Genome-Wide Association Analysis Identifies Susceptibility Loci

© 2012 Nature America, Inc. All rights reserved. A A full list of affiliations appears at the end of the paper. migraine (IHGC) Consortium Genetics Headache International to close 8q22, chromosome on locus (GWAS) studies susceptibility a migraine association wide identified aura symptoms neurological with aura). (migraine Previous genome- up to one-third of affected individuals, attacks may be associated with In aura). without (migraine phonophobia and photo- and vomiting nausea, and headache unilateral throbbing severe, by characterized population general the of 12% ing affect disorder brain neurovascular episodic is a disabling Migraine debilitating migraine and at P ( and combined 1 at controls. testing more matched individuals association associated autonomic characterized Migraine for the International Headache Consortium Genetics KubischChristian Müller-MyhsokBertram Jaakko Kaprio HofmanAlbert Marta Vila-Pueyo Mark A Louter Willebrordus P J van Oosterhout Gisela M Terwindt Tobias Freilinger loci for migraine without aura Genome-wide association analysis identifies susceptibility Nature Ge Nature Received 13 December 2011; accepted 3 May 2012; published online 10 June 2012; P 0

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© 2012 Nature America, Inc. All rights reserved. migraine, neuronal activity–dependent migraine, activation neuronal of activity–dependent MEF2D restricts neurons formed newly of survival the supporting by differentiation neuronal regulates MEF2D brain. in expressed highly is that factor transcription a is protein MEF2D 3 the within and (intronic 2D) factor enhancer myocyte the within SNPs located were associated All (OR = 1.20), respectively) ( meta-analysis (overall replicated successfully were associations and stage, lication rep the to forward taken were SNPs rs3790455 and rs1050316 The analysis and that were all in close linkage disequilibrium (LD; ( significance genome-wide in SNPs shown any the of for eQTLs tissues and cell lines of sets data SNPs available in associated the to mapped that (eQTLs) we extend our for analyses, searched expression quantitative trait loci To further genders. both in (ORs) ratios odds similar relatively had 2 Table ( SNPs reported the for analysis interaction gender performed we women, in prevalent more is migraine Because cated. repli at were and convincingly 2q37 12q13 of the associations which cohort. each in cases of number the indicate parentheses in Numbers scale. a logarithmic on plotted are ratios Odds intervals. confidence 95% the represent lines horizontal gray the cohorts; individual the for ratios odds the represent squares black plots, forest the In lines. blue light as shown are 22 release (CEU) ancestry European Western and Northern of residents Utah HapMap from rates recombination SNP. that with LD of Estimated extent the to according colored are SNPs other the diamond; P –log against region specific the in SNPs of 36) Build (NCBI position chromosomal the ( rs9349379 ( rs3790455 for shown are results locus; respective the at association highest the with SNP the for plot a forest with together loci migraine ( 1 Figure s r e t t e l synapses excitatory of number the a values. The SNP with the highest association signal at each locus is represented as a purple a purple as represented is locus each at signal association highest the with SNP The values. – Study reference The locus at 1q22 contained six SNPs that were associated with with associated were that SNPs six contained 1q22 at locus The d Plotted ( Fixed effectmeta-analysi Norway Finland The Netherlands Spain Discovery sample a n SNPs ) Regional association plots (generated using LocusZoom) are shown for the four new new four the for shown are LocusZoom) using (generated plots association ) Regional =4,834) –log10 (P value) 10 0 2 4 6 8 (

). No significant interactions were observed, and all SNPs SNPs all and observed, were interactions significant No ). n LMNA ( Regional and forest plots for the newly identified loci associated with migraine. migraine. with associated loci identified newly the for plots forest and Regional ( =837) n SEMA4A n 154. =871) =245) SLC25A44 PHACTR1 PMF1 4 BGLAP PAQR6 P ( SMG5 n ( values of 3.21 × 10 × 3.21 of values C1orf8 n =555) VHLL C1orf182 Position onchr.1(Mb) =2,326) CCT3 154.6 5 RHBG rs3790455 7 s , 8 ) ) ( C1orf6 MIR9–1 , , but we did not consistently observe significant 1.00 MEF2D c 1 MEF2D ) and rs6478241 ( rs6478241 ) and 154. Fig. Fig. APOA1BP IQGAP3

1.12 TTC24 P HAPLN2 GPATCH4 < 5 × 10 8 Odds rati BCAN 1 1.26 , , NES Table C1orf6 CRABP2 ISG20L2 155.0 MRPL24 9 HDGF −10 6 . Perhaps even more relevant to to relevant more even Perhaps . PRCC 1 o 1.41 Table 0 −8 SH2D2A r . As the brain in migraine is is migraine in brain the As . NTRK1 (OR = 1.19) and 7.06 × 10 × 7.06 and 1.19) = (OR 2 0. 0. 0. 0. 1 ) in the discovery stage of stage the ) in the discovery INSRR 2 4 6 8 and 1.58 ASTN2 0 20 40 60 80 100 1

