No Association of Coronary Artery Disease with X-Chromosomal Variants in Comprehensive International Meta-Analysis

1 Scien i ic RepoR s | 6:35278 | DOI: 10.1038/s ep35278 www.na u e.com/scien i ic epo s No Associa ion o Co ona y A e y Disease wi h X-Ch omosomal Va ian s in Comp ehensi e In e na ional Me a-Analysis Ch is ina Loley1,2, Ma is Al e 3,4, Themis ocles L. Assimes5, And ew Bjonnes6, Anuj Goel7,8, S e an Gus a sson9, Jussi He nesniemi10,11, Jemma C. Hopewell12, S a oula Kanoni13, Ma cus E. Klebe 14, King Wai Lau12, Yingchang Lu15, Leo-Pekka Lyy ikäinen10,16, Ch is ophe P. Nelson17,18, Majid Nikpay19, Liming Qu20, Elias Sal a i5, Ma kus Scholz21,22, Ta u Tukiainen23,24, Ch is ina Willenbo g2,25, Hong-Hee Won26, Lingyao Zeng27,28, Weihua Zhang29,30, Sonia S. Anand31, F ank Beu ne 22,32, E win P. Bo inge 15, Robe Cla ke12, Geo ge Dedoussis33, Ron Do15,34,35,36, Tõnu Esko3,37, Ma kku Eskola11, Ma in Fa all7,8, Dominique Gauguie 38, Vilman as Gied ai is39, Ch is ophe B. G ange 40, Alis ai S. Hall41, Ande s Hams en42, S anley L. Hazen43, Jie Huang44, Mika Kähönen45,46, Theodosios Ky iakou7,8, Reijo Laaksonen10,16,47, La s Lind48, Cecilia Lindg en8,49, Pa ik K. E. Magnusson50, Ei ini Ma ouli13, E elin Mihailo 3, And ew P. Mo is8,51, Kjell Nikus11, Nancy Pede sen50, Loukianos Rallidis52, Veikko Salomaa53, S a i H. Shah40, Alexand e F. R. S ewa 19, John R. Thompson54, Pie e A. Zalloua55,56, John C. Chambe s30,31,57, Ro y Collins12, E ik Ingelsson8,9, Ca los I iba en58, Pekka J. Ka hunen10,59, Jaspal S. Koone 31,57,60, Te ho Leh imäki10,16, Ru h J. F. Loos15,61, Win ied Mä z14,62,63, Ru h McPhe son19, And es Me spalu3,4, Mu edach P. Reilly64, Samuli Ripa i58,65,66, Dha ambi K. Sanghe a67,68,69, Joachim Thie y22,70, Hugh Wa kins7,8, Panos Deloukas13,71,72, Seka Ka hi esan6,24,37,73, Nilesh J. Samani17,18, He ibe Schunke 28,29, Jeane e E dmann2,25 & Inke R. König1,2 In ecen yea s, genome-wide associa ion s udies ha e iden i ied 58 independen isk loci o co ona y a e y disease (CAD) on he au osome. Howe e , due o he sex-speci ic da a s uc u e o he X ch omosome, i has been excluded om mos o hese analyses. While emales ha e 2 copies o ch omosome X, males ha e only one. Also, one o he emale X ch omosomes may be inac i a ed. The e o e, special es s a is ics and quali y con ol p ocedu es a e equi ed. Thus, li le is known abou he ole o X-ch omosomal a ian s in CAD. To ill his gap, we conduc ed a comp ehensi e X-ch omosome-wide me a-analysis including mo e han 43,000 CAD cases and 58,000 con ols om 35 in e na ional s udy coho s. Fo quali y con ol, sex-speci ic il e s we e used o adequa ely ake he special s uc u e o X-ch omosomal da a in o accoun . Fo single s udy analyses, se e al logis ic eg ession models we e calcula ed allowing o inac i a ion o one emale X-ch omosome, adjus ing o sex and in es iga ing in e ac ions be ween sex and gene ic a ian s. Then, me a-analyses including all 35 s udies we e conduc ed using andom e ec s models. None o he in es iga ed models e ealed genome-wide signi ican associa ions o any a ian . Al hough we analyzed he la ges - o-da e sample, cu en ly a ailable me hods we e no able o de ec any associa ions o X-ch omosomal a ian s wi h CAD. ecei ed: 27 May 2016 accep ed: 26 Sep embe 2016 Published: 12 Oc obe 2016 OPEN 1Ins i u ü Medizinische Biome ie und S a is ik, Uni e si ä zu Lübeck, Uni e si ä sklinikum Schleswig-Hols ein, Campus Lübeck, Lübeck, Ge many. 2DZHK (Ge man Cen e o Ca dio ascula Resea ch), pa ne si e Hambu g– Lübeck–Kiel, Lübeck, Ge many. 3Es onian Genome Cen e , Uni e si y o Ta u, Ta u, Es onia. 4Ins i u e o Molecula and Cell Biology, Ta u, Es onia. 5Depa men o Medicine, Di ision o Ca dio ascula Medicine, S an o d Uni e si y School o Medicine S an o d, S and o d, Cali o nia, USA. 6Cen e o Human Gene ic Resea ch, Massachuse s Gene al Hospi al, Bos on, Massachuse s, USA. 7Di ision o Ca dio ascula Medicine, Radcli e www.na u e.com/scien i ic epo s/ 2 Scien i ic RepoR s | 6:35278 | DOI: 10.1038/s ep35278 In he las yea s, genome-wide associa ion s udies (GWAS) ha e unco e ed nume ous ch omosomal isk loci o a ious complex diseases. Speci ically, o co ona y a e y disease (CAD), 58 independen isk loci ha e been iden i ied and e i ied in independen eplica ion da ase s1. Howe e , a la ge pa o he es ima ed he i abili y o CAD is no ye explained. This could be due pa ly o he ac ha he X ch omosome has ou inely been excluded om GWAS. One eason o his is ha he da a has a di e en , sex-speci ic s uc u e and, he e o e, equi es special analy ical ools including special quali y con ol and es s a is ics2. Thus, despi e he p o ound e ec s o gende on he mani es a ion o CAD, no sys ema ic associa ion analyses o X-ch omosomal a ian s wi h CAD Depa men o Medicine, Uni e si y o Ox o d, Ox o d, UK. 8Wellcome T us Cen e o Human Gene ics, Uni e si y o Ox o d, Ox o d, UK. 9Depa men o Medical Sciences, Molecula Epidemiology and Science o Li e Labo a o y, Uppsala Uni e si y, Uppsala, Sweden. 10Depa men o Clinical Chemis y, Fimlab Labo a o ies, Tampe e, Finland. 11Depa men o Ca diology, Hea Hospi al and Uni e si y o Tampe e School o Medicine, Tampe e, Finland. 12CTSU, Nu ield Depa men o Popula ion Heal h, Uni e si y o Ox o d, Ox o d, UK. 13William Ha ey Resea ch Ins i u e, Ba s and The London School o Medicine and Den is y, Queen Ma y Uni e si y o London, London, UK. 14V h Depa men o Medicine, Medical Facul y Mannheim, Heidelbe g Uni e si y, Mannheim, Ge many. 15The Cha les B on man Ins i u e o Pe sonalized Medicine, Icahn School o Medicine a Moun Sinai, New Yo k, USA. 16Depa men o Clinical Chemis y, Uni e si y o Tampe e School o Medicine, Tampe e, Finland. 17Depa men o Ca dio ascula Sciences, Uni e si y o Leices e , Leices e , UK. 18NIHR Leices e Ca dio ascula Biomedical Resea ch Uni , Glen ield Hospi al, Leices e , UK. 19Ruddy Canadian Ca dio ascula Gene ics Cen e Uni e si y o O awa Hea Ins i u e, O awa, Canada. 