Genome-wide association study identifies loci on 12q24 and 13q32 associated with Tetralogy of Fallot.

0.36 MB·English

by  Cordell, Heather J.

Human Molecular Genetics, 2013, Vol. 22, No. 7 1473–1481
doi:10.1093/hmg/dds552
Advance Access published on January 7, 2013
Genome-wide association study identifies loci on
12q24 and 13q32 associated with Tetralogy of Fallot
Heather J. Cordell1, Ana To¨pf1, Chrysovalanto Mamasoula1, Alex V. Postma2, Jamie Bentham3,
Diana Zelenika4,5, Simon Heath4,6, Gillian Blue7, Catherine Cosgrove3, Javier Granados
Riveron8, Rebecca Darlay1, Rachel Soemedi1, Ian J. Wilson1, Kristin L. Ayers1, Thahira J.
Rahman1, Darroch Hall1, Barbara J.M. Mulder2, Aelko H. Zwinderman2, Klaartje van Engelen2,
J. David Brook8, Kerry Setchfield8, Frances A. Bu’Lock9, Chris Thornborough9, John
O’Sullivan10, A. Graham Stuart11, Jonathan Parsons12, Shoumo Bhattacharya3, David Winlaw7,
Seema Mital13, Marc Gewillig14, Jeroen Breckpot15, Koen Devriendt15, Antoon F.M. Moorman2,
Anita Rauch16, G. Mark Lathrop4,5, Bernard D. Keavney1,∗,{ and Judith A. Goodship1,{
1InstituteofGeneticMedicine,NewcastleUniversity,NewcastleuponTyneNE13BZ,UK,2AcademicMedicalCenter,
Amsterdam, The Netherlands, 3Department of Cardiovascular Medicine, Oxford University, Oxford OX1 2JD, UK,
4InstitutGenomique,CentreNationaldeGenotypage,Commisariata` l’e´nergieAtomique(CEA),Evry,France,5Centre
d’Etude du Polymorphisme Humain, Fondation Jean Dausset, Paris 75010, France, 6Centro Nacional de Ana´lisis
Geno´mico, Barcelona, Spain, 7The Children’s Hospital at Westmead, Westmead, New South Wales, Australia,
8InstituteofGenetics,NottinghamUniversity,NottinghamNG72RD,UK,9UniversityHospitalsofLeicesterNHSTrust,
Leicester, UK, 10Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle, UK, 11Bristol Royal Hospital for
Children, Bristol, UK, 12Leeds Teaching Hospitals NHS Trust, Leeds, UK, 13Hospital for Sick Children, Toronto,
Canada, 14Department of Pediatric Cardiology, 15Center for Human Genetics, University of Leuven, Leuven 3000,
Belgium and 16Institute of Medical Genetics, University of Zurich, Zurich 8006, Switzerland
ReceivedSeptember12,2012;RevisedDecember17,2012;AcceptedDecember24,2012
Weconductedagenome-wideassociationstudytosearchforriskallelesassociatedwithTetralogyofFallot
(TOF), using a northern European discovery set of 835 cases and 5159 controls. A region on chromosome
12q24 was associated (P51.431027) and replicated convincingly (P53.931025) in 798 cases and 2931
controls [per allele odds ratio (OR)51.27 in replication cohort, P57.7310211 in combined populations].
Single nucleotide polymorphisms in the glypican 5 gene on chromosome 13q32 were also associated
(P51.731027) and replicated convincingly (P51.231025) in 789 cases and 2927 controls (per allele
OR51.31inreplicationcohort,P53.03310211incombinedpopulations).Fouradditionalregionsonchro-
mosomes10,15and16showedsuggestiveassociationaccompaniedbynominalreplication.Thisstudy,the
first genome-wide association study of a congenital heart malformation phenotype, provides evidence that
common genetic variation influences the risk of TOF.
