Genetic and nongenetic sources of variation in phospholipid transfer protein activity
2009; Elsevier BV; Volume: 51; Issue: 5 Linguagem: Inglês
10.1194/jlr.m000125
ISSN1539-7262
AutoresGail P. Jarvik, Ramakrishnan Rajagopalan, Elisabeth A. Rosenthal, Gertrud Wolfbauer, Laura McKinstry, Aditya Vaze, John D. Brunzell, Arno G. Motulsky, Deborah A. Nickerson, Patrick J. Heagerty, Ellen M. Wijsman, John J. Albers,
Tópico(s)Diabetes, Cardiovascular Risks, and Lipoproteins
ResumoPhospholipid transfer protein (PLTP) belongs to the lipid transfer/lipopolysaccharide-binding protein gene family. Expression of PLTP has been implicated in the development of atherosclerosis. We evaluated the effects of PLTP region tagging single nucleotide polymorphisms (SNPs) on the prediction of both carotid artery disease (CAAD) and PLTP activity. CAAD effects were evaluated in 442 Caucasian male subjects with severe CAAD and 497 vascular disease-free controls. SNP prediction of PLTP transfer activity was evaluated in both a subsample of 87 subjects enriched for an allele of interest and in a confirmation sample of 210 Caucasian males and females. Hemoglobin A1c or insulin level predicted 11–14% of age- and sex-adjusted PLTP activity. PLTP SNPs that predicted ∼11–30% of adjusted PLTP activity variance were identified in the two cohorts. For rs6065904, the allele that was associated with CAAD was also associated with elevated PLTP activity in both cohorts. SNPs associated with PLTP activity also predicted variation in LDL-cholesterol and LDL-B level only in the replication cohort. These results demonstrate that PLTP activity is strongly influenced by PLTP region polymorphisms and metabolic factors. Phospholipid transfer protein (PLTP) belongs to the lipid transfer/lipopolysaccharide-binding protein gene family. Expression of PLTP has been implicated in the development of atherosclerosis. We evaluated the effects of PLTP region tagging single nucleotide polymorphisms (SNPs) on the prediction of both carotid artery disease (CAAD) and PLTP activity. CAAD effects were evaluated in 442 Caucasian male subjects with severe CAAD and 497 vascular disease-free controls. SNP prediction of PLTP transfer activity was evaluated in both a subsample of 87 subjects enriched for an allele of interest and in a confirmation sample of 210 Caucasian males and females. Hemoglobin A1c or insulin level predicted 11–14% of age- and sex-adjusted PLTP activity. PLTP SNPs that predicted ∼11–30% of adjusted PLTP activity variance were identified in the two cohorts. For rs6065904, the allele that was associated with CAAD was also associated with elevated PLTP activity in both cohorts. SNPs associated with PLTP activity also predicted variation in LDL-cholesterol and LDL-B level only in the replication cohort. These results demonstrate that PLTP activity is strongly influenced by PLTP region polymorphisms and metabolic factors. Phospholipid transfer protein (PLTP) belongs to the lipid transfer/lipopolysacharide binding protein gene family. It is expressed in the liver and in macrophages (1Laffitte B.A. Joseph S.B. Chen M. Castrillo A. Repa J. Wilpitz D. Mangelsdorf D. Tontonoz P. The phospholipid transfer protein gene is a liver X receptor target expressed by macrophages in atherosclerotic lesions.Mol. Cell. Biol. 2003; 23: 2182-2191Crossref PubMed Scopus (133) Google Scholar). PLTP is responsible for most of the transfer of phospholipids from plasma VLDL to HDL (2Tall A.R. Krumholz S. Olivecrona T. Deckelbaum R.J. Plasma phospholipid transfer protein enhances transfer and exchange of phospholipids between very low density lipoproteins and high density lipoproteins during lipolysis.J. Lipid Res. 1985; 26: 842-851Abstract Full Text PDF PubMed Google Scholar, 3Tollefson J.H. Ravnik S. Albers J.J. Isolation and characterization of a phospholipid transfer protein (LTP-II) from human plasma.J. Lipid Res. 1988; 29: 1593-1602Abstract Full Text PDF PubMed Google Scholar, 4Jiang X.C. Bruce C. Mar J. Lin M. Ji Y. Francone O.L. Tall A.R. Targeted mutation of plasma phospholipid transfer protein gene markedly reduces high-density lipoprotein levels.J. Clin. Invest. 