research article the association between the lpa gene...

Research ArticleThe Association between the LPA Gene Polymorphismand Coronary Artery Disease in Chinese Han Population

Zi-Kai Song Hai-Di Wu Hong-Yan Cao and Ling Qin

Department of Cardiology The First Hospital of Jilin University 3302 JiLin Street Changchun 130031 China

Correspondence should be addressed to Ling Qin qinling1958aliyuncom

Received 23 January 2014 Accepted 26 February 2014 Published 25 March 2014

Academic Editor Cristiano Capurso

Copyright copy 2014 Zi-Kai Song et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Lp(a) has been well known as an independent risk factor for coronary artery disease (CAD) The LPA gene as it encodes apo(a) ofthe Lp(a) lipoprotein particle was associated with increased risk of CADThe purpose of this study was to analyze the relationshipbetween the polymorphisms of LPA gene and CAD in Chinese Han population Five SNPs (rs1367211 rs3127596 rs6415085rs9347438 and rs9364559) in the LPA gene were genotyped using SequenomMassARRAY time-of-flight mass spectrometer (TOF)in 560 CADpatients as case group and 531 non-CAD subjects as control groupThe numbers of these two groups were fromChineseHan ancestry The results showed that allele (119875 = 0046) and genotype (119875 = 0026) of rs9364559 in the LPA gene was associatedwith CAD The frequency of rs9364559 minor allele (G) in case group was obviously higher than that in control group Resultsof haplotype analysis showed that 4 haplotypes which contained rs9364559-G were associated with increased risk of CAD in thispopulation This study explored rs9364559 in the LPA gene may be associated with the pathogenesis of CAD and the risk of CADmight be higher in the population carrying 4 haplotypes of different blocks in the LPA gene

1 Introduction

Coronary artery disease (CAD) has become a major cause ofdeath and disability accounting for up to 40 of all lethalevents [1] However the pathogenesis of CAD is not fullyunderstood yet Many studies showed that CAD is mainlycaused by genetic and environmental factors [2] Manygenomewide association studies (GWAS) have identified sev-eral novel susceptibility gene loci for CAD [3ndash6] Variants oflipid metabolism-related genes have received the widespreadattention of scientists

Elevated Lp(a) levels has been considered to be anindependent risk factor for CAD [7ndash10] In 1963 Berg firstdiscovered the lipoprotein (a) (Lp(a)) in the separation ofplasma lipoprotein [11] Lp(a) is produced in the liver [12]and circulates in the plasma which is an LDL-like particlethat consists of an apolipoprotein (a) (apo(a)) moiety linkedto one molecule of apolipoprotein B100 via a disulfide bond[13] The study by Boerwinkle et al shown that the plasmaLp(a) concentration was mainly affected by apo(a) genepolymorphism which accounts for 91 of the variation [14]

A genome wide association study showed that a clusterof genes-solute carrier family 22 member 3 (SLC22A3)lipoprotein (a)-like 2 (LPAL2) and lipoprotein (a) (LPA) onchromosome 6q26-27 was strongly associated with CAD risk[15] LPA gene in the 6q26-27 region encodes apo(a) of theLp(a) lipoprotein particle [9] Several LPA gene polymor-phisms have been identified that have significant associationswith an elevated Lp(a) level and a reduced copy number ofK4 repeats [16] However the relationship between LPA genepolymorphisms and CAD varies across racesethnicities asthere are large differences between minor allele frequenciesIn the study by Clarke et al rs3798220 and rs10455872 arestrongly associated with serum Lp(a) levels in Caucasians [9]However the variant allele frequency (G allele) of rs10455872is less than 1 in Chinese people without CAD AnotherLPA SNP rs6415084 within the same haplotype block asthe KIV-2 variation was significantly associated with bothLp(a) concentration and KIV-2 copy number in the samedirection in all 3 ethnicities Therefore the objective of ourstudy was to investigate a possible association between LPAgene polymorphisms and the risk of CAD in a case-controlstudy of the Chinese Han population

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 370670 6 pageshttpdxdoiorg1011552014370670

2 BioMed Research International

2 Materials and Methods

21 Study Subjects All subjects included 560 CAD patients(308 males and 252 females) and 531 controls (270 malesand 261 females) in order to undertake a genetic analysis forassociation between the LPA gene polymorphisms and CADAll the subjects including the case group and the controlgroup used for this study were Chinese of Han descent Thecase group was collected from the hospitalized patients withCAD in the cardiology department of the First Hospital ofJilin University from June 2009 to September 2012

