DisciplineofHumanGenetics,FacultyofMedicine Associationof MTHFR and9p21withIschemicStrokeintheChineseHanPopulation

AssociationofMTHFRand 9p21 with Ischemic Stroke in the ChineseHan Population

by

©ShuoLi

A Thesis submittedto the School of GraduateStudies in partialfulfillment of the requirements for thedegree of

Masterof Science

Disciplineof Human Genetics,Faculty of Medicine

MemorialUnive rsityof Newfoundland Augus t2012

St. John's Newfoundland

Previo usstudieshaverevealed inconsistentresultsin examining the associatio nof MTHFRc.677C>T,c.1298A>Cvariantsand9p2 1 locus with ischemic stroke.Thisthesis includestwocasecont rol studies by enrolling 1,429ischemic stroke patients and 1,197 control individualsfrom theChinese Hanpopulation. Onestudyvalidates the association betweenthe candidate9p2 1 locus and ischemi c stroke bothat the SNPand Haplotype levelsby genotyping four commonvariants encompassing a44kbcandidate riskregion on9p2 1. Moreover,thehaplotypeanalysisreducesthe candidate riskregionon9p2 1 locusto 28 kb. Theotherstudy indicatesthattheMTHFRlocus is associated with ischemi c stroke in the Chinese Hanpopulation. TheMTHFRc.677C>Tvariant is arisk factor,andits effectcould bemodi fiedby geneticorenvironmentalfactorswhichvary among differentethnicpopulations.TheMTHFRc.1298A>C variantismorelikelyto playtheroleof a genetic marker thana causativevariantinassociatio nwith ischem ic stroke.Combinedanalysissuggests the9p21 locus andlvrrHFRrisk allelesconferan additiveco-effec t forincreasedriskinischemicstroke.

ACKNOWLEDGEMENTS

First and foremos t, [would [ike to expressmy gratitudetomy superviso rs, Drs.Yagang Xie and FeiyuHan,for providingtheprecious opportunity to accept my applicatio nas a graduatestudent.Thisofferopeneda whole new chapter in mylife,from China to Canada,fromclinicalwork tobiomedical research,from a spoiledgirl to an indepe ndent individ ual.No wordscanexpress my gratitudeforyoursupport, patience andguidance bothin my academicand personallife.

[would [iketo extend thanks tomy committee memb ers,Drs. Edwa rd Randell and Rodger Green,forproviding generous help in my researchand thesis writing. [would also [iketo extend thanks for course instructors,Drs. SevtapSavas,and MichaelWoods, for help ingmeincoursestudy,presentation skilland proposal writing.Yourhelp gave me confidence to completethecourse requ irementsingenetics.

[would liketo extend thank s fortheKeith Griffithsfamily,for announcing methe award of theKeithGriffiths Memorialheart andstrokefoundatio nscholarship in20 [I.The awardenlightened mylifein lab work,espec iallyaftera seriesofunexpecteddifficulties in the experiment.

Inthe deepofmyheart,there is a warm homein Dr. YumingXu'team in neurology departme ntof thefirst affiliated hospital ofZhengZhouUniversity.Iwould [iketo iii

Wang. Thankyouforyour help in mytwo-yearperiodin clinicalneurology,which is a warm memoryI cannot forget.

Toallmy friends,both inChinaandCanada,thankyouforyouraccompany.Icherished all themoments we shared together.

To Mom andDad,thank youforyourunderstand ing,patience andsupportever sinceI wasborn.Thankyou for always being there forme.Ilove you.

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Tableof Contents

Abstract. i

Acknow ledgements iii

Tableof Contents vi

ListofTables vii

List ofFigures viii

Chapter I StrokeIntroduction 1

1.1Stroke Definition andClinical Feature s 2

1.2Stroke Subtypes 2

1.3 StrokeEpidemiology 6

1.4CascadeofIschemic Stroke 9

1.5Risk Factorsfor Ischemic Stroke 10

1.5.I Modifi ableRisk Factors 10

1.5.2 Non-modifia ble RiskFactors 21

Chapter2CandidateGenesfor IschemicStroke 25

2.1Single NucleotidePolymorphi sm,Haplotypeand LinkageDisequilibrium 26 2.2Candidate GeneApproachStudy of Ischemic Stroke 27

2.3Candidate GeneticVariantsinIschemicStroke 28

2.4Genomewide Associat ion Study of Stroke 31

2.5Candidate GeneticVariants in thePresent Study 31

2.5.2Chromoso me9p2 1 Locus 34 2.6TheGeneticComplexityofthe Chinese HanPopulation 39

Chapter3Materials and Methods... .40

3.1 Rationale .41

3.2Objec tives .41

3.3Materials and Methods .42

Chapter 4 Reduction ofChromosome 9p21 Locu sIntervalAssoci atedwith Risk for IschemicStrokein the Chinese HanPopulation .... . . .45 Chapter 5The Methylenet etrahydrofolat eReductase(MTHFR)isAssociatedwith

Ischemi c Stroke in the Chinese HanPopul ation 59

Chapter6 Conclusion 72

6.1CombinedAnalysisonAssociationofMTHFRand9p2 1Variantswith Ischemic

Stroke 73

References 76

vi

Listof Tables

Table2.1Candidategenes forischemic stroke.. . 30

Table 4.1 Genotype distributi ons ofrs l 0116277,rsI07 57274,rs2383207,and rsl 333049

in ischemic strokeand controlsubjec ts 53

Table 4.2Distribution of haplotypefrequency estimation forrs10757274 and rs2383207

in ischemic strokeandcontrolsubjec ts 55

Table5.1Genotype distributi onsofMT HFRc.677C>Tandc.1298A>Cvariants in

ischemic stroke and control subjec ts 66

Table5.2Distributi on ofhaplotypefrequencies forMTHFRc.677C>T andc.1298A>C variants in ischemic stroke and control subjects 67

Table6.1 Distributi on ofcombinedgenotypesofMTHFRc.677C>T andrs2303207 in

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ischemicstrokeand control subjec ts.... . 74

Figure1.1 Compariso nof thefrequencyof foursubtypesof strokeinChinaduring1991

and 2000 .

Figure 4.1Candidateregion on9p21for ischem icstroke .

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ix

Chapter 1

Stroke Introduction

1.1Stroke Definition and Clinical Features

Stroke,acco rding totheWorldHealth Organization(WHO),israpidl ydevelopedfocal or globalneuro logica l deficits,persistingmorethan 24 hours orinterrupted byinterventi on ordeath within24 hours, withapresumed vascularcause(WHO 1990). The clinical symptoms of strokevarydepend ing on lesion sizeand location in thebrain. The common symptoms include hemipl egia,hemianesthesia,aphasia,vision fielddefects,dysarth ria, headache,consciousimpairments,dysphonia andspatial neglects (Adamsand Victoria Principl esof Neurol ogy,

s"

Adva nces in neuroim agingtechnique shavehelped in facilitatingthediagno sisofstroke.

X-ray computed tomograph y (CT) scans candifferenti atehemorrh agicfromischemic stroke immediately afteronset. Magneticresonanceimaging (MRI) perfu sion weighted imag ing(PWI) and diffusion weighted imaging (OWl) mismatch scanscan identi fythe penum bra areawhere the affected brain tissuecan betreatedbythrombolysisincasesof earlyischemicstroke.MRIscanscan identifythe lesionlocation andsizein thebrain.

Magnetic resonance angiog raphy(MRA)scansor computedtomographyangiogra phy (CTA) scans canrevealthemorphologicalchangesincerebralvasculature(Warac het al.

1995;Baird etal. 1998;Peter etal.2003).

1.2 Stroke Subtypes

Based oncurrent neuroim agin gexaminations,the etiologyof stroke iscategor izedinto foursubtypes: ischemic,intracranial hemorrh agic,subarac hnoid hemorrhagic and

oremboligeneratedin thecardiovascularsystem, mainlydueto atherosclerosis andatrial fibrillation. Intracranialhemorrhagerefersto situations inwhich bloodleaks from cerebral arteriesintobrain tissue.Subarachnoid hemorrhageis caused byrupture ofan aneurysm in theprimarybranches from the circleof Willis,and bloodburstsintothe subarachnoidspace. Theundeterminedtype ofstrokeoccurswhenaneuroimaging examination isunavailable (Adamsand VictoriaPrinciple ofNeurology,

s"

Thefrequenciesofstrokesubtypes havebeeninvestigated worldwide. Ischemi c stroke is theleading subtypeaccording to most studies.Feiginet al.(2003) pooledtenpopulation- based studiesacross European,AustralianandCaribbeanareas. Ischemic strokewasthe most frequentin these studies,whiehconstituted67.3%-80.5%ofallstrokeevents.

Intracranialhemorrhage,subarachnoid hemorrhage and unknowntypes accounted for 6.5%-19.6%,0.8% -7.0%,and2.0% -14.5%,respectively.A similarfrequencywas alsoobserved in the Chinese population.Alarge comprehensivestudywasconductedby the China Multicenter CollaborativeStudyofCardiovascularEpidemiology(Zhangetal.

2003).Atotalof16,031first-time stroke patients(~25years old) wereregisteredin 17

communities from 1991to2000. Acomparison ofthefrequencydistribution ofstroke subtypes in 1991and2000areshown inFigure 1.1.Theischemic stroke rateincreased from the second(30.2%)in 1991 to themost commonsubtype(6 1.9%) in2000.

Intracranialhemorrhage accounted for 28.5%ofcasesin2000,which wasmuch higher

than 16.8%, as recorded in 1991. The reason for thisdifference lies in the implementation ofneuroimagingtechniquesduringthe study period.In2000,91.4%of patientsreceived CT scans,compared tothat of 49.4%in 1991.