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General SNP information Discovery samples All replications Overall meta-analysis ADVANCE ONLINE PUBLICATION ONLINE ADVANCE Minor Minor allele Sequence OR OR OR r 2 with SNP Chr. Positiona Location Geneb allele frequency typec P value (95% CI) I 2 P value (95% CI) I 2 P value (95% CI) I2 top SNP Locus 1 rs1050316 1 154701327 Intragenic MEF2D G 0.34 Imputed 2.59 × 10−8 1.24 0.00 1.15 × 10−3 1.14 0.00 3.21 × 10−10 1.19 0.00 0.987 (1.15–1.33) (1.06–1.24) (1.13–1.26)

rs2274316 1 154712866 Intragenic MEF2D C 0.35 Genotyped 3.60 × 10−8 1.23 0.00 – – – – – – 0.986 (1.14–1.33)

rs1925950 1 154717364 Intragenic MEF2D G 0.35 Imputed 2.97 × 10−8 1.24 0.00 – – – – – – 0.988 (1.15–1.33)

rs3790455 1 154722925 Intragenic MEF2D C 0.34 Genotyped 1.71 × 10−8 1.24 0.00 4.85 × 10−4 1.16 0.00 7.06 × 10−11 1.20 0.00 –

(1.15–1.34) (1.07–1.26) (1.14–1.27)

rs3790459 1 154728331 Intragenic MEF2D A 0.35 Imputed 2.85 × 10−8 1.24 0.00 – – – – – – 0.989 (1.15–1.33)

rs12136856 1 154739738 Intergenic MEF2D C 0.34 Imputed 3.90 × 10−8 1.23 0.00 – – – – – – 0.985 (1.15–1.33)

Locus 2 rs7640543 3 30437407 Intergenic TGFBR2 A 0.32 Genotyped 2.72 × 10−6 1.20 0.00 1.02 × 10−4 1.18 0.51 1.17 × 10−9 1.19 0.19 – (1.11–1.30) (1.09–1.29) (1.13–1.26)

Locus 3 rs9349379 6 13011943 Intragenic PHACTR1 G 0.38 Genotyped 2.06 × 10−7 0.82 0.00 0.01 0.90 0.00 3.20 × 10−8 0.86 0.10 – (0.77–0.89) (0.83–0.98) (0.81–0.91)

Locus 4 rs6478241 9 118292450 Intragenic ASTN2 A 0.38 Genotyped 1.14 × 10−7 1.22 0.00 0.02 1.10 0.57 3.86 × 10−8 1.16 0.56 – (1.13–1.31) (1.02–1.19) (1.10–1.23)

Locus 5 rs10166942 2 234489832 Intergenic TRPM8 C 0.18 Genotyped 1.32 × 10−5 0.82 0.00 5.62 × 10−9 0.74 0.00 9.83 × 10−13 0.78 0.00 – (0.74–0.89) (0.67–0.82) (0.73–0.84)

rs17862920 2 234492734 Intragenic TRPM8 T 0.10 Genotyped 2.19 × 10−5 0.78 0.00 6.44 × 10−5 0.75 0.00 5.97 × 10−9 0.77 0.00 0.520 (0.69–0.87) (0.66–0.87) (0.70–0.84)

Locus 6 rs11172113 12 55813550 Intragenic LRP1 C 0.40 Genotyped 3.38 × 10−5 0.86 0.00 2.33 × 10−4 0.86 0.67 2.97 × 10−8 0.86 0.45 – (0.80–0.92) (0.79–0.93) (0.81–0.91)

