Genetic variation of PLTP modulates lipoprotein profiles in hypoalphalipoproteinemia

Phospholipid transfer protein (PLTP) participates in key processes in lipoprotein metabolism, including interparticle phospholipid transfer, remodeling of HDL, cholesterol and phospholipid efflux from peripheral tissues, and the production of hepatic VLDL. The impact of PLTP on reverse cholesterol transport suggests that the gene may harbor sequence anomalies that contribute to disorders of HDL metabolism. The human PLTP gene was screened for sequence anomalies by DNA melting analysis in 276 subjects with hypoalphalipoproteinemia (HA) and 364 controls. The association with plasma lipid parameters was evaluated. We discovered 18 sequence variations, including four missense mutations and a novel polymorphism (c.-34G>C). In healthy controls, the c.-34G>C minor allele was associated with higher high density lipoprotein-cholesterol (HDL-C) and was depleted in subjects with HA. Linear regression models predict that possession of the rare allele decreases plasma triglyceride (TG) and TG/HDL-C and increases HDL-C independent of TG. Decreased PLTP activity was observed in one (p.R235W) of four (p.E72G, p.S119A, p.S124Y, and p.R235W) mutations in an in vitro activity assay. These findings indicate that PLTP gene variation is an important determinant of plasma lipoproteins and affects disorders of HDL metabolism. Phospholipid transfer protein (PLTP) participates in key processes in lipoprotein metabolism, including interparticle phospholipid transfer, remodeling of HDL, cholesterol and phospholipid efflux from peripheral tissues, and the production of hepatic VLDL. The impact of PLTP on reverse cholesterol transport suggests that the gene may harbor sequence anomalies that contribute to disorders of HDL metabolism. The human PLTP gene was screened for sequence anomalies by DNA melting analysis in 276 subjects with hypoalphalipoproteinemia (HA) and 364 controls. The association with plasma lipid parameters was evaluated. We discovered 18 sequence variations, including four missense mutations and a novel polymorphism (c.-34G>C). In healthy controls, the c.-34G>C minor allele was associated with higher high density lipoprotein-cholesterol (HDL-C) and was depleted in subjects with HA. Linear regression models predict that possession of the rare allele decreases plasma triglyceride (TG) and TG/HDL-C and increases HDL-C independent of TG. Decreased PLTP activity was observed in one (p.R235W) of four (p.E72G, p.S119A, p.S124Y, and p.R235W) mutations in an in vitro activity assay. These findings indicate that PLTP gene variation is an important determinant of plasma lipoproteins and affects disorders of HDL metabolism. Hypoalphalipoproteinemia [HA (Online Mendelian Inheritance in Man, entry: 604091)] refers to a category of HDL deficiency (less than the population-based 10th percentile) at risk for premature coronary artery disease (1Ordovas J.M. Schaefer E.J. Coronary artery disease, lipid disorders and genetic polymorphisms.Ann. Biol. Clin. (Paris). 1988; 46: 24-29PubMed Google Scholar). Whereas ablative mutations in several genes (e.g., ABCA1, APOA1, APOC3, LCAT, and LPL) have been characterized in discrete disorders of HDL metabolism, as a group they explain only a fraction of the cases of primary high density lipoprotein-cholesterol (HDL-C) deficiency (2Miller M. Zhan M. Genetic determinants of low high-density lipoprotein cholesterol.Curr. Opin. Cardiol. 2004; 19: 380-384Crossref PubMed Scopus (34) Google Scholar). Given the estimated heritability of HDL-C levels (∼50%) (3Austin M.A. King M.C. Bawol R.D. Hulley S.B. Friedman G.D. Risk factors for coronary heart disease in adult female twins. Genetic heritability and shared environmental influences.Am. J. Epidemiol. 1987; 125: 308-318Crossref PubMed Scopus (184) Google Scholar), the search for the primary genetic determinants is not only desirable, but tenable. A combination of in vivo and in vitro studies has demonstrated the participation of phospholipid transfer protein (PLTP) in several key processes in lipoprotein metabolism, including the transfer of phospholipids from triglyceride (TG)-rich lipoproteins to HDL particles (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 (321) Google Scholar), the remodeling of HDL (5Settasatian N. Duong M. Curtiss L.K. Ehnholm C. Jauhiainen M. Huuskonen J. Rye K.A. The mechanism of the remodeling of high density lipoproteins by phospholipid transfer protein.J. Biol. Chem. 2001; 276: 26898-26905Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), the ABCA1-mediated efflux of cholesterol