Figure1.1Comparison ofthe frequencyof four subtypesofstroke inChina during1991 and2000(Adapted from Zhangetal. 2003)(IS:ischemic stroke,ICH:intracranial hemorrh age,SAH:subarachnoid hemorrhage; UNO:undeterm ined)

1.3Stroke Epidemiology

Prevalence: 9.0millionindividualsare estimatedto sufferfrom strokeworldwideeach year,whileapprox imately 1.5 to 2.0million patients resideinChina(Liuetal.2007;

Meretoja2011).Strokeoccurs in individualsfrom allage groups,withan increasing trend after 30 yearsold.About95%ofstrokeeventsaffect individuals older than45 yearsold, and two thirdsoccur inindividualsover 65 yearsold(Senelicketal. 1994).InChina,the mean onset for strokeis 63.9±8.4years old for malesand64.9±7.0years old forfemal es

(Zhaoetal.2008).

Incidence: Theincidenceof strokeshowedan increasingtrendin the pastthreedecadesin China.Zhao et al.(2008) observed 14,585 consec utiveacutestrokeevents in Beij ing from 1984to 2004,confined topatients aged25through74 yearsold.Theannual incidenceincreasedby 6.7%and 8.7%for total andischemicstrokeevents, respectively.

The increasing incidencewas associated with rapiddevelopmentduringthethree decades,which hasledto a growing number of seniorindividualswith prolongedlife expectancy.Also,a shift to a westernized lifestyle inChinesesociety,espec ially incities, may contribute to increase datherosc lerotic riskfactors.Thisstudyalso recordedthe increas ing intakeof fatandcholestero l duringthestudy period. Fat intake increasedfrom 88.1g1dto97.4g/d,while cholestero l intakeincreasedfrom 334.5g1dto 488.4g/d from 198 3to 2002.The prevalence oftype 2diabetesmellitu s andobesityalso increasedby 97%and85% in rura l areas,respective ly(Liuetal.2007;Zhaoetal.2008).

relateddeaths showedan upwardtrendduringthetwodecades,increasingfrom4.4 millionin 1990to5.7 millionin2004,whichapproxi matelyconstituted9.7%of total deaths worldwide(Murrayetal. 1997;Bonita et al. 2004;Geneva:World Health Organization.2000,2004; Donnan etal.2008; Mathers et al.2009).The World Health Organiza tion(2000) estimatesthat strokeandcoronaryartery disease will rank asthetop causefor losthealth ylife-yearsbytheyear 2020.According to areport from theWorld Health Organization,85% ofstroke-related deaths occur in low and middle-income countries(Matherset al.2009).Vasculardisease,includingmyocardialinfarction and stroke,isthepredominant cause foradult deathinChina.Thestroke mortali tyrate varied from 116.63per100,000per yearfor urban areas to111.74per100,000per yearin suburbanareasinChina.Itconstitutes 40% of allstroke-related deaths in develop ing countries(Reddyetal. 1998;Wuetal.200 I; Zhaoetal.2008).

Impact on healthcare: Stroke remains a substantial financial challenge forpublichealth care services.Stroke isthemaincauseoflong-termdisabil ityworldwid e, and 50%- 75%

of survivo rs livewithstroke-related disabilitie s (Foulkesetal. 1988 ; Geddesetal. 1996;

Bonitaetal.1997;O' Mahony etal. 1999).Garoet al.(2000) perform ed ameta- anal ysis, enro lling 1,446ischemic stroke patientsfrom13developed countriesin the1990s. This studyshowe d thatthemeantotal costof healthcarewas US$14,000 perindividual in the firstthreemonth s after astroke,and the estimated lifelong cost per survivo r rangedfrom

US$59, 800 to US$230,000 in developedcountri es. Atotal of US$65.5 billionis estimated to coverthetotal stroke-relatedcosts in the UnitedStates for the year2008(Rosa mondet al.2008).TheEuropean Heart Network(2008) reportedthat atotal of E27billion was devoted to stroke-related fieldsin27Europeancountrieseachyear.

Geographical variation ofstroke in China:One interesti ng phenom enon isthe geographicalvariationof stroke incidenceinChina.TheSINO -MON ICA project registered strokeevents in individualsaged between 25 to 64yearsold in 16 provinc esin Chinafrom 1987 to 1993.Thehighest incidence wasreportedin the northernprovinceof Heilongjiang (337.7and 553.3per100,000per year for maleand female,respecti vely), while thelowest was insouthern provinces suchas Anhui(33.0per100,000per year for male) andFujia n(29.7 per100,000per yearforfemale)(Wuetal.200 1).Other epidemiologystudiesalso documentedthenorth-southgradie nt(Li etal. 1985;Wang et al. 1985;Chenetal.1993;Chengetat.1995).

The reasonforthe geographical diff erentiationremainsunknown,butgeneticfactors might playarole.Theanalysisof genetic markers on theYchromoso meand mitochondrialDNAs reveal thatthe Chinese Hanpopulationcanbedifferentiatedinto north ernHan andsouthern Han, geographicallyseparated bytheYangtzeRiver (Du etal.

1997;Wen etal. 2004).Recent genome-widestudiessubdivided the Chinese Haninto north ern, centralandsouthern Han subgroups(Xu etat.2009;Chenet at.2009).The Chinese Han population occupied Northern Chinaareaapproximately 4,700 yearsago,

fromNorthern to SouthernChinaoccurred during the westernJin Dynasty (AD265-3 16), the TangDynasty (AD618-907)and the SouthernSong Dynasty (AD 1127-1279 ), respectively(Wenetal.2004).The Han cultureexpanded into southernChina,and merged withsouthern natives (Feietal. 1999, Geetal. 1997). ThesouthernChinese Han popul ation subsequently developed (Wenetal.2004).Thegenetic differenti ation of the Chinese Hanpopul ation will befurtherdescribedinChapter2.

1.4Cascade of Ischemic Stroke

Theavailabilityof glucoseandoxygenis crucia l formaintainin gnorm alneurol ogical function. Inischemicstroke, the occlusionof cerebralarteries leadstoaloss ofblood supply.The depletion of glucoseandoxygen causesfunctional and metab oli cimpairm ent in the affected tissue.Incases whereoxygenatedbloodis entirelyabsent, theneurons will undergonecrosis withineight minutes;however,in mostischemic strokes,collateral vesselswillcomplementarilysupply bloodtothelesiontissue, and theneuroncells in marginallesionlocations cansurvivefor up tofive orsixhoursbeforeirreversibl e damage occurs(Adamsand Victoria Neurology 8th).

Animalexperiments (gerbil and ratpenumbramodels)indicate that thenormaland criticalcerebralbloodflowfor brainmetabolism and function are 0.55 L/g/minand 0.23 L/g/mi n, respecti vely (I-Iossmannetal. 1994).Oncecerebral bloodflowfallsbelow 0.23

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L/g/minin theischemic area,acascadeof multiple functionaland metabolic changeswill occurat themolecularand cellularlevels. These include ATP depletion, eft1uxof potassium,influx ofsodiumandcalcium,releaseofglutamate,accumulationoffree fatty acids,prostaglandin s,leukotrienes and free radica ls, degeneration ofproteins and membr anedisrupti on. All these changes finallytriggercell edema, necro sis andapoptos is afterseveral hours if effec tive intervention isnotprovided (Dimag letal. 1999).When cerebral bloodflowisreduc edbelow0.12 L/glmin, thehistologicalinfarctionlesion can beidentifi ed(Hossmannetal. 1994).

1.5 Risk factors for IschemicStroke

According totheprimaryprevention guidelinesestablished bythe American Heart Association!AmericanStrokeAssociationStrokeCouncil(Goldsteineetal.2006),risk factorsfor ischem ic stroke canbedivided into modifiable and non-m od ifiable categories . Mod ifiablerisk factorscan becont rolled by ahealth ylife styleor medication,while non- modifia ble risk factors include age,sex, genetic predisposition andethnicity.Currently, knownrisk factorsaccount for only60%of stroke,and the etiologyof the remainin g40%

of cases remains undetermin ed (Whisnatet al. 1997;Donnan etal.2008).

1.5.1 Modifiable RiskFactors

Hypertension

Hypertension, defined as systolic blood pressure (SBP) 140 mmHg or diastolicBP~ 90 mmHg, is an independent risk factor for stroke (Rosamond et al. 2007).The number of adults with hypertension was approximately 972 million worldwide in 2000, and is estimated to reach 1.56 billion by the year 2025 (Kearney et al. 2005). Persistent hypertension can lead to vascular endothelial damage,oxidative stress, altered vascular tone and permeability, and the degeneration of vascular smooth muscle cells, all of which contribute to stroke by aggravating atherosclerosis (Johansson et al. 1999). The severity of hypertension is directly correlated with stroke risk (Lewington et al. 2002).One epidemiology survey estimated that 18.8%of adult Chinese (160 million individuals) are hypertensive (Wang et al. 2005;Fang et al. 2006). A prospective observational survey followed up on 5,092 male Chinese steelworkers for an average of 13.5years. About 31%

of these participants were hypertensive, and a total of 87 (1.7%) individuals developed ischemic stroke.Logistic regression analysis revealed that,for every ten mmHg rise in systolic or diastolic blood pressure,there was an associated 1.4- or 1.8-foldhigher risk for stroke, respectively (Zhang et al. 2004).

Diabetes mellitus

Diabetes mellitus refers to systematic metabolic syndromes characterized by persistently increased plasma glucose levels due to a lack of insulin secretion or increased resistance to insulin.According to the WHO (2006), a diagnosis of diabetes mellitus is made if any

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ofthe followingfoursituationsismet:I) fasting plasma glucose2:7.0mmol/L;2)plasma glucose2:11.1mmol/Ltwohours aftera75goral glucose load;3) symptomsof hyperglycemia andcasual plasma glucose level2:11.1mmol/L;and 4) glycog lated hemoglobin2:6.5%.Ameta-analysisincluding 39 countriesestimated that171 million individuals havebeendiagnosed with diabetesmell itus worldwi de.This number is expectedtoriseto 366 million bythe year 2030 (Wildetai. 2004).