Diagnose was carried independently by at least two well-trained physicians based on the following criteria All patientswere identified with CAD by coronary computed tomo-graphic angiography (SIEMNS Somatom Definition AS+128row spiral CT) CAD was defined by ge50 stenosis in anymajor coronary artery All recruited patients had evidence ofCAD documented by unstable angina or myocardial infarc-tion Unstable angina and myocardial infarction were con-firmed by Chinese guidelines (Chinese Medical CardiologySubcommittee Chinese Editorial Committee of CardiologyJournal 2010 Chinese Medical Cardiology SubcommitteeChinese Editorial Committee of Cardiology Journal 2007)Patients with nonatherosclerotic vascular diseases congen-ital heart disease cardiomyopathy valvular disease renalor hepatic disease and cancer were excluded All controlsubjects had ECG chest X-ray and serum analysisTheywereclassified as healthy subjects based on their normal physicalexamination results coupled with the absence of personal orfamily history and reasons for being suspected CAD

The presence of cardiovascular risk factors includingdiabetes mellitus (fasting blood glucose ge70mmolL andorusing glucose-loweringmedication including insulin) bloodpressure and cigarette smoking were obtained from allparticipants Hypertension was defined according to seatedblood pressure readings of 14090mmHg and higher andorsubjectsrsquo receiving antihypertensivemedication In this studyhypercholesterolemiawas defined as a serum total cholesterollevel of 200mgdL or more and a smoking habit was definedas a daily intake of gt10 cigarettes [17]

All the subjects have written informed consent for thestudy which was approved by ethics committee of JilinUniversity Changchun China

22 Laboratory Examination Before starting the study allparticipants underwent an initial screening assessment thatincluded medical history vital signs a 12-lead electrocar-diogram and measurement of lipid variables and novel riskfactors Venous blood was collected in the morning afteran overnight (8ndash12 hours) fast Serumplasma samples werefrozen and stored at minus80∘C prior to analysis All measure-ments were performed in a central laboratory

23 SNP Selection Identification and Genotyping TaggingSNPs were chosen from genotyped SNPs in ChineseHan population (CHB) of the HapMap project (Phase Idatabase) The candidate SNPs were restricted to minorallele frequency bigger than 15 in HAPMAP-CHB database(httpwwwhapmaporg) Genomic DNA used for PCR

amplification was extracted from the whole blood sampleusing a DNA extraction kit (Takara China) Primers ofamplification and extension were used AssayDesigner31software Amplification and extension primers sequences offive loci in LPA genewere in Table 1 Genotypes were assignedreal time using Typer 40 software (Sequenom) As qualitycontrols 5ndash10 of the samples were genotyped in duplicateNo inconsistencies were observed Controls distributed ineach 384 well plates were also consistent Cluster plots weremade of the signals from the low and the high mass allele

24 Statistical Analysis Data were expressed as percentagesof total for categorical variables or mean plusmn SDThe statisticalanalyses on the characteristics of the subjects were performedwith Pearson 1205942 test for the categorical variables such assex smokers drinkers hypertension and diabetes and withStudentrsquos 119905-test for the continuous variable of age TC and TGwith normal distribution SPSS 160 was used for the aboveanalyses

TheHardy-Weinberg equilibrium for the genotypic distri-butions of SNPs was tested by the chi-square (1205942) goodness-of-fit test The Haploview program (version 41) was appliedto estimate the linkage disequilibrium (LD)measures (1198631015840 and1199032) between paired SNPs Allelic genotypic and haplotypeanalyses were performed with SHEsis software Softwarewebsite is httpanalysisbio-xcn Results are expressed asodds ratio (OR) and 95 confidence intervals (CI) 119875 lt 005was used as the criterion of statistical significance and allstatistical tests were two sided

3 Results

31 Characteristics of Participants In Table 2 the demo-graphic and clinical characteristics of the 560 CAD patientsand 531 control subjects have been listed There was nosignificant difference of mean ages sex BMI and serum TGlevel between case and control groups However comparedwith control group CAD group had more smokers and moreindividuals with hypertension and diabetes Additionallycompared with control group CAD group had higher levelof serum TC

32 Allele and Genotype Analysis The distributions of allelesand genotypes of five loci among participants were presentedin Tables 3 and 4 respectively Analysis with the SHEsissoftware showed that genotype and allele frequencies ofrs9364559 were significantly higher in case group than thatin control group respectively (1205942 = 7302 119875 = 0026 1205942 =3981119875 = 0046)However for another four SNPs therewereno differences in genotype and allele frequencies between twogroups