∗Towhomcorrespondenceshouldbeaddressedat:InstituteofGeneticMedicine,NewcastleUniversity,InternationalCentreforLife,CentralParkway,
NewcastleuponTyneNE13BZ,UK.Tel: [phone];Fax: [phone];Email:[email protected]
†Theseauthorscontributedequallytothiswork.
# The Author 2013. Published by Oxford University Press.
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1474 Human Molecular Genetics, 2013, Vol. 22, No. 7
INTRODUCTION RESULTS
Congenitalheartdisease(CHD)affects(cid:3)1%oflivebirthsand Figure 1 shows the genome-wide association results obtained
with two complementary approaches, case/control analysis in
isamajorsourceofmorbidityandmortalityinchildhood.Ap-
PLINK and family-based analysis in estimation of maternal,
proximately20%ofCHDoccursinthesettingofchromosomal
imprintingandinteractioneffectssoftware(EMIM)(seeMate-
conditions or multisystem malformation syndromes. Family
rialsandMethods).WithPLINK,thestrongestsignalofasso-
studiesintheremaining80%of‘sporadic’casesindicateasig-
ciation occurred at a group of SNPs on chromosome 12q24
nificantcomplexgeneticcomponenttothedisease(1).Previous
(top SNP rs11065987, P¼4.6×1028), with several other
studieshaveindicatedthatrareanddenovocopynumbervari-
regions, including SNPs on chromosomes 3, 10, 13 and 16
ationinthehumangenomecontribute5–10%ofthepopulation
risk of sporadic CHD (2–5), but genome-wide association reaching more modest, but suggestive levels of significance
studies (GWAS) assessing the relationship between common (P≤1×1025). Quantile–quantile (QQ) plots (Supplemen-
single nucleotide polymorphism (SNP) and CHD risk are yet tary Material, Fig. S1A) indicated a slight inflation in the
to be reported. Tetralogy of Fallot (TOF) is the commonest genome-widedistributionofteststatistics(genomiccontrolin-
formofcyanoticCHD,affecting (cid:3)3per10000newborns(6). flationfactorl¼1.076)(9),possiblyduetounmodelledpopu-
AlthoughTOFisusuallyrepairedininfancywithlowmortality, lation substructure. Correction using the top 10 principal
thereissubstantiallatemorbidity,inparticularfrompulmonary components from EIGENSOFT reduced the genomic control
valvular insufficiency and atrial and ventricular arrhythmias. inflation factor slightly (l¼1.037, Supplementary Material,
Population studies suggest a substantial familial recurrence Fig. S1B) without affecting the overall results substantially
risk in sporadic, non-syndromic TOF (1,7). We, therefore, (rs11065987remained thetopSNP,P¼6.0×1028).Correc-
undertookaGWAStoidentifycommongeneticriskfactorspre- tion using GenABEL resulted in a reduction in the genomic
disposingtoTOF,giventhehighsuccessofthisdesignoverthe control factor lto 0.996, with no systematic departure from
past5years(8). the expected line in the resulting QQ plot (Supplementary
Figure1.Manhattanplotsofthegenome-wideassociationresults.Thetoppanel(PLINKresults)showsthe–log10P-valuesfromtheCochran–Armitagetrend
test(forautosomalSNPs)orlogisticregressionallowingforgenderasacovariate(forX-chromosomalSNPs).Thebottompanel(EMIMresults)showsthe
2log10P-valuesfromthechildtrendtest(autosomalSNPsonly).Dashedlinesareshownatsignificancethresholdsforsuggestive(P¼1×1025)andsignifi-
cant(P¼5×1028)association,respectively.
Human Molecular Genetics, 2013, Vol. 22, No. 7 1475
Material, Fig. S1C). The 12q24 region remained the strongest
s
signal of association (Table 1, Supplementary Material, ult
M es
Table S1) with a slight decrease in overall significance MI nr
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nominal significance P≤1×1025 (Table 1, Supplementary P 3.4.1.0.9.5.1.2.