Diabetesmellitusis anindependent risk factorforfirst-ever strokeand accountsfor 9.1%

of recurre ntstroke(Stamleretai. 1993;Burchfiel etai. 1994;Petty etai. 1998). Chronic diabetes mellitus facilitates atheroscleros isby inducing endothelial dysfun ction, inhibi ting nitric oxide production andenhancing theproliferation of vascularsmooth musclecells(Brown leeet ai. 200I).About 15%to 33% of stroke patientshavediabetes mell itus(Hieretai. 1991;Woo etai.1999;Hillen etal.2003;Megherbi et al.2003;

Karapanayiot idesetal.2004).Ameta-analysis of sevenstudies identifi edthatdiabetes mell itus conferreda1.7-foldhigherrisk for ischemic strokein patients withatria l fibrillatio n(95%CI:1.4-2.0). Theannualstrokeincidencevariedfrom2.0%to 3.5%in individualswith diabetesmellitus andatrialfibrillatio n(Stroke Risk in Atria lFibrillation WorkingGroup,2007).Diabetes mellitu sis alsoassociatedwithincrease dstroke mortalityand morbidity. Jia etal.(2011)registered 14,526hospitalizedpatients with acute ischemicstrokeandfollowed upon them forsixmonths inChina.Theyfoundthat 27.0%ofstrokepatientshaddiabetesmellitus. Amultivariatelogisticregression analysis illustratedthattheprevalence of diabetesmellitus independently predictedhigher riskfor

deathor livingdepend ency afterstrokeonsetin the Chinese popul ation (OR:1.23; 95%

CI: 1.10 to 1.37).

AtrialFibrillation

Atrial fibrillationdenotes cardiac arrhythm ia withchaoticelectrica lsignalsgenerated from the atria.Atrialfibrillation,whichcan enhanceemboli form ation viaabnormal hemostasis, endothelial dysfunction and increasedplatelet activation,acco unts for approxi mately20%ofischemic stroke(Savelievaetal. 2007; AtrialFibrillation Investigators1999).A cross-sectionalstudyof1.89million adults inCaliforniascreened 17,974 adultsage~20 years oldwithatrial fibrillation.Itestimated that 2.3 million adults

have atrial fibrillation in the US, and 60,000 of these patientsdevelopischemi c stroke annually(Go et al.200 1) The Framinghamstudy followedupon5,070 individu alsfor vascularevents for 34years. Atotal of 572stroke eventswererecorded.Compared to individualswithoutatrial fibrillation, individuals with this condition possessed a 2.6- 4.5 foldhigherriskfor strokeafteradj ustmentforothercardiovas cular riskfactors,suchas coronary heartdisease,heart failureand hypertension(P<O.OOI)(Wolfetal. 1991).

Dyslipidemia

Dyslipidem iadenotesderangementin lipidcomponents,suchas elevated total cholesterol and triglycerid es, andimbalance in lipoproteins,i.e.,increasedlow-densitylipoprot ein

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cholestero l (LDL-C) or reduced high-density lipoprotein cholesterol (HDL-C) . Dyslipidemiatriggersthe atheros clero tic processesbyinducingreleaseofinflammatory factors,proliferatio nof smooth muscle cells,and dysfunction of endotheliumand plaqu e formation(Gauetat.2006). Elevatedtotalcholesterol,LDL-C,and triglycerid eshave been reporte d tobe associatedwith increasedrisk forischemicstroke,especially forthe large arteryatheros cleroticstrokesubtype (Iso etat. 1989;Leppala etat. 1999; Ebrahim etat. 2006;Bansal etat.2007;Freibergetat. 2008; Bang etat.2008).Theassociatio nsof fatandcholesterol intake with ischemic stroke were assessed in the Chinese I-Ian population.Zhaoetat.(2008) reportedthatischemic strokeevents increasedby8.7%

annually in theBeijing areafrom 1984to 2004.Dyslipid emia was suggested tocontribut e tothisupwardtrend.Duringthe study period, fat intakeincreased from88. 1g/din 1983 to 97.4 g/din2002, and cholestero l intake from334.5g/din 1983to488.4g/din2002.

Studiesin other populations alsoconfirmedthisassociation. Koren-M orag etat.(2002) followe d upon 11,177patients in Israel withcoronaryartery disease for eight years.A total of 487individuals developed ischemicstrokeor transientischemic attac ks(TIA).

Logistic regression analysisshowed thatreducedHDL cholestero l(OR:0.89, 95%CI:

0.8 1-0.98),elevate d totalcholesterollevel (OR: 1.12, 95%CI: 1.03-1.23) andLDL cholestero l(OR:1.14, 95%CI:1.00-1.26) wereassociated with ischemic strokeor transientischemi c attacks(Koren-Moragetat.2002).

AsymptomaticCarotidStenos is

Asymptomaticcarotidstenosis(ACS)is the presence of atheroscleroticplaquewithout clinical manife stations . Embolican break off fromtheplaque and blockcerebralblood.

Stroke developsinsuch caseswhenpersistentischemi a occurs in the affected region.Ina prospective observationalstudy,Norrisetal.(199 1) followed 696 patients with asymptomatic carotid stenos is. Annual stroke rateswere1.3% and3.3% for patient s with carotidocclusion<75% and2:75%,respectively.Acohort study inToronto foll owed 106patientsdiagnosedwithasymptomaticcarotidstenosisfor ten years.Forty-eig ht patientsshowe d moderat e carotidstenosis<50%,while58 patients showedsevere stenos is(2:50%) .The ten-yearriskofstroke was 5.7% in patientswith moderate stenos is, and9.3%in patientswith severestenosis(Nadareishvilietal.2002).

Hyperhomocysteinemia

Hom ocysteineisan intermedi ateproduct formed in methioninemetabolism.Methi onin e fi rst fo rms S-adenosy lmethionine (SAM) by receiving adenosine from adenosi ne triphosphate (ATP), then SAM demethylatesto S-adenosy lhomocys teine(SAH) , which

donates adenosine to generate homocysteine.Homocystienehastwodestini es in humans:

a) reversible rernethylationtomethione viafolateandvitamin 812, or b)irreversible tran ssul furationto cystathionine byVitamin 86 (Selhubetal. 1999 ).The norm alplasma homocysteine concentration in humansis<15umol/L. (HandyandLoscalzo2003).

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Thecontributionof hyperhomocysteinemiato vascular disease wasfirstproposedby McCull y in 1969, who reported a deathfrom homocystinemia,cystathioninemiaand meth ylmalon ic acidemia dueto adefectincobalamin metabolism (McC ully 1969).

Arte rioscle rosiswas foundatautopsy,whichsugges ted that elevated homocysteinecould induce vascularendothelium damage. Elevated plasmahomocysteinelevels can induce vascular damagethroughthefollowingpathways:1) facilitationof the atherosc lerostic processbyincreasing vascular inflamm atorymediator s (Hofmann etal.200 I; Zhouet al.

2001 );2)impaired endothelialvasomotorfunction(Eberhardtetal.2000; Lentz etal.

2000;Dayal etal.200 1);3) reduced bioavailability of nitric oxide(NO)byinducing vascularoxidativestressandelevating plasma asymmetric dimethylarginine (ADMA) levels (Vallanceetal.200 1;Weiss etal.200 1;Weissetal.2002;Ungva ri etal.2003;

Boger etal.2003);and 4)dysregulation oflipidmetabolism,inflammationandapoptos is byinducingproteinmodi ficationand endoplasmicreticulum stress(Lentzetal. 1991;

Lentzet al. 1993;Jakub owskiet al.2000;Austinetal.2004).

Mild to moderate hyperhomocysteinemia (Hey=15- 100 umol/L)hasbeenrecognized as anindependentriskfactorforvascular disease,includingischemic stroke(Grahamet al. 1997;Welch et al. 1998;Rozen etal.2000;Bersano etal.2008).TheFraminghan study measured the totalplasmahomocysteinelevels of 1,947 US part icipant s (mea nage

±SD:70±7years)andfollowed up for 9.9years.One hundredathero thromboticstroke

eventsoccurred.Logisticregression analysis docum entedthe associatio n between

hyperhomocysteinemia (greaterthan 14.2umol/L) andantherothromboticstroke(OR:

1.90, 95%CI: 1.02- 3.51, P<0.001)afteradjustment forconventional vascular risk factors (Bostometal.2003).Otherstudiesalsoconfirmed thecontribution of hyperhomocys teinemia to ischemicstroke.Eikerlboometal.(2000) compared theplasma homocysteinelevelbetween 219 first-ever ischemicstroke patientsand205 age and gender matchedhealth yindividuals. The meanplasmahomocysteinelevelswere14.1 umol/Lin patient swith large-arteryischemi c strokeand 12.7umol/Lfor thosewith sma ll-artery ischemic stroke, bothofwhichwere significantly higherthanthat found in the controlgroup (10.5umol/L).Logisticregressionanalysisrevealedthat theupper quart ile oftheplasmahomocysteinelevel (greater than13.8l!mol/L)was associated with increasedriskforischem ic stroke(OR:2.2, 95%CI:1.1to4.2) compared tothelowest quart ile group(homocysteine level lessthan 9.0 umol/L) afteradj ustment for conve ntionalvascular risk factors.Theassociationofhomocysteinelevels with thelarge artery subtype of ischemic strokeexhibitedadose-dependentpattern . Comparedwith the lowest quartileofhomocysteine (lessthan 9.0umol/L),the ORs forlarge arterystroke riskof theother threequart ile groups were 3.0(95%CI:0.8 to 10.8), 5.6 (95%CI: 1.6to 20)and 8.7 (95%CI:2.4 to 32),correspondin gto the second quartile (9.0 umol/Lto11.2 umol/L),the thirdquartil e(11.3umol/Lto13.8umol/L) and the fourth quartile (beyo nd 13.8umol/L),respectively.Asimilar trend wasobservedin the Chinese Hanpopulation.