1999; 103: 907-914Crossref PubMed Scopus (319) Google Scholar). Mouse studies support the hypothesis that increased PLTP level is positively associated with atherosclerotic risk. Increased PLTP expression in mice has been associated with atherosclerosis (5Lie J. de Crom R. van Gent T. van Haperen R. Scheek L. Sadeghi-Niaraki F. van Tol A. Elevation of plasma phospholipid transfer protein increases the risk of atherosclerosis despite lower apolipoprotein B-containing lipoproteins.J. Lipid Res. 2004; 45: 805-811Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 6Moerland M. Samyn H. van Gent T. van Haperen R. Dallinga-Thie G. Grosveld F. van Tol A. de Crom R. Acute elevation of plasma PLTP activity strongly increases pre-existing atherosclerosis.Arterioscler. Thromb. Vasc. Biol. 2008; 28: 1277-1282Crossref PubMed Scopus (31) Google Scholar, 7Samyn H. Moerland M. van Gent T. van Haperen R. Grosveld F. van Tol A. de Crom R. Elevation of systemic PLTP, but not macrophage-PLTP, impairs macrophage reverse cholesterol transport in transgenic mice.Atherosclerosis. 2009; 204: 429-434Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), impaired reverse cholesterol transport (7Samyn H. Moerland M. van Gent T. van Haperen R. Grosveld F. van Tol A. de Crom R. Elevation of systemic PLTP, but not macrophage-PLTP, impairs macrophage reverse cholesterol transport in transgenic mice.Atherosclerosis. 2009; 204: 429-434Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), and decreased HDL, LDL, and VLDL levels (5Lie J. de Crom R. van Gent T. van Haperen R. Scheek L. Sadeghi-Niaraki F. van Tol A. Elevation of plasma phospholipid transfer protein increases the risk of atherosclerosis despite lower apolipoprotein B-containing lipoproteins.J. Lipid Res. 2004; 45: 805-811Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Elevated PLTP may lower HDL by increased hapatic uptake in mice (8van Haperen R. Samyn H. van Gent T. Zonneveld A.J. Moerland M. Grosveld F. Jansen H. Dallinga-Thie G.M. van Tol A. de Crom R. Novel roles of hepatic lipase and phospholipid transfer protein in VLDL as well as HDL metabolism.Biochim Biophys Acta. 2009; 1791: 1031-1036Crossref PubMed Scopus (16) Google Scholar). Mice with PLTP deficiency also absorbed less dietary cholesterol (9Liu R. Iqbal J. Yeang C. Wang D.Q. Hussain M.M. Jiang X.C. Phospholipid transfer protein-deficient mice absorb less cholesterol.Arterioscler. Thromb. Vasc. Biol. 2007; 27: 2014-2021Crossref PubMed Scopus (38) Google Scholar). Macrophage cholesterol efflux is impaired in PLTP transgenic mice, which may promote atherosclerosis (7Samyn H. Moerland M. van Gent T. van Haperen R. Grosveld F. van Tol A. de Crom R. Elevation of systemic PLTP, but not macrophage-PLTP, impairs macrophage reverse cholesterol transport in transgenic mice.Atherosclerosis. 2009; 204: 429-434Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Acute elevation of PLTP was found to increase both atherosclerotic lesions size and macrophage content in transgenic mice (6Moerland M. Samyn H. van Gent T. van Haperen R. Dallinga-Thie G. Grosveld F. van Tol A. de Crom R. Acute elevation of plasma PLTP activity strongly increases pre-existing atherosclerosis.Arterioscler. Thromb. Vasc. Biol. 2008; 28: 1277-1282Crossref PubMed Scopus (31) Google Scholar). Also, transplantation of bone marrow from PTLP-deficient mice into irradiated LDL receptor-deficient