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European ancestry. The chromosome 12q24 region strongly 70942602
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replicated (P¼9.7×1026 at rs233722; P¼3.9×1025 at U9 1.41.31.31.31.41.41.41.4
rs11065987; combined discovery and replication P¼7.7× 20732812
10211 at rs11065987) with a per allele odds ratio (OR) L95 1.131.091.111.081.151.121.161.15
for TOF of 1.27 [95% CI (1.13–1.42)] in the replication 76877822
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r0s.102056923a2t2r3s)2a2n2d861368)(Pan¼d0o.n03c3hraotmrso6s4o9m9e1s001)5. (P¼0.043 at PLINK ORfor 1.2661.2181.2501.2111.2891.2631.3111.302
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orMswsing2iaten3htea3iSrnl7iNa2tclh2Po,euhT(lLdarasDdb1ll1aebb0relgo6eSec5n2lk9y).8,in7bcs)eulugrdaegecteadcsiotniaunesndgteanadgocaomfiovniranraitblahytaestiag(tSnhsiueifinpcgpaallsneescmoevceianaorttinaiaocrnynet SNPs(replicati A1A2 72424GA86818GA10714AG06415AG31474GA39943GA88323AC94509AC StandardError.
1wtosbhh2feieoeTtqhiwn2aomis4nessp,defotuuehcwtnrseietaiedhgat5eintocrSMioanfiNrurbcrserPaiifiergsntednontglep(ayiSologuutwnshepttnaecprsooielmentnasymgstupprsepueeoerpdntdcaotiataaarSisrrtotNesyoinooduPncnMb,iidnayrastsiirtr1goteseh11rnnsei01aua6l0lltdt,h56siaa95sFtfn89criog7o8chv..m7hae.rSdroaAy4mln)at,oculhrosmoneohuoabmodgneryheert Table1.Topreplicating CHRSNPBP 12rs11065987112012rs17696736112412rs11066188112612rs11066320112912rs233722113012rs233716113013rs798267792913rs4771856929 CHR,Chromosome;SE,
1476 Human Molecular Genetics, 2013, Vol. 22, No. 7
Figure2.LocusZoomplotsforthereplicatinglocionchromosomes12(PLINKresults)and13(EMIMresults).ThecolourcodingindicatesthedegreeofLDof
eachSNPwiththenamedindexSNP(shownasapurplediamond),asestimatedfromtheHapMapCEUpopulation.
Human Molecular Genetics, 2013, Vol. 22, No. 7 1477
DISCUSSION haplotype isunknown,althoughgiventheroleofgenesinthe
region in T-cell function, it is thought likely to relate to
Ourstudyprovidescompellingevidencethatcommongenetic enhancedresistancetoinfectiousdiseaseatatimeofincreased
variationinfluencestheriskofCHD.ThetopSNPsonchromo- human population density in Europe (13). Our data on a
some 12 implicated by our discovery and replication analyses childhood phenotype that until recently was highly lethal
(P¼7.7×10211) are located within a large LD block on illustrate the impact of immunity as a selective force in
12q24. This region has previously been associated with a human evolution—as has been recently shown for the effects
varietyofcomplexgeneticconditions,includingtype1diabetes ofYchromosomehaplogroupsonimmunefunctionandathero-
(11),celiacdisease(12),coronaryarterydisease(13),rheuma- sclerosisrisk(19).