33 Linkage Disequilibrium (LD) Analysis Rs1367211 andrs6415085 rs1367211 and rs9347438 and rs3127596 andrs9364559 are all located in different LD block on 6q26-27region (1198631015840 = 1000 1199032 = 0085 1198631015840 = 1000 1199032 = 0149 1198631015840 =0999 1199032 = 0082)

BioMed Research International 3

Table1Amplificatio

nandextensionprim

erssequences

offivelociinLP

Agene

SNPs

Forw

ardprim

er(51015840

-31015840

)Re

versep

rimer

(51015840

-31015840

)Ex

tensionprim

er(51015840

-31015840

)rs1367211

ACGTT

GGAT

GGTG

GAT

TAGGTT

CAGAAT

GC

ACGTT

GGAT

GGCT

CTAT

GCT

GGAAAAC

TGG

ATGGGAAAC

TGGAT

TATT

GAAC

AGGCA

Crs3127596

ACGTT

GGAT

GTA

TGGGAT

GCC

ATCC

TTCT

CAC

GTT

GGAT

GAAG

CACT

GCA

GAT

GCT

TGAG

AGTA

ATAT

GCT

CATA

AGTT

CCC

rs9364

559

ACGTT

GGAT

GTT

TTGTC

CATG

TACC

TGCC

CAC

GTT

GGAT

GAG

GAG

AGGAAG

AGCA

AAAG

CAT

GAG

AAT

TAGGAAG

TAAAC

AGAC

rs64

15085

ACGTT

GGAT

GAC

TTTA

GCA

TATG

TAGAG

GAC

GTT

GGAT

GCA

CTTC

CAAT

TATT

CCCC

ACTT

ATTC

CCCA

CATG

ATTT

AGG

rs9347438

ACGTT

GGAT

GGTT

CTAG

CCTT

TGGGTG

TAG

ACGTT

GGAT

GTT

CAGCC

CATG

GAAAC

TAGG

CCAT

GGAAAC

TAGGAT

GTA

GA


Table 2 Base characteristics of case group and control group

Variable Case group (119899 = 560) Control group (119899 = 531) 119875 valueAge (year) 6239 plusmn 1090 6182 plusmn 1277 0447Sex () 550 508 0170Smoking () 383 233 0000Drinking () 223 227 0903Hypertension () 561 307 0000Diabetes mellitus () 240 121 0000TC (mmolL) 522 plusmn 428 462 plusmn 124 0002TG (mmolL) 176 plusmn 122 174 plusmn 120 0962BMI (kgm2) 2419 plusmn 268 2401 plusmn 331 0431

Table 3 SNPs loci allelic frequency distribution of LPA gene andthe relationship with coronary heart disease

SNPs Controls Cases1205942

119875A G A G

rs1367211 204 842 216 860 0109 0741rs3127596 889 157 914 170 0186 0667rs9364559 738 306 716 358 3981 0046

G T G Trs6415085 769 257 796 280 0262 0609

C T C Trs9347438 395 653 407 671 0001 0976

Table 4 SNPs loci genotype frequency distribution of LPA gene andthe relationship with coronary heart disease


119875AA GA GG AA GA GG

rs1367211 23 158 342 20 176 342 0967 0616rs3127596 379 131 13 387 140 15 0186 0911rs9364559 266 206 50 231 254 52 7302 0026

GG GT TT GG GT TTrs6415085 346 77 90 352 92 94 1574 0455

CC TC TT CC TC TTrs9347438 75 245 204 77 253 209 0004 0998

34 Haplotype Association Analysis All haplotypes with afrequency above 3 were included in the following analysisIn the haplotype association analysis one haplotype wastreated as a single variant and all the other haplotypes werecollapsed into the alternative allele to test its associationwith CAD Four haplotypes formed by different blocks wereassociated with increased risk of CAD in this population(Table 5) GATG (whichwasmade up by rs1367211 rs3127596rs6415085 and rs9364559) ATG (which was made up byrs3127596 rs9347438 and rs9364559) GTG (whichwasmadeup by rs1367211 rs9347438 and rs9364559) and TG (whichwasmade up by rs6415085 and rs9364559) reached the single-point significance level (119875 = 0034 0036 0049 and 0039)