mice reduced atherosclerosis compared with mice possessing functional macrophage PLTP (10Vikstedt R. Ye D. Metso J. Hildebrand R.B. Van Berkel T.J. Ehnholm C. Jauhiainen M. Van Eck M. Macrophage phospholipid transfer protein contributes significantly to total plasma phospholipid transfer activity and its deficiency leads to diminished atherosclerotic lesion development.Arterioscler. Thromb. Vasc. Biol. 2007; 27: 578-586Crossref PubMed Scopus (57) Google Scholar). In contrast, macrophage PLTP expression introduced through bone marrow transplantation in double LDL receptor and PLTP-deficient mice decreased atherosclerotic lesions and total cholesterol, while increasing HDL (11Valenta D.T. Bulgrien J.J. Bonnet D.J. Curtiss L.K. Macrophage PLTP is atheroprotective in LDLr-deficient mice with systemic PLTP deficiency.J. Lipid Res. 2008; 49: 24-32Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Similarly, bone marrow knockouts of PLTP have also been reported to have lower atherosclerosis on an LDL receptor background (11Valenta D.T. Bulgrien J.J. Bonnet D.J. Curtiss L.K. Macrophage PLTP is atheroprotective in LDLr-deficient mice with systemic PLTP deficiency.J. Lipid Res. 2008; 49: 24-32Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar).In humans, PLTP has been reported to influence the size, density, and levels of VLDL and HDL (12Tu A-Y. Nishida H.I. Nishida T. High density lipoprotein conversion mediated by human plasma phospholipid transfer protein.J. Biol. Chem. 1993; 268: 23098-23105Abstract Full Text PDF PubMed Google Scholar, 13Jauhiainen M. Metso J. Pahlman R. Blomqvist S. van Tol A. Ehnholm C. Human plasma phospholipid transfer protein causes high density lipoprotein conversion.J. Biol. Chem. 1993; 268: 4032-4036Abstract Full Text PDF PubMed Google Scholar, 14Albers J.J. Wolfbauer J.G. Cheung M.C. Day J.R. Ching A.F.T. Lok S. Tu A-Y. Functional expression of human and mouse plasma phospholipid transfer protein: effect of recombinant and plasma PLTP on HDL subspecies.Biochim. Biophys. Acta. 1995; 1258: 27-34Crossref PubMed Scopus (121) Google Scholar, 15Murdoch S.J. Carr M.C. Hokanson J.E. Brunzell J.D. Albers J.J. PLTP activity in premenopausal women: relationship with lipoprotein lipase, HDL, LDL, body fat, and insulin resistance.J. Lipid Res. 2000; 41: 237-244Abstract Full Text Full Text PDF PubMed Google Scholar) as well as LDL (15Murdoch S.J. Carr M.C. Hokanson J.E. Brunzell J.D. Albers J.J. PLTP activity in premenopausal women: relationship with lipoprotein lipase, HDL, LDL, body fat, and insulin resistance.J. Lipid Res. 2000; 41: 237-244Abstract Full Text Full Text PDF PubMed Google Scholar, 16Murdoch S.J. Carr M.C. Kennedy H. Brunzell J.D. Albers J.J. Selective and independent associations of phospholipid transfer protein and hepatic lipase with the LDL subfraction distribution.J. Lipid Res. 2002; 43: 1256-1263Abstract Full Text Full Text PDF PubMed Google Scholar). We recently described the relationship between PLTP mass, activity, and lipid levels. PLTP mass and activity were poorly correlated, but PLTP mass was correlated with HDL size and concentration, while PLTP activity was correlated with VLDL size, total cholesterol, and triglyceride levels. These correlations differ by gender (17Cheung M.C. Wolfbauer G. Deguchi H. Fernandez J.A. Griffin J.H. Albers J.J. Human plasma phospholipid transfer protein specific activity is correlated with HDL size: implications for lipoprotein physiology.Biochim. Biophys. Acta. 2009; 1791: 206-211Crossref PubMed Scopus (21) Google Scholar).The relationship of PLTP activity and expression to vascular disease in humans requires further investigation. While studies have associated elevated PLTP with coronary artery disease (18Schlitt A. Bickel C. Thumma P. Blankenberg S. Rupprecht H.J. Meyer J. Jiang X.C. High plasma phospholipid transfer protein levels as a risk factor for coronary artery disease.Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1857-1862Crossref PubMed Scopus (115) Google Scholar), left ventricular dysfunction (19Cavusoglu E. Marmur J.D. Chhabra S. Chopra V. Eng C. Jiang X.C. Relation of baseline plasma phospholipid transfer protein (PLTP) activity to left ventricular systolic dysfunction in patients referred for coronary angiography.Atherosclerosis. 2009; 207: 261-265Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar), and coronary artery events in subjects on statins (20Schlitt A. Blankenberg S. Bickel C. Lackner K.J. Heine G.H. Buerke M. Werdan K. Maegdefessel L. Raaz U. Rupprecht H.J. et al.PLTP activity is a risk factor for subsequent cardiovascular events in CAD patients under statin therapy: the AtheroGene study.J. Lipid Res. 2009; 50: 723-729Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), the opposite result was reported for peripheral artery disease. Peripheral artery vascular disease has been associated with lower PLTP (21Schgoer W. Mueller T. Jauhiainen M. Wehinger A. Gander R. Tancevski I. Salzmann K. Eller P. Ritsch A. Haltmayer M. et al.Low phospholipid transfer protein (PLTP) is a risk factor for peripheral atherosclerosis.Atherosclerosis. 2008; 196: 219-226Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), while carotida intima-media thickness (22de Vries R. Dallinga-Thie G.M. Smit A.J. Wolffenbuttel B.H. van Tol A. Dullaart R.P. Elevated plasma phospholipid transfer protein activity is a determinant of carotid intima-media thickness in type 2 diabetes mellitus.Diabetologia. 2006; 49: 398-404Crossref PubMed Scopus (67) Google Scholar) has been associated with raised PLTP in humans. PLTP expression in macrophages in the vessel wall has been proposed to be induced in the conversion of macrophages to foam cells, thus impacting atherosclerotic plaque progression (1Laffitte B.A. Joseph S.B. Chen M. Castrillo A. Repa J. Wilpitz D. Mangelsdorf D. Tontonoz P. The phospholipid transfer protein gene is a liver X receptor target expressed by macrophages in atherosclerotic lesions.Mol. Cell. Biol. 2003; 23: 2182-2191Crossref PubMed Scopus (133) Google Scholar, 23O'Brien K.D. Vuletic S. McDonald T.O. Wolfbauer G. Lewis K. Tu A.Y. Marcovina S. Wight T.N. Chait A. Albers J.J. Cell-associated and extracellular phospholipid transfer protein in human coronary atherosclerosis.Circulation. 2003; 108: 270-274Crossref PubMed Scopus (74) Google Scholar, 24Desrumaux C.M. Mak P.A. Boisvert W.A. Masson D. Stupack D. Jauhiainen M. Ehnholm C. Curtiss L.K. Phospholipid transfer protein is present in human atherosclerotic lesions and is expressed by macrophages and foam cells.J. Lipid Res. 2003; 44: 1453-1461Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar).Prior investigation identified genetic variation associated with PLTP level and with lipids. PLTP single nucleotide polymorphism (SNP) rs7679 was found to predict serum triglyceride level as well as RNA expression of PLTP in liver (25Kathiresan S. Voight B.F. Purcell S. Musunuru K. Ardissino D. Mannucci P.M. Anand S. Engert J.C. Samani N.J. Schunkert H. et al.Genome-wide association of early-onset myocardial infarction with single nucleotide polymorphisms and copy number variants.Nat. Genet. 2009; 41: 334-341Crossref PubMed Scopus (903) Google Scholar). In that report, the rs7679 T allele was associated with increased PLTP expression, increased HDL level, and decreased triglyceride level. SNP rs2294213 has been reported to be associated with raised HDL, lowered triglycerides, and higher PLTP activity (26Engler M.B. Pullinger C.R. Malloy M.J. Natanzon Y. Kulkarni M.V. Song J. Eng C. Huuskonen J. Rivera C. Poon A. et al.Genetic variation in phospholipid transfer protein modulates lipoprotein profiles in hyperalphalipoproteinemia.Metabolism. 