Liet al.(2003) measuredtheplasmahomocysteinelevelin 807 cerebral thrombosis patients,513sma llarterystrokecases and 1,832 ageandsex- matchedcontrols.The med iantotal plasma homocyste inelevels were 14.7 umol/L inatherothromboticstroke

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and14.8umol/L insma llarterystro ke, respectively.Both ofthese weresignifica ntly higherthanthemedian valueof12.8umol/L found inthe controls.Logistic regression ana lysisindicated thatthe elevated homocysteinelevel wasindepend entl y associatedwith athero thro mbot icstroke(OR:1.72, 95%CI:1.39to 2. 12, P<O.OOI) andsma llartery disease afteradjustingforconventionalvascular risk factors(OR:1.89, 95%CI:1.50to 2.40,P<O.OOI) (Lietat.2003).

Lifestyle: cigarett e smoking, nutrition,obesity and physicalinactivity

Unhea lthy mod em life styles,includingsmo king, poordiet , obes ity and physical inacti vity, can impairthe cardiovasc ular systemand increase stroke risk.

Smoki ng, including exposu re to a smo kingenviro nme ntandactivecigarettesmo king, has been recognized as a significa nt risk factorfor ischemi c stroke(Wo lfetat. 1988;Mast et at. 1998;Bon ita etat. 1999;Heuschm ann etat.2007).Cigare ttesmo kingcanenha nce vascularinflammation,thromb osis andoxidatio nof LDLcho lestero l(Ambroseand Baru a 2004). In a large,popul ation-based cohortstudy, Mannam i etat.(2004)observe d 19,78 2male s and21,500 fem ales aged from40 to 59 yearsold,allof whomhad no history of vascular disea se. Theirsmo kingstatus and inciden ce ofstroke wererecorded from 1990to2001. A totalof3 27and 176 ischemicstrokeeventsoccurred in male and femaleindividual s,respectivel y. Compared tostroke patient swithout ahistory of smo king,logisticregressionanalysisrevealedthat currentsmo kersshowe da1.66- fold

(95%CI:1.25to 2.20) higherrisk for ischemicstroke independ ent ofconventiona l risk factors. Asimilar trend was observed inotherstudies.Another prospective studyenro lled 118,539 US female participants aged from 30 to 55 yearsoldandwithoutahistory of vascular disease,andwerefollowed upon for eightyears.Atotal of122thromboemb olic strokeeventswererecorded.Data analysis indicatedthat currentsmokersconsuming morethan15 cigarettesperdayconferreda 2.7-fold higherriskfor ischemic stroke compared tonon-smokers (Colditzetal. 1988).

Excess intake ofsodiumcan induce water retentionin thecardiovascularsystem which increasesthe riskforhypertension (He and MacGregor2004 ).Recomm end edintakefor sodiumand potassium are :52.3 g/dand ;:::4.7 g/d,respectively (Goldsteinetal.2006) .A

19-year cohortstudy,including2,688overweightindividualsaged25to74yearsold, revealedthatadailyintake of extra 100mmolpredicted a1.32-foldhigherrisk forstroke (95%CI: 1.07- 1.64,P<0.01)(He et al. 1999).Another populat ion-b ased study enro lling 43,783middle-aged USmaleindividualsreported apositive associa tion betwee n potassium supplementsandareducedriskforstrokeaftereightyearsfollow- up (RR:0.36,95%C I:0.18-0.72) (Ascherioetal. 1998).

Obesity,defin ed asbodymassindex (8 MI)>30 kg/rrr' ,contributes to ischemi c stroke by inducing endothelial damage and theproliferation ofinflammato ryfactorsin vessel s (Poirieret al.2006;Strazz ulloetal. 2010).Kurthetal.(2002) enro lled21,4 14USmale physicians freeof cardiovasc ular diseasein 1982.Atotal of 631(5.5%)ischemic stroke

20

events were recordedin thesubsequent 12.5years.Compared to those with norm al weight(8 MI<23),obeseindividualswitha8MI>30 showeda1.95-foldhigherrisk (95%CI: 1.39to 2.73, P<0.00I) for ischemic stroke.Inaddition tomales, obesityalso confersarisk forischemic stroke in females. Aprospective cohortstudyenro lled 116,7 59 USfemalenurses aged30to 55 years old,whowerefreeof cardiovas cular diseases, and followe d upfor 16 years.Compared tonormal weighted participants (8 MI<27 kg/rrr'), therelativerisk ratios for ischemicstroke inoverweight individual swere 1.75 (95%C I:

1.17-2.59), 1.90(95%CI: 1.28-2.82)and2.37(95%C I: 1.60-3.50)for individual s with27 kg/rn''S;8MIS;28.9kg/m²,29kg/m²S;8MIS;31.9kg/m²,and 8MI~ 32kg/m²,

respecti vely (P<0.00I) (Rexrodeetal. 1997).

Physical inactivitycontributes toischemic stroke by affectingvasc ular riskfactors (Lee et al. 2003).Inalargepopulation-based cohort,39,315UShealthyindividuals over45 years oldwererecru ited.During11.9 years follow-up, 473 ischemicstrokeeventsoccurred.

Compared toindividuals with little physicalactivity«200kcal/week),parti cipantswith moderate (600-1,499kcal/week) and highphysical activity(~ 1, 5 00kcal/week) showed

alowerriskforischemic stroke,OR ratioswere0.66 (95%CI: 0.52to0.84) and 0.61 (95%CI:0.47 to0.80)for moderate and highphysical activity,respecti vely.Other studies alsoprovided evidence that regular physicalactivityhasbeneficial effects in reducin gthe incidenceofischemicstroke.Lee et al.(2003)analyzed23 studies from 1966to 2002, andrevealed that physical activity was associatedwithalower riskofstroke,showinga

20% reduction formoderateactivit y (RR=0.80) anda 27%reduction forhigh stre ngth activ ity(RR: 0.73,95%C I: 0.67-0.79),respectively, compared tolowlevels of activity.

1.5.2 Non-modifiable RiskFactors

Age

Adva nce dageis a signific ant risk factor for stroke, resultin gin a2-fold increasedrisk for everyten yearsafter the age of 55yearsold,with two thirdsofstroke patient sbein g greater than 65 yearsold(Wo lfetal. 1992; Senelicketal. 1994;Brown etal. 1996;).

Feiginet al.(2003), after per form ing ameta-analysis invo lvingatotal of 3.266,366 individualsfrom13 countriesworldwide, revealedthe trend toward inc rease dstro ke inciden ce withadva ncedage. Theage-standard izedstroke incidence rateranged from 0.1 to 0.3per 1,000person-years forindivi duals with an agelessthan45 years old; 4.2-6.5 per1,000person- years forindividuals agedgreater than or equalto 55 yearsold;and incide nce increasedto12.0-20.0per1,000person-years for elderlypeopl e agedgreater than 75 years old.Another meta-analysis reported thatthe stroke incid ence was 30times higher inindividualsbeyond75 yearsold,compared toindividuals aged35to44years oldin the Chinese Hanpopul at ion (Liuetal.2007).

Sex

22

Malesaremore vulnerableto stroke than females.TheChina Multicenter Collaborative Studyof Cardiovasc ularEpidemiology in17communitiesacross Chinafrom 1991 to 2000 showe dthatmen accountedfor59.0%oftotal strokeeventsamongall 16,031 registered first-eve rstroke patients(~25 years old) (Zhangetal.2003). Aneight-yea r

nationalregistryin Denm ark aimedat hospitalizedindividuals with first-everischem ic stroke reportedthatmales accounted for52.1%of40,102 individuals,and theincidence rate was obvio usly higher in malesthan femalesamong middle-agedindividu als (Andersenetal.20 I0).

Race-Ethnicity

AfricanAmericansshow higher strokeincidencethanEuropeanCaucasians(Broderic ket al. 1998). Thereasonfor thismightincludehigherprevalence of vascular risk factors suchashypertension and diabetes inAfricanAmericanscompared to Caucas ianand Hispanic populations(Schcammetal. 2010).The prospectivepopulation-based Northern Manhatta nStrokeStudy(NOMASS) examined210,000residentsin northern Manhattan area.ThestudyrevealedthatAfricanAmericansshoweda 2-3 foldhigherriskfor developin gischemic stroke per yearcompared to European descend ant s (Saccoetal.

1998). A more recentGreater CincinnatilNo rthern Kentuck ypopulation project ascert ained strokeeventsamong 1.3millionresidentsin the studyareain 1993,1999 and 2005.The race-adju stedischemic stroke incidencerateswere 303,291and294 per 100,000personsfor AfricanAmericansfor the same periods,respecti vely.The incidence ratesfortheir Europeandescendantswere 206,241and 179per100,000persons for the

year 1993,1999 and2005, respectively. Comparison betweenthetwo ethnicgro ups revealedthat AfricanAmer icans have ahigher chanceof developin g ischemicstroke than Caucas ian popul ati onsdespitethedecl inin gtrendin incidencewithin eachethnicgroup (Kleindorferetal.20I0).

Genetic pred isposition

Thegenet ic pred isposition for strokewas identifiedinanimal models.Rubattu etal.

(1996)createda stro ke-pro ne hypertension-ind ep endentratmodelbyhybr idizingmale stro ke-pro nespontaneo usly hyperten siverats and fem ale stroke-res istan tspontaneo usly hypertensive rats . The research ers observe d stroke incidence amo ng the 220 norm otensive ratsand perform ed a genome-wide linkage analysis by genotypingatotal of 1,038 genetic markers. Three quantit ati veloci onChromoso mes Iand5 showedLOD scores >3.0.Thisstudyindicated that gene ticcomponentscontr ibute to stroke, inde pende ntofhypert ension.