20Depa men o Bios a is ics and Epidemiology, Uni e si y o Pennsyl ania, Philadelphia, Pennsyl ania, USA. 21Ins i u e o Medical In o ma ics, S a is ics and Epidemiology/ Medical Facul y/Uni e si y o Leipzig, Leipzig, Ge many. 22LIFE Resea ch Cen e o Ci iliza ion Diseases, Leipzig, Ge many. 23Analy ic and T ansla ion Gene ics Uni , Massachuse s Gene al Hospi al, Bos on, Massachuse s, USA. 24P og am in Medical and Popula ion Gene ics, B oad Ins i u e, Camb idge, Massachuse s, USA. 25Ins i u ü In eg a i e und Expe imen elle Genomik, Uni e si ä zu Lübeck, Uni e si ä sklinikum Schleswig-Hols ein, Campus Lübeck, Lübeck, Ge many and Uni e si y Hea Cen e Luebeck, Campus Lübeck, Lübeck, Ge many. 26Samsung Ad anced Ins i u e o Heal h Sciences and Technology (SAIHST), Sungkyunkwan Uni e si y, Samsung Medical Cen e , Seoul, Ko ea. 27Deu sches He zzen um München, Technische Uni e si ä München, Munich, Ge many. 28DZHK (Ge man Cen e o Ca dio ascula Resea ch), pa ne si e Munich Hea Alliance, München, Ge many. 29Depa men o Epidemiology and Bios a is ics, Impe ial College London, London, UK. 30Depa men o Ca diology, Ealing Hospi al Na ional Heal h Se ice (NHS) T us , Middlesex, UK. 31Popula ion Heal h Resea ch Ins i u e, McMas e Uni e si y, Hamil on, On a io, Canada. 32Hea Cen e Leipzig, Ca diology, Uni e si y o Leipzig, Leipzig, Ge many. 33Ha okopio Uni e si y A hens, A hens, G eece. 34The Cen e o S a is ical Gene ics, Icahn School o Medicine a Moun Sinai, New Yo k, USA. 35The Icahn Ins i u e o Genomics and Mul iscale Biology, Icahn School o Medicine a Moun Sinai, New Yo k, USA. 36The Zena and Michael A. Weine Ca dio ascula Ins i u e, Icahn School o Medicine a Moun Sinai, New Yo k, USA. 37Depa men o Medicine, Ha a d Medical School, Bos on, Massachuse s, USA. 38INSERM, UMRS1138, Cen e de Reche che des Co delie s, Pa is, F ance. 39Depa men o Public Heal h and Ca ing Sciences, Ge ia ics, Uppsala Uni e si , Uppsala, Sweden. 40Duke Uni e si y School o Medicine, Du ham, No h Ca olina, USA. 41Leeds Ins i u e o Gene ics, Heal h and The apeu ics, Uni e si y o Leeds, UK. 42Ca dio ascula Gene ics and Genomics G oup, A he oscle osis Resea ch Uni , Depa men o Medicine Solna, Ka olinska Ins i u e , S ockholm, Sweden. 43Cle eland Clinic, Cle eland, Ohio, USA. 44Bos on VA Resea ch Ins i u e, Inc., Bos on, Massachuse s, USA. 45Depa men o Clinical Physiology, Tampe e Uni e si y Hospi al, Tampe e, Finland. 46Depa men o Clinical Physiology, Uni e si y o Tampe e School o Medicine, Tampe e, Finland. 47Zo a Biosciences, Espoo, Finland. 48Depa men o Medical Sciences, Ca dio ascula Epidemiology, Uppsala Uni e si y, Uppsala, Sweden. 49B oad Ins i u e o he Massachuse s Ins i u e o Technology and Ha a d Uni e si y, Camb idge, Massachuse s, USA. 50Depa men o Medical Epidemiology and Bios a is ics, Ka olinska Ins i u e , S ockholm, Sweden. 51Depa men o Bios a is ics, Uni e si y o Li e pool, Li e pool, UK. 52Second Depa men o Ca diology, Uni e si y Gene al Hospi al A ikon, A hens, G eece. 53Depa men o Ch onic Disease P e en ion, Na ional Ins i u e o Heal h and Wel a e, Helsinki, Finland. 54Depa men o Heal h Sciences, Uni e si y o Leices e , Leices e , UK. 55Lebanese Ame ican Uni e si y, School o Medicine, Bei u , Lebanon. 56Ha a d School o Public Heal h, Bos on, Massachuse s, USA. 57Impe ial College Heal hca e NHS T us , London, UK. 58Kaise Pe manen e, Di ision o Resea ch, Oakland, Cali o nia, USA. 59Depa men o Fo ensic Medicine, Uni e si y o Tampe e School o Medicine, Tampe e, Finland. 60Ca dio ascula Science, Na ional Hea and Lung Ins i u e, Impe ial College London, London, UK. 61The Mindich Child Heal h and De elopmen Ins i u e, Icahn School o Medicine a Moun Sinai, New Yo k, USA. 62Synlab Academy, Synlab Se ices GmbH, Mannheim, Ge many. 63Clinical Ins i u e o Medical and Chemical Labo a o y Diagnos ics, Medical Uni e si y o G az, G az, Aus ia. 64Ca dio ascula Ins i u e, Pe elman School o Medicine a he Uni e si y o Pennsyl ania, Philadelphia, Pennsyl ania, USA. 65Hjel Ins i u e, Uni e si y o Helsinki, Helsinki, Finland. 66Ins i u e o Molecula Medicine Finland FIMM, Uni e si y o Helsinki, Helsinki, Finland. 67Depa men o Pedia ics, College o Medicine, Uni e si y o Oklahoma Heal h Sciences Cen e , Oklahoma Ci y, Oklahoma, USA. 68Depa men o Pha maceu ical Sciences, College o Pha macy, Uni e si y o Oklahoma Heal h Sciences Cen e , Oklahoma Ci y, Oklahoma, USA. 69Oklahoma Cen e o Neu oscience, Oklahoma Ci y, Oklahoma, USA. 70Ins i u e o Labo a o y Medicine, Clinical Chemis y and Molecula Diagnos ics, Uni e si y Hospi al Leipzig, Medical Facul y, Leipzig, Ge many. 71Wellcome T us Sange Ins i u e, Hinx on, Camb idge, UK. 72P incess Al-Jawha a Al-B ahim Cen e o Excellence in Resea ch o He edi a y Diso de s (PACER-HD), King Abdulaziz Uni e si y, Jeddah, Saudi A abia. 73Ca dio ascula Resea ch Cen e , Massachuse s Gene al Hospi al, Bos on, Massachuse s, USA. Co espondence and eques s o ma e ials should be add essed o J.E. (email: [email p o ec ed]) o I.R.K. (email: Ink[email p o ec ed]) www.na u e.com/scien i ic epo s/ 3 Scien i ic RepoR s | 6:35278 | DOI: 10.1038/s ep35278 ha e been epo ed so a . The e o e, an analysis o he X-ch omosome om GWAS da a migh help o na ow he gap o missing he i abili y and help o yield new insigh s in o he gene ics o CAD. X-ch omosomal a ian s migh be expec ed o play a ole in he pa hophysiology, since sex-speci ic ea u es a e known o CAD. Speci ically, he isk o de elop CAD a ies be ween males and emales independen om o he isk ac o s. The symp oms o myoca dial in a c ion (MI) as well as he p ognosis a e MI di e be ween males and emales. Males a e mo e likely han emales o mani es CAD a young age, bu emales a e mo e likely han males o die o a i s MI. Fu he mo e, hea disease is he mos common cause o dea h o emales3. Thus, he analysis o X-ch omosomal a ian s could help o explain he sex di e ences in CAD. To comp ehensi ely in es iga e he associa ion o a ian s on ch omosome X and CAD, we collec ed da a om 35 wo ld-wide s udy coho s. All pa icipa ing s udies we e pa o he CARDIoGRAM + C4D cons o - ium1. A