and phospholipids from peripheral tissues to HDL (6Oram J.F. Wolfbauer G. Vaughan A.M. Tang C. Albers J.J. Phospholipid transfer protein interacts with and stabilizes ATP-binding cassette transporter A1 and enhances cholesterol efflux from cells.J. Biol. Chem. 2003; 278: 52379-52385Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar), and its participation in hepatic VLDL synthesis (7Lie 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, 8Jiang X.C. Li Z. Liu R. Yang X.P. Pan M. Lagrost L. Fisher E.A. Williams K.J. Phospholipid transfer protein deficiency impairs apolipoprotein-B secretion from hepatocytes by stimulating a proteolytic pathway through a relative deficiency of vitamin E and an increase in intracellular oxidants.J. Biol. Chem. 2005; 280: 18336-18340Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 9van Haperen R. van Tol A. van Gent T. Scheek L. Visser P. van der Kamp A. Grosveld F. de Crom R. Increased risk of atherosclerosis by elevated plasma levels of phospholipid transfer protein.J. Biol. Chem. 2002; 277: 48938-48943Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Thus, the analysis of human genetic variation within the PLTP gene may provide important clues to the importance of PLTP in HDL-associated comorbidities [e.g., dyslipidemia, insulin resistance, and obesity (10Bosse Y. Bouchard L. Despres J.P. Bouchard C. Perusse L. Vohl M.C. Haplotypes in the phospholipid transfer protein gene are associated with obesity-related phenotypes: the Quebec Family Study.Int. J. Obes. (Lond.). 2005; 29: 1338-1345Crossref PubMed Scopus (14) Google Scholar)] and outcomes (e.g., type 2 diabetes, metabolic syndrome, and myocardial infarction). Although the PLTP gene was previously studied in French Canadian (10Bosse Y. Bouchard L. Despres J.P. Bouchard C. Perusse L. Vohl M.C. Haplotypes in the phospholipid transfer protein gene are associated with obesity-related phenotypes: the Quebec Family Study.Int. J. Obes. (Lond.). 2005; 29: 1338-1345Crossref PubMed Scopus (14) Google Scholar) and Finnish (11Tahvanainen E. Jauhiainen M. Funke H. Vartiainen E. Sundvall J. Ehnholm C. Serum phospholipid transfer protein activity and genetic variation of the PLTP gene.Atherosclerosis. 1999; 146: 107-115Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) samples, the inclusion of only 19 and 24 subjects, respectively, for resequencing efforts would be likely to reveal only polymorphisms that are relatively frequent. Although 48 alleles (11Tahvanainen E. Jauhiainen M. Funke H. Vartiainen E. Sundvall J. Ehnholm C. Serum phospholipid transfer protein activity and genetic variation of the PLTP gene.Atherosclerosis. 1999; 146: 107-115Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) are in theory sufficient to identify variations with 1% frequency (e.g., 1 heterozygote in 24), sampling error can easily mask such discoveries. We chose to conduct a broader mutation detection effort in an attempt to identify both rare and common sequence anomalies within the transcribed region and exon-intron boundaries of PLTP. The aims of this study, therefore, were to screen a case-control cohort for sequence anomalies in the PLTP gene and to test for genetic association with HA and/or biochemical measurements. An additional aim was to test for significant differences in biochemical properties, including lipoprotein compartments, associated with sequence variations. These analyses were conducted in two groups: one with HDL-C less than the 10th percentile for gender, and a healthy control group. We report a number of new sequence anomalies within the PLTP transcribed region and provide evidence consistent with a role for variation in the PLTP gene in regulating HDL-C. Importantly, functional studies of four newly discovered missense mutations resulted in the identification of a missense mutant (p.R235W) with altered PLTP activity and support the recent observation that both common polymorphisms (c.-34G>C) and rare functional sequence anomalies are independent sources of HDL deficiency. This study was a retrospective analysis using a racially and ethnically diverse study population sample. The study population was selected from the University of California San Francisco (UCSF) Genomic Resource in Arteriosclerosis (GRA). Subjects with HA (n = 276) were identified from the GRA as individuals with HDL-C less than the 19th percentile for their age and gender (approximately <35 mg/dl and <45 mg/dl for men and women, respectively). To control for HA secondary to increased transfer of cholesteryl ester to TG-rich lipoproteins in this group, individuals with TG > 3 SD from the mean were excluded. Healthy controls (n = 364) were selected from the GRA as individuals without a primary form of dyslipidemia (e.g., HA). Subjects were of non-Hispanic Caucasian (European) ancestry. All subjects gave informed consent, and the UCSF Committee on Human Research approved the study protocol. Genomic DNA was prepared from whole blood obtained from patients in the GRA population of UCSF (12Pullinger C.R. Hennessy L.K. Chatterton J.E. Liu W. Love J.A. Mendel C.M. Frost P.H. Malloy M.J. Schumaker V.N. Kane J.P. Familial ligand-defective apolipoprotein B. Identification of a new mutation that decreases LDL receptor binding affinity.J. Clin. Invest. 