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Association of single nucleotide polymorphisms in the LPA gene region with serum Lp(a) levels and myocardial infarction [Elektronische Ressource] / vorgelegt von Katharina Neureuther

88 pages
AssociationofsinglenucleotidepolymorphismsintheLPAgeneregionwithserumLp(a)levelsandmyocardialinfarctionDISSERTATIONZURERLANGUNGDESDOKTORGRADESDERNATURWISSENSCHAFTEN(DR.RER.NAT.)DERNATURWISSENSCHAFTLICHEN¨ ¨FAKULTATIII-BIOLOGIEUNDVORKLINISCHEMEDIZIN-DERUNIVERSITATREGENSBURGvorgelegt vonKatharina Neureutheraus RegensburgFebruar 2008Association of single nucleotide polymorphisms in the LPAgene region with serum Lp(a) levels and myocardial infarctionDissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer.nat.) der naturwissenschaftlichen Fakult¨at III - Biologie und vorklinische Medizin -der Universit¨at Regensburgvorgelegt vonKatharina Neureutheraus RegensburgFebruar 2008Promotionsgesuch eingereicht am: 06.02.2008Die Arbeit wurde angeleitet von: PD Dr. Thomas LangmanPrufungsau¨ schuss:Vorsitzender: Prof. Dr. Stephan SchneuwlyErstgutachter: PD Dr. Thomas LangmannZweitgutachter: Prof. Dr. med. Christian HengstenbergDritter Prufer:¨ Prof. Dr. Gernot Langst¨Quidquid agis prudenter agas et respice finem.
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Association of single nucleotide polymorphisms in theLPAgene region with serum Lp(a) levels and myocardial infarction
DISSERTATION ZUR ERLANGUNG DES DOKTORGRADES DER NATURWISSENSCHAFTEN (DR. RER. NAT.) DER NATURWISSENSCHAFTLICHEN ¨ ¨ FAKULTAT III - BIOLOGIE UND VORKLINISCHE MEDIZIN - DER UNIVERSITAT REGENSBURG
vorgelegt von
Katharina Neureuther
aus Regensburg
Februar 2008
Association of single nucleotide polymorphisms in theLPA gene region with serum Lp(a) levels and myocardial infarction
Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.)dernaturwissenschaftlichenFakult¨atIII-BiologieundvorklinischeMedizin-derUniversit¨atRegensburg
vorgelegt von Katharina Neureuther
aus Regensburg Februar 2008
Promotionsgesuch eingereicht am: 06.02.2008 Die Arbeit wurde angeleitet von: PD Dr. Thomas Langman
P ¨fungsauschuss: ru
Vorsitzender:
Erstgutachter:
Zweitgutachter:
Dritter Prufer: ¨
Prof. Dr. Stephan Schneuwly
PD Dr. Thomas Langmann
Prof. Dr. med. Christian Hengstenberg
Prof.Dr.GernotL¨angst
Quidquid agis prudenter agas et respice finem.
List
AA apo(a) asPCR BMI bp CAD CHD CI cM Da dbSNP EDTA FAM FBAT GWA HDL HW KIV-2 KV LD LDL LOD MAF MGB MI NCBI OD ON OR PCR QTL RefSeq SD SNP Taq TDT Tm TRIS UCSC VIC WHO
of
abbreviations
amino acid apolipoprotein (a) allele-specific PCR body mass index base pair coronary artery disease coronary heart disease confidence interval centiMorgan Dalton public single nucleotide database Ethylendiamintetraacetate 6-carboxyfluoresceine Family-Based Association Tests in genetic analyses genome-wide association high density lipoprotein Hardy-Weinberg kringle IV-like type 2 kringle V-like
linkage disequilibrium low density lipoprotein logarithm of the odds minor allele frequency minor groove binder
myocardial infarction National Center for Biotechnology Information optical density oligonucleotide, primer odds ratio
polymerase chain reaction quantitative trait locus reference sequence standard deviation single (or: simple) nucleotide polymorphism Thermus Aquaticus Transmission Disequilibrium Test melting temperature
Tris(hydroxymethyl)aminomethane University of California Santa Cruz a proprietary fluorescent dye produced by Applied Biosystems World Health Organization
I
List
1.1 1.2 1.3
2.1 2.2 2.3
2.4 2.5 2.6 2.7
3.1 3.2
3.3
3.4
3.5
3.6
3.7 3.8
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Figures
Structural composition of the Lp(a) particle . . . . . . . . . . . . . . Kringle structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structures of apo(a) . . . . . . . . . . . . . . . . . . . . . . . . . . .