each s udy si e, quali y con ol on subjec le el was pe o med, da a we e impu ed on he basis o he 1000 genomes e e ence panel, and X ch omosome-adap ed associa ion es s we e calcula ed. A e his, da a we e analyzed cen ally a he Uni e si y o Lübeck, whe e u he quali y con ol and he me a-analysis o all 35 s udies we e conduc ed. In he ollowing, we will p esen he esul s o he associa ion analysis o abou 200,000 X-ch omosomal single nucleo ide polymo phisms (SNPs) wi h CAD on a sample o mo e han 100,000 subjec s including mo e han 43,000 cases and 58,000 con ols. Resul s De ails on he in es iga ed s udies a e summa ized in Table1. Fo each o he 35 s udies, logis ic eg ession models wi h addi i e sco ing o he SNP we e used. To accoun o he sex-speci ic s uc u e o X-ch omosomal S udy Ances y Cases (% emales) Con ols (% emales) % MI* (in cases) ADVANCE Whi e Eu opean 275 (59.9) 311 (60.7) 100.0 BioMe_A Am A ican Ame ican 362 (66.5) 2778 (70.9) 36.0 BioMe_Eu Am Eu opean Ame ican 487 (35.0) 1382 (61.6) 30.4 BioMe_HisAm Hispanic Ame ican 758 (55.1) 3338 (71.3) 36.7 Ca diogenics Whi e Eu opean 391 (13.7) 410 (60.8) 12.5 CATHGEN Whi e Eu opean 1191 (31.4) 646 (58.9) 48.1 CCGB_2 Whi e Eu opean 1547 (21.8) 344 (45.0) 60.3 EGCUT Whi e Eu opean 658 (49.6) 5841 (56.1) 19.6 FGENTCARD Libanese 1802 (25.0) 466 (50.8) 16.0 FINCAVAS Finnish Eu opean 774 (21.2) 647 (44.8) 12.4 FINRISK/P edic CVD Finnish Eu opean 677 (30.9) 1200 (42.7) 40.0 Ge MIFSI Whi e Eu opean 637 (33.9) 1644 (51.2) 100.0 Ge MIFSII Whi e Eu opean 1222 (21.0) 1298 (48.5) 100.0 Ge MIFSIII Whi e Eu opean 1096 (20.5) 1509 (52.2) 100.0 Ge MIFSIV Whi e Eu opean 1002 (36.0) 1147 (61.9) 100.0 Ge MIFSV Whi e Eu opean 2459 (24.5) 1611 (53.0) 100.0 HPS Whi e Eu opean 2700 (23.9) 2748 (72.5) 65.0 HSDS Whi e Eu opean 206 (0.0) 258 (0.0) 45.6 ITH Whi e Eu opean 402 (29.4) 449 (32.9) 100.0 LIFE-Hea Whi e Eu opean 1531 (24.8) 768 (50.3) 44.0 LOLIPOP Indian Asian 2791 (18.5) 3757 (14.1) 43.9 LURIC Whi e Eu opean 2347 (25.5) 621 (48.0) 62.9 MEDSTAR Whi e Eu opean 875 (34.4) 447 (55.9) 100.0 MIGEN Whi e Eu opean 2905 (24.8) 2998 (26.9) 100.0 OHGS_A2 Whi e Eu opean 921 (24.9) 1001 (50.5) 64.3 OHGS_B2 Whi e Eu opean 1183 (22.4) 1391 (49.5) 55.6 OHGS_C2 Whi e Eu opean 833 (6.4) 317 (66.7) 44.3 PENNCATH Whi e Eu opean 933 (29.2) 468 (58.8) 100.0 PIVUS Whi e Eu opean 119 (24.3) 830 (54.7) 77.3 PROCARDIS Whi e Eu opean 5719 (29.2) 6526 (62.5) 80.0 SDS Indian Asian 176 (29.5) 1421 (49.0) 100.0 THISEAS Whi e Eu opean 423 (21.3) 593 (56.3) 60.1 TWINGENE Whi e Eu opean 814 (29.1) 5999 (55.8) 70.5 ULSAM Whi e Eu opean 322 (0.0) 857 (0.0) 84.2 WTCCC Whi e Eu opean 1922 (20.9) 2930 (51.2) 71.5 To al —43120 (28.2) 58291 (50.0) 66.7 Table 1. Coho desc ip i es o he 35 s udies pa icipa ing in he 1000G co ona y a e y disease me a- analysis o he X-ch omosome. *MI = myoca dial in a c ion. www.na u e.com/scien i ic epo s/ 4 Scien i ic RepoR s | 6:35278 | DOI: 10.1038/s ep35278 da a, sex was always included as a co a ia e. In addi ion, in e ac ions be ween SNP and sex we e in es iga ed. Whe e app op ia e, u he co a ia es could be included. Since one o he wo emale X-ch omosomes may o may no be inac i a ed a a speci ic locus, models we e calcula ed ha assumed inac i a ion as well as no assum- ing inac i a ion. The s udy-wise numbe s o SNPs excluded due o quali y con ol a e gi en in Supplemen a y Tables S1 and S2. The inspec ion o in la ion ac o s4 and Q-Q-plo s (see also Supplemen a y Fig. S1) did no e eal any sys ema ic in la ion o speci ic s udies. Thus, all s udies we e included in he me a-analysis. None o he s a is ical models used o he me a-analysis e ealed a genome-wide signi ican associa ion wi h CAD o any SNP. Associa ion esul s o he model wi hou inac i a ion assump ion and wi hou SNP*sex in e ac ion a e p esen ed in Fig.1. Resul s o he o he models a e compa able and p esen ed in Supplemen a y Figs S2–S6. Subg oup and sensi i i y analyses. To in es iga e possible sex-speci ic e ec s, we conduc ed subg oup analyses o males and emales sepa a ely. Associa ion plo s o hese models a e p esen ed in Supplemen a y Figs S7 and S8. Again, no genome-wide signi ican associa ions we e o be obse ed. To exclude a possible bias in oduced by including s udy coho s o non-Eu opean ances y, we pe o med a subg oup analysis including only he 31 s udies wi h Eu opean backg ound. Excluding non-Eu opean s udies did no show addi ional associ- a ions ei he (Supplemen a y Figs S9–S14). Finally, o elimina e possible in luences o he quali y con ol pa am- e e s, we a ied ou c i e ia on missing equencies and impu a ion quali y. Pe o ming s ic e quali y con ol educed he numbe o SNPs a ailable o me a-analysis o abou 90,000 (depending on model, be ween 90,658 and 96,502). Resul s o hese analyses we e compa able o he esul s om he p ima y analyses. Thus, none o he sensi i i y analyses did e eal no el associa ions, suppo ing he null indings o he main analyses. Powe . We es ima ed he powe o ou s udy o e all as well as in he smalles subg oup analyzed, i.e. he sub- g oup o emales, as unc ions o he odds a io and he e ec allele equency (EAF) (Fig.2). In he en i e sample o males and emales, odds a ios o 1.1 o 1.11 would ha e eliably been de ec ed wi h an EAF o 0.1 o highe . E en in he emale subg oup, odds a ios o 1.15 and highe would ha e been de ec able wi h a su icien ly la ge p obabili y. Discussion Al hough he p esen ed me a-analyses included mo e han 100,000 subjec s ( he la ges o da e), wi h 43,120 CAD cases (28.2% women) and 58,291 con ols (50% women), no genome-wide signi ican associa ions could be de ec ed. This nega i e inding was independen o he model chosen o analysis. The e we e no sex-speci ic associa ions o CAD. S ic e quali y con ol o excluding non-Eu opean s udies did no e eal any di e en indings. The NHGRI GWAS ca alog5,6 epo s 52 genome-wide signi ican associa ions o a ian s on ch omosome X wi h mo e han 600 ai s. All o he epo ed s udies a e o much smalle sample size (less han 50,000 sam- ples) and