A multilocus genotyping assay for candidate markers of cardiovascular disease risk.

A number of chronic diseases, including cardiovascular disease, appear to have a multifactorial genetic risk component. Consequently, techniques are needed to facilitate evaluation of complex genetic risk factors in large cohorts. We have designed a prototype assay for genotyping a panel of 35 biallelic sites that represent variation within 15 genes from biochemical pathways implicated in the development and progression of cardiovascular disease. Each DNA sample is amplified using two multiplex polymerase chain reactions, and the alleles are genotyped simultaneously using an array of immobilized, sequence-specific oligonucleotide probes. This multilocus assay was applied to two types of cohorts. Population frequencies for the markers were estimated using 496 unrelated individuals from a family-based cohort, and the observed values were consistent with previous reports. Linkage disequilibrium between consecutive pairs of markers within the apoCIII, LPL, and ELAM genes was also estimated. A preliminary analysis of single and pairwise locus associations with severity of atherosclerosis was performed using a composite cohort of 142 individuals for whom quantitative angiography data were available; evaluation of the potentially interesting associations observed will require analysis of an independent and larger cohort. This assay format provides a research tool for studies of multilocus genetic risk factors in large cardiovascular disease cohorts, and for the subsequent development of diagnostic tests.

[1]  S. Grundy,et al.  Task force 3. Spectrum of risk factors for coronary heart disease , 1996 .

[2]  J. Hoeg.  Evaluating coronary heart disease risk. Tiles in the mosaic. , 1997, JAMA.

[3]  P. de Knijff,et al.  Genetic heterogeneity of apolipoprotein E and its influence on plasma lipid and lipoprotein levels , 1994, Human mutation.

[4]  A. Tall,et al.  Organization of the human cholesteryl ester transfer protein gene. , 1990, Biochemistry.

[5]  S. Humphries,et al.  Gene-Environment Interaction in the Determination of Levels of Haemostatic Variables Involved in Thrombosis and Fibrinolysis , 1997, Thrombosis and Haemostasis.

[6]  A. Zwinderman,et al.  The Asp9 Asn mutation in the lipoprotein lipase gene is associated with increased progression of coronary atherosclerosis. REGRESS Study Group, Interuniversity Cardiology Institute, Utrecht, The Netherlands. Regression Growth Evaluation Statin Study. , 1996, Circulation.

[7]  Shirley A. Miller,et al.  A simple salting out procedure for extracting DNA from human nucleated cells. , 1988, Nucleic acids research.

[8]  S. Grundy,et al.  Association between a specific apolipoprotein B mutation and familial defective apolipoprotein B-100. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[9]  Philippe Amouyel,et al.  Deletion polymorphism in the gene for angiotensin-converting enzyme is a potent risk factor for myocardial infarction , 1992, Nature.

[10]  F. Soubrier,et al.  PCR detection of the insertion/deletion polymorphism of the human angiotensin converting enzyme gene (DCP1) (dipeptidyl carboxypeptidase 1). , 1992, Nucleic acids research.

[11]  J. Boer,et al.  Ser447stop mutation in lipoprotein lipase is associated with elevated HDL cholesterol levels in normolipidemic males. , 1997, Arteriosclerosis, thrombosis, and vascular biology.

[12]  Chun-Fang Xu,et al.  Association between genetic variation at the APO AI‐CIII‐AIV gene cluster and familial combined hyperlipidaemia , 1994, Clinical genetics.

[13]  D. Sanghera,et al.  Genetic polymorphism of paraoxonase and the risk of coronary heart disease. , 1997, Arteriosclerosis, thrombosis, and vascular biology.

[14]  M. Carrington,et al.  Discordant patterns of linkage disequilibrium of the peptide-transporter loci within the HLA class II region. , 1995, American journal of human genetics.

[15]  R. Matthews,et al.  A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase , 1995, Nature Genetics.

[16]  P. Ridker,et al.  PIA1/A2 polymorphism of platelet glycoprotein IIIa and risks of myocardial infarction, stroke, and venous thrombosis , 1997, The Lancet.

[17]  K. Mullis,et al.  Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. , 1987, Methods in enzymology.

[18]  L. Tiret,et al.  Angiotensin II type 1 receptor gene polymorphisms in human essential hypertension. , 1994, Hypertension.

[19]  H A Erlich,et al.  Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[20]  E. Boerwinkle,et al.  Haplotype structure and population genetic inferences from nucleotide-sequence variation in human lipoprotein lipase. , 1998, American journal of human genetics.

[21]  A. Nurden.  Polymorphisms of Human Platelet Membrane Glycoproteins: Structure and Clinical Significance , 1995, Thrombosis and Haemostasis.