4 Discussion

Based on our results we found the association betweenrs9364559 in the LPA gene and risk of CAD in ChineseHan population haplotypes GATG (which was made up byrs1367211 rs3127596 rs6415085 and rs9364559) ATG (whichwas made up by rs3127596 rs9347438 and rs9364559) GTG(which wasmade up by rs1367211 rs9347438 and rs9364559)and TG (which was made up by rs6415085 and rs9364559)were risk haplotypes for CAD in Chinese Han populationTherefore rs9364559 in the LPA gene has played a significantrole in the pathogenesis of CAD in Chinese Han popula-tion This result agrees with the previous results in otherpopulations [9 18] However the result that rs9364559 wasassociated with CAD in Chinese Han population is reportedfor the first time

High level of Lp(a) in plasma has been confirmed tobe associated with an increased cardiovascular risk [9 19ndash23] which is predominantly determined by apo(a) gene [14]Apo(a) size polymorphism and nonsize polymorphism in theLPA gene all affect Lp(a) level [24 25] Rs3798220 located inthe protease-like domain of apo(a) and rs10455872 whichmaps to intron 25 have repeatedly been associated with anincreased Lp(a) level and a reduced copy number of K4repeats inCaucasians [9]These variants were both associatedwith plasma Lp(a) levels and the association between thesegene variants and CHD was abolished when plasma Lp(a)levels were entered into the model [9] However the studyby Lamon-Fava et al showed that rs3798220 was significantlyassociated with Lp(a) levels and it was not a significantpredictor of CHD [26] And the association between apo(a)gene polymorphism and CAD varies among races Both ofthese SNPs had no significant effect on serum Lp(a) levels inChinese Han population [27] Our study found that genotypeand allele frequencies of rs9364559 in the LPA gene weresignificantly higher in case group than that in control grouprespectively Therefore rs9364559 might affect risk of CADIn this study 4 haplotypes formed by different blocks in theLPA gene were risk haplotypes for CAD in Chinese Hanpopulation and these haplotypes all contain rs9364559-GConsequently the above results further proved that LPA geneis associated with CAD which is consistent with previousstudies


Table 5 Haplotype analysis of all blocks between two groups

Haplotype case control 1205942

119875 OR 95 CIrs1367211 rs3127596 rs6415085 rs9364559

GATG 7295 (0069) 4780 (0047) 4486 0034 1497 1028ndash2179rs3127596 rs9347438 rs9364559

ATG 17652 (0166) 13894 (0133) 4384 0036 1293 1016ndash1645rs1367211 rs9347438 rs9364559

GTG 4238 (0040) 2566 (0025) 3890 0049 1643 0998ndash2703rs6415085 rs9364559

TG 7508 (0071) 5013 (0049) 4278 0039 1472 1019ndash2127Frequency lt003 in both control and casegroups has been dropped

However in haplotype association analysis there is nodifference for haplotype frequencies formed by five loci in thetwo groups Less number of samplesmay be themain cause ofthe problemTherefore further research conducted in a lagersample size in different race is necessary

In conclusion our case-control study explored the asso-ciation between five SNPs (rs1367211 rs3127596 rs9347438rs6415085 and rs9364559) in the LPA gene and CAD inChinese Han population for the first time Rs9364559 in theLPA gene may be associated with risk of CAD in ChineseHan population and with 4 haplotypes population formedby different blocks in the LPA gene may be associated withincreased risk of CAD in Chinese Han populationThereforeLPA gene is strongly associatedwith CAD in the ChineseHanpopulation which agrees with previous study

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Zi-Kai Song carried out the molecular genetic studies par-ticipated in the sequence alignment and drafted the paperHong-YanCao andHai-DiWu carried out the immunoassaysand participated in the sequence alignment Zi-Kai Songand Hai-Di Wu participated in the design of the study andperformed the statistical analysis Ling Qin conceived of thestudy and participated in its design and coordination andhelped to draft the paper All authors read and approved thepaper

Acknowledgments

The authors thank all participants for their supports andparticipation Special thanks are due to the support from theHepatology Department Institute of Translational Medicinethe First Hospital Jilin University Changchun China

References

[1] M Franchini F Peyvandi and P M Mannucci ldquoThe geneticbasis of coronary artery disease from candidate genes to whole

genome analysisrdquoTrends in CardiovascularMedicine vol 18 no5 pp 157ndash162 2008

[2] Q Wang ldquoMolecular genetics of coronary artery diseaserdquoCurrent Opinion in Cardiology vol 20 no 3 pp 182ndash188 2005

[3] P R Burton D G Clayton L R Cardon et al ldquoGenome-wideassociation study of 14000 cases of seven common diseases and3000 shared controlsrdquo Nature vol 447 no 7145 pp 661ndash6782007