used ewe me hods han ou analyses. Ye , hey success ully disco e ed associa ions. Howe e , no genome-wide signi ican associa ions wi h CAD o any o he co ela ed pheno ype ha e been epo ed on he X ch omosome. The only gene epo ed o CAD on ch omosome X is CHRDL1 wi h a p- alue o 9 · 10−7 o s59430577, bu his did no eplica e in ou analyses (p = 0.0172 o he model wi hou inac i a ion assump ion and wi hou SNP*sex in e ac ion). As ou powe es ima es indica ed (Fig.2), in such a la ge da ase , he s a is ical powe o de ec medium o la ge e ec s is high, and only small e ec s a e likely o ha e been missed. The e o e, he mos na u al explana ion o he nega i e inding o his me a-analysis is ha he e a e no subs an ial associa- ions o X-ch omosomal a ian s wi h CAD. Howe e , since he p og ession and he symp oms o CAD, as well as he p ognosis a e MI, a e sex-speci ic, i may be ha he gene ics o ch omosome X a e mo e complex han p e iously assumed. Fo example, he inac i a ion pa e ns a e no ye unde s ood comple ely, and i has been shown ha inac i a ion o he emale X-ch omosome can be cell-speci ic. The silenced X-ch omosome is no necessa ily chosen andomly, and silenced egions can di e be ween emales8,9. Al hough we e alua ed models wi h and wi hou he assump ion o inac i a ion, di e en inac i a ion pa e ns be ween emales a one locus we e no aken in o accoun . No was Figu e 1. Ch omosome-wide associa ion esul s. The s a is ical model assumes no inac i a ion and no SNP*sex in e ac ion. Shown a e loga i hmized andom e ec s p- alues o all 184,673 quali y con olled SNPs in o de o physical posi ion in mega base pai s (mbp). www.na u e.com/scien i ic epo s/ 5 Scien i ic RepoR s | 6:35278 | DOI: 10.1038/s ep35278 non- andom inac i a ion inco po a ed in o he model. This could a ec he powe o he es s a is ics ha we e used and migh esul in an analysis ha is less powe ul han es ima ed. Fu he , i migh be a gued ha he use o o he s a is ical me hods could ha e yielded signi ican indings. Speci ically, mos o he s udies (e.g. e s 10 and 11) a e based on a sepa a e analysis o males and emales wi h a subsequen summa y by a classical o sex-speci ic12 me a-analy ic me hod. In con as , we ollowed he app oach o di ec ly compu e join es s a is ics o males and emales, aking he di e en s uc u e o ch omosome X in he sexes in o accoun . Howe e , examples o bo h me hodological app oaches ha e been compa ed in simula ions13,14 wi h he o e all esul ha he join es s ha e g ea e powe unless he e a e ele an di e ences in he e ec sizes be ween males and emales, which is no o be expec ed gi en ou sex-speci ic subg oup analyses. Ano he po en ial limi a ion could be he lowe co e age o ch omosome X han au osomal ch omosomes2,15. Speci ically, depending on he speci ic geno yping chip, he dis ance be ween wo known a ia ions16 a e ages oughly 700 o 10,000 bp on all ch omosomes bu abou 1400 o 22,500 bp on ch omosome X. Acco dingly, he median dis ance in ou s udies is 11,300 bp, so ha he co e age is less han op imal bu compa able o he use o an olde geno yping a ay in gene al. Mo e gene ally, he use o hese echnologies is es ic ed o inding associa ions wi h SNPs only; he e ec o o he s uc u al a ian s o e en ha ing XX ins ead o XY canno hus be de ec ed. Ano he explana ion o he lack o signi ican associa ions could be p oblems wi h he impu a ion. Using he IMPUTE2 algo i hm, as mos o he s udy si es did, a mix u e o wo popula ions, males and emales, is impu ed oge he . Pe haps his leads o a bias ha has no been aken adequa ely in o accoun . F om a clinical pe spec i e, co ona y disease and i s mani es a ions di e in women, including a la ge p opo ion o younge women wi h myoca dial in a c ion ha ing he dis inc pa hophysiology o co ona y dissec ion, which could complica e he dissec ion o gene ic in luence acco ding o sex. Al hough we ha e analyzed he la ges sample o da e, we we e no able o de ec genome-wide signi ican associa ions be ween ch omosome X a ian s and CAD wi h cu en ly a ailable me hods. Due o his lack o signi ican associa ions, he sex-speci ic di e ences in CAD a e s ill unexplained. The gene ics o ch omosome X may be mo e complex han has been assumed, so ha mo e sophis ica ed es s a is ics which allow o hese com- plex biological p ocesses would be equi ed o de ec associa ions o a ian s on he X-ch omosome wi h CAD. Ma e ials and Me hods Sex-speci ic s uc u e o ch omosome X. One eason why ch omosome X is usually excluded om GWAS is ha he da a has a di e en , sex-speci ic s uc u e and, he e o e, equi es special analy ical ools15,17. While he e a e wo copies o each au osomal ch omosome, males ca y only one copy o he X ch omosome whe eas emales, again, ca y wo copies. The e o e, a each SNP, emales can ca y one o h ee possible gen- o ypes; ha is, hey can ha e 0, 1 o 2 copies o a speci ic allele. In con as o his, he e a e only wo possible geno ypes o males, co esponding o 0 o 1 copies o a speci ic allele. Only o he so-called pseudo-au osomal Figu e 2. Es ima ed powe . The powe o de ec an e ec was es ima ed in dependence o he odds a io (OR) and he e ec allele equency (EAF) using so wa e Quan o ( e sion 1.2.4 om May 2009). Pa ame e s used o simula ion: Bina y (disease) pheno ype, signi icance le el α = 5·10−8, disease p e alence kP = 0.1, log-addi i e gene ic model, no gene-en i onmen in e ac ion. (A) E ec i e Ncases = 27,640, 1.5817 e ec i e con ols pe e ec i e case (co esponding o 43,718 e ec i e con ols), (B) Female Ncases = 12,160, 2.3968 emale con ols pe emale case (co esponding o 29,145 emale con ols). www.na u e.com/scien i ic epo s/ 6 Scien i ic RepoR s | 6:35278 | DOI: 10.1038/s ep35278 egions, he e exis homologous loci on he Y ch omosome, and males can ha e up o 2 copies o a speci ic allele. In addi ion, one o he wo emale X ch omosomes migh be inac i a ed. In each cell, one o he wo emale X ch omosomes is andomly selec ed o be silenced18. This means ha he exp ession le els o his ch omosome a e much lowe han o he second ch omosome in he cell. This mechanism