[22]  P. Frosst,et al.  Seven novel mutations in the methylenetetrahydrofolate reductase gene and genotype/phenotype correlations in severe methylenetetrahydrofolate reductase deficiency. , 1995, American journal of human genetics.

[23]  E. Boerwinkle,et al.  DNA sequence diversity in a 9.7-kb region of the human lipoprotein lipase gene , 1998, Nature Genetics.

[24]  F. Cambien,et al.  Polymorphisms of the angiotensin-converting-enzyme gene in subjects who die from coronary heart disease. , 1994, The Quarterly journal of medicine.

[25]  H. Dietz,et al.  Single-strand conformation polymorphism analysis is a rapid and effective method for the identification of mutations and polymorphisms in the gene for glycoprotein IIIa. , 1993, Blood.

[26]  P. Frosst,et al.  Correlation of a common mutation in the methylenetetrahydrofolate reductase gene with plasma homocysteine in patients with premature coronary artery disease. , 1997, Arteriosclerosis, thrombosis, and vascular biology.

[27]  Steven C. Hunt,et al.  Molecular basis of human hypertension: Role of angiotensinogen , 1992, Cell.

[28]  D. Adler,et al.  The molecular basis of the human serum paraoxonase activity polymorphism , 1993, Nature Genetics.

[29]  P. Ridker,et al.  Mutation in the gene coding for coagulation factor V and the risk of myocardial infarction, stroke, and venous thrombosis in apparently healthy men. , 1995, The New England journal of medicine.

[30]  M. Hayden,et al.  Ethnic variation and in vivo effects of the -93t-->g promoter variant in the lipoprotein lipase gene. , 1997, Arteriosclerosis, thrombosis, and vascular biology.

[31]  W. Rutter,et al.  Polymorphic DNA region adjacent to the 5' end of the human insulin gene. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[32]  M. Hayden,et al.  Patients with apoE3 deficiency (E2/2, E3/2, and E4/2) who manifest with hyperlipidemia have increased frequency of an Asn 291-->Ser mutation in the human LPL gene. , 1995, Arteriosclerosis, thrombosis, and vascular biology.

[33]  J. Hixson.  Apolipoprotein E polymorphisms affect atherosclerosis in young males. Pathobiological Determinants of Atherosclerosis in Youth (PDAY) Research Group. , 1991, Arteriosclerosis and thrombosis : a journal of vascular biology.

[34]  M. Tsai,et al.  High prevalence of a mutation in the cystathionine beta-synthase gene. , 1996, American journal of human genetics.

[35]  N. Kalsheker,et al.  Angiotensinogen: molecular biology, biochemistry and physiology. , 1996, The international journal of biochemistry & cell biology.

[36]  P. Durrington,et al.  Alloenzymes of paraoxonase and effectiveness of high-density lipoproteins in protecting low-density lipoprotein against lipid peroxidation , 1997, The Lancet.

[37]  A. Marian,et al.  A variant of human paraoxonase/arylesterase (HUMPONA) gene is a risk factor for coronary artery disease. , 1995, The Journal of clinical investigation.

[38]  S. Young,et al.  An ApaLI restriction site polymorphism is associated with the MB19 polymorphism in apolipoprotein B. , 1989, Journal of lipid research.

[39]  V. Ramesh,et al.  Molecular basis of cystathionine beta-synthase deficiency in pyridoxine responsive and nonresponsive homocystinuria. , 1993, Human molecular genetics.

[40]  J. Kraus,et al.  Screening for mutations by expressing patient cDNA segments in E. coli: Homocystinuria due to cystathionine β‐synthase deficiency , 1992, Human Mutation.

[41]  V. Gudnason,et al.  Spectrum of LDL receptor gene mutations in heterozygous familial hypercholesterolemia , 1997, Human mutation.

[42]  B. Hainque,et al.  Familial hypertrophic cardiomyopathy: from mutations to functional defects. , 1998, Circulation research.

[43]  S. Stein,et al.  Preparation of oligonucleotide-peptide conjugates. , 1991, Bioconjugate chemistry.

[44]  M J Malloy,et al.  A Multilocus Genotyping Assay for Cardiovascular Disease , 1998, Clinical chemistry and laboratory medicine.

[45]  P. Mannucci,et al.  A common mutation in the methylenetetrahydrofolate reductase gene (C677T) increases the risk for deep-vein thrombosis in patients with mutant factor V (factor V:Q506). , 1997, Arteriosclerosis, thrombosis, and vascular biology.

[46]  R Kreutz,et al.  A prospective evaluation of an angiotensin-converting-enzyme gene polymorphism and the risk of ischemic heart disease. , 1995, The New England journal of medicine.