[4] R McPherson A Pertsemlidis N Kavaslar et al ldquoA commonallele on chromosome 9 associatedwith coronary heart diseaserdquoScience vol 316 no 5830 pp 1488ndash1491 2007

[5] AHelgadottir GThorleifsson AManolescu et al ldquoA commonvariant on chromosome 9p21 affects the risk of myocardialinfarctionrdquo Science vol 316 no 5830 pp 1491ndash1493 2007

[6] N J Samani J Erdmann A S Hall et al ldquoGenomewide associ-ation analysis of coronary artery diseaserdquo New England Journalof Medicine vol 357 pp 443ndash453 2007

[7] S Erqou S Kaptoge P L Perry et al ldquoLipoprotein(a) con-centration and the risk of coronary heart disease strokeand nonvascular mortalityrdquo Journal of the American MedicalAssociation vol 302 no 4 pp 412ndash423 2009

[8] B G Nordestgaard M J Chapman K Ray et al ldquoLipopro-tein(a) as a cardiovascular risk factor current statusrdquo EuropeanHeart Journal vol 31 no 23 pp 2844ndash2853 2010

[9] R Clarke J F Peden J C Hopewell et al ldquoGenetic variantsassociated with Lp(a) lipoprotein level and coronary diseaserdquoNewEngland Journal ofMedicine vol 361 no 26 pp 2518ndash25282009

[10] GHDahlen andH Stenlund ldquoLp(a) lipoprotein is amajor riskfactor for cardiovascular disease pathogenic mechanisms andclinical significancerdquo Clinical Genetics vol 52 no 5 pp 272ndash280 1997

[11] K Berg ldquoA new serum type systerm in man the Lp systermrdquoActa Pathologica Microbiologica et Immunologica Scandinav-ica vol 59 pp 369ndash382 1963

[12] H G Kraft H J Menzel F Hoppichler W Vogel and GUtermann ldquoChanges of genetic apolipoprotein phenotypescaused by liver transplantation Implications for apolipoproteinsynthesisrdquo Journal of Clinical Investigation vol 83 no 1 pp 137ndash142 1989

[13] H H Hobbs and A L White ldquoLipoprotein(a) intrigues andinsightsrdquo Current Opinion in Lipidology vol 10 no 3 pp 225ndash236 1999

[14] E Boerwinkle C C Leffert J Lin C Lackner G Chiesa andH H Hobbs ldquoApolipoprotein(a) gene accounts for greater than90 of the variation in plasma lipoprotein(a) concentrationsrdquoJournal of Clinical Investigation vol 90 no 1 pp 52ndash60 1992


[15] D-A Tregouet I R Konig J Erdmann et al ldquoGenome-widehaplotype association study identifies the SLC22A3-LPAL2-LPA gene cluster as a risk locus for coronary artery diseaserdquoNature Genetics vol 41 no 3 pp 283ndash285 2009

[16] M M Luke J P Kane D M Liu et al ldquoA polymorphism inthe protease-like domain of apolipoprotein(a) is associated withsevere coronary artery diseaserdquo Arteriosclerosis Thrombosisand Vascular Biology vol 27 no 9 pp 2030ndash2036 2007

[17] Y-H Chen J-M Liu R-J Hsu et al ldquoAngiotensin convertingenzyme DD genotype is associated with acute coronary syn-drome severity and sudden cardiac death in Taiwan a case-control emergency room studyrdquoBMCCardiovascular Disordersvol 12 article 6 2012

[18] L Qi J Ma Q Qi J Hartiala H Allayee and H CamposldquoGenetic risk score and risk of myocardial infarction in hispan-icsrdquo Circulation vol 123 no 4 pp 374ndash380 2011

[19] J Danesh R Collins and R Peto ldquoLipoprotein(a) and coronaryheart disease meta-analysis of prospective studiesrdquoCirculationvol 102 no 10 pp 1082ndash1085 2000

[20] J S Danik N Rifai J E Buring and P M Ridker ldquoLipopro-tein(a) measured with an assay independent of apolipopro-tein(a) isoform size and risk of future cardiovascular eventsamong initially healthy womenrdquo Journal of the American Medi-cal Association vol 296 no 11 pp 1363ndash1370 2006

[21] P M Ridker J E Buring N Rifai and N R Cook ldquoDevelop-ment and validation of improved algorithms for the assessmentof global cardiovascular risk in women The Reynolds RiskScorerdquo Journal of the AmericanMedical Association vol 297 no6 pp 611ndash619 2007