toidarthritis (14),systemic lupus erythematosus (15)multiple Ourfindingsonchromosome13[rs7982677at13q32,OR¼
sclerosis (16) and with blood platelet count (13). The risk 1.31, 95% CI¼(1.16–1.48), P¼3.03×10211] and in two
alleles at the top SNPs in our analysis all lie on a common separate regions of chromosome 10 [rs2388896 at 10p14,
(40% frequency in individuals of White European ancestry) OR¼0.83, 95% CI¼(0.74, 0.93), P¼8.55×1028 and
1.6Mbhaplotypeatchromosome12q24,containing15genes, rs2228638 at 10p11.2, OR¼1.28, 95% CI¼(1.07, 1.53),
that spans our associated interval (Fig. 2 and Supplementary P¼2.05×1027] are also significant. The top SNPs in the
Material, Table S3). A previous analysis (13) provided strong 13q32 region lie in intron 7 of the GPC5 gene that encodes
evidencethatthehaplotypehasundergonerecentpositiveselec- for glypican 5. Glypicans are heparan sulphate proteoglycans
tioninindividualsofEuropeanancestry.Forallconditionsasso- that are bound to the outer surface of the plasma membrane
ciatedwiththishaplotypedescribedthusfar,includingTOF,the by a glycosyl-phosphatidylinositol anchor. There are six
allelesconferringincreasedriskattheassociatedSNPsarethe members of the glypican family in mammals, and they serve
derivedalleles,whosefrequencieshaveincreasedinEuropeans asregulatorsofkeydevelopmentalsignallingpathways,includ-
by a selective sweep (starting around 3000 years ago). Our ingtheWnt,Hedgehog,fibroblastgrowthfactorandbonemor-
chromosome12resultsaddtotheremarkablediseasepleiotropy phogenetic protein pathways (20). Glypicans are, therefore,
associated with variation in the 12q24 region of the human strong candidate genes for involvement in cellular growth
genome. control and morphogenesis during heart development. GPC5
Imputation analysis in our discovery cohort indicated that, has eight exons and occupies (cid:3)1.42Mb at chromosome
although our association signal could be driven by any one 13p32. The seventh intron, which harbours our top SNPs, is
of a number of imputed SNPs, none show significantly stron- 735Kblongandcontainsthreeputativenon-codingtranscripts,
gerassociation than isalreadyseen atourtopgenotyped SNP noneofwhichareoverlappedbyourtopSNPs.SNPsinGPC5
(rs11065987). Further interrogation of this region, ideally in havebeenassociatedwiththeriskofnephroticsyndrome(21),
non-European cohorts, showing varying LD patterns, will be withprotectionfromsuddencardiacarrest(22)andwithhigher
required to further refine the association signal. Within the risks of lung cancer and multiple sclerosis (23,24). GPC5 is
12q24 region, the strongest candidate gene is tyrosine-protein amongthegenesdeletedinasubsetofpatientswith13qdeletion
phosphatase non-receptor type 11 (PTPN11), a regulator of syndrome, a multisystem disorder (typically characterized by
Ras/mitogen-associated protein kinase signalling. Activating holoprosencephaly) in which CHD can occur. In a French
mutations of PTPN11 are a cause of Noonan’s syndrome cohort of 12 patients with 13q deletion, 2 had TOF, and
(17),aMendelianmultisystemdisorderinwhichmalformation GPC5wasdeletedinbothofthesepatients(25);singlepatients
of the cardiac outflow tract is a typical feature. Association with 13q deletion and TOF have been reported by two other
betweena SNPinPTPN11 (rs11066320)andTOFwas previ- groups (26,27). Defects in genes encoding for other members
ously demonstrated (18) in a candidate gene study using a oftheglypicanproteinfamilyhavebeenlinkedtocardiacmal-
family-based association analysis approach on a set of 754 formation syndromes. CHD is common in Simpson–Golabi–
TOFcasesthatpartiallyoverlapwiththesetofcasesdescribed Behmel syndrome, the condition associated withGPC3 muta-
here. Further research will be necessary to investigate the hy- tion(28),andhasbeenreportedinomodysplasia,thecondition
pothesis that the SNP association we have observed in this associatedwithGPC6mutation(29).Giventhelargesizeofthe
region results from upregulation of the activity of PTPN11 genomicintervalspannedbytheGPC5gene,itseemslikelythat
and the magnitude of effect compared with that of the theassociationwehaveobservedhereresultsfromaneffecton
gain-of-function mutations that lead to Noonan’s syndrome. thefunctionofGPC5itself,ratherthananeighbouringgene.