1995; 95: 1225-1234Crossref PubMed Scopus (164) Google Scholar). Blood was drawn after a 10 h fast, and lipoprotein quantification was carried out using standard protocols (13Rush R.L. Leon L. Turrell J. Advances in Automated Analysis, Technicon International Congress 1970.Vol. 1. Thurman Assoc., Miami, FL1971: 503Google Scholar, 14Warnick 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 (1811) Google Scholar, 15Kane J.P. Malloy M.J. Ports T.A. Phillips N.R. Diehl J.C. Havel R.J. Regression of coronary atherosclerosis during treatment of familial hypercholesterolemia with combined drug regimens.J. Am. Med. Assoc. 1990; 264: 3007-3012Crossref PubMed Scopus (700) Google Scholar, 16Havel R.J. Eder H.A. Bragdon J.H. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum.J. Clin. Invest. 1955; 34: 1345-1353Crossref PubMed Scopus (6476) Google Scholar). Standards were provided by the Centers for Disease Control and Prevention (Atlanta, GA). Baseline lipoprotein measurements were obtained when patients had received no lipid-lowering medication for at least 1 month, a standard “wash-out” period for such medications (12Pullinger C.R. Hennessy L.K. Chatterton J.E. Liu W. Love J.A. Mendel C.M. Frost P.H. Malloy M.J. Schumaker V.N. Kane J.P. Familial ligand-defective apolipoprotein B. Identification of a new mutation that decreases LDL receptor binding affinity.J. Clin. Invest. 1995; 95: 1225-1234Crossref PubMed Scopus (164) Google Scholar). A combination of denaturing high-performance liquid chromatography (dHPLC) (17O'Donovan M.C. Oefner P.J. Roberts S.C. Austin J. Hoogendoorn B. Guy C. Speight G. Upadhyaya M. Sommer S.S. McGuffin P. Blind analysis of denaturing high-performance liquid chromatography as a tool for mutation detection.Genomics. 1998; 52: 44-49Crossref PubMed Scopus (289) Google Scholar) and denaturing gradient gel electrophoresis (DGGE) (18Fischer S.G. Lerman L.S. DNA fragments differing by single base-pair substitutions are separated in denaturing gradient gels: correspondence with melting theory.Proc. Natl. Acad. Sci. USA. 1983; 80: 1579-1583Crossref PubMed Scopus (741) Google Scholar) mutation detection was used to scan the 10 amplicons that spanned the 16 exons encoding the full-length PLTP transcript (BC01984) using standard protocols. Exon 5, which is absent in one of the two known PLTP splice variants (19Schneider M. Verges B. Klein A. Miller E.R. Deckert V. Desrumaux C. Masson D. Gambert P. J.M. J. in plasma vitamin E distribution in type 2 patients with elevated plasma phospholipid transfer protein 2004; PubMed Scopus Google Scholar), was in the (dHPLC) electrophoresis (DGGE) consistent with sequence were studied by DNA to identify the DNA variations. for and sequence are at A was used for the of and mutant PLTP PLTP in was a from Ehnholm was carried out using the were in with and were on the The were using to the and in were after to and used for PLTP transfer activity and PLTP activity was using a M. J. R. van Tol A. Ehnholm C. Human plasma phospholipid transfer protein high density lipoprotein Biol. Chem. Full Text PDF PubMed Google Scholar, J. J. G. of and human plasma high density of a plasma using a transfer 1982; PubMed Scopus Google Scholar). In this the of PLTP to transfer phospholipids from to ultracentrifugally particles was PLTP was obtained from were using separated on and using the J. Jauhiainen M. Ehnholm C. and secretion of human plasma phospholipid transfer protein.J. Lipid Res. 1998; Full Text Full Text PDF PubMed Google Scholar) from Jauhiainen and Ehnholm PLTP with the type was obtained by and quantitation of the PLTP PLTP activity was obtained by the activity by and is relative to the PLTP PLTP activity was and was to a of All mutations were in four and both and controls. analyses were conducted using the for for standard Scholar). and were by the for in controls, and association in cases controls, were by of of used the independent was to be for the of of of