 38.79205.10 ± 56.88205.22 ± 46.360.0008LDL-C121.40 ± 33.90120.70 ± 40.80116.40 ± 35.500.0017HDL-C51.53 ± 16.3555.52 ± 17.9944.89 ± 12.64<0.0001TG193.59 ± 103.58145.45 ± 90.87234.18 ± 176.99<0.0001ApoE5.16 ± 1.655.31 ± 2.205.43 ± 2.220.0014ApoB106.65 ± 23.12102.59 ± 25.95108.54 ± 26.30<0.0001ApoA-I157.47 ± 31.36158.83 ± 32.26144.91 ± 25.67<0.0001Glucose100.11 ± 28.84111.90 ± 49.09152.82 ± 71.90<0.0001Lipid-lowering medication21.38%7.6%19.77%Anti-hypertensive medication68.88%66.8%49.84%Anti-diabetic medication8.28%21.98%62.66%GENOA, Genetic Epidemiology Network of Arteriopathy study; ApoE, apolipoprotein E; BMI, body mass index; W/H, waist-to-hip ratio; TG, triglycerides, HDL-C, HDL cholesterol; LDL-C, LDL cholesterol; TC, total cholesterol. Plasma levels of lipids, lipoproteins, apolipoproteins, and glucose are measured in mg/dl. Data are given as mean ± SD. Percentages of individuals taking medications were calculated for 991 African Americans, 1,196 Mexican Americans, and 1,038 European Americans. Open table in a new tab Single SNP associationsFor association studies, we assigned plasma lipoprotein and apolipoprotein measures according to three cholesterol transport pathways including HCT (HCT includes TC, LDL-C, and apoB), DCT (DCT includes TG and apoE), and RCT (RCT includes HDL-C and apoA-I). These groupings reflect involvement in common transport pathways and known correlations among traits (e.g., LDL-C and apoB, HDL-C and apoA-I). However, these assignments are not exact, because some traits are components of overlapping transport pathways. Figure 1shows FBAT results for association of individual APOH SNPs with apolipoprotein components of the three cholesterol transport groupings. Table 3shows P values for APOH SNPs that showed point-wise significant associations (unadjusted P < 0.05) for all traits in all races, as well as experiment-wise P values adjusted for multiple testing based on FDRs. supplementary Table I shows the means of lipoprotein and apolipoprotein levels for APOH SNP genotypes that showed point-wise significance (unadjusted P < 0.05).Fig. 1Association of individual APOH single-nucleotide polymorphisms (SNPs) with plasma apolipoprotein levels in GENOA. Each APOH SNP is listed on the x axis, and numbered relative to the translational start site. The underlined SNPs are polymorphisms located in the coding sequence of the gene. The y axis shows the point-wise –log P values (not adjusted for multiple testing) for associations (P = 0.05 corresponds to –log P value of 2.3, solid horizontal line). The solid curved lines show associations with apolipoprotein B (apoB) levels (hepatic cholesterol transport), the short dashed curves show associations with apoE levels (dietary cholesterol transport), and the long dashed curves show associations with apoA-I levels (reverse cholesterol transport). Asterisks below the graph mark APOH SNPs that show point-wise significant associations (unadjusted P > 0.05) with each apolipoprotein measure. A: Associations for African American families. B: Associations for Mexican Americans. C: Associations for European Americans.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 1Association of individual APOH single-nucleotide polymorphisms (SNPs) with plasma apolipoprotein levels in GENOA. Each APOH SNP is listed on the x axis, and numbered relative to the translational start site. The underlined SNPs are polymorphisms located in the coding sequence of the gene. The y axis shows the point-wise –log P values (not adjusted for multiple testing) for associations (P = 0.05 corresponds to –log P value of 2.3, solid horizontal line). The solid curved lines show associations with apolipoprotein B (apoB) levels (hepatic cholesterol transport), the short dashed curves show associations with apoE levels (dietary cholesterol transport), and the long dashed curves show associations with apoA-I levels (reverse cholesterol transport). Asterisks below the graph mark APOH SNPs that show point-wise significant associations (unadjusted P > 0.05) with each apolipoprotein measure. A: Associations for African American families. B: Associations for Mexican Americans. C: Associations for European Americans.