The observation of association between TOF and an evolu- On chromosome 10, rs2388896 lies in a gene desert [the
tionarily selected haplotype at 12q24 raises some intriguing nearest gene, GATA-binding transcription factor protein
questions.Incontrastwithconditionsofadultonset,suchascor- family,liessome0.8Mbdistant],whereasrs2228638isanon-
onaryarterydiseaseandrheumatoidarthritis,previouslyshown synonymouscodingSNPthatresultsinthesubstitutionofIso-
tobe associated with 12q24SNPs,thecondition onwhichwe leucine for Valine at position 733 of the neuropilin-1 protein,
have focussed carries substantial early mortality that signifi- whichisencodedbythegeneNRP1.Neuropilin-1isareceptor
cantly impacts reproductive fitness. Prior to the modern for ligands from the vascular endothelial growth factor and
cardiac surgical era, mortality from TOF before the age of 10 semaphorin families that is essential for septation of the
years was around 80%. Given this, it might be expected that cardiacoutflowtract(30).Neuropilin-1hasanimportantfunc-
any variant conferring even a modest additional risk of TOF tioninnormalarteriovenouspatterning(31),anddisruptionof
would have been eliminated, or driven to very low allele fre- endothelial neuropilin-1 leads to cardiac outflow tract defects
quency,bynaturalselection.Thenatureoftheselectionevent similar to those seen in human DiGeorge syndrome (in which
responsible for the emergence of the pleiotropic 12q24 risk context TOF is a frequent occurrence) (32). Further research
1478 Human Molecular Genetics, 2013, Vol. 22, No. 7
willberequiredtoidentifywhethertheassociationisduetoa assistedlaserdesorption/ionization—timeofflight.Additional
functionaleffectofrs2228638orofsomeothervariantinLD. UK population-based control data for the replication were
In conclusion, our study has identified two regions (12q24 obtained from the TwinsUK resource (http://www.twinsuk.a
and 13q32) that are strongly and replicably associated with c.uk),anadulttwinregistrycomprising12000(predominantly
TOF. As far as we are aware, this is the first genome-wide female) twins. Genotype data for 3512 twin individuals (gen-
study focussing on SNP associations to have been reported otypedusingtheIllumina610Karray)wereobtainedfromthe
in CHD. Pooling patient resources from several international Department of Twin Research and Genetic Epidemiology at
groups was required to achieve sufficient numbers of cases King’s College, London. Only the first twin from each pair
for adequate power to detect and replicate a number of of genotyped twins (2603 unrelated individuals) was used in
genetic effects; however, studies of commoner diseases have the current study.
emphasized the importance of very large cohorts of cases
and controls. The present relatively small GWAS cohort has
Statistical analysis
in all likelihood detected only the strongest genome-wide
influences on TOF risk. Our results suggest that larger inter- Following stringent quality control (QC) procedures (see
national collaborative studies have the potential to discover below), the final discovery dataset for analysis consisted of
additional significantly associated loci. 835 unrelated cases and 5159 controls (plus 717 additional
Complete summary association results from the GWAS family members, including both parents for 293 of the
component of our study (the EIGENSOFT corrected results) cases), genotyped at 516131 autosomal and X-chromosomal
areavailabletointerestedresearchersbycontactingthecorre- SNPs.Theprimaryanalysisperformedwasacase/controlana-
sponding author. lysisofthe835unrelatedcasesand5159controlsusingeither
the Cochran–Armitage trend test (for autosomal SNPs) or lo-
gistic regression, allowing for gender as a covariate (for X-
MATERIALS AND METHODS chromosomal SNPs), using the software PLINK (36). We
also used two alternative approaches designed to correct for
Study subjects and genotyping
possible population stratification: Firstly, we used the
Fortheinitial(discovery)phase,individualswithTOFofnorth- ‘smartpca’routineoftheEIGENSOFTpackage (37)tocalcu-
ernEuropeanancestry,together withtheir parentsandaffected latethetop10principalcomponentsthatwereenteredascov-
siblings (when available), were recruited from multiplecentres ariates into a logistic regression analysis of each SNP across
in Newcastle, Leeds, Bristol, Liverpool, Oxford, Nottingham the genome. Secondly, we analysed each SNP using a score
and Leicester (all UK), Leuven (Belgium), Erlangen test from a linear mixed model (38), allowing for possible re-
(Germany) and Sydney (Australia). Ethical approval was latedness between individuals via consideration of their
obtained from the local institutional review boards at each of genome-wide estimated kinship coefficients, using the
the participatingcentres priortoblood or salivasamplecollec- ‘mmscore’functionintheRpackageGenABEL(39).Thisap-
tions, and informed consent was obtained from all subjects, or proachhasbeenpreviouslyproposedasameansofcorrecting
fromtheirparents/legalguardians,ifthepatientwasachildtoo for unknown population structure between apparently unre-
young to provide consent him/herself. Patients who exhibited lated individuals in a genome-wide association study (40).