[22] P R Kamstrup M Benn A Tybjaeligrg-Hansen and B GNordestgaard ldquoExtreme lipoprotein(a) levels and risk ofmyocardial infarction in the general population The Copen-hagen City Heart Studyrdquo Circulation vol 117 no 2 pp 176ndash1842008

[23] S J Nicholls W H W Tang H Scoffone et al ldquoLipoprotein(a)levels and long-term cardiovascular risk in the contemporaryera of statin therapyrdquo Journal of Lipid Research vol 51 no 10pp 3055ndash3061 2010

[24] J Rubin J K Han T A Pearson S Holleran R Ramakrish-nan and L Berglund ldquoApo[a] size and PNR explain AfricanAmerican-Caucasian differences in allele-specific apo[a] levelsfor small but not large apo[a]rdquo Journal of Lipid Research vol 47no 5 pp 982ndash989 2006

[25] B Enkhmaa E Anuurad W Zhang T Tran and L BerglundldquoLipoprotein(a) genotype-phenotype relationship and impacton atherogenic riskrdquoMetabolic Syndrome and RelatedDisordersvol 9 no 6 pp 411ndash418 2011

[26] S Lamon-Fava S M Marcovina J J Albers et al ldquoLipopro-tein(a) levels apo(a) isoform size and coronary heart diseaserisk in the Framingham Offspring Studyrdquo Journal of LipidResearch vol 52 no 6 pp 1181ndash1187 2011

[27] Z G Li G Li and Y L Zhou ldquoLack of association betweenlipoprotein(a) genetic variants and subsequent cardiovascularevents in Chinese Han patients with coronary artery diseaseafter percutaneous coronary interventionrdquo Lipids in Health andDisease vol 12 article 127 2013

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Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


2 Materials and Methods

21 Study Subjects All subjects included 560 CAD patients(308 males and 252 females) and 531 controls (270 malesand 261 females) in order to undertake a genetic analysis forassociation between the LPA gene polymorphisms and CADAll the subjects including the case group and the controlgroup used for this study were Chinese of Han descent Thecase group was collected from the hospitalized patients withCAD in the cardiology department of the First Hospital ofJilin University from June 2009 to September 2012

Diagnose was carried independently by at least two well-trained physicians based on the following criteria All patientswere identified with CAD by coronary computed tomo-graphic angiography (SIEMNS Somatom Definition AS+128row spiral CT) CAD was defined by ge50 stenosis in anymajor coronary artery All recruited patients had evidence ofCAD documented by unstable angina or myocardial infarc-tion Unstable angina and myocardial infarction were con-firmed by Chinese guidelines (Chinese Medical CardiologySubcommittee Chinese Editorial Committee of CardiologyJournal 2010 Chinese Medical Cardiology SubcommitteeChinese Editorial Committee of Cardiology Journal 2007)Patients with nonatherosclerotic vascular diseases congen-ital heart disease cardiomyopathy valvular disease renalor hepatic disease and cancer were excluded All controlsubjects had ECG chest X-ray and serum analysisTheywereclassified as healthy subjects based on their normal physicalexamination results coupled with the absence of personal orfamily history and reasons for being suspected CAD

The presence of cardiovascular risk factors includingdiabetes mellitus (fasting blood glucose ge70mmolL andorusing glucose-loweringmedication including insulin) bloodpressure and cigarette smoking were obtained from allparticipants Hypertension was defined according to seatedblood pressure readings of 14090mmHg and higher andorsubjectsrsquo receiving antihypertensivemedication In this studyhypercholesterolemiawas defined as a serum total cholesterollevel of 200mgdL or more and a smoking habit was definedas a daily intake of gt10 cigarettes [17]

All the subjects have written informed consent for thestudy which was approved by ethics committee of JilinUniversity Changchun China

22 Laboratory Examination Before starting the study allparticipants underwent an initial screening assessment thatincluded medical history vital signs a 12-lead electrocar-diogram and measurement of lipid variables and novel riskfactors Venous blood was collected in the morning afteran overnight (8ndash12 hours) fast Serumplasma samples werefrozen and stored at minus80∘C prior to analysis All measure-ments were performed in a central laboratory

23 SNP Selection Identification and Genotyping TaggingSNPs were chosen from genotyped SNPs in ChineseHan population (CHB) of the HapMap project (Phase Idatabase) The candidate SNPs were restricted to minorallele frequency bigger than 15 in HAPMAP-CHB database(httpwwwhapmaporg) Genomic DNA used for PCR

amplification was extracted from the whole blood sampleusing a DNA extraction kit (Takara China) Primers ofamplification and extension were used AssayDesigner31software Amplification and extension primers sequences offive loci in LPA genewere in Table 1 Genotypes were assignedreal time using Typer 40 software (Sequenom) As qualitycontrols 5ndash10 of the samples were genotyped in duplicateNo inconsistencies were observed Controls distributed ineach 384 well plates were also consistent Cluster plots weremade of the signals from the low and the high mass allele

