presentation for phd

HeritabilityanddetectionofexpressionQTL(eQTL)

byperformingGenome-Wide-Associationstudies

AbhilashKannanMscAnalyticalGenomicsUniversityofBirmingham

(c)AbhilashKannan- tobeusedonly foreducationalpurposes

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Overview

• WhatiseQTL?• PerformingGenome-Wide-Association(GWAS)studiesin

eQTLanalysis• Importantthingstobeaddressedbeforeperforming

GWAS.• IssueofMissingHeritability• Aimofthestudy• Approachestaken• Someresultsobtainedsofar….


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eQTL

It is a particular site or Position in the Genome where the variation in thenucleotide sequences between two genotypes leads to a significantdifference in the gene expression levels between the individuals with thesegenotypes

eQTL analysis aims to combine the variation in the DNA sequence to theindividualdifferences in gene expression.

MeasuredbyMicroarrayGenotypedMarkerscoveringtheentiregenome


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TypesofeQTL…..

based on their physical distance from the regulated gene.

cis-linkage - if the eQTL is located near the target gene itself. (depends on thegene size).

Trans-linkage - if the eQTL is located on a different gene.


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Thingstobeaddressed………ProblemofPopulationStructure

systematic difference in the allele frequency between subpopulations in a studydue to ancestry difference between study subjects.

In case of unrelated individuals, Population stratification inflates the test statistics- type 1 error. If unnoticed population structure can result in false associationsignals.

Most notable methods for correcting the Structure:• Principal Components Analysis• Genomic Control

Missing heritability

only a small proportion of genetic variation - explained by any one expression trait

Large proportion – attributed to gene*gene or gene*environment interactions


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PCAplot MDSplot

CCEU

EV=5.81279

EV=12.269

CCEU

CYRICYRI CCHB+JPTCCHB+JPT


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MainObjectiveofthestudy……• To detect cis- and trans- acting expression QTLs (eQTLs) in a mixture of

HapMap populations

• To estimate the heritability of the gene expression traits

• to explore the relationship between the heritability of the gene’s expressionlevel and the power to identify cis- and trans-acting eQTLs.

Samples Used for the study:

210 unrelated HapMap individuals – 60 European founders (CEU)45 unrelated Chinese (CHB)45 unrelated Japanese (JPT)60 Africans (Nigeria – YRI)

Normalized Gene Expression Values from all the 210 individuals (illuminaBeadarray).

More than 2 million markers covering the entire genome (obtained from HapMapPhase 2 & 3).


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Approachtaken….

1. Selectingthegenestostudy------------ BLATsearch,UniqueRefSeqnumber.AvoidinggeneswithalternateSpliceformsandmultipletranscriptionsites.

2. InvestigationandcorrectionofthePopulationStructure(PlinkandGoldenHelix)---------- UsingPCAapproach

3. TestforAssociationsbetweentheGeneexpressiontraitsandSNPmarkergenotypes(R(GenABEL),PlinkandGoldenhelix)-------------- PerformLinearRegression/correlation

3. Calculatingthemeasureofheritability(GeneticRelationshipmatrixandassociatingthemwiththegeneexpressionphenotype– Program- GCTA)


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SomepromisingResults…….

POMZP3Cis- regulation

FDRpValue=1.153e-057

EV1=12.269EV2=5.8129

45Significantlyassociatedmarkers


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PEX6Cis- regulation

55Significantlyassociatedmarkers

EV1=12.269EV2=5.8129

FDRpValue=3.38e-005


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Q-QplotforGene‘hmm28636’ afterPopulationstratificationcorrection

majorityof thep-valuesisinagreementwithrandomexpectation

λ =1


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LocalizationofCis-eQTLsignalsrelativetoTSS


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PhaseI

PhaseII

7SNPsabovethe threshold

NoSignificantAssociation


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NoSignificantAssociationNoSignificantAssociation


ManySNPssignificantlyassociatedwiththegene

MultipopulationanalysisvsSinglePopulationAnalysis


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ComparisonofAdjustedR2 values

R2 =0.30- 1 R2 =0.35- 1

R2 =0.43- 1 R2 =0.28- 1

R2 =0.12- 0.73R2 =0.12–0.45

CiseffectsinMultipopulationmuchsmaller


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ExampleofTrans-eQTL

• Gene‘hmm26702’belongstoChromosome20• ShowssignificantassociationswithSNPspresentonchromosome9


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Trans–associationsvs.Cis-associations

• TransassociationsweremuchweakercomparedtoCis-associations• MajorityoftheCisassociations(60%)hada–logPscoregreaterthan10


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HeritabilityofGeneExpressiontraits

• Mostofthegeneshaveheritabilitybetween0.15to0.60• 507genes(83%)hadheritabilityhigherthan0.2• VeryFewgeneswithheritabilitygreaterthat0.5• 112genes(18%)hadheritabilityhigherthat0.5


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CanwerelyonHeritabilitytodetecteQTLs?????