o dose compensa ion should esul in compa able exp ession le els o males and emales despi e he di e en numbe o ch omosome copies. Howe e , his inac i a ion is incomple e: while some genes o egions will be comple ely inac i a ed, some genes migh show exp ession le els ha a e educed only sligh ly o no a all8. The e o e, o analyze X-ch omosomal da a, special quali y con ol and es s a is ics a e equi ed2. Mos o he quali y con ol needs o be done sepa- a ely o males and emales, and es s a is ics o ch omosome X should ake in o accoun he di e en da a s uc u e o males and emales, o example by including sex as a co a ia e in o he model. The choice o he bes s a is ical es depends on he unde lying gene ic model and he inac i a ion pa e ns a a speci ic locus13. S udy coho s. The me a-analysis includes da a om 43,120 cases wi h CAD and 58,291 con ols om 35 s udies wi h 28.2% emale cases and 50.0% emale con ols ( o de ails, see Table1). A subjec was ega ded as a CAD case i he/she had an inclusi e CAD diagnosis, e.g. MI, acu e co ona y synd ome, ch onic s able angina, o co ona y s enosis > 50%. Mo e de ailed in o ma ion on he s udy coho s can be ound in he me a-analysis o au osomal a ian s o he CARDIoGRAMplusC4D Conso ium1 which included mos o he samples p esen ed he e. Thi y-one o he 35 s udies consis o subjec s wi h Eu opean ances y, wo s udy coho s a e o Asian ances y, one o Hispanic and one o A ican ances y. Geno yping and impu a ion o 1000G da a. De ails on he geno yping a ays used o each s udy coho ha e been published be o e1. A each s udy si e, un yped SNPs we e impu ed on he basis o he 1000 genomes phase 1 e sion 3 e e ence panel19. Al hough his e e ence panel includes inse ion and dele ion a i- an s (indels), hese we e excluded om u he analyses. P io o any quali y con ol, he e we e 1,193,934 SNPs a ailable o he non-pseudo-au osomal egion o ch omosome X. Quali y con ol. Quali y con ol a subjec le el and a a ian le el was pe o med a each s udy si e p io o impu a ion and associa ion analysis as desc ibed p e iously1. In addi ion o quali y c i e ia ypically used o analyses o au osomal SNPs20,21, all subjec s o whom he geno ypic and epo ed sex could no be assigned unambiguously we e excluded. Pos -impu a ion quali y con ol a SNP le el was done cen ally and in he same manne o all con ibu ing s udies. He e, SNPs we e excluded i one o he ollowing c i e ia was ul illed: (1) ≥ 25% missing geno ypes in ei he emale o male cases o con ols, (2) de ia ion om Ha dy-Weinbe g equilib ium in emale con ols wi h p < 0.0001, (3) mino allele equency < 1% in ei he males o emales, and (4) impu a ion quali y sco e (INFO o IMPUTE222–25 and 2 o Minimac22,26) < 0.5. Fo sensi i i y analysis, we addi ionally applied s ic e c i e ia o impu a ion quali y (INFO > 0.7) and missing geno ypes (< 2% in ei he emale o male cases o con ols). S udy-wise associa ion analysis. S udy-wise associa ion analyses we e calcula ed a each s udy si e. Logis ic eg ession models wi h addi i e sco ing o he SNP we e used. The sex-speci ic s uc u e o X-ch omosomal da a implies di e en a iances in male and emale sub-samples. To accoun o his, sex needs o be included as a co a ia e in he model. In addi ion, in e ac ions be ween SNP and sex we e in es iga ed. Whe e app op ia e, addi ional co a ia es o adjus o popula ion s a i ica ion ha e been included in he model, o example in he o m o a iables calcula ed om a p incipal componen analysis o a iables desc ibing he e hnic backg ound o he subjec s. Since one o he wo emale X-ch omosomes may o may no be inac i a ed a a speci ic locus, models we e calcula ed ha assumed inac i a ion as well as no assuming inac i a ion. I inac i a ion is p esen a a locus, wo isk alleles o a emale subjec should show simila exp ession le els as compa ed o one isk allele in a male indi idual. The e o e, while he emale geno ypes o such a SNP a e coded 0, 1 o 2, acco ding o 0, 1 o 2 alleles, he geno ypes o males should be coded 0 o 2 acco ding o 0 o 1 alleles. I no inac i a ion occu s, he exp ession le els o one allele in emales should be he same as one allele in males. The e o e, while he coding o emale geno ypes is unchanged, male geno ypes should now be coded 0 o 1, acco ding o 0 o 1 alleles. As an al e na i e o assuming comple e o no inac i a ion app oach, Wang e al.27 p oposed likelihood a io es s o he si ua ion o non- andom o skewed inac i a ion, which can be mo e powe ul in he case o non- andom inac i a ion. Howe e , gi en ha he gain in powe is small and ha hese es s a e a ailable in Ma lab28 only, which was no a ailable o many o he pa icipa ing s udy si es, we e ained om using his pa icula app oach. These con- side a ions esul ed in ou models being in es iga ed (Table2). Pos -hoc quali y con ol. A e calcula ion o he associa ion analyses o each single s udy, pos -hoc qual- i y con ol was applied o all s udies. To con ol o popula ion s a i ica ion o o he sou ces o in la ion o p- alues, in la ion ac o s acco ding o De lin and Roede 4 we e calcula ed, and plo s o expec ed e sus obse ed es s a is ics we e inspec ed isually. In addi ion, o each SNP, mean EAFs o e all s udies we e calcula ed and compa ed o he s udy-wise EAFs. SNPs wi h ex eme de ia ions (> 0.1, co esponding o mo e han 4 s anda d de ia ions) om he mean EAF we e excluded om u he analyses. Finally, only SNPs o which a leas hal o he s udies we e a ailable we e included in he me a-analyses. Me a-analyses. Fo he me a-analyses o he 35 s udies, andom e ec models we e calcula ed o each o he ou models de ined in Table2. In he same way, me a-analyses o he e ec es ima es o he SNP*sex in e ac ion we e pe o med. In all o hese analyses, ou lie analyses acco ding o P euß e al.20 we e pe o med o exclude s udies wi h ex emely in la ed e ec es ima es. www.na u e.com/scien i ic epo s/ 7 Scien i ic RepoR s | 6:35278 | DOI: 10.1038/s ep35278 Sensi i i y and subg oup analyses. We conduc ed he ollowing sensi i i y and subg oup analyses: (1) Subg oup analyses o males and emales sepa a ely wi