Popul at ion studiessugges tthatgeneticcomponent play a substantialrolein isch emic strokeinhumans.The nationwidetwin study in Denm arkrecru ited 351 mon ozygoti c and 639 dizygotic twin sbornfrom1870 to1952. Atthe initia lscreening,one twin fromeach pair was verifiedforstroke death,the causeofdeath in the other twin was the n rec ord ed fromthe Register of CausesofDeath or theDan ish National Disch argeRegister.It showed thatmon ozygotictwin shad ahigher concordancerate(35pairs,10% ) of stro ke

24

death compared todizygotictwins (34 pairs,5%) (OR:2.1,95%CI:1.3-3.3)(Ba ketal.

2002).Thisstudy provides evidence for genetic predi sposit ionto stro ke-re lated death.

TheSahlgrenskaAcade mystudyon the Europea nCaucas ian popul ation inSweden registered 600 consecutiveischemicstroke patien ts withonsetage<70yearsoldand 600 age- andsex- matched contro ls.Compar ison betweencases and cont rol s showed thatthe first-degree family history of stroke was associatedwithincreased risk ofisch emi c stroke (OR:1.75,95%CI: 1.26- 2.43).Statist icalsignifica nce rem ained whe n performin g similaranalysis in three subtypesofischemic stroke.Theoddratioswere 1.88,1.79 and 1.70 forlarge-vessel stroke(95%CI:1.02-3.44),sma ll-vesse lstroke(95%CI: 1.13- 2.84)andcryptoge nicstro ke(95%CI:1.13-2.56),respectively(Jood etal.2003).

The mech anisms invo lvedingeneticpredisposition for strokeareprop osedtoinvolvethe following path ways:I) geneticcomponentsfacilitatethe developm ent of conve ntio nal vascularrisk factors,suc hashypertension and hyperh omocysteinem ia, which increased stro keriskinvulnerable individu als; 2) genetic comp onentsinteract withenviro nmental factors to enhancestroke risk;and3) genet iccomponentscontribute to inter mediate pheno types,suchas atherosclerosis,whichcan leadto stroke(Dichga nsetal.2007;

Stan kovicet al.20I0).Duringthepasttwodecades,dozens of candidategenes have been reporte d tobe associatedwithstroke;however,few ofthese showconsis ten t result s in the studied popul ati ons.Thecandidategenestudyof ischemi c strokewill bediscussedin cha pter2.

Chapter 2

Candidate Genes for Ischemic Stroke

26

2.1Single Nucleotide Polymorphism,Haplotype and Linkage Disequilibrium

Single nucleotid epolym orph isms (SNP)referto variation in DNA sequencecause d by a single nucleot ide (adeni ne, thymin e,cytosineandgua nine)variatio natspec ific loci.The human genomeisestimated to contai n6.0million commonSNPs(minoralle1efrequency

>5%)(Danaetal.2005).Approxima tely2.3million SNPsdifferbetw eentwounrelated indiv iduals,i.e.,I nucleotid e variation per1,000-2,000 basepairs (The Intern ational SNP MapWorkingGroup200 1).WhenSNPsoccurin theprotein- cod ingregion ,they are categorized intotwotypes,dependingontheireffectson theresultin gpol ypeptid e seque nces:(I) synonymo usvariation, inwhich thenucleotide variatio ndoesnot change amino acid sequence;and(2)non- synonymous variation,which result s inaminoacid changeorpremature terminatio n.SNPs areusuallybiallel ic.The frequ ency of alle lescan vary geographica lly betweendifferent ethnicpopulations:the maj or alle leata certa in locus inapopul ation canbe the minor onein another(Taras et al.20 10).

Ingeneticassociationstudies,haplotyperefer sto SNPson differentloci on the same chromosome statis tica llyassociated with eachother(The International HapM ap Consort ium2003,2005).Linkage disequil ibrium describes thenon-r and om associationof alleleson different loci (Falconeret al.1996).Genetic linkagedisequ ilibrium can help identify candidate loci contributingtodiseasesincasethatthetested genet ic marker and thediseasecausingvariantareclosely linked (Briscoe etal. 1994Plomin etal. 1994;

Jorde et al. 1995;Kaplan etal.1995;Chap manand Wijsman1998).Fortwolocinot

tightl y linked, linkagedisequilibrium willgradually decay overgenerations for recom binati on occurs betweenthetwoloci.For those closelylinked loci,linkage disequ ilibriumbetweendisease and markerloc i will remainstrongthrough generatio ns.

Theseassumptions providethebasis forgeneticassociationstudiesidentifyingcandida te geneticvar iantsfordiseases.

The International HapM ap Project cate gori zes the human genome into linkage disequilibrium (LD) blocks,i.e.,certainchromosom eregion swhereallelic asso ciation between SNPs showsfewhistoricalrecombination events (Tailon-Mill eretal. 2000;Daly et al.200 1). Theidentificationof LD blocksprovidesanefficient tool ingenetic associationstudies.Testing tagSNP s,which repr esenttheseLD blocks,canavoid the need to genotypeallSNPswithin thecandidate region (Rischetal.200 0;Zha ngetal.

2002).TheLD pattern s ofmorethan 4.0 million SNPsacrossthe wholegenome have been characterized in different ethnic popul ationsfollowingthecompl eti on of the Intern ationalHapM apProj ectphaseIand II in2005 and 2007 (Cheeetal.20 I0).

2.2 Candidate Gene Approach Study of Ischemic Stroke

Thecandidategeneapproachstudy hasbeenwidelyusedin investigatin g gene tic risk factorsfor compl exdisease(Matarinet al. 2009).SNPs are often used as genetic variants in these associationstudies.These SNPs are genotyped insubjects with the target phenotype (cases)andage-andsex- match edhealth y indiv iduals(co ntrols).The

28

association betweenthe candidatevariantsand targeteddisease can be assessed bythe

X:

test analyzing the frequencydifference between case andcontrolgroups.Oddsratiosare used to indicate the relative risk of the tested variantsondisease (Sanja20I0). A reliable geneticassociationstudy requ ires alarge samplesize (>1,000)withadjustmen t for age and sex (Li etal.2003).

2.3 Candidate Genetic Variantsin IschemicStroke

Geneticassociationstudies havebeenwidelyappliedtomapcommon geneticvariants (mi norallele frequency>5%) conferring risk forstroke(Matarinetal.2009) .Todate.

studies haverevealedpathoph ysiologicalprocess of ischemicstrokeare involved in the following pathways:I.haemostasis system:factorV,factorVII,factorXII,factorXIII.

factor II (prothrombin), fibrinogen, plasminogen activator inhibitor- l, platelet glycoprotei n receptors,vonWillebrandfactor,tissueplasmi nogenactiva tor,thrombin activable fibrinolysis inhibitor,thrornbomod ulin,protein Z,andannex inAS ; 2.

homocysteine metabolism: methylenetetrahydrofolatereductase, cystathione beta- synthase,and methionine synthase;3. lipidmetabolism : apolipoproteins, lipoprotein receptors,and key enzymesofplasmalipoproteinmetabolism (Bersanoetal. 2008, Stankov icetal.20 10).Thecandidategeneticvariantsfor ischemic strokearesummarized in table 2.1.Theassociation betweenthesecandid ate geneticvariantsand strokeremain s inco nsistent in different ethnic populations (Matarinetal.2009). The reason accounting

for thisphenom enonmaylie inselection bias, sma llsamplesize, and heterogen eit yof stro ke.There fore,furtherstudiesareneededtohelp resolve thisdebat e.

30

Table 2.1 Candidate genes for ischemic stroke (Adapted from Bernaso et at. 2008 and Stankovic et at. 2010)

Path ogen etic syste ms I-1acm ostasis

Homocysteinemetab oli sm

Lipid metaboli sm

Candidategenes

F2, F5,FGA/FGB,F7. FI2A!,VWF, F12, SERPINE I, ITGB 3, IT GA2B , ITGA 2,GPIBA,ACE,ACT

MTHFR,CBS, MTR

APOf:2,d,f:4,LPL,PON 1/2/3, CETP, ABCA I

2.4 Genomewide Association Study of Stroke

A genome-wideassociationstudy(OWAS) is amethodby which largenumbers ofSN Ps evenly scattered throughoutthewholegenomearegenotyped.Itis expected todiscover novelgenetic variants byprovidin g ahypothesis-freemethodto inves tigatecommo n geneticvariants with moderate effectson complex disease (Ku Ietal.20I0).Sofar, OWAShave identifiedseveral novel candidategenetic loci for ischemicstroke,including 4q25(PlTX2gene)(Gretarsdottiretal.2008),16q22 (ZFHX3 gene)(Gudbja rtsso netal.

2008) and 12pl 3(N/NJ2gene) (Ikram et al.2009).However, subsequentindependent validationstudies have failed toreproducethese OWASfindings (Hege leet al.2010).In addition,geneticvariantsforischemicstrokeidentifie dbythe candidategeneapproac h didnot achievethestatistica lsignificance level ingenome-wideassociationstudies (Ikrametal.2009).Up tonow, OW AShavenotrevolutionized thegeneticstudyof strokeas expected.

2.5Candidate Genetic Vari ant sin thePresentStudy

2.5.1 MTHFRc.677C>Tand c.l298A> C variants

Theassociation betweenhyperhomocysteinemi a andstroke isdescribedinchapter I.

5,10-Me thylenetera hydrofo late reductase (MTHFR), the key enzyme regulating homocysteinemetabolism, remethylates homocysteinebacktomethionineby catalyzi ng

32

the reductionof5,10-meth ylenetetrahydrofol ateto5-methyltetrahydrofo late(Ueland et al.2000, 2001;Champe etal.2008).Impaired MTHFRactivity interrupt sthe remeth ylation process ofhomocysteineto methionine,and the plasma level of homocysteine subsequentlyaccumulates leadin gtohyperhomocysteinemia.