Principle of allele-specific PCR . . . . . . . . . . . . . . . . . . . . . Amplification plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Distribution of SNPs in theLPAgene region investigated by asPCR on DNA pools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5’ nuclease assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Allelic discrimination . . . . . . . . . . . . . . . . . . . . . . . . . . . Cluster plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . GeneChip Human Mapping 500K Array Set from Affymetrix . . . . .
Schematic representation of theLPAgene structure and its LD pattern Comparison of mean Lp(a) levels between control and MI/CAD study
populations and influence of KIV-2 repeat number on Lp(a) levels in each study population . . . . . . . . . . . . . . . . . . . . . . . . . . Interaction between KIV-2 repeat number and rs11751605 genotype
and the influence on Lp(a) levels . . . . . . . . . . . . . . . . . . . . Odds ratios from logistic regression analysis for MI and CAD accord-
ing to rs11751605 genotypes . . . . . . . . . . . . . . . . . . . . . . . Results from the 500K genome-wide association with square-root trans-
formed Lp(a) levels using PLINK . . . . . . . . . . . . . . . . . . . . Results of association for markers of theLPAgene region from the 500K chip with square-root transformed Lp(a) levels using PLINK . .
Distribution of tagging SNPs . . . . . . . . . . . . . . . . . . . . . . . Results for chromosome 6 from genome-wide association of Lp(a) lev-els, adjusted forLPA. . . . . . . . . . . . . . . . . . . tagging SNPs
II
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List of Figures
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Results from genome-wide asso LPA . . . . . . .tagging SNPs
ciation . . . .
Lp . .
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(a) . .
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List
2.1 2.2 2.3 2.4
3.1 3.2 3.3 3.4 3.5
3.6
3.7 3.8 3.9
3.10 3.11
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Tables
Oligonucleotides for allele-specific PCR . . . . . . . . . . Standard asPCR reaction . . . . . . . . . . . . . . . . . Thermocycling conditions of asPCR runs on DNA pools SNP characteristics . . . . . . . . . . . . . . . . . . . . .
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Results from asPCR on contributor and non-contributor pools . . . . SNP characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . Clinical characteristics of the family sample . . . . . . . . . . . . . . Distribution of genotypes in the family sample . . . . . . . . . . . . . Results from family-based association testing with square-root trans-formed Lp(a) levels as a quantitative trait . . . . . . . . . . . . . . . Lp(a) QTL linkage analysis on chromosome 6q27 with SNP rs11751605 and/or KIV-2 included as covariates . . . . . . . . . . . . . . . . . . . Distribution of genotypes in case-control samples . . . . . . . . . . . Clinical characteristics of case-control study populations . . . . . . . SNP genotypes and the corresponding median Lp(a) levels (mg/dl) with 25% to 75% quantile range in all populations . . . . . . . . . . . Clinical characteristics of the Cardiogenics sub-sample . . . . . . . . List of tagging SNPs . . . . . . . . . . . . . . . . . . . . . . . . . . .
IV
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Analyzing the PCR data .
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Genotyping with TaqMan Assays
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Selection and pooling of DNA samples
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asPCR procedures . . . .
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Allele-specific PCR . . . . . . . . . . . . . .
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Selection of SNP markers for allele-specific PCR
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Allele-specific PCR on pooled DNA . . . . . . . . .
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Quantitative real-time PCR . . . . . . . . .