h subsequen me a-analyses o gain u he insigh in o sex-speci ic e ec s; (2) Subg oup me a-analysis including only he 31 s udies wi h Eu opean backg ound; (3) Me a-analysis o SNPs ul illing s ic e quali y c i e ia as desc ibed abo e. Powe es ima ion. Using he so wa e Quan o, e sion 1.2.429, we es ima ed he powe o ou analyses in wo ways. Fi s ly, o ake in o accoun ou en i e sample o males and emales, a simple combina ion o he da a is no possible due o he sex-speci ic s uc u e o X ch omosomal a ian s. We he e o e ollowed Clay on30 in assuming ha he a iance in males has wice he size o ha in emales in he addi i e model assuming inac i a ion. The e o e, we assumed ha he e ec i e sample size in males is hal ed, and his was added o he emale sample size. Powe was hen es ima ed as a unc ion o he odds a io and he EAF a a signi icance le el o α = 5·10−8, a disease p e alence kP = 0.1, and a log-addi i e gene ic model. Secondly, we es ima ed he powe o he smalles subg oup analyzed, i.e. he subg oup o emales. The pa ame e s in Quan o we e se o he same alues as be o e. Re e ences 1. CARDIoGRAMplusC4D-Conso ium. A comp ehensi e 1000 Genomes-based genome-wide associa ion me a-analysis o co ona y a e y disease. Na . Gene . 47, 1121–1130 (2015). 2. König, I. R., Loley, C., E dmann, J. & Ziegle , A. How o include ch omosome X in you genome-wide associa ion s udy. Gene . Epidemiol. 38, 97–103 (2014). 3. Solimene, M. C. Co ona y hea disease in women: a challenge o he 21s cen u y. Clinics (Sao Paulo) 65, 99–106 (2010). 4. De lin, B. & Roede , K. Genomic con ol o associa ion s udies. Biome ics 55, 997–1004 (1999). 5. Hindo , L. e al. A Ca alog o Published Genome-Wide Associa ion S udies www.genome.go /gwas udies (2014). 6. Wel e , D. e al. The NHGRI GWAS Ca alog, a cu a ed esou ce o SNP- ai associa ions. Nucleic Acids Res. 42, D1001–D1006 (2014). 7. Co ona y A e y Disease Gene ics Conso ium. A genome-wide associa ion s udy in Eu opeans and Sou h Asians iden i ies i e new loci o co ona y a e y disease. Na . Gene . 43, 339–344 (2011). 8. Ca el, L. & Willa d, H. F. X-inac i a ion p o ile e eals ex ensi e a iabili y in X-linked gene exp ession in emales. Na u e 434, 400–404 (2005). 9. Wong, C. C. e al. A longi udinal win s udy o skewed X ch omosome-inac i a ion. PLoS One 6, e17873 (2011). 10. Tukiainen, T. e al. Ch omosome X-wide associa ion s udy iden i ies Loci o as ing insulin and heigh and e idence o incomple e dosage compensa ion. PLoS Gene . 10, e1004127 (2014). 11. de V ies, P. S. e al. A me a-analysis o 120 246 indi iduals iden i ies 18 new loci o ib inogen concen a ion. Hum. Mol. Gene . 25, 358–370 (2016). 12. Mägi, R., Lindg en, C. M. & Mo is, A. P. Me a-analysis o sex-speci ic genome-wide associa ion s udies. Gene . Epidemiol. 34, 846–853 (2010). 13. Loley, C., Ziegle , A. & König, I. R. Associa ion es s o X-ch omosomal ma ke s–a compa ison o di e en es s a is ics. Hum. He ed. 71, 23–36 (2011). 14. Chang, D. e al. Accoun ing o eXen ici ies: analysis o he X ch omosome in GWAS e eals X-linked genes implica ed in au oimmune diseases. PLoS One 9, e113684 (2014). 15. Wise, A. L., Gyi, L. & Manolio, T. A. eXclusion: owa d in eg a ing he X ch omosome in genome-wide associa ion analyses. Am. J. Hum. Gene . 92, 643–647 (2013). 16. The 1000 Genomes P ojec Conso ium. An in eg a ed map o gene ic a ia ion om 1,092 human genomes. Na u e 491, 56–65 (2012). 17. Winham, S. J., de And ade, M. & Mille , V. M. Gene ics o ca dio ascula disease: Impo ance o sex and e hnici y. A he oscle osis 241, 219–228 (2015). 18. Chow, J. C., Yen, Z., Ziesche, S. M. & B own, C. J. Silencing o he mammalian X ch omosome. Annu. Re . Genomics Hum. Gene . 6, 69–92 (2005). 19. Howie, B. & Ma chini, J. 1,000 Genomes haplo ypes – Phase I in eg a ed a ian se elease ( 3) in NCBI build 37 (hg19) coo dina es h ps://ma hgen.s a s.ox.ac.uk/impu e/da a_download_1000G_phase1_in eg a ed.h ml (2012). 20. P euss, M. e al. Design o he Co ona y AR e y DIsease Genome-Wide Replica ion And Me a-Analysis (CARDIoGRAM) S udy: A Genome-wide associa ion me a-analysis in ol ing mo e han 22 000 cases and 60 000 con ols. Ci c. Ca dio asc. Gene . 3, 475–483 (2010). 21. Ziegle , A. Genome-wide associa ion s udies: quali y con ol and popula ion-based measu es. Gene . Epidemiol. 33 Suppl 1, S45–S50 (2009). 22. Howie, B., Fuchsbe ge , C., S ephens, M., Ma chini, J. & Abecasis, G. R. Fas and accu a e geno ype impu a ion in genome-wide associa ion s udies h ough p e-phasing. Na . Gene . 44, 955–959 (2012). 23. Howie, B. & Ma chini, J. IMPUTE2 h ps://ma hgen.s a s.ox.ac.uk/impu e/impu e_ 2.h ml (2014). 24. Howie, B., Ma chini, J. & S ephens, M. Geno ype impu a ion wi h housands o genomes. G3 (Be hesda) 1, 457–470 (2011). 25. Ma chini, J. & Howie, B. Geno ype impu a ion o genome-wide associa ion s udies. Na . Re . Gene . 11, 499–511 (2010). 26. Fuchsbe ge , C., Abecasis, G. R. & Hinds, D. A. minimac2: as e geno ype impu a ion. Bioin o ma ics 31, 782–784 (2015). 27. Wang, J., Yu, R. & She e, S. X-ch omosome gene ic associa ion es accoun ing o X-inac i a ion, skewed X-inac i a ion, and escape om X-inac i a ion. Gene . Epidemiol. 38, 483–493 (2014). 28. Ma hwo ks. Ma lab h p://www.ma hwo ks.com/p oduc s/ma lab/ (2016). 29. Mo is, J. & Gaude man, J. So wa e by USC Bios a s h p://bios a s.usc.edu/so wa e (2016). 