used the The procedure was used for regression of were to the of the the and of the for levels of factors were using the with were in the are as The of subjects with HA and healthy controls are in 1. with healthy controls, HA subjects increased = and = In HA subjects increased cholesterol = = and = The findings are in by the in the of subjects the UCSF GRA to the UCSF Lipid as a of increased Given their in the of HA the of the differences in TG and HDL-C was not an of insulin and a important risk were to be increased in HA was increased in HA Subjects in the HA group not with to age of the study = 1 = = = = = = = = = of not not in a new of not not analysis of the 16 exons of the PLTP gene in both the HA and healthy control resulted in the identification of several sequence including a single polymorphism in 1 missense mutations were identified in four subjects, of which are novel (p.E72G, and mutation and a splice mutation were identified in two subjects with HA and and sequence anomalies identified in the PLTP sequence variations are to Human allele allele of the number phospholipid transfer sequence anomalies are using standard single sequence variations are to Human allele number in a new phospholipid transfer sequence anomalies are using standard single mutations of PLTP were in was carried by of PLTP functional and for protein to study the in lipid transfer of mutant of the mutations transfer activity to that of protein one mutation in activity in We were to for cholesterol efflux studies from this A of the was screened for the mutation by and was to be All of the mutations were and from the as as the the transfer activity of the mutant is not the of and secretion but a functional of the PLTP activity in the four PLTP > = with PLTP activity are mean from four independent and are as of the activity of = with PLTP activity in a new The are mean from four independent and are as of the activity of PLTP. of the of the c.-34G>C polymorphism on lipoprotein using lipoprotein as a metabolic significant in mean of plasma lipoprotein were In the control population of the allele increased HDL-C were in HA subjects evidence of an association with increased in of the allele was observed in healthy controls but not in HA subjects lipid in the control by = = = = = = = = not Lipid measurements are in in a new lipid in the HA by is not provided for this used in the of is not provided for this used in the of not Lipid measurements are in A is not provided for this used in the of in a new not Lipid measurements are in not Lipid measurements are in and for the c.-34G>C in cases controls are in The c.-34G>C distribution not from = 1 of = with healthy controls, the frequency of allele was in HA subjects = 1 of = = significant gender differences were observed with to c.-34G>C allele The observed c.-34G>C minor allele frequency was and in controls and and in cases for and c.-34G>C variation allele and and were by gene in a new and were by gene for known to be of lipoprotein (e.g., gender, and increased the observed c.-34G>C and HDL-C and TG/HDL-C and provided evidence of an association with TG. In for the observed association with Regression models for lipid the polymorphism and of the minor The of the gender, and category healthy were population and The models in which a polymorphism not are not In the models that the polymorphism was and is in to PLTP c.-34G>C = age = = age = TG = = measurements are in in the were at the in the = age = = age = TG = = in a new Lipid measurements are in in the were at the in the plasma TG (n = for the selected selected category = category = and age = the for the was In c.-34G>C minor allele the in TG was HDL-C (n = for the selected selected category gender = age = category = = and the of TG = the for the was In healthy controls, the in HDL-C of the allele and was In HA subjects, the the two was the TG/HDL-C (n = for the selected selected category gender = and = the for the was In c.-34G>C minor allele the in TG/HDL-C was role in phospholipid transfer from TG-rich phospholipid and cholesterol from peripheral and participation in hepatic synthesis of VLDL indicate that PLTP is a of HDL metabolism. In this study, discovered a polymorphism in 1 of PLTP that is associated with in lipoprotein in subjects with HA healthy controls. in possession of the c.