• heritabilitymeasuresshowareasonablecorrelation(r=0.2)withthecis-associationsignificanceBut

• ButGeneswithverylowheritabilityestimates(heritability<0.1)showsignificantcisassociations

• maximum–log10PvaluesoftheseassociatedSNPs>–log10PvaluesofsomeoftheSNPsshowingstrongcis- associationswiththegenehavinghighheritability(heritability>0.5).


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HeritabilityestimatesofCis–associatedgenesvs.HeritabilityestimatesofTrans–associatedgenes

• Nosignificantdifferencebetweenthemwithrespecttotheirheritabilityestimates• p-value=0.2792


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eQTLheritability

• ThemeanheritabilityoftheSNPthatwasstronglyassociatedwitheachgeneexpressiontraitwas0.16

• meanheritabilityfortheoverallgeneexpressiontraitwas0.36.• about44%oftheheritabilityinthegeneexpressioncanbeexplainedby

thepeakSNP

The proportion ofvariation explained bythe cis eQTLs (cis eQTLheritability) issignificantly higherthan the variationexplained by transeQTLs (trans eQTLheritability).(p-value = 1.190e-10)

Only2trans-eQTLshaveheritability>0.3


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Summaryofresults• Thefourdifferentpopulationusedinthisanalysisgaverisetoasignificant

stratification• LargenumberofCis- regulatoryvariantswereobtainedfromtheanalysis• weakerregulatoryeffectssharedacrossthefourpopulationgroupscould

beidentifiedfromtheMultipopulationanalysis.• associationsignalsinsomeofgenescouldeasilypassthestringent

significancethresholdwhenthePhaseIIgenotypeswereused• ThepresenceofmajorityoftheCisassociatedSNPsneartheTSS.• ComparedtoCiseffectsveryfewgeneshadsignificanttransassociations

withtheSNPs• transeffectswerenotasstrongascis.• medianheritabilityofthegenesexpressiontraitshavingeQTLswas34.5%

whichisconsistentwiththepreviousstudies.• geneswithheritabilityestimatesofbelow0.2weresignificantlyassociated

withtheSNPsandhadeQTLs.• cis-eQTLshavehigherestimatesofheritabilitycomparedtothetrans-

eQTLs


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NotableObservations

• 579geneshadsignificantCisassociationsand32geneshadsignificanttransassociations

• Thesensitivityoftheanalysiswasgreatlyimprovedbyusingthepooledsampleoffourpopulationgroups(multipopulation).

• Becauseofthesmallsamplesizeinthesinglepopulationanalysis,thegeneticeffectscapturedbytheassociationtestingwashigh.

• slowdecayofLinkageDisequilibriumwiththecausalvariantsinPhaseIIHapMap.

• mostofthevariantswithciseffectspresentingenicorimmediateintergenicregionsinthehumangenome.

• Inspiteoftheweaktranseffects,severaldistantassociationsbetweentheSNPsandgeneexpressionphenotypeswereobserved.

• SNPsexplainedaslowas0%toashighas86%variationamongthephenotypes

• heritabilityestimateofthegeneexpressiontraitsdoesnotnecessarilydetermineaboutthepresenceofabsenceofeQTLsforaparticulargene


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Challenges• theanalysiswasmainlyfocussedonasinglecelltype• limitedpoweroftheanalysis.• transregulationsareconsideredtomoreindirect(generegulatedbya

largenoofSNPs,whichfailtopasssignificantthresold)• Useofsmallsamplesize– lesspowertodetectmanytransassociation

signals• Humancell- aminutepartofthewholeorganism.Thereforemajorityof

thetranseffectsmediatedbyintercellulareventscanbedifficulttodetect.

• useofstrictsignificancethresholdtoavoidfalsepositives• causalvariantscouldstillhaveverylowminorallelefrequency(MAF)and

apoorLDwiththeassayedSNPs- hencelowHeritability• manycausalvariantsforaparticulargene- onlyasmallpercentageof

varianceisexplainedbythemajorityofthecausalvariants.• Heritabilityestimatesdependsonthestateofthecell/Tissue.• locihavingsmalleffectsbecomedifficulttobecapturedthanthosewith

largeeffects


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FutureDirections

• Insomeofthediseaseslikecancer,Singlegeneisnotthecausativefactors.

• FunctionalSNPsaffectingasinglegene/allelemayhavelittleeffectoftheirown.

• HighThroughputsequencingtechnologies– largedatasets,clearlydefineSNPsandLDstructure.

• Detailedmappingandannotations,Largecollectionofsamples.• Integrationofresequenceddatawithindifferentethnicgroups.• InclusionofvariantswithMAF<0.001• Establishthegeneticnetwork.• Prioritizethe(QuantitativeTraitGenes)QTGs.• Canbeappliedtothedatafrommethylation,miRNAandother

experimentstocompletelycharacterizemolecularbasisofcomplextraits.


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Valuableinsightsfrom

ProfZ.Luo,UniversityofBirminghamMinghuiWang,UniversityofBirminghamNingJiang,UniversityofBirmingham

Thankyou……..


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presentation for phd

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