30. Clay on, D. Tes ing o associa ion on he X ch omosome. Bios a is ics 9, 593–600 (2008). Model Ma hema ical desc ip ionaSNP*sex in e ac ion Coding o SNP Inac i a ion ILogi (CAD) = β0 + β1 · SNP + β2 · sex No Females: 0, 1, o 2; Males: 0 o 1 No II Logi (CAD) = β0 + β1 · SNP + β2 · sex No Females: 0, 1, o 2; Males: 0 o 2 Yes III Logi (CAD) = β0 + β1 · SNP + β2 · sex + β3 · SNP*sex Yes Females: 0, 1, o 2; Males: 0 o 1 No IV Logi (CAD) = β0 + β1 · SNP + β2 · sex + β3 · SNP*sex Yes Females: 0, 1, o 2; Males: 0 o 2 Yes Table 2. Associa ion models o ch omosome X. aFo each s udy, u he co a ia es o adjus o popula ion s a i ica ion ha e been added o he model whe e app op ia e. www.na u e.com/scien i ic epo s/ 8 Scien i ic RepoR s | 6:35278 | DOI: 10.1038/s ep35278 Acknowledgemen s This wo k was suppo ed by he Ge man Fede al Minis y o Educa ion and Resea ch (BMBF) wi hin he amewo k o he e:Med esea ch and unding concep (g an 01ZX1313A-2014). The ADVANCE s udy was suppo ed by a g an om he Reynold's Founda ion andNHLBI g an HL087647. Sample collec ion in he Ca diogenics Conso ium (h p://www.ca diogenics.eu/web/) was unded by he 6 h F amewo k P og am o he Eu opean Union (LSHM-CT-2006-037593). We hank all he pa icipan s and clinicians in ol ed in he ec ui men p ocess a Camb idge and Leices e (UK), Luebeck and Regensbu g (Ge many), and Pa is (F ance). CATHGEN was suppo ed by NIH g an s HL095987 and HL101621. The Cle eland Clinic Gene Bank s udy was unded by P01HL076491 ( o S.L.H). EGCUT was suppo ed by Es onian Resea ch Council g an no. IUT20-60 and Resea ch Roadmap g an no. 3.2.0304.11-0312 and by Uni e si y Ta u g an no. ARENG SP1GV. The FGENTCARD-Func ional Genomic diagnos ic ools o co ona y a e y disease p ojec was unded by an EU FP6 awa d. We hank he pa ien s o ag eeing o pa icipa e in he s udy. We hank Sonia Youhanna, Nou Moukalled and Ba iaa Khalil o hei help wi h subjec ec ui men and da a collec ion. The wo k o FINCAVAS was suppo ed by he Compe i i e Resea ch Funding o he Tampe e Uni e si y Hospi al (G an 9M048 and 9N035), he Finnish Cul u al Founda ion, he Finnish Founda ion o Ca dio ascula Resea ch, he Emil Aal onen Founda ion, Finland, and he Tampe e Tube culosis Founda ion. The au ho s hank he s a o he Depa men o Clinical Physiology o collec ing he exe cise es da a. The Ge MIFS s udies we e suppo ed by g an s om he Ge man Fede al Minis y o Educa ion and Resea ch (BMBF) wi hin he amewo k o NGFN and NGFN-plus (A he ogenomics) and e:Med esea ch and unding concep (e:A he oSysMed, g an 01ZX1313A-2014), he Fonda ion Leducq (CADgenomics: Unde s anding CAD Genes, 12CVD02), and he Eu opean Union Six h F amewo k P og amme FP6 (unde g an ag eemen FP6-LIFESCIHEALTH (Ca diogenics)) and he Se en h F amewo k P og amme FP7/2007-2013 unde g an ag eemen n° HEALTH-F2-2013-601456 (CVgenes-a - a ge ). The Hea P o ec ion S udy (HPS) (ISRCTN48489393) was suppo ed by he UK Medical Resea ch Council (MRC), B i ish Hea Founda ion, Me ck and Co (manu ac u e s o sim as a in), and Roche Vi amins L d (manu ac u e s o i amins). Geno yping was suppo ed by a g an o Ox o d Uni e si y and CNG om Me ck and Co. Jemma C. Hopewell acknowledges suppo om he B i ish Hea Founda ion (FS/14/55/30806). HPS acknowledges he Na ional Blood Se ice (NBS) dono s and UK Twin s udy o using as popula ion con ols. A ull lis o he in es iga o s who con ibu ed o he gene a ion o he NBS da a is a ailable om www.w ccc.o g.uk. Funding o he p ojec was p o ided by he Wellcome T us unde awa d 07611. The UK Twin s udy was unded by he Wellcome T us ; Eu opean Communi y‟ s Se en h F amewo k P og amme (FP7/2007–2013). The Helsinki Sudden Dea h S udy (HSDS) was inancially suppo ed by EU’s 7 h F amewo k P og amme (g an no. 201668 o A he oRemo), he Tampe e Uni e si y Founda ion, he Tampe e Uni e si y Hospi al Medical Funds (g an s X51001, 9M048 and 9N035 o Te ho Leh imäki, he Emil Aal onen Founda ion (Te ho Leh imäki, he Finnish Founda ion o Ca dio ascula Resea ch (Te ho Leh imäki, Pekka J. Ka hunen), he Pi kanmaa Regional Fund o he Finnish Cul u al Founda ion, he Y jö Jahnsson Founda ion, and he Tampe e Tube culosis Founda ion (Te ho Leh imäki). LIFE-Hea is a pa o he LIFE – Leipzig Resea ch Cen e o Ci iliza ion Diseases, Uni e si ä Leipzig. LIFE is unded by means o he Eu opean Union, by he Eu opean Regional De elopmen Fund (ERDF) and by means o he F ee S a e o Saxony wi hin he amewo k o he excellence ini ia i e. The LOLIPOP s udy is suppo ed by he Na ional Ins i u e o Heal h Resea ch (NIHR) Comp ehensi e Biomedical Resea ch Cen e Impe ial College Heal hca e NHS T us , he B i ish Hea Founda ion (SP/04/002), he Medical Resea ch Council (G0601966, G0700931), he Wellcome T us (084723/Z/08/Z), he NIHR (RP-PG-0407-10371), Eu opean Union FP7 (EpiMig an , 279143) and Ac ion on Hea ing (G51). We hank he pa icipan s and esea ch s a who made he s udy possible. LURIC was suppo ed by he 7 h F amewo k P og am (in eg a ed p ojec A he oRemo, g an ag eemen numbe 201668 and RiskyCAD, g an ag eemen numbe 305739) o he Eu opean Union and by he INTERREG IV Obe hein P og am (P ojec A28, Gene ic mechanisms o ca dio ascula diseases) wi h suppo om he Eu opean Regional De elopmen Fund (ERDF) and he Wissenscha so ensi e TMO. We ex end ou app ecia ion o he pa icipan s o he LURIC s udy and hank he LURIC s udy eam who we e ei he empo a ily o pe manen ly in ol ed in pa ien ec ui men as well as sample and da a handling, in addi ion o he labo a o y s a a he Ludwigsha en Gene al Hospi al and he Uni e si ies o F eibu g and Ulm, Ge many. The MIGen s udy was unded by R01HL087676 om he US Na ional Hea , Lung, and Blood Ins i u e. The Moun Sinai IPM Biobank P og am is suppo ed by The And ea and Cha les B on man Philan h opies. I was in pa suppo ed by NHGRI U01HG007417. OHGS_A2, OHGS_B2, and OHGS_C2 we e unded by Canadian Ins i u es o Heal h Resea ch (# MOP-2380941 o R.M.), (#MOP82810, MOP77682 o R.R., A.F.S. & R.M.); Canada Founda ion o Inno a ion (#11966 o R.R., R.M. & A.F.S.; Hea & S oke Founda ion o Canada (#NA6001, #NA6650 o R.M). PIVUS was suppo ed by Knu and Alice Wallenbe g Founda ion (Wallenbe g Academy Fellow), Eu opean Resea ch Council (ERC S a ing G an ), Swedish Diabe es Founda ion (g an no. 2013-024), Swedish Resea ch Council (g an no. 2012-1397), and Swedish Hea -Lung Founda ion (20120197). We hank he SNP&SEQ Technology Pla o m in Uppsala (www.geno yping.se) o excellen geno yping. The compu a ions we e pe o med on esou ces p o ided by SNIC h ough Uppsala Mul idisciplina y Cen e o Ad anced Compu a ional Science (UPPMAX) unde P ojec b2011036. PROCARDIS was suppo ed by he Eu opean Communi y Six h F amewo k P og am (LSHM-CT- 2007-037273), As aZeneca, he B i ish Hea