TheMTHF Rgene,which islocated onchromoso me I p36.3 andencodes the MTI-IFR enzy me, hasbeen wide ly investigatedforits contributiontocardiovasculardisease (Goye tte et al. 1994;Bersano et al.2008; Stankovic etal.20 10). Themost studied variant istheMTHFRc.677C>T(p.Ala222Val)vairant.In vitroexperimen tsindicatedthat heterozygous CT and homozygous TT genotypesreduceMTHFRactivity to 65% and 35%compared tothehomozygous CC genotype.The homozygous TTgenotype leadsto enzyme thermoliability andincreases plasmahomocysteinelevelsby 25%(Frosstetal.

1995).Van der Putetal. (1998)confirmed this phenomenonby comparing the homocysteinelevel amongdifferentgenotypesof theilvfTHFRe.677C>Tvariantin186 individua ls.Thenumbe rof individ ualswithl\r rHFRc.677C>Tgenotypeswas44.0%

(n=82),41.9%(n=78)and 14.0%(n=26)for CC, CT andTT genotypes. respectively.The mean plasma homocysteinelevel was 18.4umol/Lin theTl'genotype.whichwas significa ntly higherthanthat oftheCC genotype(13.1umol/L). Thissugges ts thatthe MTHF Rc.677C>T variant may confer risk to stroke viainf1uence on plasma homocysteinelevels.

Theassociation betweenMTHFRc.677C>Tand ischemi c strokeremains inconsistent.

Croninet al.(2005) analyzed 22 studies, recruit ing4,740 ischemicstrokecases and 7,486 contro ls,andrevealeda dose-dependentassociation betweentheMTHFRc.677C>T variant andischemicstroke.Compared to CC genotype,oddratioswere 1.18for heterozygous CT genotype(95%CI: 1.09 to1.29,P<O.OOI)and 1.48for homozygous1'1' genoty pe(95%C I:1.22to 1.8,P<O.OOI).Lietal.(2003) recruit ed807ischemi c stroke patientsand 1,832 contro ls. Themedianplasmahomocysteinelevel was significantly higherincases (14.7 umol/L) compared to controls(12.8 urnol/L), and increasedriskfor ischemic strokein the Chinese Hanpopulation (OR: 1.72,95%CI:1.39-2.12, P<0.(01).

Inaddition,thehomozygous1'1'genotype was foundin24.5%ofcases, versus21.7%in contro ls(OR: 1.37, 95%CI: 1.06to 1.78).Thisindica testhattheTT genotype is associatedwith increased risk forischemic stroke. However,anotherassociationstudyin the SingaporeChinese populationdidnot confirmthisassociation(Low et al.20II).A recentmeta-analysis, including six meta-analyses and ten case-contro lstudies,failed to confi rm the associationbetween theAfTHFRc.677C>Tvariantand ischemic stroke (Bersanoet al.2(09).

Anothercommonvariant,A1THFRc.1298A>C (p.Glu429A la), was identifiedby sequencing the codingregionofAfTHFRin 86individualswith anopen neural-tub e defect and 403 controlsubjects(Van der Putetal.1998).Theyestablishedanassociation between AC/CCgenotypesand reduced MTHFRactivityin isolatedhumanlymph ocytes.

However,the effectofMTHFRc.1298A>Cvariantonplasmahomocysteinelevels

34

remainsuncertain.Studies reportedthat this varianteither did notaffectthehomocysteine levels, orwasassociatedwithelevatedorevenreducedhomocysteinelevels (van derPut etal.1998, Friedmanetal. 1999,Lieversetal.2001,Castroetal.2003).Sazcietal.

(2006) genotypedtheMTHFRc.1298A>Cvariant in 92 ischemicstroke patients and259 healthy contro lsfroma TurkishCaucasian population.TheCC genotypedistributed to 20.7%of cases, whichwas significantly higherthan8.1%inthecontrolgroup(OR:2.950, 95%CI:1.504-5.786, P=0.001).Han etal. (20 10) perform ed a similarstudyon264 silentbrain infarctionpatients and234 controlsin theKoreanpopulation .Heterozygous AC genotype possessed 30.3%ofcases compared to21.8%of cont rolindividuals, and increasedrisk (OR: 1.734, 95%CI: 1.13-2.66,P<0.05)for silent braininfarcti on.

2.5.2Chromosome 9p21 Locus

Thechro moso me9p2 1 locushasbeen identified tobe associatedwithabroadrange of vascular diseases.Rs1333049,anSNPwithin the chromoso me9p2 1 locus, wasreported tobe associatedwith increasedrisk ofcoronary heart disease in thelargeWellcome Trust Case Contro lConsortium(WTCCC) study(1,926 cases and2,938 cont rols, OR=1.37, 95%CI:1.26to1.48,P=1.80xI0-¹\ andwas validatedsubsequently in the Germa n Myocardi alInfarction Family Study (875cases and 1,644cont rols,OR=1.33, 95%CI:

1.18 to1.51,P=3.40xI0-⁶)(Samani et al. 2007). Ameta-an alysisinvolving12,004 patientswithcoronary heartdisease and28,949controlsconfirme d the association betweenrs1333049 and coronaryheartdiseasein the Caucasian populati on (OR: 1.14,

95%CI: 1.20- 1.29, P=6.04xI0-1o)(Schunkertetat.2008).Saxena etat.(2007) genotype d386,73 1SNPsin 1,464patient s withType2 diabetes mellitu s and 1,467 age andgende r-ma tchedcontrols,andfound thatthe T alle leofrsI08 1166 1onchro moso me 9p21 locusdistribut ed significa ntly higherincasesthanin control s. (OR:1.37, 95%CI:

1.18-1.59,P=3.6x10-⁵) .A subsequentcohortof 3,167Chinese Han individu als repli catedthe enhanced risk ofthe variant rsl0811 661 conferring to Type2diab etes mellitu s (OR:1.23, 95%C I:1.03to1.47,P=0.02)(Chengetat.20 11).Alarge case controlstudysugges ted thattheGalle le of rs107 57278 onchromosome9p2 1 locu sis assoc iated withabdominalaorticaneurysm(2,836 patient s and 16,732controls,OR:1.31, 95%C I:1.22 - 1.42,P=1.2x10-¹²)and intracrani al ane urysm (1,134patient s and 15,481 contro l individu als, OR:1.29, 95%CI:1.16- 1.43,P=2.5x10-⁶)(Helgadottiretat.2008).

Theassociationbetwee n9p2 1 locus and ischemi c stroke represent s oneof themost intriguing discoveri esin the geneticstudyof ischemicstroke for the year 20I0 (Meschia 2011).Matarinet at.(2008) recruit ed 249 ischem ic stroke pat ients and268contro ls from a Caucas ian popul at ion.Theyidentified that SNPs rsI011 6277,rs13 33040,rsl 33304 2 andrs2383207,whichenco mpasse da 44-kb region on9p2 1,were associatedwith increasedriskfor ischemi c stroke(all P<0.05).The association betweenthislocus and isch emi c strokeremai ns weakin the Chinese Hanpopul ati on.Huetat.(2009) registered 355ischemi c stroke patients and 430health ycontrolsin the Beijingarea. Bygenoty ping SNPsrs23832 06and rsI 0757278,this study identified thatthe AG/GGgenoty pesof rs2383206 conferreda1.51-foldhigherriskfor ischem ic stro ke(95%CI: 1.11to 2.05, P=

36

0.009),espec iallysignifica nt in large vesselstroke(OR:2.09,95%C[:1.30to 3.37, P= 0.002).However,thehaplotype analysisdid notreach significance level(I'=0.067).This suggestedthat9p2 [confersa genetic risk forischemic strokein the Chinese Han population. Ding etal. (2009) genoty ped [7tagSNPs on chromosom e 9p2 [in558 patientswithischemicstroke, 510patients withcoronaryartery disease and557contro l individuals free ofthese conditions in the Chinese Hanpopulation. Theirstudyshowe d thatrs2383206 (OR:1.35,95%C[: 1.11to1.64,I'=0.006), rsl004638 (OR:0.65, 95%C[:0.53to0.81,I'=0.001) and rs10757278 (OR: 1.39,95%CI:1.15to1.69,p=

0.002)distribut ed differentlybetweencoronaryarterydiseasepatient sandcontro l individuals.However,none of thetested SNPsreachedstatisticalsignific ance levelfor ischemicstroke in the study. Haplotype analysis revealedthat the AITAhaplotype of tag SNPsrs2383206,rs1004638,rsl 776 1442 and rs10757278distributed 0.7%inischemic strokepatients, significantlylowerthan incontrolsubj ects (4. 1%)(OR:0.78,95%CI:

0.67to 0.87,P=4.1 x10"").Subsequentvalidationstudies bythe same research group recruited 442 ischemicstrokepatientsand502 contro l individuals. TheabovefourSNJ>s on9p2 1candidate locus weregenotyped.Statisticalanalysisconfirmedtheabove associatio nat thehaplotypelevel (OR:0.85,95%CI:0.77-0.95,1'=0.003).However,no statistica lsignifica ncewas reached at the SNP level (Dinget al.2009).More investigation s are needed on the association between chromoso me9p2 1 locus and ischemic stroke in the Chinese Han population.

The9p2 1 locus alsoconferred riskfor several non-vasculardiseases, suchasopen-an gle glauco ma(Ramdamsetal. 20I0),melanoma (Bishopet al.20I0), childhoodacute lymph ob last icleukemia (Sherborneetal. 2010),glioma(Sheteetal.2009),basal cell carcinoma(Stacey etal.2009) and breast cance r(Turnbulletal.2010).