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 3P values for significant apoH SNP associations (unadjusted P < 0.05) for all traits in all races, including P values adjusted for multiple testing based on FDRAAMAEAPositionTraitP < 0.05P (FDR)P < 0.05P (FDR)P < 0.05P (FDR)5956ApoB0.0390.1466482ApoB0.0090.1468643aSNPs that determine known apoH protein charge isoforms.ApoB0.0230.1468682aSNPs that determine known apoH protein charge isoforms.ApoB0.040.1469926ApoB0.0350.14613255ApoB0.0490.4230.0430.14615957ApoB0.0280.14616219ApoB0.030.4230.0290.146−1054LDL-C0.0470.9156482LDL-C0.0130.2245956TC0.0450.2086482TC0.0210.2088643aSNPs that determine known apoH protein charge isoforms.TC0.0310.208−32ApoE0.0340.2573333aSNPs that determine known apoH protein charge isoforms.ApoE0.0390.5590.0480.2575956ApoE0.0490.2578627ApoE0.0350.2578682aSNPs that determine known apoH protein charge isoforms.ApoE0.0410.25714917ApoE<0.0010.0078700aSNPs that determine known apoH protein charge isoforms.TG0.0270.1809926TG0.040.21713255TG0.0440.21714917TG0.0010.05215937TG0.0120.14315957TG0.0190.16818368TG0.0040.11218774TG0.0060.11214969HDL-C0.0310.770FDR, false discovery rate.a SNPs that determine known apoH protein charge isoforms. Open table in a new tab For HCT in EAs, APOH associations reflected the high correlations among HCT traits (Fig. 1C). For example, intronic SNP 6482 A/G showed point-wise highly significant associations with plasma apoB levels, as well as for TC and LDL-C (Table 3). Four other intronic SNPs (9926 T/C, 13255 G/A, 15957 C/A, 16219 C/G) also showed associations with apoB levels in EAs. These 4 intronic SNPs are strongly correlated (r2 > 0.8). In addition, 2 correlated nonsynonymous SNPs that distinguish the H*3 protein isoform were associated with apoB levels (8643 T/C, 8682 G/A), and TC (8643 T/C). In AAs (Fig. 1A), 2 SNPs showed associations with apoB levels, including 13255 G/A and 16219 C/G, which were also associated with apoB levels in EAs (Table 3). Overall, the less common alleles for these SNPs were associated with increased plasma apoB levels in both EAs and AAs (see supplementary Table I). In addition, SNP −1054 T/G in the promoter region was associated with LDL-C levels in AAs. No associations of APOH variants and HCT traits were observed in MAs at the P < 0.05 level.For DCT in MAs, a nonsynonymous SNP, 14917 T/G (Cys306Gly), showed point-wise highly significant associations for both apoE levels (Fig. 1B) and TG levels (Table 3). This association remained statistically significant after adjustment for multiple testing using FDRs (P = 0.007; Table 3). In addition, a nonsynonymous SNP that distinguishes the H*1 protein isoform (3333 G/A encoding Ser88Asn) was associated with apoE levels in MAs (and EAs). In EAs, 5 correlated SNPs (r2 > 0.8) showed point-wise significant associations with plasma apoE levels, including promoter SNP −32 C/A, intronic SNPs 5956 A/G and 8627 A/C, and nonsynonymous SNPs 3333 G/A (Ser88Asn) and 8682 G/A (Arg135His) (Fig. 1C). All of these correlated SNPs showed point-wise significant associations with apoE levels at P < 0.05 (Table 3). For all of these SNPs, the less common alleles were associated with reduced apoE levels in EAs (see supplementary Table I). In AAs, the strongest associations for DCT were with TG levels for 2 correlated SNPs in the 3′ flanking region (18368 G/A and 18774 T/G) an