SNPs showing significant association either in the discovery stage (locus 1) or after meta-analysis of the discovery and replication samples (loci 2–6). Genome-wide significant P values and successful replications are shown in boldface. s r e t t e l ORs are reported for the minor allele. Chr., chromosome; I 2, heterogeneity index. r 2 indicates the LD between the SNP and the top SNP at the respective locus. aChromosomal positions are based on NCBI Build 36. bFor intragenic SNPs, the gene is listed in which the SNP is located, whereas, for intergenic SNPs, the nearest gene is listed. cIndicates whether a SNP was genotyped or imputed. © 2012 Nature America, Inc. All rights reserved. a TGF- a PHACTR1 may also be migraine through involved disease in vascular systemic with linked be to seemed all infarction myocardial and disease cardiovascular dysfunction, endothelial or as transmission, synaptic aberrant through neuronal, be i endothelial cell function binding actin 1 and phosphatase protein of regulation through morphology synapse and activity synaptic controls which family, PHACTR/scapinin the of member a is protein encoded The 1). regulator actin and phatase Table 10 and discovery and the from ( data samples the replication combining when significance dissection artery cervical for risk increased twofold a have to seemed migraineurs that observation the with fit of 14 mutation carriers in a large multigenerational family in 11 headaches but migrainous caused also dissection aortic familial monogenic, seemingly caused only not substitution p.Arg460His a in mutation missense the as migraine, for gene production matrix extracellular as as well in and differentiation, of proliferation cell in regulation the factor located ~200 kb upstream of ( OR = 1.19) ( samples replication and discovery the of analysis ( 10 headache migraine the of origin presumed activation trigeminovascular nitroglycerol-induced aura without migraine with persons in attacks migraine-like may trigger transmission, synaptic excitatory modulates and genes target MEF2D activity-regulated of cases in each cohort. scale. Numbers in parentheses indicate the number intervals. Odds ratios are plotted on a logarithmic horizontal lines represent the 95% confidence the odds ratios for the individual cohorts; the gray lines. In the forest plots, black squares represent HapMap CEU release 22 are shown as light blue with this SNP. Estimated recombination rates from SNPs are colored according to the extent of LD locus is represented as a purple diamond; the other SNP with the highest association signal in each in the specific region against –log the chromosomal position (NCBI Build 36) of SNPs rs11172113 ( results are shown for rs10166942 ( the SNP with the highest association at these loci; in the current study together with forest plots for reported migraine loci using LocusZoom) are shown for the two previously study. ( that replicate in the current migraine without aura previously reported loci associated with migraine Figure 2 s r e t t e l wide significance in the meta-analysis of the discovery and n P farction = 1.02 × 10 −6 −5 The locus at 9p33 with the top SNP rs6478241 reached genome- reached rs6478241 SNP top the with 9p33 at locus The genome-wide reached SNP rs9349379 the 6p24, at locus the For The locus at 3p24 contained the top SNP rs7640543 (discovery , OR = 1.20). This SNP showed strong replication of association association of replication strong showed SNP This 1.20). = OR , ) were taken forward to the replication stage ( stage replication the to forward taken were ) Supplementary Fig. 3 Fig. Supplementary

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=4,834) –log10 (P value) 10 0 2 4 6 8 d HSD17B ( n PRIM1 ( ( =837) n n =871 =245) 6 2 −8 P 9 . Although structural abnormalities abnormalities structural Although . 32– = 5.97 × 10 × 5.97 = , OR = 1.16) ( 1.16) = OR , ) 55.6 ( ). The rs10166942 SNP is located located is SNP rs10166942 The ). n SDR9C7 ( 3 n =555) RDH1 4 =2,326) GPR182 Position onchr.12(Mb) , which is defined as pain due due pain as defined is which , ZBTB3 6 TAC3 r MYO1 2 TMEM194A rs11172113 0.63 9 NAB2 05) n h migraine- the in 0.52) = A 55.8 LRP1 STAT 0.71 6 LRP1 MIR1228 I −9 2 NXPH4 SHMT2 ASTN2 = 0.57) in the repli the in 0.57) =

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© 2012 Nature America, Inc. All rights reserved. E.W. in the context of the German National Genome Research Network, (NGFN-2 Education and Research (BMBF) (grant 01GS08121 to M.D., along with support to participation. This work was supported by the German ofFederal Ministry We wish to thank all individuals in the respective cohorts for their generous Note: Supplementary information is available in the the pape the of in version available are references associated any and Methods Me gov/gte Browser, eQTL (GTEx) Expression Tissue h SNPTEST, ncbi.nlm.n URLs. disorder. brain debilitating common this for targets treatment putative identify to order in ways path molecular underlying exact the to dissect are necessary studies Functional study. aura without migraine clinic-based this in licated identified loci associated with migraine ( In two loci. of addition, the four susceptibility previously as migraine relevance their test to needed are studies additional and weaker, was the for Replication meta-analysis. bined the replication stage levels and in the significance genome-wide com ( loci Two loci. the most associated common and type, several migraine identify headache. to than aura to susceptibility more GWAS migraine GWAS present the ( in gene susceptibility migraine putative the GWAS aura with migraine IHGC the in 8q22 at SNP rs1835740 the contrast, In migraine. of forms various in role scan) OR = in initial the 0.86 also showed association in the WGHS migraine study rs10166942 ( Notably, set. data aura with migraine the in 0.82) = OR 10 × 2.19 (rs1786920: tion studies. both The in direction same the in were ORs and set, data aura showed loci all 3p24, at set data GWAS study aura out entities the top tested SNPs with from from migraine the six the loci current disease different represent aura without migraine function. vascular for LRP1 in can migraine be of envisaged because its neuronal and/or muscle cells and modulates smooth synaptic transmission vascular of proliferation the in involved is it environment: LRP1 is a receptor cell surface that acts as a sensor of the extracellular vasculature. and neurons including tissues, multiple in expressed is protein 1, a protein that receptor–related lipoprotein the low-density of intron first the within is located rs11172113 10 overall the overall (rs11172113: in meta-analysis aura without migraine significance genome-wide reached 6) (ref. 12q13 at Nature Ge Nature and NGFN-plus) for the Heinz Nixdorf Study);Recall the Spanish of Ministry IMPUTE2, IMPUTE2, A t cknowledgments m −8 In conclusion, we present the first GWAS of migraine without aura, and aura with migraine whether of question the Addressing locus migraine-associated reported previously at top SNP The the t l O = .6 ( 0.86) = OR , TRPM8 ; LocusZoom, LocusZoom, ; ho GWAMA, x / ds t e MEF2D h i s n h t t t locus showed the most significant significant most the showed locus / . etics h p g G t : o / t T v / p in silico in 5 w E 6 / http://www.w ( : ; HapMap 3 data,HapMap 3 ; ( / X w and and