-34G>C minor allele higher HDL-C. The minor allele was associated with lower plasma TG and a lower TG/HDL-C Importantly, PLTP activity was observed in one (p.R235W) of four (p.E72G, p.S119A, p.S124Y, and p.R235W) missense mutations in an in vitro activity assay. Although of the missense mutations was common in this study they have the to of PLTP and the impact of missense mutations on PLTP can be within the of the by Desrumaux and C. C. A. J. J. M. F. A at the of the human plasma phospholipid transfer protein is for activity on high density lipoproteins.J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). The mutation to a of and this mutant is to be when by This is by and analysis of PLTP using of the at likely to be important in binding to lipid G. and functional determinants of human plasma phospholipid transfer protein activity as by of 2003; PubMed Scopus Google Scholar). The mutant low binding for HDL and phospholipid transfer activity G. and functional determinants of human plasma phospholipid transfer protein activity as by of 2003; PubMed Scopus Google Scholar). In to the in secretion observed for the PLTP mutant studied by and G. and functional determinants of human plasma phospholipid transfer protein activity as by of 2003; PubMed Scopus Google Scholar), the PLTP mutant no such This the that are no the secretion of the mutant the mutation affects the of the studies are to the functional impact of is likely that is in with a in the Although the two polymorphisms previously (11Tahvanainen E. Jauhiainen M. Funke H. Vartiainen E. Sundvall J. Ehnholm C. Serum phospholipid transfer protein activity and genetic variation of the PLTP gene.Atherosclerosis. 1999; 146: 107-115Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) were not in the 1 in this study, the polymorphisms and c.-34G>C be in The c.-34G>C minor allele was associated with in and their is that the of this polymorphism was in HA the of this can only be in this of this in an independent and is TG and low HDL-C is a in individuals A. for an independent insulin and plasma triglyceride and insulin Med. PubMed Scopus Google Scholar). Importantly, an increased TG/HDL-C was to be as least as a of heart disease as high J. P. F. of high HDL cholesterol and LDL cholesterol to the of heart An in the Biol. PubMed Scopus Google Scholar). findings have been in cohort studies G. H. The importance of from the J. Epidemiol. PubMed Scopus Google Scholar, of a from J. Cardiol. Full Text PDF PubMed Scopus Google Scholar). Whereas studies Yang of genetic and heritability of triglyceride high density lipoprotein cholesterol in two 2003; PubMed Scopus Google Scholar) and A.M. J.M. Schaefer E.J. Y. for a gene the TG/HDL-C on a scan in the PubMed Scopus Google Scholar) have provided evidence for genetic determinants gene studies at the variants are Given the of PLTP in both hepatic VLDL production and HDL in (5Settasatian N. Duong M. Curtiss L.K. Ehnholm C. Jauhiainen M. Huuskonen J. Rye K.A. The mechanism of the remodeling of high density lipoproteins by phospholipid transfer protein.J. Biol. Chem. 2001; 276: 26898-26905Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, J.F. Wolfbauer G. Vaughan A.M. Tang C. Albers J.J. Phospholipid transfer protein interacts with and stabilizes ATP-binding cassette transporter A1 and enhances cholesterol efflux from cells.J. Biol. Chem. 2003; 278: 52379-52385Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 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, 9van Haperen R. van Tol A. van Gent T. Scheek L. Visser P. van der Kamp A. Grosveld F. de Crom R. Increased risk of atherosclerosis by elevated plasma levels of phospholipid transfer protein.J. Biol. Chem. 2002; 277: 48938-48943Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar), the of PLTP gene variation on and their demonstrated a of and suggests that the role of PLTP in dyslipidemia and risk study a of of primary HA. The UCSF Lipid is a to which individuals are for increased which explain the increased levels observed in this Although both and have detection R. University Scholar), is the that a of sequence variants were not using the mutation detection Although PLTP activity have on the phospholipid transfer an of the role of PLTP c.-34G>C in this of PLTP would have been of The HDL-C and risk of coronary heart disease is The role of PLTP in HDL and the that increased PLTP activity to be J.J. M.C. for phospholipid transfer protein in lipid and lipoprotein Opin. 2004; PubMed Scopus Google Scholar) an for This report the sequence analysis of the PLTP gene in and a novel polymorphism associated with in lipoprotein of this polymorphism in independent studies and of the functional impact of the missense mutations the in the study of PLTP gene variation in lipid metabolism. The the subjects for in this of in of and This was by from and and

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