[47]  M. Baur,et al.  Analysis of Multilocus Pedigree Data by Computer , 1984 .

[48]  A. Tall,et al.  Increased coronary heart disease in Japanese-American men with mutation in the cholesteryl ester transfer protein gene despite increased HDL levels. , 1996, The Journal of clinical investigation.

[49]  P. Mannucci.  Recent progress in the pathophysiology of fibrinogen. , 1995, European heart journal.

[50]  D. Ubben,et al.  Fibrinogen as a major risk factor in cardiovascular disease. , 1990, Trends in pharmacological sciences.

[51]  K. Mullis,et al.  Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. , 1988, Science.

[52]  B. Haldane.  THE ESTIMATION AND SIGNIFICANCE OF THE LOGARITHM OF A RATIO OF FREQUENCIES , 1956, Annals of human genetics.

[53]  M. P. Sperandeo,et al.  A 68-bp insertion found in a homocystinuric patient is a common variant and is skipped by alternative splicing of the cystathionine beta-synthase mRNA. , 1996, American journal of human genetics.

[54]  H. Mcgill,et al.  Determinants of atherosclerosis in the young , 1998 .

[55]  M. Emi,et al.  Direct detection and automated sequencing of individual alleles after electrophoretic strand separation: identification of a common nonsense mutation in exon 9 of the human lipoprotein lipase gene. , 1990, Nucleic acids research.

[56]  J W Jukema,et al.  The role of a common variant of the cholesteryl ester transfer protein gene in the progression of coronary atherosclerosis. The Regression Growth Evaluation Statin Study Group. , 1998, The New England journal of medicine.

[57]  P. Frosst,et al.  Molecular genetic analysis in mild hyperhomocysteinemia: a common mutation in the methylenetetrahydrofolate reductase gene is a genetic risk factor for cardiovascular disease. , 1996, American journal of human genetics.

[58]  B. Guy-grand,et al.  Preferential cholesteryl ester acceptors among triglyceride-rich lipoproteins during alimentary lipemia in normolipidemic subjects. , 1998, Arteriosclerosis, thrombosis, and vascular biology.

[59]  K. Ishimura-Oka,et al.  Structure and polymorphic map of human lipoprotein lipase gene. , 1990, Biochimica et biophysica acta.

[60]  G. Siest,et al.  Combined effects of lipoprotein lipase and apolipoprotein E polymorphisms on lipid and lipoprotein levels in the Stanislas cohort. , 1997, Journal of lipid research.

[61]  Pieter H. Reitsma,et al.  Mutation in blood coagulation factor V associated with resistance to activated protein C , 1994, Nature.

[62]  J. Hartley,et al.  Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. , 1990, Gene.

[63]  D. Valle,et al.  Characterization of a cystathionine beta-synthase allele with three mutations in cis in a patient with B6 nonresponsive homocystinuria. , 1994, Human molecular genetics.

[64]  G. Baumann,et al.  E-selectin polymorphism and atherosclerosis: an association study. , 1994, Human molecular genetics.

[65]  R. Lewontin.  The Interaction of Selection and Linkage. I. General Considerations; Heterotic Models. , 1964, Genetics.

[66]  D. Kromhout,et al.  A frequently occurring mutation in the lipoprotein lipase gene (Asn291Ser) contributes to the expression of familial combined hyperlipidemia. , 1995, Human molecular genetics.

[67]  P Ducimetière,et al.  Synergistic effects of angiotensin-converting enzyme and angiotensin-II type 1 receptor gene polymorphisms on risk of myocardial infarction , 1994, The Lancet.

[68]  J. Weiss,et al.  A polymorphism of a platelet glycoprotein receptor as an inherited risk factor for coronary thrombosis. , 1996, The New England journal of medicine.

[69]  JoAnn E. Manson,et al.  Design of the Women's Health Initiative clinical trial and observational study. The Women's Health Initiative Study Group. , 1998, Controlled clinical trials.

[70]  K. Mann,et al.  Factor VLeiden and thrombophilia. , 1997, Arteriosclerosis, thrombosis, and vascular biology.

[71]  S. Young,et al.  Apolipoprotein B allotypes MB19(1) and MB19(2) in subjects with coronary artery disease and hypercholesterolemia. , 1987, Arteriosclerosis.

[72]  R. Mahley,et al.  Apolipoprotein E: cholesterol transport protein with expanding role in cell biology. , 1988, Science.

[73]  J. Kraus,et al.  Molecular defect in a patient with pyridoxine-responsive homocystinuria. , 1993, Human molecular genetics.

[74]  G Siest,et al.  Objectives, Design and Recruitment of a Familial and Longitudinal Cohort for Studying Gene-Environment Interactions in the Field of Cardiovascular Risk: The Stanislas Cohort , 1998, Clinical chemistry and laboratory medicine.