2008; 57: 1719-1724Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). The lipid profiles would suggest that increased expression might be cardioprotective. This is inconsistent with elevated PLTP predicting atherosclerosis in humans.Our goal was to evaluate the relationship of common genetic variation in PLTP with both carotid artery disease (CAAD) and with PLTP activity using a tagging SNP (tagSNP) approach. A cohort of subjects with and without severe carotid artery disease was considered, and a subset was selected for the labor intensive PLTP activity measure. An additional replication cohort was available for evaluation of PLTP activity.METHODSSampleThe sample population included 939 unrelated Caucasian males from the CLEAR study of carotid artery disease risk. This study has been previously described in detail elsewhere (27Crawford D.C. Nord A.S. Badzioch M.D. Ranchalis J. McKinstry L.A. Ahearn M. Bertucci C. Shephard C. Wong M. Rieder M.J. et al.A common VLDLR polymorphism interacts with APOE genotype in the prediction of carotid artery disease risk.J. Lipid Res. 2008; 49: 588-596Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Briefly, the study gains power per sampled individual from using a tails-of-the-distribution sampling scheme where cases (n = 442) had >80% stenosis of one or both internal carotid arteries, and controls (n = 497) had <15% stenosis bilaterally on duplex ultrasound. Additionally, controls had no known atherosclerotic vascular disease, had normal ankle-arm indices measures for lower extremity vascular disease, and were age distribution matched with the cases based on the age of onset of disease (censored age). Use of medications was ascertained by report and reconciled with review of pharmacy and medical records. Self-reported race was confirmed by analysis with STRUCTURE (28Pritchard J.K. Stephens M. Donnelly P. Inference of population structure using multilocus genotype data.Genetics. 2000; 155: 945-959Crossref PubMed Scopus (24773) Google Scholar), incorporating HapMap ethnicity groups. One inconsistent subject was removed from the analysis. Height and weight were measured, with medical records or self-report used to complete missing data. The PLTP activity assay has not been adapted to high throughput; hence, 87 genotyped subjects were selected for phenotyping. To minimize confounding with disease status and medication, these subjects were all controls who were not on statin medications. To maximize power to detect effects on PLTP activity, the selection of subjects was enriched for the rare genotype for a SNP associated with CAAD. As shown on Table 1, this strategy raised the minor allele frequency (MAF) of the SNP-predicting CAAD from 0.02 to 0.12. The study was approved by the University of Washington, the Veterans Affairs Puget Sound Health Care System, and the Virginia Mason Medical Center human subject review processes. Subjects gave written informed consent. Demographics are given in supplementary Table A.TABLE 1Effects of genotyped PLTP tagSNPs on prediction of severe carotid artery disease and on PLTP activity leveln = 939n = 87 Discoveryn = 210 ConfirmationrsChr 20. BPLocationMAF-AllMinor AlleleORp-CAADMAF-Discoveryβ-PLTPap-PLTPaMAF-Confβ-PLTPap-PLTPars173649343964041Intron 40.069G1.521.53E-02∗0.1380.091.11E-02∗0.093−0.032.76E-01rs55335943964245Intron 40.394C0.861.65E-010.465−0.058.29E-03∗0.370−0.034.61E-02∗rs1156964943964824Intron 60.024C0.662.12E-010.023−0.073.59E-010.045−0.077.93E-02rs1108698543965662Intron 60.381A0.989.95E-010.4080.071.14E-03∗0.3250.023.18E-01rs1156967143965887Intron 