/ upeetr Tbe 3 Table Supplementary r P h m 2 . w ADVANCE ONLINE PUBLICATION ONLINE ADVANCE Fig. Fig. = 0.22). This may suggest that that suggest may This 0.22). = t / t . a g s p t P t t h : TGFBR2 a e in our previous IHGC migraine with aura aura with migraine IHGC previous our in / = 0.70) nor in the population-based WGHS population-based the in nor 0.70) = t x g / s P c 2 . e . c s o values below 0.05 in the migraine with with migraine the in 0.05 below values n , , g −5 g x . i 6 Table Table . s . . s , OR = 0.78; rs10166942: 1.32 × 10 × 1.32 rs10166942: 0.78; = OR , a . This suggests that that . suggests This t ell.ox.ac.uk/gwam p c a h . t u ) showed convincing association in in association convincing showed ) s . u . k o m / x h ~ 1 . i t and and m a c t p c h a . : . u r TRPM8 / e c / PHACTR1 k d 5 h online version of the pape h did not show association association show not did / u upeetr Fg 3 Fig. Supplementary a i http://www. i n / p m ). Except for the locus locus the for Except ). l i m 5 o / p pointing at at pointing s cuszoom LRP1 a o u a and p /; NCBI, P f t 35 TRPM8 t e . value for associa for value w n , / 3 i c and and a 6 m

LRP1 , which encodes encodes , which b r . . A possible role MTDH P e i p . / = 2.30 × 10 n ncbi.nlm.nih. g u / l ; Genotype- ; w ASTN2 t h may have a m e ) ) were rep ttp://www. P a MTDH _ . s n = 2.97 × = 2.97 v 37 / confers confers i s 2 h , online online n r 3 . . . h 8 p g loci loci , we we , t t o m e −5 as as −7 v s ). ). / t l - - - - - , . , ; ;

Genetics (grantsGenetics 213506 and 129680 to A.P. and J.K.); the NorwaySouth-Eastern to M.W.); the Academy of Finland, Center of Excellence in Complex Disease (grant 098051 to A.P.); the Academy of Finland (grant 251704 to A.P. and 139795 (ARC) Future Fellowship schemes (FT0991022) (to D.R.N.); the Wellcome Trust (NHMRC) Research Fellowship (613674) and the Australian Research Council for Environmental Health; the National Health and Medical Research Council which was initiated by the Helmholtz Center Munich, German Research Center (MC Sciences Health) as part of LMUinnovativ) for the KORA research platform, Research and the State of Bavaria, supported within the Munich Center of Health 01GS08120 and 01GS1103 to C.K.); the German ofFederal Ministry Education and the framework of the National Genome Research Network (NGFN-Plus; grants RIDE2); the German ofFederal Ministry Education and Research (BMBF) within Aging (NCHA) and the Research Institute for in Diseases the Elderly (014-93-015; and Sports), as well as the NGI-sponsored Netherlands Consortium for Healthy Rotterdam and the Ministries of Education, Culture and Health,Science, Welfare and by(funded 911-03-012) the Erasmus Medical Center, Erasmus University management of GWAS genotype data for the Rotterdam Study (175.010.2005.011 M.D.F. and A.M.J.M.v.d.M. and to 050-060-810 C.M.v.D.) and the generation and for Research Scientific (NGI/NWO) (project to 050-060-409 C.M.v.D., R.R.F., established in the Netherlands Genomics Initiative/Netherlands Organisation (2009) grants (to M.D.F.); the Center for Medical Systems (CMSB) Biology Organisation for Research Scientific (NWO) VICI and(918.56.602) Spinoza and(90700217) VIDI (ZonMw) (91711319 to G.M.T.); the Netherlands the Netherlands Organization for Health Research and Development (ZonMw) grantunrestricted of the Vascular Dementia Research Foundation (to M.D.); (grants 2009SGR78 and to2009SGR971 A.M. and B.C., respectively); an B.C.); the Agència de d’AjutsGestió Universitaris i de Recerca (AGAUR) andScience Innovation (grant to SAF2009-13182-C03 A.M. and 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. reprints/index.html. http://www.nature.com/ at online available is information permissions and Reprints Published online at The authors declare no competing interests.financial in review ofcritical the manuscript for intellectual content. M.W., R.R.F., J.-A.Z., C.K., A.P., M.D. and A.M.J.M.v.d.M. All authors participated and B.M.-M. The manuscript was written by T.F., V. Anttila, B.d.V., D.R.N., B.C., performed by V. Anttila, B.d.V., B.W.,R.M., D.R.N., E.H., A.G.U., F.R., M.W., T.M. C.M.v.D., J.K., B.C., T.M., J.-A.Z., M.F. and A.M. Analysis and genotyping were J.S., O.R., T.L., M.V.-P., H.G., E.W., C.S., A.G.U., A. Heinze, A. Hoffman., E.T., T.F., M.K., G.M.T., P.P.-R., B.W., W.P.J.v.O., C.K., V.A.P., M.D.Artto, U.T.,and A.M.J.M.v.d.M. Cohorts andwere phenotyped bysupervised J.F.-M., M.A.L., M.A.K., study Overall design was guided by T.F., V. Anttila, B.d.V., D.R.N.,R.M., J.-A.Z., Funding was obtained by M.D., M.D.F., A.P., A.M.J.M.v.d.M., C.K. and C.M.v.D. University HospitalCentral (to M.K., V. Artto and M.F.). the Folkhälsan Research Foundation, Helsinki (to M.W.); and the Helsinki Systems (grant 242167 to A.P.); the Sigrid Juselius Foundation, Finland (to A.P.); Consortium, (grant agreement HEALTH-F4-2007- 201413); EU/SYNSYS–Synaptic Community’s Seventh Framework Programme the ENGAGE(FP7/2007-2013), Vetlesen Medical Fund (to B.W.) and the fund Ullevaal (to B.W.); the European Regional Health Authority (2010075 and 2011083 to B.W. and J.-A.Z.), the Unger- COMPETING FINANCIAL INTERESTS FINANCIAL COMPETING AUTHOR CONTRIBUTIONS