Thecandidate locus on9p2 1 isdevoid of any known gene.Since these associatedSNPs identifi ed in previous studieswerecommonSNPs(minorallele frequency>5%among the studied population )from the initialGWASSNP panel, scientists proposedthatinter- frequencyvariants(minorallele frequenc y variesat2%-5%),or thosecommonSNPs notincludedin thepublisheddata,might conferahigherriskforthesephenotypes.Shea etal.(2011) then sequenceda 240-kb region on9p2 1 in 47 individualsandestablished 536novel SNPs includingboth common andinter-frequencyvariants.Two associatio n studieswere performed on diabetes mellitus(1,000type 2 diabetes patients and 1,048 contro ls)and myocard ial infarct ion(1,274cases and1,407 contro ls) by genotyping these variants.However,none of these newlyestablishedSNPsshowe d higher odds ratios comparedwith thepublished GWAS SNPsignals.

Functionstudies have examined the effectofthislocus ongeneexpressio n.Twotumor suppresso rgenes,cyclin-dependent kinaseinhibitor 2A(CDKN2A)andCDKN2Breside approxim ately100 kbaway from thecorevascular diseaseriskregion.Theyare involved in the regulationof cell cycle, aging,senescence,andapoptosis(KimandSharpless2006;

Giland Peters 2006).ANRILisa non-codin gRNA with its exons 13-19 overlapping the

38

corevascularriskregionon 9p21, andistranscribedin theoppositedirection tothe CDKN2Bgene.ANRILis expressed invascular tissues, suchascoronary smoo th muscle, vascular endothelial cells, human monocyte-derived macroph ages, carotid endarterec tomyspecimens,andabdominalaneurysm(Cunnington and Keavne y 20 11).

Todate,function studies haverevealedinconsistentresults. Liuetal.(2009) identifi edthe association betweenrs10757278 on9p2 1andreducedexpress ionofCDKN2A,CDKN2B andANRILin periphe ralblood Tlymphocytes from 170 healthy volunteers.Cunnington etal.(2010)validateda similarassociation between chromosome9p2 1 locus and decreased ANRILtranscripti on in periph eral blood extracted from 487 health y indiv iduals.Thesestudiessugges ted that chromoso me9p2 1 locusmay contribute to vascular diseasebyregulating expressio nofCDNKN2A, CDKN2BandANRILinvascular tissues.However,otherstudiesfailed to confirm this association(Michaelet al.20 11).

Hold etal.(20 10) revealedthat chromoso me9p21 was associatedwithreducedANRIL andCDKN2Bexpressio nin mononucl ear cellsfrom 1,098 coronaryartery patient s.

However,this correlation diminished when testingtheassociation inatheros clero tic plaqu e speci mens.Ananimalstudyknockedouta70kb onchromoso me 4in mice,which was estimated tobe orthologousto 9p2 1inhumans. These mice showed reduced expressio nofCDKN2AandCDKN2Band proliferation of aortic smooth muscle cells.

However,theydid not showapredispositionto vascular disease (Viseletal.20 I0).

Anotherstudysugges ted that 9p2 1 risk allelescontribute to vascular diseasebyincreasin g plateletreactivity (Musunuruetal2010).Musunuru etat(20 10) identifi edthat12 SNPs inthe 9p2 1regionshowedanassociationwith increasedplateletreactivity among 1,402

asymptomatic participant s(P<O.OOI).Theyalsovalidatedthis associationin the 2,363 participants from the Framingham heart study cohort.Theexact mechanismunderlying the associatio n between 9p2 1andvasculardiseases needs furtherelucidatio n.

2.6 The Genetic Complexityof theChinese Han Population

Chinahasthelargestpopul ation worldwide,with 1.3billionindividu als from56 ethnic groups.About92% Chinese belongtotheHan Chineseethnicgroup,whichconstitutes 20%ofthe world'sentire population. Geneticstudiesanalyzing Ychro moso meand mitochondri alDNA variationssugges t thatthe Chinese Hanpopulationcouldbe sub- catego rizedinto NorthernHan andSouthern Han,geographically dividedbytheYangtze River, the largest river running from westto east acrossChina(Xiaoetal.2000;Wen et al.2004).Arecent genomewi destudy,which mapped 160,000SNPs in 1,700individu als from22 regionsacross Chinaindicated thatthe Chinese Han population could be further differentiatedintothree subgroups:theNorthern,CentralandSouthernHan(Xuetal.

2009).Another larger study,genotyping350,000SNPs inover 6,000participants sampledfrom tenprovinces,confirmed these geneticsubgroupswithin the Chinese Han population.Theyfurtherindicated thatthe Chinese HanBeijing (CHB) population in the internationalHapMapdatabase could representthe Chinese Han from north ern and centralareas(Chenetal.2009).Oursamplewas collectedsolelyfrom Henanprovince, which islocatedin cent ral Chinaamong34admin istrative regionsinChina.

40

Chapter 3

Materials and Methods

3.1Rationale

Strokeisthe second most commoncauseof adult death andisthe leadin g reason forlong- term adult disability worldwide(DonnanetaL2008;Bonita etal.2004).Assessment of genetic predisposition will facilitate prevention andindividualized treatment ofstroke.

The associationofMTHFRc.677C>T,c.1298A>Cvariantsand9p2 1 locus with ischemi c strokeis inconsistent in publisheddata. The reasonsforthese inconsiste nt reportsmight liein theheterogeneity ofstroke,geneticcomplexity between ethnicgroups,or limits of thestudy design.

TheChineseHanpopul ation,a seemingly homogenous ethnic popul ation,hasbeen classifiedinto three geographicsubregions :theNorth Han, Central HanandSouth Han (Chenetal.2009, XuetaL2009).Geneticvariantsassociated withcomplex disease mightbediff erentinthese subpopulations;therefore,sampleselectionshould be area specific.The present study focuses on the Chinese Hanpopulation from Henanprovince, thecentralChina.

3.2Objectives

In the presentstudy,weperformedtwo case controlstudies,with thefollowing aims:

To assesstheassociation between two commonvariants,MTHFRc.677C>Tand

42

c.1298A>C,andischemic stroke intheChinese Hanpopulation from Henan province ;

To investigate the genetic associationbetweenthe candidat echromosome9p21 locusand ischemic stroke in the Chinese Hanpopulation from Henanprovince.

3.3Materials and Methods

FromFebruary 2006 toMarch2007,1,429consecuti vepatients(Meanage±SO: 62.7 1±

11.80,male :n=868,60.7%),diagnosed with ischemi c stroke from 18 hospitalsin Henan province,were enrolledin thepresent study.The inclusion criteria were:I) stroke diagnosis according toWHO criteria,and2) an MRIorCTscanindicating theischemic lesion corresponding tothe neurol ogicaldeficit s.Patientswith hemorrhagic stroke, subarachnoid hemor rhage,transientischemicattack andseveresystemic diseaseswere excludedfrom thisstudy.The controlgroupconsistedof 1,197 (Meanage±SO: 58.51± 9.237,male:n=760, 63.5%)ethnic matchedindividuals withoutahistory of myocardial infarction orstrokewhowereadmitted tothesehospital sforroutinehealth examinations.

Inform ed consent wasobtained from all participant s.Samples werecollected bythe Genetics departmentof ZhengzhouUniversity.Thestudy protocolon the collaboration between Zhengz houUnive rsityand Department of Laborator ymedicinein Health SciencesCenters, Newfoundland andLabrador ,was approved byinstitution alboards.

SNPGenotyping

Genomic DNA samples were obtained by salt extractionprotocol(Miller etal 1988 ).In theMTHFRstudy,twovariants were tested,includingMTHFRc.677C>T(rs I8 0 1133, C_1202883_20)andMTHFRc.1298A>C(rs I 801131,C_850486_20)variants.For the 9p2 1study, four SNPs wereselected fromChromosome 9p21.3locu s:including rsl3330 49 (C_175466 6_10), rs2383207 (C_157890 10_10), rs10757274 (C_265058 12_1O),and rs1011 6277 (C_2999 1625_20),whichencompasse d the44-kb candidate region on chromosome 9p2 1(the positionmappedtochr9.22071 397 and chr9.22 115503) .All tested SNPswere genotyped using TaqManSNPgenotyping kit (ABI;FosterCity,CA) on real-tim ePCRplatform (ABI Prism7000sequence detection system).The reactionvolum eincluded:2.0 IIIgenomic DNA(100 ng/ul),2.5111TaqMan universalPCRmastermix,0.125III primerand probes, 0.375 III ddH20.The standard reaction condition included activationof uracil-N-gl ycolase (UNG)(2min,50°C), polymeraseactivation(lOmin;95°C),40cyclesof denaturation(l5s;95°C),annealing andextension(Imin; 60°C) .

Statistica lAnalysis

Thegenotypefrequency dataforeach variant in the control groupwastestedfor deviation fromtheHard y-Weinb erg equilibrium using onlinesoftware(http://ihg.gsf.de/cgi- bin/hw/hwal.pl).Haplotype frequencieswereestimated usingthePowerMa rker V3.25

44

Software.Genotypeand haplotypedistributi onbetween caseand contr ol groupswere testedbythe X²testunderrecessive,dominant andco-dominant modelsby SPSS 16.0 software package (SPSS Inc.).Statistical power was calculated usingQUANTOV1.2.3 software.Differenc es with P<0.05(two-tailed) were consideredstatisticallysignificant.

Bonferronicorrectionwasnotperfo rmedin thetwo studies duetothefoll owin greasons:

I) fortheMTHFRstudy, twotested variantsof theMTHFRgeneare in linkage disequil ibrium ; 2)forthe 9p2 1study,thefour variantsarefrom onecandid ateriskregion, wherers107 57274and rs2383207 are in one LD block,and two othervariantsadjacentto thisblock; 3) Bonferronicorrectionistoo conservati ve.