60.027A1.503.76E-010.042−0.045.07E-010.039−0.034.73E-01rs607395043966517Intron 60.333T1.006.23E-010.3210.063.02E-03∗0.2390.014.87E-01rs606590443968058Intron 80.214A0.762.30E-02∗0.339−0.082.55E-05∗0.279−0.081.80E-06∗rs607395243970339Intron 110.194A0.851.16E-010.264−0.103.39E-06∗0.275−0.095.51E-07∗rs37811443971834Intron 120.272A0.862.71E-010.2180.105.29E-05∗0.1570.047.22E-02rs1156963343973142Intron 130.012A1.487.83E-010.0170.019.28E-010.0050.084.73E-01rs39464343973585Intron 150.461A0.792.40E-02∗0.471−0.022.85E-010.445−0.051.58E-03∗rs11569668439762395′ (-2046)0.024A0.308.70E-03∗0.1210.101.56E-02∗0.0220.018.16E-01rs4810479439784555′ (-4262)0.249G0.884.15E-010.391−0.074.93E-04∗0.331−0.098.51E-08∗rs7679440099095′ (-35716)0.182C0.811.20E-010.242−0.115.39E-07∗0.261−0.093.62E-07∗rs is the number that uniquely identifies the polymorphism; BP is the base pair position on chromosome 20 for the SNP consistent with dbSNP for the 3′ strand; location refers to the placement of the SNP, either in the listed PLTP intron or 5′ of PLTP at the number of listed base pairs (−) from the initiation site per dbSNP build 130; MAF-all is the minor allele frequency for the case control cohort; OR is the odds ratio for the two less common genotype groups pooled relative to the most common group in the prediction of CAAD; p-CAAD is the P value for the test of the SNP effect on CAAD prediction (see Methods); MAF-discovery is the minor allele frequency in the subsample phenotyped for PLTP activity, β-PLTP is the coefficient for the minor allele's regression on PLTP activity; and p-PLTPα is the P value for that test. ∗ P < 0.05. Open table in a new tab A separate cohort from a family study was evaluated for confirmation of the SNP PLTP activity effects. This cohort consisted of 210 adult Caucasian subjects, 100 male and 110 female, ages 11–89 (mean age 47 years, eight minors) in four large families ascertained for familial combined hyperlipidemia (29Badzioch M.D. Igo Jr., R.P. Gagnon F. Brunzell J.D. Krauss R.M. Motulsky A.G. Wijsman E.M. Jarvik G.P. Low-density lipoprotein particle size loci in familial combined hyperlipidemia: evidence for multiple loci from a genome scan.Arterioscler. Thromb. Vasc. Biol. 2004; 24: 1942-1950Crossref PubMed Scopus (36) Google Scholar). The families were originally ascertained in the 1980s and recollected from 2003–2009. All data included here are from recent collection. Lipid and PLTP measures are as described. Height and weight were from survey data. Body mass indexes (BMIs) for 17 subjects were imputed based on the regression of age, sex, age by sex interaction, and natural log-transformed C-reactive protein level on BMI. The family study was approved by the University of Washington human subject review processes. Subjects gave written informed consent or assent with parental consent.GenotypingDNA was prepared from buffy coat preparations by a modification of the procedure of Miller, Dykes, and Polesky (30Miller S.A. Dykes D.D. Polesky H.F. A simple salting out procedure for extracting DNA from human nucleated cells.Nucleic Acids Res. 1988; 16: 1215Crossref PubMed Scopus (17698) Google Scholar) using Puregene reagents (Gentra, Minneapolis, MN). Genotyping (except rs7679) used the Illumina CVD chip of ∼2,100 loci with possible effects in vascular disease, including inflammation, lipid, and oxidation pathways. The effects of PLTP were prespecified as a locus of interest based on our longstanding investigation of this trait. TagSNPs use the correlations among SNPs to type SNPs that represent most common variation in the gene. As the tag SNPs were selected for a multiethnic cohort, some were uncommon and others redundant in Caucasians. Only one SNP's call rate was <99%: rs4810482 had a call rate of 97.4%. Genotype distributions were tested for departure