clinicalfeaturesofrecognizedmalformationsyndromes,devel- Inadditiontothecase/controlanalyses,wealsoperformeda
opmental abnormalities or learning difficulties were excluded family-basedassociationanalysis(ofautosomalSNPsonly)of
from the study. All samples were screened for the known all cases, their relatives and controls using the software
22q11.2 deletion associated with TOF (33,34) using multiplex package EMIM (41). EMIM is primarily designed for the in-
ligation-dependent probe amplification (MRC-Holland) and vestigation of complex genetic effects, including maternal
excluded, if the deletion was present. SNP genotyping was genotype effects, maternal–fetal interactions and imprinting
carriedoutattheCentreNationaldeGenotypage(EvryCedex, (41). In this instance, we used the ‘child trend’ model in
France)usingtheIllumina660wQUADarray,andthegenotypes EMIM to model only an effect of the child’s (case’s) own
werecomparedwithgenotypedataforUKpopulation-basedcon- genotype. The resulting analysis was, thus, conceptually
trols (5667 individuals genotyped on the Illumina 1.2M chip) similar to the case/control analysis we had performed in
obtained from the Wellcome Trust Case Control Consortium 2 PLINK, but with the advantage of allowing additional infor-
(WTCCC2)(https://ccc.wtccc.org.uk/ccc2). mation from parental genotypes to be incorporated, where
For the replication phase, individuals with TOF of self- available.
reportedCaucasianancestrywererecruitedfromOxford,Not- Thereplicationcohort(afterQC)consistedof798casesand
tingham, Newcastle, The Netherlands and Canada. Five 2931controls.Genotypeswere analysedusinglogisticregres-
hundred and fifteen Dutch TOF cases were identified from sioninPLINK,allowingfortwolevelsofnationality(British/
the Netherlands national registry and DNA bank of patients DutchorCanadian)asacovariate. P-valuesforassociation in
with CHD (CONCOR), the design of which has been previ- thecombined(discoveryand replication) datasets were calcu-
ously described (35). One hundred and forty-four Canadian latedusingFisher’strendapproachforcombiningP-valuesas
TOF cases, together with Canadian controls of self-reported implemented in the online software MetaP (http://www.svap
Caucasian ancestry, were obtained from the SickKids Heart roject.org/metap.php).