i, . Sa, . Blet RF Te xrsin f E2 ee i ipiae in implicated is genes MEF2 of expression The R.F. Bulleit, & S. Shah, X., Lin, Nica, A.C. A.S. Dimas, D.I.Chasman, V.Anttila, headache of Funding B. Jönsson, & P. Andlin-Sobocki, I., Lekander, J., Olesen, L. Stovner, of Epidemiology J. Pascual, Terwindt,& K., G.M. Hagen, J.A., Zwart, Stovner,L.J., Launer, L.J., Terwindt, G.M. & Ferrari, M.D. The prevalence and characteristics of migraine CNS neuronal differentiation. neuronal CNS study. MuTHER the tissues: manner. type–dependent population. general the (2011). in migraine common for 8q22.1. on variant susceptibility Europe. in research worldwide. disability and prevalence Europe. in headache population-basedain cohort: theGEMstudy. lvl, S.W. Flavell, suppresses excitatory synapse number. synapse excitatory suppresses et al. et al. et t al. et et al. et t al. et et al. et The architecture of gene regulatory variation across multiple human Genome-wide association study of migraine implicates a common a implicates migraine of study association Genome-wide http://www.nature.com/doifinder/10.1038/ng.230 h goa bre o haah: dcmnain f headache of documentation a headache: of burden global The Common regulatory variation impacts gene expression in a cell a in expression gene impacts variation regulatory Common Genome-wide association study reveals three susceptibilitythreelocireveals Genome-wide associationstudy ciiydpnet euain f E2 rncito factors transcription MEF2 of regulation Activity-dependent Cephalalgia Eur. J. Neurol. J. Eur. Science PLoS Genet. PLoS Brain Res. Mol. Brain Res. Brain Mol. Res. Brain

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Institute Institute of Human Genetics, Helmholtz Zentrum München, Neuherberg, Germany. Biomedical Network Research Centre on Rare Diseases (CIBERER), Barcelona, Spain. Pediatric Pediatric Neurology Research Group, Vall d’Hebron Research Institute, Universitat Autònoma de Barcelona, Barcelona, Spain. PACAP38 induces migraine-like attacks in patients with migraine 321 60 Genome-wide analysis of MEF2 transcriptional program reveals program transcriptional MEF2 of analysis Genome-wide 33 β

Pituitary adenylate cyclase–activating polypeptide plays a key a plays polypeptide cyclase–activating adenylate Pituitary rgl X etl eadto poen s eurd o synapse for required is protein retardation mental X Fragile dniyn ats lc ad ee b taig eet shared recent tracing by genes and loci autism Identifying 132 type 2 receptor, a functional transmembrane serine/threonine transmembrane functional receptor,a 2 type , 1022–1038 (2008). 1022–1038 , Department Department of Medical Genetics, University of Helsinki, Helsinki, Finland. , 218–223 (2008). 218–223 , , 16–25 (2009). 16–25 , 35