24 Statistical Analysis Data were expressed as percentagesof total for categorical variables or mean plusmn SDThe statisticalanalyses on the characteristics of the subjects were performedwith Pearson 1205942 test for the categorical variables such assex smokers drinkers hypertension and diabetes and withStudentrsquos 119905-test for the continuous variable of age TC and TGwith normal distribution SPSS 160 was used for the aboveanalyses

TheHardy-Weinberg equilibrium for the genotypic distri-butions of SNPs was tested by the chi-square (1205942) goodness-of-fit test The Haploview program (version 41) was appliedto estimate the linkage disequilibrium (LD)measures (1198631015840 and1199032) between paired SNPs Allelic genotypic and haplotypeanalyses were performed with SHEsis software Softwarewebsite is httpanalysisbio-xcn Results are expressed asodds ratio (OR) and 95 confidence intervals (CI) 119875 lt 005was used as the criterion of statistical significance and allstatistical tests were two sided

3 Results

31 Characteristics of Participants In Table 2 the demo-graphic and clinical characteristics of the 560 CAD patientsand 531 control subjects have been listed There was nosignificant difference of mean ages sex BMI and serum TGlevel between case and control groups However comparedwith control group CAD group had more smokers and moreindividuals with hypertension and diabetes Additionallycompared with control group CAD group had higher levelof serum TC

32 Allele and Genotype Analysis The distributions of allelesand genotypes of five loci among participants were presentedin Tables 3 and 4 respectively Analysis with the SHEsissoftware showed that genotype and allele frequencies ofrs9364559 were significantly higher in case group than thatin control group respectively (1205942 = 7302 119875 = 0026 1205942 =3981119875 = 0046)However for another four SNPs therewereno differences in genotype and allele frequencies between twogroups

33 Linkage Disequilibrium (LD) Analysis Rs1367211 andrs6415085 rs1367211 and rs9347438 and rs3127596 andrs9364559 are all located in different LD block on 6q26-27region (1198631015840 = 1000 1199032 = 0085 1198631015840 = 1000 1199032 = 0149 1198631015840 =0999 1199032 = 0082)


Table1Amplificatio

nandextensionprim

erssequences

offivelociinLP

Agene

SNPs

Forw

ardprim

er(51015840

-31015840

)Re

versep

rimer

(51015840

-31015840

)Ex

tensionprim

er(51015840

-31015840

)rs1367211

ACGTT

GGAT

GGTG

GAT

TAGGTT

CAGAAT

GC

ACGTT

GGAT

GGCT

CTAT

GCT

GGAAAAC

TGG

ATGGGAAAC

TGGAT

TATT

GAAC

AGGCA

Crs3127596

ACGTT

GGAT

GTA

TGGGAT

GCC

ATCC

TTCT

CAC

GTT

GGAT

GAAG

CACT

GCA

GAT

GCT

TGAG

AGTA

ATAT

GCT

CATA

AGTT

CCC

rs9364

559

ACGTT

GGAT

GTT

TTGTC

CATG

TACC

TGCC

CAC

GTT

GGAT

GAG

GAG

AGGAAG

AGCA

AAAG

CAT

GAG

AAT

TAGGAAG

TAAAC

AGAC

rs64

15085

ACGTT

GGAT

GAC

TTTA

GCA

TATG

TAGAG

GAC

GTT

GGAT

GCA

CTTC

CAAT

TATT

CCCC

ACTT

ATTC

CCCA

CATG

ATTT

AGG

rs9347438

ACGTT

GGAT

GGTT

CTAG

CCTT

TGGGTG

TAG

ACGTT

GGAT

GTT

CAGCC

CATG

GAAAC

TAGG

CCAT

GGAAAC

TAGGAT

GTA

GA






119875A G A G

rs1367211 204 842 216 860 0109 0741rs3127596 889 157 914 170 0186 0667rs9364559 738 306 716 358 3981 0046

G T G Trs6415085 769 257 796 280 0262 0609

C T C Trs9347438 395 653 407 671 0001 0976




rs1367211 23 158 342 20 176 342 0967 0616rs3127596 379 131 13 387 140 15 0186 0911rs9364559 266 206 50 231 254 52 7302 0026