Centre Biobank, an Ontario province-wide biorepository for To further refine the association signal at chromosome
CHD. These replication samples were genotyped at the 12q24, we carried out imputation in the discovery cohort
Centre National de Genotypage using Sequenom matrix- within the 5Mb region centred around rs11065987. We used
Human Molecular Genetics, 2013, Vol. 22, No. 7 1479
the program IMPUTE version 2 (42) with data from the 1000 components analysis using the ‘smartpca’ routine of the
GenomesProject(PhaseIinterimdata,releasedJune2011)as EIGENSOFT package (37).
a reference panel. Data at 11208 SNPs passing post- Within each of the case and control cohorts, we excluded
imputation QC (from an original 56637 imputed SNPs) any SNPs with minor allele frequencies ,0.01 that were suc-
wereanalysedusingtheprogramSNPTESTtotestforassoci- cessfully genotyped in ,95% of individuals or that had a
ation with disease status. Hardy–Weinberg equilibrium test P-value,1028. Within
thetwocontrolcohorts,wealsoimplementedSNPexclusions
recommended by WTCCC2 relating to a measure of the stat-
QC procedures
isticalinformationinthegenotypedataaboutallelefrequency
Discovery cohort (exclude if ,0.975), missingness (exclude if .2% missing
We used stringent QC checks to ensure that only high-quality genotypes) and plate effects (exclude if P-value from an
data were included in the final analysis. QC procedures were n-degree of freedom test of plate association ,1×1025).
carriedoutinPLINKversion1.07(36)withvisualizationper- Within the TOF case cohort (for which a number of family
formedinR(http://www.r-project.org/).Forthecurrentstudy, members had been genotyped), we also excluded SNPs
genotype data were generated at 557124 SNPs across the showing .10% Mendelian inheritance errors.
genome for1733individuals (comprising913TOF casesplus Following an initial association analysis, visual inspection
a number of unaffected relatives). We excluded individuals of intensity cluster plots (46) was performed for all SNPs
withgenotypecallrates,98.78%andaverageheterozygosities showing nominal P-value,1025, and only those SNPs for
outside the range (0.310, 0.336) (based on consideration of which the genotype calls appeared reliable (well clustered
540241autosomalSNPspassinglooseQC,namelysuccessful- into three distinct groups) were taken forward for replication.
lygenotypedin.95%ofindividualsandwithaHardy–Wein- All SNPs reported here passed this visual inspection of
berg equilibrium test P-value.1028). These exclusion intensity cluster plots in the discovery cohort, indicating that
thresholds were chosen based on visual inspection of the call genotypedataand,thus,resultsattheseSNPscouldbeconsid-
rates and heterozygosities (Supplementary Material, Fig. S5) ered reliable.
toretainthemajorityofindividuals,whereasexcludingoutlying
individuals.
Wegeneratedasmallersetof41692autosomalSNPs(suc- Replication cohort
cessfully genotyped in .95% of individuals, with a Hardy–
QC was also performed on the genotype data from the
Weinberg equilibrium test P-value.1028, with minor allele
TwinsUK replication sample. From the 2603 first twins con-
frequencies .0.4 and pruned to show low levels of LD using
sidered, we excluded 43 showing genotype call rates ,99%
the PLINK command ‘–indep 50 5 2’) that were used to
and average heterozygosities outside the range (0.312,
check relationships/sample duplications and ethnicities.
0.331) (based on the consideration of 576610 autosomal
Genome-wide identity-by-descent (IBD) sharing was calcu-
SNPs passing loose QC, namely: successfully genotyped in
lated using the‘–Z-genome’ commandinPLINK, andoneof
.95%ofindividualsandwithaHardy–Weinbergequilibrium
each pair of related individuals (mean proportion of alleles
test P-value.1028). These exclusion thresholds were chosen
shared IBD .0.1)wasexcluded. Multidimensional scalingof
based on visual inspection of the call rates and heterozygos-
our samples together with 210 unrelated Phase II HapMap
ities. We carried out testing of relationships/sample duplica-
(43)individualsfrom4populations[Utahresidentswithances-
tions and ethnicity using the same approach as described
try from northern and western Europe (CEU), Japanese in
above for the TOF cohort and excluded first twins who did
Tokyo, Japan, Han Chinese in Beijing, China and Yoruba in
not cluster with the CEU HapMap samples and one of each
Ibadan, Nigeria] (genotyped at same set of 41692 autosomal
pair of first twins who showed high IBD sharing (mean pro-
SNPs) was performed and identified 33 individuals in our
portion of alleles IBD .0.05). This resulted in a final set of
study who did not cluster with the CEU samples, suggesting
2547 TwinsUK controls to be used in the replication study.
non-European ancestry (Supplementary Material, Fig. S6).