A A full list of members and affiliations is provided in the 101 5 346 Department Department of Human Genetics, Leiden University Medical Centre, Leiden, The Netherlands. 20 , 7187–7192 (2004). 7187–7192 , , 257–270 (2002). 257–270 , J. Med. Genet. Med. J. Institute Institute of Epidemiology, Helmholtz Zentrum München, Neuherberg, Germany. 24 Department Department of Epidemiology, Erasmus University Medical Center, Rotterdam, The Netherlands. v 10 2.1 knockin migraine mice. migraine knockin 2.1 Department Department of Neurology, Oslo University Hospital and University of Oslo, Oslo, Norway. 7 Department Department of Neurology, Leiden University Medical Centre, Leiden, The Netherlands. Neurology

43 31 , 908–916 (2006). 908–916 , Max Max Planck Institute of Psychiatry, Munich, Germany.

40 , 1582–1586 (1990). 1582–1586 , Cephalalgia Neurobiol. Dis. Neurobiol. 26 Neuron Neuron Cephalalgia 14 Department Department of Public Health, University of Helsinki, Helsinki, Finland. 17 Folkhälsan Folkhälsan Research Center, Helsinki, Finland. Department Department of Clinical Chemistry, Tampere University Hospital and University of Tampere, 12

31 66 61 Institute Institute of Human Genetics, University of Ulm, Ulm, Germany. , , ,

37. 36. 35. 34. 33. 32. 31. 30. 29. 28. 27. 26. 25. 38. S upplementary upplementary Note

usl, .. Rsusn BK, egr K & lsn J Mgan wtot aura without Migraine J. Olesen, & K. Fenger, B.K., Rasmussen, M.B., Russell, LDL D.K. Strickland, May,P. & J.E. Murphy-Ullrich, L.B., Duyn, van A.P., Lillis, d’Agostino, V.C., Francia, E., Licursi, V. & Cerbo, R. Clinical and personality features Caspani,O., Zurborg, S., Labuz, D. &Heppenstall, P.A. The contribution of TRPM8 and C.J. Proudfoot, the M.C. Kruit, in Thickening N. Hadjikhani, & J. Snyder, C., Granziera, A.F., DaSilva, M.E. Hatten, Y.& Fang, R.H., P.M.,Fryer, Wilson, vasculopathy. systemic a as Migraine G.E. Tietjen, early-onset of association Genome-wide Consortium. Genetics Infarction Myocardial Jarray,R. Greengard, P., Allen, P.B. & Nairn, A.C. Beyond the dopamine receptor: the DARPP- Kallela, M., Wessman, M., Havanka, H., Palotie, A. & Färkkilä, M. Familial migraine eighty-four male and female migraineurs from the general population. general the from migraineurs female and and hundred male eighty-four four of study a entities: clinical distinct are aura with migraine and requirednormalformotor function mice.in studies. knockout byselective gene revealed functions tissue-specific unique 1: protein receptor–related migraine. allodynic of TRPA1channels coldtoallodynia andneuropathic pain. pain. neuropathic Assoc. Med. migraine. with patients (2007). of cortex somatosensory glial-guided during ASTN1 of trafficking migration. neuronal the regulates family, gene astrotactin (2009). number copy and polymorphisms nucleotide variants. single with infarction myocardial 93 cells abolishes tube formation and induces cell death receptor apoptosis. cascade. phosphatase-1 32/protein 8 co-occurrence. and characteristics clinical aura: without and with 16 9 , 441–449 (2001). 441–449 , Headache Headache Research Group, Vall d’Hebron Research Institute, Universitat , 1668–1675 (2011). 1668–1675 , , 239–245 (1996). 239–245 , et al.et Nat. Genet. Nat. 28 et al. et 34 2 Institut Institut de Biomedicina de la Universitat de Barcelona (IBUB),

t al. et Department Department of Medical Genetics, Helsinki University Central Neuronal LRP1functionally associates withpostsynaptic proteins andis Department Department of Neurology, Klinikum der Universität München, 291 Depletion of the novel protein PHACTR-1 from human endothelial human from PHACTR-1 protein novel the of Depletion 30 4 et al. et a Institute Institute for Molecular Medicine Finland (FIMM), University , 427–434 (2004). 427–434 , DVANCE ONLINE PUBLICATION ONLINE DVANCE irie s rs fco fr uciia ban lesions. brain subclinical for factor risk a as Migraine Institute Institute of Human Genetics, Klinikum Rechts der Isar, . . Curr. Biol. Curr. 23 36 J. Neurosci. J.