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DNA preparation for pooled DNA samples .
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DNA preparation for individual genotyping .
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Isolating genomic DNA from blood . . . . .
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DNA processing . . . .
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Materials and methods
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Lp(a) components
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1.9 Aim of this investigation . . . . . . . . . . . . . . .
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Heritability of Lp(a) levels . . . . . . . . . . . . . . .
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Genetic polymorphisms of theLPAgene . . . . . . .
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Repeat polymorphisms . . . . . . . . . . . . .
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Single nucleotide polymorphisms . . . . . .
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Metabolism of Lp(a) . . . . . . . . . . . . . . . . . .
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Elevated Lp(a) levels as a cardiovascular risk factor .
1.4
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Evolution of Lp(a) . . . . . . . .
Function of Lp(a) . . . . . . . . .
1.2
Structure-function relationships in
1.1.2 The apo(a) component .
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1.1.1 The LDL component . .
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Introduction
1.1
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Structures of the Lp(a) particle
Contents
3
2.4 2.5
2.6
2.3.1 The 5’ nuclease assay . . . . . . . . . . . . . . . . . . . . . . . 2.3.2 SNP selection and genotyping of individual DNA samples . . . 2.3.3 Standard TaqMan Assay . . . . . . . . . . . . . . . . . . . . . Genotyping with microarrays for genome-wide association . . . . . . .
Sample selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5.1 Measurement of Lp(a) levels . . . . . . . . . . . . . . . . . . . 2.5.2 Phenotypic apo(a) information and information on the pen-tanucleotide repeat polymorphism . . . . . . . . . . . . . . . . 2.5.3 Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6.1 Family-based association testing . . . . . . . . . . . . . . . . .
2.6.2 2.6.3
Case-control studies . . . . . . . . . . . . . . . . . . . . . . . . Genome-wide association testing . . . . . . . . . . . . . . . . .
Results 3.1 Allele-specific PCR on pooled DNA samples . . . . . . . . . . . . . . 3.1.1 SNP selection and genotyping of individual DNA samples . . . 3.2 Results from individual genotyping in the MI family study . . . . . . 3.2.1 Characteristics of the family sample . . . . . . . . . . . . . . . 3.2.2 Family-based association testing . . . . . . . . . . . . . . . . . 3.2.3 Contribution to the evidence of linkage . . . . . . . . . . . . . 3.3 Case-control association samples . . . . . . . . . . . . . . . . . . . . . 3.3.1 Genotyping in the population-based sample . . . . . . . . . . 3.3.2 Individual genotyping in the KORA MI sample . . . . . . . . 3.3.3 Genotype distribution in the subsamples from the family sam-ple for association testing . . . . . . . . . . . . . . . . . . . . 3.3.4 Clinical characteristics of the case-control study populations . 3.3.5 Association of individual SNP markers with Lp(a) levels . . . 3.3.6 Interaction between SNP markers and KIV-2 repeats on Lp(a) levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.7 Association of SNP markers with MI . . . . . . . . . . . . . . 3.4 Linkage disequilibrium analysis and Hardy-Weinberg proportions . . . 3.5 Genome-wide association testing . . . . . . . . . . . . . . . . . . . . . 3.5.1 Characteristics of the subsample with GWA data . . . . . . . 3.5.2 Quantitative association analysis . . . . . . . . . . . . . . . .
VI
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Contents
4
5
3.5.3 3.5.4
3.5.5
Results for the SNPs from theLPA . . . . . . .gene region . Adjustment of the GWA data for the significant SNPs from theLPA. . . . . . . . . . . . . . . . . . . . . .  .gene region Regions of interest for further association analysis . . . . . . .
Discussion 4.1 Association of single SNP markers with MI and Lp(a) levels . . . . . 4.1.1 Influence of previously described rare SNP markers . . . . . . 4.1.2 Possible influences of rs11751605 on functional properties of Lp(a) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genome-wide association data . . . . . . . . . . . . . . . . . . . . . .
4.2
Summary
Bibliography
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