Risk with Associated Interval

Chapter 4

Reduction of Chromosome 9p21 Locus for Ischemic Stroke in the Chinese Han Population

ShuoLil,2, Yu-MingXu5,Edward Randell',Hong Zheng", Hai- Zhen gWang", Guang Sun",Fei-Yu Hanl,2,and Ya-GangXic l,2,3,4

Discipl inesof 'LaboratoryMedicin e, 2Genetics, 3pediatrics,and"Medicine, Memori al Unive rsityof Newf oundland,St. John ' s,NL,Canada,

Departm ent s of5Neurologyof the FirstAffiliated hospital,and6Genetics ,Zhengz ho u Unive rsity,Henan,P,R,China,

This manuscripthasnot yet been submitted/or publication.

46

Abstract

Background:Chromosome 9p21commonvariants are stronglyassociated withcoronary arterial disease (CAD). This association hasbeen expended to other vasculardisord ers including ischemi c stroke inCaucasians. However,the association between 9p2 1 commonvariants and ischemic strokein the Chinese Hanpopulation lacks strong evidence. Method:Fourcommonvariants, rs1333049,rs2383207 ,rsI0757274 , and rsI0116277were selectedfrom the44 kbcandidate region onchromoso me9p2 1 locus.

We genotyped these SNPs in 1,429ischemic stroke patients and 1,191 controls fromthe Chinese Hanpopulationbyusingthe TaqManSNPgenotyping technology on areal-time PCRplatform.Results:Heterozyous AG and homozygous GG genotypesof rs2383207 conferreda increase d riskforischemic strokein thepresent study(P<0.05). Haplotype analysisof rs10757274 and rs2383207 showed thatthe G-Ahaplotypewas associated withsignificantly lower riskwith ischemic stroke(OR:0.359, 95%CI:0.256to0.504, P<O.OOI).Conclusion: Acandid ateregion on9p2 1 locus is assoc iated with ischemic stroke in the Chinese Hanpopu lationboth atSNPand Haplo typelevels.Ourstudy reducesthe candid ateregionto 28 kb in the Chinese Hanpopulation.

Introdu ction

Strokeisdefined asacutefocalorglobal neurologi caldeficitspersistin gmore than 24 hours, orinterrupted by interventionor deathwithin24 hours,withapresumed vascular cause(WHO 1990 ).9 millionindividuals areestimated tosuffer fromstrokeeachyear, andapproximately 1.5-2 millionof thesepatientsresideinChina(Liueta12 007,Zhao et al2008,Meretoja 20 11).Strokeisthe second mostcomm on causeofadult death worldwi de, andabout 5.4 millionindividualsareestimated todieofstroke-relatedevents eachyear,accounting for approximately 10%of totaldeathsworldw ide (Murrayetal 1997,Donn anet ai,2008 Bonita etal,2004).Two-third s of stroke relateddeathsoccur in developin gcountr ies,with approximately40%occurringinChina(Geneva:World HealthOrganization. 1998,Reddy et al 1998,Feiginetal2003,).

Strokeis acomplexdiseaseto which both geneticandenvironmental factorscontribute. Commonvariants ina candidatechromoso me 9p2l locushaverecentl ybeenassociated with increasedriskforcoronary arterial disease(CAD ). The 9p21locu swasfirst identifiedas a genetic riskfactorinCAD byfour parallel genome-wideassociation studies(The Wellcome TrustCase ControlConsortium 2007;Helgadottir etal.2007;

McPherson etal.2007;Samani etal.2007).Subsequently, genome-wideassociation studies havealsodemon stratedthe association betweenthislocus andothervascular diseases, including diabetesmellitus,abdominalaorticaneurysm,and intracrani al ane urism(Saxe naet al 2008,Helgadottir et al.2008).Matarin et al.(2008) docum ented thatcommonvariants from a44-kb core candidate region on9p2 1 were associated with ischemi c stroke ina Caucas ian population.However,replication studies in the Chinese

48

Han population haverevealed weakassociatio n between 9p2 1and ischem ic stroke(I-Iuet al. 2009,Dinget al.2009).This may be due to arelatively sma llsamplesizeandgenetic heterogeneity among theChineseHanpopulation.In thepresentstudy, weperformed a large case controlstudyto replicate the association betwee nthecorecandidate9p21 region andischemicstroke inthe Chinese Han population.

Materials andMethods Subjects

FromFebruary2006 toMarch 2007, 1,429 consecutive patients (Mea nage±SO:62.7± 11.8,male:n=868, 60.7%), diagnosed withischemicstrokefrom 18hospitalsin Henan province,wereenro lledin thepresent study.The inclusion criteriawere:1) stroke diagnosisaccordingto WHOcriteria,and2) MRIorCT scan indicatingtheischemic lesion corresponding to the neurologicaldeficits.Patients withhemorrhagicstroke, subarachnoidhemorrhage,transientischemicattack and severe systemicdiseases were excluded from this study. Thecontrolgroupconsistedof1,197 (Meanage±SO:58.5± 9.2 male:n=760, 63.5%)ethnically-matchedindividualswithout history ofmyocardial infarction or strokewhowere adm ittedtothesehospitals forroutine health examinations.

Inform edconsentwas obtainedfromallparticipants.Peripheralblood sampleswere collectedfrom patients andcontrols bythe Genetics Department of Zhengz houUniversity.

Thestudy protocol on the collaboration between ZhengzhouUniversityand Departm ent oflaboratorymedicine,Health SciencesCenters,Newfoundlandand Labrador was approved by institutionalboards.

SNPGenotyping

Genomic DNAwas extracted from periph eralblood samples byusing a standardsalt precip itationmethod (Milleret al. 1988). FourSNPs, rs1333049(C_1754666_l0), rs2383207 (C_l 5789010_10), rs107 57274 (C_265058 I2_10) and rs1011 6277 (C_29991625_20) were selectedfroma44-kb candid ateregiononchromosome9p2 l (chr9.2207 1397-chr 9.22115 503). SNPGenotyping wasperform edusin gthe TaqMan SNPgenotyping technolo gyonareal-timePCRplatform (ABI Prism700sequence detection system) . The reaction volumeincluded: 2.0IIIgenomic DNA(l OOng/ill),2.5III TaqMan universalPCRmastermix,0.125III primerand probes, and 0.375III ddH20.

Thestandard reaction condition included theactivationof uracil-N-glycolase (UNG)(2 min;50 °C),polymerase activation(I Omin; 95°C), 40cycles of denaturation (15s;95°C), annea lingand extension (I min;60 °C).

StatisticalAnalysis

Thegenotype frequ encydatafor eachvariant in the contr ol group wastestedfordeviation from theHardy-Weinber g equilibrium using onlinesoftware(http://ihg.osCde/cgi- bin/hw/hwa 1.pl).Haplotypefrequ enciesand associationofhaplotypesfor case and controlstatus were estimated usingthePowerMarkerV3.25 Software.Genotype frequenciesand haplotypedistribut ionbetweencase andcontrolgroups weretestedby the X²testunderrecessive,dominant andco-dominant modelsbythe SPSS 16.0software

50

package (SPSS Inc.). Statistical powerwas calculated usingQUANTaV1.2.3 software, Differenceswith P<0.05(two-tailed)wereconsideredstatistically significant.

Theminor allelefrequencies(MAF) ofthe fourtested SNPs ranged from0.3 1 to 0.49, according toHapMap CHB(Chinese HanBeij ing)data, withameanMAF of0.40forthe fourSNPs.Given thediseaseprevalence at0.5%,1,429patientsand 1,197 controls yieldeda statistica l power>0.8todetect an ORof1.50fortheMAF of 0.40for the tested SNPs at asignificance level of 0.05(two-tailed)underrecessive,dominant and additive models.The software QUANTa version 1.2.3wasusedfor the calculation of statistica l powe r.

Results

The physical locationsof thefourstudiedgeneticvariants(rs1333049,rs238320 7, rs107572 74,andrs10116277)withinthe44 kbcorecandidate regionfor strokearegive n inFigure4.1.Fourselectedgeneticvariantsweregenotyped in 1,429patients and 1,191 controls.Thedistributionsof all possible genotypesfor eachvariantaregiveninTable 4.1. Thegenotype frequenci es ofall four geneticvariant s in the controlsubjects were all undertheHard y-Weinb ergequilibrium(all P>0.05).

The result showed thatthe AG/GG genotypesofSNP rs2383207were associatedwith increase d riskforischem ic stroke(OR: 1.417, 95%CI: 1.123to1.786,P=0.003),with

ORat 1.382 (95%CI:1.081-1.766) and 1.417 (95%CI:1.123-1.786)forheterozygous AG and homozygous GG genotypes,respect ively.Accordingtothedatafrom HapMap CHB(Chinese HanBeijin g),thers10757274 and thers2383207are withinoneLD block (r²>0.8),and thers 1333049 and thers10116277 arescattered intotwoflankin g LD blocks, respectively. Noneofthe other three SNPsachieveda signifi cance level in the present study, whichindicates a smaller interval ofthe candidate region «28 kb)in the Chinese Hanpopulati on compared with theone(44 kb)previouslyreportedinCaucas ians (Matarin etal.2008).

In thehaplotypefrequency analysis, onlyrsI0757274andrs2383207 were included becausethesetwo variantsarein the same LD blocks. The distributi ons ofall four possiblehaplotype frequencies forrs 10757274 and rs2383207 aregiven inTable 4.2.

Among thefourpossiblehaplotypes,theG-Ahaplotypewas associatedwithsignificantly lowerrisk forischemic stroke(OR:0.359, 95%CI:0.256to 0.504, P<O.OOI), which suggestsa protective haplotype inthe studied population.

iCrs13333049 (Chr9.22125503)

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