[75]  J. Viikari,et al.  Apolipoprotein B polymorphism and altered apolipoprotein B and low density lipoprotein cholesterol concentrations in Finnish children , 1988, British medical journal.

[76]  T. Dawber,et al.  Epidemiological approaches to heart disease: the Framingham Study. , 1951, American journal of public health and the nation's health.

[77]  R. Hegele,et al.  A polymorphism of the paraoxonase gene associated with variation in plasma lipoproteins in a genetic isolate. , 1995, Arteriosclerosis, thrombosis, and vascular biology.

[78]  P. Talmud,et al.  Genetic factors affecting the consistency and magnitude of changes in plasma cholesterol in response to dietary challenge. , 1996, QJM : monthly journal of the Association of Physicians.

[79]  S. Yamashita,et al.  A missense mutation in the cholesteryl ester transfer protein gene with possible dominant effects on plasma high density lipoproteins. , 1993, The Journal of clinical investigation.

[80]  M. Blombäck,et al.  The Role of β-Fibrinogen Genotype in Determining Plasma Fibrinogen Levels in Young Survivors of Myocardial Infarction and Healthy Controls from Sweden , 1993, Thrombosis and Haemostasis.

[81]  H. Hobbs,et al.  Molecular genetics of the LDL receptor gene in familial hypercholesterolemia , 1992, Human mutation.

[82]  M. P. Sperandeo,et al.  The molecular basis of homocystinuria due to cystathionine beta-synthase deficiency in Italian families, and report of four novel mutations. , 1995, American journal of human genetics.

[83]  S. Deeb,et al.  A mutation in the promoter of the lipoprotein lipase (LPL) gene in a patient with familial combined hyperlipidemia and low LPL activity. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[84]  V. Gudnason,et al.  Interaction of the cholesteryl ester transfer protein I405V polymorphism with alcohol consumption in smoking and non‐smoking healthy men, and the effect on plasma HDL cholesterol and apoAI concentration , 1997, Clinical genetics.

[85]  E. Boerwinkle,et al.  Genetic Variant Showing a Positive Interaction With β-Blocking Agents With a Beneficial Influence on Lipoprotein Lipase Activity, HDL Cholesterol, and Triglyceride Levels in Coronary Artery Disease Patients: The Ser447-Stop Substitution in the Lipoprotein Lipase Gene , 1997 .

[86]  P. Talmud,et al.  A common mutation in the lipoprotein lipase gene promoter, -93T/G, is associated with lower plasma triglyceride levels and increased promoter activity in vitro. , 1997, Arteriosclerosis, thrombosis, and vascular biology.

[87]  S. Humphries,et al.  A common variant in the gene for lipoprotein lipase (Asp9→Asn) : functional implications and prevalence in normal and hyperlipidemic subjects , 1995 .

[88]  L. Peltonen,et al.  Array-based multiplex analysis of candidate genes reveals two independent and additive genetic risk factors for myocardial infarction in the Finnish population. , 1998, Human molecular genetics.

[89]  D. Levy,et al.  Prediction of coronary heart disease using risk factor categories. , 1998, Circulation.

[90]  J. Wetmur.  DNA probes: applications of the principles of nucleic acid hybridization. , 1991, Critical reviews in biochemistry and molecular biology.

[91]  P. Talmud,et al.  Hyperlipidaemia associated with genetic variation in the apolipoprotein B gene. , 1995, Current opinion in lipidology.

[92]  D. T. Vernier,et al.  Restriction isotyping of human apolipoprotein E by gene amplification and cleavage with HhaI. , 1990, Journal of lipid research.

[93]  Weiqun,et al.  Familial ligand-defective apolipoprotein B. Identification of a new mutation that decreases LDL receptor binding affinity. , 1995, The Journal of clinical investigation.

[94]  A. Tall.  Plasma lipid transfer proteins. , 1995, Annual Review of Biochemistry.

[95]  J M Lalouel,et al.  Angiotensin-converting enzyme gene polymorphism is associated with myocardial infarction but not with development of coronary stenosis. , 1995, Circulation.

[96]  R. Aster,et al.  The human platelet alloantigens, PlA1 and PlA2, are associated with a leucine33/proline33 amino acid polymorphism in membrane glycoprotein IIIa, and are distinguishable by DNA typing. , 1989, The Journal of clinical investigation.

[97]  J. Breslow,et al.  An apolipoprotein CIII haplotype protective against hypertriglyceridemia is specified by promoter and 3' untranslated region polymorphisms. , 1993, Proceedings of the National Academy of Sciences of the United States of America.