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© 2012 Nature America, Inc. All rights reserved. those with cryptic relatedness and those considered to be heterozygosity out to heterozygosity be considered and those relatedness cryptic with those ( other each to related closely were who rate and of subjects <97% a genotyping with Subjects were excluded. of <97% rates call SNPs with Further, controls. and of cases set combined the for used also was latter the and populations), controls both and in cases independently the genotyped were (as independently controls and cases for used were 10 × 1 and frequency allele minor for 1% of cutoffs analysis, of types both In imputation. after and study were subjected to per-SNP and per-sample quality control analysis before control. Quality stage. replication the in included were ( loci migraine-associated reported previously four the from SNPs top the addition, In heterogeneity. possible cover robustly to locus at this follow-up for five) of total a (for SNPs additional three chose we stage, discovery the in alleles minor for directions effect both observed we as ( v1 610-Quad Human Illumina the using München Zentrum Helmholtz at Center Analysis Genome the at performed was GWASsample German the of Genotyping protocols. standard to according samples blood genotyping. Discovery-stage (Norway). Ethics Institute (Spain) and the Regional Committee for Medical and HealthResearch Research Valld’Hebron the (Finland), Hospital Central University Helsinki the Netherlands), (The Centre Medical Leiden of University the (Germany), Gro ethics Klinikum research the local of respective committees the by approved was study the and pants, aspects. Ethical 1 Fig. Supplementary ( (Leiden) (Trondheim) and Norway The Netherlands (Barcelona), Spain (Helsinki), Finland from samples aura without migraine clinic-based smaller, four in replication for tested were the meta-analysis the Subsequently, from SNPs top sets. data two the for performed then and was samples, both meta-analysis in independently performed was analysis regression tic logis genome-wide step, As initial an panel. a as reference 39) 2 (ref. release 3 HapMap using SNPs million 1.4 approximately to imputed were data raw the in given are controls and of cohorts study (details data genotyping existing with from studies recruited ( (Leiden) Netherlands The and Kiel) and (Munich Germany in clinics headache from of analysis design. study Overall O doi:10.1038/ng.2307 In liers. were outliers population by excluded addition, from manual selection using Illumina Gencall data analyses software. Genotyping of the complete complete the of Genotyping software. analyses data Gencall Illumina using II was Infinium protocol performed from calling the manufacturer. Genotype v1 ( Human 660W-Quad Illumina from the remaining loci ( loci remaining the from ( convincing most to be considered loci the from Two selected were SNPs each P evaluated. were calls doubtful all Clusters were to used the manually,call checked genotypes. automatically was and manufacturer the by supplied software Spectrocaller respectively. tion, contamina and performance assay for check to controls negative and DNA) 384-well (CEPH) Humain Each du Polymorphism d’Etude (Centre positive contained plate instructions. manufacturer’s and the on reactions based were Amplification parameters methodology. MassARRAY Sequenom with mass spectr (MALDI-TOF) time-of-flight desorption/ionization laser matrix-assisted using Wellcome at the TrustInstitute Sanger genotyped were genotyping. Replication-stage using performed was software calling Illuminus Genotype technology. 660W Illumina using Institute Sanger TrustWellcome the at performed was GWAS sample Dutch MEF2D < 1 × 10 × 1 < NLIN In the replication stage, we selected all loci with at least two SNPs with with SNPs two least at with loci all selected we stage, replication the In , , E E PHACTR1 −5 de de novo M upeetr Fg 1 Fig. Supplementary in the discovery stage for follow-up ( follow-up for stage discovery the in E T Written informed consent was obtained from all partici all from obtained was consent informed Written HO To ensure high data quality, the data sets from the primary primary the from sets data the quality, data Tohigh ensure genotyping in two large migraine without aura without in sample sets two genotyping large migraine 4 0 , , near . DS −6 ). The discovery stage of the study was based on an an on based was study the of stage discovery The for Hardy-Weinberg equilibrium (HWE) (HWE) equilibrium Hardy-Weinberg for TGFBR2 Supplementary Table 1 Table Supplementary Genomic DNA was extracted from peripheral peripheral from extracted DNAwas Genomic Supplementary Note Supplementary For the replication study, all cases and controls n and β = 391) SNP microarrays according to the according SNP = microarrays 391) hadern, Ludwig-Maximilian-University Ludwig-Maximilian-University hadern, ). Population-matched controls were were controls Population-matched ). FHL5 π _hat > 0.15) were removed, as were were as removed, were 0.15) > _hat ), ), and one SNP each was selected ). At the the At ). Supplementary Table 1 Table Supplementary Supplementary Table1 Supplementary ). For both sample sets, sets, sample both For ). PHACTR1 n = 838) or or 838) = Fig. Fig. P o values values locus, locus, metry metry 1 and ). ). - - - - )