Theseindividualswereexcluded.
Weusedthe‘–check-sex’optioninPLINKtocheck(based
Post-imputation QC
ontheaverageXchromosomalheterozygosity)thatthegender
of our samples matched its expected value and excluded WeusedtheprogramIMPUTEversion2(42)tocarryoutim-
samples for which we were unable to resolve inconsistencies. putation in the discovery cohort within the 5Mb region
Following QC, we were left with 835 unrelated TOF cases centred around rs11065987. Data from the 1000 Genomes
(plus 717 additional family members), whose genotypes were Project (47) (Phase I interim data, released June 2011) were
comparedwithgenotypedatafrom5159UKpopulation-based used as a reference panel. Post-imputation QC involved ex-
controls obtained from the WTCCC2 (https://ccc.wtccc.org. cluding any SNPs likely to be poorly imputed (specifically
uk/ccc2). These controls comprised 2673 samples from the thosewithan‘info’score ,0.5orwithminorallelefrequency
1958 British Birth Cohort study (58C) and 2486 National in controls ,0.01). Data at 11208 SNPs passing post-
Blood Service (NBS) samples (selected from an initially gen- imputation QC (from an original 56637 imputed SNPs)
otypedsetof 2930 58C samples and2737 NBS samples). We were analysed in the program SNPTEST version 2.1.1 (46),
excluded the same controls as had been excluded in the using the ‘-method ml’ option (aNewton–Raphson algorithm
WTCCC2 (44) and WTCCC3 (45) studies, plus an additional that maximizes the missing data likelihood) to account for
fourcontrolsthatwefoundtobeoutliers,followingaprincipal genotype uncertainty.
1480 Human Molecular Genetics, 2013, Vol. 22, No. 7
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the National Institute for Health Research, through the North- J.H.,Howson,J.M.,Stevens,H.,McManus,R.,Wijmenga,C.etal.
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ThisworkwassupportedbytheBritishHeartFoundation,the Li,Y.,Kurreeman,F.,Zhernakova,A.,Hinks,A.etal.(2010)
European Community’s 7th Framework Programme contract Genome-wideassociationstudymeta-analysisidentifiessevennew
‘CHeartED’ (grant number HEALTH-F2-2008-223040) the rheumatoidarthritisriskloci.Nat.Genet.,42,508–514.
15. Gateva,V.,Sandling,J.K.,Hom,G.,Taylor,K.E.,Chung,S.A.,Sun,X.,
Wellcome Trust (grant number 087436), the Heart and
Ortmann,W.,Kosoy,R.,Ferreira,R.C.,Nordmark,G.etal.(2009)A
Stroke Foundation of Ontario, Ontario Ministry of Research
large-scalereplicationstudyidentifiesTNIP1,PRDM1,JAZF1,
and Innovation, McLaughlin Centre, Labatt Family Heart UHRF1BP1andIL10asrisklociforsystemiclupuserythematosus.Nat.
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WTCCC2 project was provided by the Wellcome Trust
(2010)Theautoimmunedisease-associatedKIF5A,CD226andSH2B3
under award 085475. The TwinsUK cohort acknowledges
genevariantsconfersusceptibilityformultiplesclerosis.GenesImmun.,
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(2001)MutationsinPTPN11,encodingtheproteintyrosinephosphatase
Programme (GenomEUtwin Project QLG2-CT-2002-01254),
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BBSRC project grant G20234 and the National Eye Institute
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via an NIH/CIDR genotyping project (PI: Terri Young). T.J.,Glen,E.,Tan,H.,Doza,J.P.,Relton,C.N.etal.(2012)Acommon
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