from Hardy-Weinberg equilibrium (HWE) proportions. SNP rs4810482 and rs6073950 genotype distributions were weakly inconsistent with HWE (P values 0.01 to 0.05). Rs7679 was not included on the Illumina chip. It was genotyped by Taqman using Assay by Design. The Taqman genotypes were contrasted with imputed genotypes.While all SNPs listed in Table 1 were genotyped, we also imputed all chromosome 20 regional SNPs identified in the HapMap spanning base pair 43,907,883 to 44,027,635 for the unrelated discovery sample in an effort to determine which is the best predictor of PLTP activity. SNPs were imputed in BIMBAM, based on linkage disequilibrium (LD) in the HapMap phased genotypes (build 36; Caucasian population; www.hapmap.org), were analyzed both by a Bayesian procedure that allowed weighting by imputed genotype confidence as well as analyzed using the most likely genotype as estimated by PLINK. SNP rs7679 was both imputed and genotyped. Ten of 932 rs7679 genotypes were discordant. Repeat reading by a second blinded technician supported the molecular versus the inferred genotype. SNP rs7679 was consistent with HWE and had a 100% call rate in both samples. All genotyping was blinded to all phenotypes.PLTP activityPlasma phospholipid transfer activity mediated by PLTP was determined on fasting whole plasma by measuring the transfer of 14C-phosphatidycholine from phospholipid liposomes to HDL using an established radioassay (31Tishkoff S.A. Dietzsch E. Speed W. Pakstis A.J. Kidd J.R. Cheung K. Bonne-Tamir B. Santachiara-Benerecetti A.S. Moral P. Krings M. Global patterns of linkage disequilibrium at the CD4 locus and modern human origins.Science. 1996; 271: 1380-1387Crossref PubMed Scopus (485) Google Scholar). Briefly, each assay tube contained 50 µl HDL3 with 150 nmol phospholipids, 50 µl 14C-phosphatidycholine-labeled liposomes containing 50 nmol phosphatidylcholine, 50 µl of plasma samples prediluted 1:50 with Tris buffer (0.01 mM Tris, 150 mM NaCl, 1 mM EDTA, and 0.01% sodium azide), and 250 µl Tris buffer to bring the total assay volume to 400 µl. The HDL3(d 1.125–1.21) was isolated from pooled plasma from fasting individuals by sequential ultracentrifugation and subsequently passed over a heparin Sepharose column to remove apolipoprotein E (apoE) and residual PLTP activity. Three separate dilutions of each plasma sample were assayed. All samples were incubated at 37°C for 15 min, a condition when the rate of transfer of phospholipids from liposomes to HDL is linear. Donor and acceptor particles were separated by precipitation with dextran sulfate and magnesium chloride (32Warnick G.R. Benderson J. Albers J.J. Dextran sulfate-Mg2+ precipitation procedure for quantitation of high-density-lipoprotein cholesterol.Clin. Chem. 1982; 28: 1379-1388Crossref PubMed Scopus (1807) Google Scholar). Plasma PLTP activity was calculated as the percentage of total radioactivity per assay tube transferred to HDL minus background transfer (tubes without plasma). Three plasma samples stored at −70°C until use were included in each assay to control for interassay variation. PLTP activity phenotypes in both the CLEAR and family cohorts were determined in the Albers lab, blinded to all other subject data.Lipids, hemoglobin A1c, and insulinLipid measurements were performed on fasting whole plasma, as detailed elsewhere (33Jarvik G.P. Rozek L.S. Brophy V.H. Hatsukami T.S. Richter R.J. Schellenberg G.D. Furlong C.E. Paraoxonase (PON1) phenotype is a better predictor of vascular disease than is PON1(192) or PON1(55) genotype.Arterioscler. Thromb. Vasc. Biol. 2000; 20: 2441-2447Crossref PubMed Scopus (287) Google Scholar). Hemoglobin A1c (HbA1c) was measured using the standard HPLC methods. Insulin was measured by double antibody radioimmunoassay. Each