primary reason for not using age as a covariate was that age information information age that was covariate a as age using not for reason primary GWAS aura with an migraine clinic-based assumed previous our and with In accordance model. phenotype additive binary a as statistics. migraine of summary presence the used We population-specific generate to URLs) (ver see software 2.2.0; SNPTEST sion with data dosage allele imputed the analyzed we analysis. Statistical analysis. control quality of basis the on excluded were SNPs 1,411,821 From 165,433 quality. imputation, data after SNPs imputation high ensure to cutoffs as >0.6 I(A) of measure info imputation, and >0.9 of After probability kb). posterior 250 call of individual used instead we kb (500 samples Dutch the for size states copying of numbers ( different used we that exception the with tion, imputa for parameters recommended the We followed URLs). (see website ( samples 2009) 2 February release Dutch samples) for (v2.1.0 using IMPUTE2 the samples German and performed for v2.1.2 the Imputation. set. data Dutch the in were controls) 2,016 and cases (1,118 viduals controls) were 2,564 in SNPsdata and set, the German 494,760 for indi 3,134 PLINK. in matrix distance pairwise (IBS) identity-by-state genome-wide the of plot scaling multidimensional a we explored the US National Institutes of Health (NIH) Genotype-Tissue Genotype-Tissue (NIH) Health of Institutes National US the explored we relative values to genotypes minimum observed the by comparing assessed was Significance interest. of SNP the surrounding window 2-Mb a within genes all for correlation rank Spearman using analyzed were expression gene and SNPs of interesting most the genotypes the between association publications, studies eQTL published two eQTL analysis. between component interaction model. the in included was genotype and gender additional an where analysis a from regression those to results the compared we addition, In analyses. female-only adjustment and male- from results output the with and compared identity covariate for population with and effects additive assuming model regression PLINK in SNPs the We analyzed loci. identified newly the of each at SNPs genotyped directly the for analysis association the in mation SNPs 1,246,388 of data resulting ( the from generated were plots quantile model random-effects a using performed also with ated larger or (25–50%) moderate by indicated was heterogeneity (effect) Cochran’s the using examined observed was inflation genomic ( Reasonable meta-analysis. all included were overall the samples) replication, in replication After four and discovery structure. (two LD sets study six varying to due markers common signals of association in differences some of expectation the reflect to chosen het having for filtered that and measure SNPs retained erogeneity only were sets data meta-analysis, both in sample present were discovery the In results. primary GWAMA using population- results. the for specific samples discovery the for using used was analyzed as were model same markers the genotyped studies, replication the missing For handle to data. used was option likelihood score data missing The ples. sam case our to similar age, working of were cohorts the in individuals the of majority the However, cohorts. control the of some for available not was κ Supplementary Fig. 2 Fig. Supplementary λ = 60 for the German and 80 for the Dutch the for 80 and German the for 60 = = 1.095, 1.095, = Before imputation, 452,154 SNPs for 3,772 individuals (1,208 cases and and cases (1,208 individuals 3,772 for SNPs 452,154 imputation, Before In gender effects analysis, we analyzed the effect of including gender infor of gender including the effect we analyzed analysis, In effects gender conducted was statistics summary the of meta-analysis fixed-effect A P P λ

5 values from each of the 10,000 permutations of the expression expression the of permutations 10,000 the of each from values ≤ Imputation of the German and Dutch discovery samples was was samples discovery Dutch and German the of Imputation , association analysis was not corrected for age or gender. The The gender. or age for corrected not was analysis association , 1000 1 × 10 × 1 In the eQTL analysis, we assessed publicly available data In publicly available from we the eQTL analysis, assessed 4 4 1 = 1.031). Consistency of allelic effects across studies was was studies across effects allelic of Consistency 1.031). = 3 . . For haploid the panel, we reference phased HapMapused 3 (see URLs), first on the two discovery samples for the the for samples discovery two the on first URLs), (see For the GWAS data from the two initial study samples, samples, study initial two the from GWAS data the For I −5 2 of <0.5. This moderately high threshold for for threshold high moderately This <0.5. of and showing evidence of effect heterogeneity was was heterogeneity effect of evidence showing and ). 7 , 7 8 , , 8 4 . In these data sets, as described in the original original in the as described sets, data . In these 9 . . In an approach additional eQTLs, to identify Q I 2 4 values 4 n and and = 1,011) I P 4 2 Q value at a 0.001 threshold with the with at threshold value a 0.001 6 (ref. (ref. . Meta-analysis of SNPs associ SNPs of Meta-analysis . -statistic -statistic 4 2 as provided by the IMPUTE2 IMPUTE2 by the provided as

4 samples) and a larger buffer buffer larger a and samples) 45) metrics. Between-study Between-study metrics. 45) 7 . Manhattan and quantile- and Manhattan . P values ( values Nature Ge Nature 4 8 using a logistic logistic a using P

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© 2012 Nature America, Inc. All rights reserved. 42. 41. 40. 39. shown). not (data expression gene and genotypes between tion Expression (GTEx) database. GTEx data did not show any evidence of associa Nature Ge Nature

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