GG GT TT GG GT TTrs6415085 346 77 90 352 92 94 1574 0455

CC TC TT CC TC TTrs9347438 75 245 204 77 253 209 0004 0998


4 Discussion






119875 OR 95 CIrs1367211 rs3127596 rs6415085 rs9364559


ATG 17652 (0166) 13894 (0133) 4384 0036 1293 1016ndash1645rs1367211 rs9347438 rs9364559

GTG 4238 (0040) 2566 (0025) 3890 0049 1643 0998ndash2703rs6415085 rs9364559








Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table1Amplificatio

nandextensionprim

erssequences

offivelociinLP

Agene

SNPs

Forw

ardprim

er(51015840

-31015840

)Re

versep

rimer

(51015840

-31015840

)Ex

tensionprim

er(51015840

-31015840

)rs1367211

ACGTT

GGAT

GGTG

GAT

TAGGTT

CAGAAT

GC

ACGTT

GGAT

GGCT

CTAT

GCT

GGAAAAC

TGG

ATGGGAAAC

TGGAT

TATT

GAAC

AGGCA

Crs3127596

ACGTT

GGAT

GTA

TGGGAT

GCC

ATCC

TTCT

CAC

GTT

GGAT

GAAG

CACT

GCA

GAT

GCT

TGAG

AGTA

ATAT

GCT

CATA

AGTT

CCC

rs9364

559

ACGTT

GGAT

GTT

TTGTC

CATG

TACC

TGCC

CAC

GTT

GGAT

GAG

GAG

AGGAAG

AGCA

AAAG

CAT

GAG

AAT

TAGGAAG

TAAAC

AGAC

rs64

15085

ACGTT

GGAT

GAC

TTTA

GCA

TATG

TAGAG

GAC

GTT

GGAT

GCA

CTTC

CAAT

TATT

CCCC

ACTT

ATTC

CCCA

CATG

ATTT

AGG

rs9347438

ACGTT

GGAT

GGTT

CTAG

CCTT

TGGGTG

TAG

ACGTT

GGAT

GTT

CAGCC

CATG

GAAAC

TAGG

CCAT

GGAAAC

TAGGAT

GTA

GA






119875A G A G

rs1367211 204 842 216 860 0109 0741rs3127596 889 157 914 170 0186 0667rs9364559 738 306 716 358 3981 0046

G T G Trs6415085 769 257 796 280 0262 0609

C T C Trs9347438 395 653 407 671 0001 0976




rs1367211 23 158 342 20 176 342 0967 0616rs3127596 379 131 13 387 140 15 0186 0911rs9364559 266 206 50 231 254 52 7302 0026

GG GT TT GG GT TTrs6415085 346 77 90 352 92 94 1574 0455

CC TC TT CC TC TTrs9347438 75 245 204 77 253 209 0004 0998


4 Discussion






119875 OR 95 CIrs1367211 rs3127596 rs6415085 rs9364559


ATG 17652 (0166) 13894 (0133) 4384 0036 1293 1016ndash1645rs1367211 rs9347438 rs9364559

GTG 4238 (0040) 2566 (0025) 3890 0049 1643 0998ndash2703rs6415085 rs9364559








Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















119875A G A G

rs1367211 204 842 216 860 0109 0741rs3127596 889 157 914 170 0186 0667rs9364559 738 306 716 358 3981 0046

G T G Trs6415085 769 257 796 280 0262 0609

C T C Trs9347438 395 653 407 671 0001 0976




rs1367211 23 158 342 20 176 342 0967 0616rs3127596 379 131 13 387 140 15 0186 0911rs9364559 266 206 50 231 254 52 7302 0026

GG GT TT GG GT TTrs6415085 346 77 90 352 92 94 1574 0455

CC TC TT CC TC TTrs9347438 75 245 204 77 253 209 0004 0998


4 Discussion






119875 OR 95 CIrs1367211 rs3127596 rs6415085 rs9364559


ATG 17652 (0166) 13894 (0133) 4384 0036 1293 1016ndash1645rs1367211 rs9347438 rs9364559

GTG 4238 (0040) 2566 (0025) 3890 0049 1643 0998ndash2703rs6415085 rs9364559








Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















119875 OR 95 CIrs1367211 rs3127596 rs6415085 rs9364559


ATG 17652 (0166) 13894 (0133) 4384 0036 1293 1016ndash1645rs1367211 rs9347438 rs9364559

GTG 4238 (0040) 2566 (0025) 3890 0049 1643 0998ndash2703rs6415085 rs9364559








Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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