assay includes appropriate reference samples.AnalysisLogistic regression considered an additive genetic model to predict CAAD status, with genotypes coded linearly as 0, 1, and 2. Age and current smoking were considered as covariates for CAAD prediction. Based on the SNPs that predicted CAAD, a subsample was evaluated for SNP prediction of ln-PLTP activity, considering age, BMI, and HbA1c as covariates. These covariates were selected due to prior repeats of PLTP associations with the metabolic syndrome and that glucose has been shown to regulate PLTP expression (34Tu A.Y. Albers J.J. Glucose regulates the transcription of human genes relevant to HDL metabolism: responsive elements for peroxisome proliferator-activated receptor are involved in the regulation of phospholipid transfer protein.Diabetes. 2001; 50: 1851-1856Crossref PubMed Scopus (62) Google Scholar), as discussed below. SNPS that predicted PLTP activity were then further considered for their independent effects on PLTP activity in multi-SNP models including the same covariates. Linkage disequilibrium of the SNPs was evaluated by considering the squared correlation between alleles at two sites, r2. PLTP activity was also measured on the second cohort of 210 subjects in four families ascertained for familial combined hyperlipidemia. In order to use the largest possible sample, we chose to ignore relatedness in the family cohort. This does not influence the estimated effect sizes of genotype effects but could affect the statistical tests by underestimating the variance (35Durbin J. Watson G.S. Testing for serial correlation in least squares regression. I.Biometrika. 1950; 37: 409-428PubMed Scopus (0) Google Scholar, 36Tregouet D.A. Ducimetiere P. Tiret L. Testing association between candidate-gene markers and phenotype in related individuals, by use of estimating equations.Am. J. Hum. Genet. 1997; 61: 189-199Abstract Full Text PDF PubMed Scopus (78) Google Scholar). Single SNP tests of SNPs associated with PLTP activity for effects on other phenotypes, including lipids, used the same models and covariates. Lipid tests performed on the entire cases control cohort of 939 subjects and were repeated on only 511 the subjects who were not taking lipid lowering medications. Due to correlations among SNPs and among phenotypes, it is not possible to quantitate the number of independent tests made; thus, adjustments for multiple contrasts were not made. HbA1c was not available for the family, and serum insulin levels served as a covariate instead, along with age, sex, BMI, and an age by sex interaction term. Insulin level was imputed for 20 subjects, using the regression of insulin on age, sex, BMI, and ln-triglyceride level. Individuals with isolated missing data for single SNPs or lipid phenotypes of interest were dropped from that analysis. Imputation of untyped SNPs was performed using the program BIMBAM (37Servin B. Stephens M. Imputation-based analysis of association studies: candidate regions and quantitative traits.PLoS Genet. 2007; 3: e114Crossref PubMed Scopus (378) Google Scholar, 38Guan Y. Stephens M. Practical issues in imputation-based association mapping.PLoS Genet. 2008; 4: e1000279Crossref PubMed Scopus (129) Google Scholar). Other statistical tests utilized program packages PLINK, R, and SPSS.RESULTSDemographics for the CLEAR cohort used for these analyses are reported in supplementary Table A. The PLTP region SNP effects on case status and PLTP activity in the CLEAR subsample and replication families are shown in Table 1. Four SNPs, rs6065904 (P = 0.023), rs11569668 (P = 0.009), rs1736493 (P = 0.015), and rs394643 (P = 0.024), were associated with CAAD status at the 0.05 level, without adj
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