Founda ion, he Swedish Resea ch Council, he Knu and Alice Wallenbe g Founda ion, he Swedish Hea -Lung Founda ion, he To s en and Ragna Söde be g Founda ion, he S a egic Ca dio ascula P og am o Ka olinska Ins i u e and S ockholm Coun y Council, he Founda ion o S a egic Resea ch and he S ockholm Coun y Council (560283). Resea ch in SDS was pa ly suppo ed by NIH g an s -R01DK082766 unded by he Na ional Ins i u e o Diabe es and Diges i e and Kidney Diseases and NOT-HG-11-009 unded by Na ional Genome Resea ch Ins i u e, and VPR B idge g an om Uni e si y o Oklahoma Heal h Sciences Cen e , Oklahoma Ci y, USA. Rec ui men o THISEAS was pa ially unded by a esea ch g an (PENED 2003) om he G eek Gene al Sec e a y o Resea ch and Technology; we www.na u e.com/scien i ic epo s/ 9 Scien i ic RepoR s | 6:35278 | DOI: 10.1038/s ep35278 hank all he die icians and clinicians o hei con ibu ion o he p ojec . TwinGene was suppo ed by g an s om he Minis y o Highe Educa ion, he Swedish Resea ch Council (M-2005-1112 and 2009-2298), GenomEU win (EU/QLRT-2001-01254; QLG2-CT-2002-01254), NIH g an DK U01-066134, Knu and Alice Wallenbe g Founda ion (Wallenbe g Academy Fellow), Eu opean Resea ch Council (ERC S a ing G an ), Swedish Diabe es Founda ion (g an no. 2013-024), Swedish Resea ch Council (g an no. 2012-1397), and Swedish Hea -Lung Founda ion (20120197). We hank he SNP&SEQ Technology Pla o m in Uppsala (www. geno yping.se) o excellen geno yping. The compu a ions we e pe o med on esou ces p o ided by SNIC h ough Uppsala Mul idisciplina y Cen e o Ad anced Compu a ional Science (UPPMAX) unde P ojec b2011036. ULSAM was suppo ed by Knu and Alice Wallenbe g Founda ion (Wallenbe g Academy Fellow), Eu opean Resea ch Council (ERC S a ing G an ), Swedish Diabe es Founda ion (g an no. 2013-024), Swedish Resea ch Council (g an no. 2012-1397), and Swedish Hea -Lung Founda ion (20120197). We hank he SNP&SEQ Technology Pla o m in Uppsala (www.geno yping.se) o excellen geno yping. The compu a ions we e pe o med on esou ces p o ided by SNIC h ough Uppsala Mul idisciplina y Cen e o Ad anced Compu a ional Science (UPPMAX) unde P ojec b2011036. Rec ui men o he WTCCC s udy was unded by he B i ish Hea Founda ion and geno yping by he Wellcome T us . Themis ocles L. Assimes was suppo ed by an NIDDK ca ee de elopmen awa dDK088942. Panos Deloukas’s wo k o ms pa o he esea ch hemes con ibu ing o he ansla ional esea ch po olio o Ba s Ca dio ascula Biomedical Resea ch Uni which is suppo ed and unded by he Na ional Ins i u e o Heal h Resea ch. Analysis was pa ly suppo ed by BHF g an ( o Panos Deloukas) RG/14/5/30893. Ma in Fa all and Hugh Wa kins acknowledge he suppo o he Wellcome T us co e awa d (090532/Z/09/Z) and Ma in Fa all, Hugh Wa kins and Theodosios Ky iakou, he BHF Cen e o Resea ch Excellence. Anuj Goel, Hugh Wa kins and Theodosios Ky iakou acknowledge Eu opean Union Se en h F amewo k P og amme FP7/2007-2013 unde g an ag eemen no. HEALTH-F2-2013-601456 (CVGenes@Ta ge ) & and Anuj Goel, he Wellcome T us Ins i u ional s a egic suppo und. The UK Twin s udy was unded by he Wellcome T us ; Eu opean Communi y’s Se en h F amewo k P og amme (FP7/2007- 2013). PoBI samples om he Wellcome T us unded People o he B i ish Isles p ojec . Seka Ka hi esan issuppo ed by he Dono an Family Founda ion, Fonda ion Leducq, MGH Resea ch Schola Awa d, and R01 HL107816. And ew P. Mo is is a Wellcome T us Senio Fellow in Basic Biomedical Science, unded unde g an WT098017. Ch is ophe P. Nelson and Nilesh J. Samani a e unded by he B i ish Hea Founda ion and Nilesh J. Samani is a UK NIUHR Senio In es iga o . Ch is ophe P. Nelson and Nilesh J. Samani a e unded by he B i ish Hea Founda ion and Nilesh J. Samani is a UK NIUHR Senio In es iga o . Samuli Ripa i was suppo ed by he Academy o Finland Cen e o Excellence in Complex Disease Gene ics (G an No. 213506 and 129680), Academy o Finland (G an No. 251217 and 285380), he Finnish ounda ion o Ca dio ascula Resea ch, he Sig id Juselius Founda ion and he Eu opean Communi y’s Se en h F amewo k P og amme (FP7/2007-2013) h ough he BioSHaRE-EU (Biobank S anda disa ion and Ha monisa ion o Resea ch Excellence in he Eu opean Union) p ojec , g an ag eemen 261433. Alexand e F. R. S ewa is suppo ed by ope a ing g an s om he Canadian Ins i u e o Heal h Resea ch and Na u al Sciences and Enginee ing Resea ch Council o Canada. Hong-Hee Won is suppo ed by a pos doc o al awa d om he Ame ican Hea Associa ion (15POST23280019). Au ho Con ibu ions C.L., I.R.K., J.E. and H.S. concei ed he idea and he design o he s udy. I.R.K. and J.E. ec ui ed he pa icipa ing s udies o he me a-analysis. C.L., M.A., T.L.A., A.B., A.G., S.G., J.H., J.C.H., S.K., M.E.K., K.W.L., Y.L., L.P.L., C.P.N., M.N., L.Q., E.S., M.S., T.T., C.W., H.H.W., L.Z., W.Z., S.S.A., F.B., E.P.B., R.C., G.D., R.D., T.E., M.E., M.F., D.G., V.G., C.B.G., A.S.H., A.H., S.L.H., J.H., M.K., T.K., P.R.L., L.L., C.L., P.K.E.M., E.M., E.M., A.P.M., K.N., N.P., L.R., V.S., S.H.S., A.F.R.S., J.R.T., P.A.Z., J.C.C., R.C., E.I., C.I., P.J.K., J.S.K., T.L., R.J.F.L., W.M., R.M., A.M., M.P.R., S.R., D.K.S., J.T., H.W., P.D., S.K., N.J.S., H.S., J.E. and I.R.K. con ibu ed o he da a analysis on single s udy le el. C.L. checked all single s udy da a o alidi y and did all calcula ions and p og amming o he me a- analysis including quali y con ol. C.L. and I.R.K. w o e he manusc ip . C.L., M.A., T.L.A., A.B., A.G., S.G., J.H., J.C.H., S.K., M.E.K., K.W.L., Y.L., L.P.L., C.P.N., M.N., L.Q., E.S., M.S., T.T., C.W., H.H.W., L.Z., W.Z., S.S.A., F.B., E.P.B., R.C., G.D., R.D., T.E., M.E., M.F., D.G., V.G., C.B.G., A.S.H., A.H., S.L.H., J.H., M.K., T.K., P.R.L., L.L., C.L., P.K.E.M., E.M., E.M., A.P.M., K.N., N.P., L.R., V.S., S.H.S., A.F.R.S., J.R.T., P.A.Z., J.C.C., R.C., E.I., C.I., P.J.K., J.S.K., T.L., R.J.F.L., W.M., R.M., A.M., M.P.R., S.R., D.K.S., J.T., H.W., P.D., S.K., N.J.S., H.S., J.E. and I.R.K. con ibu ed o he in e p e a ion o he esul s o he me a-analysis and e iewed he manusc ip . Addi ional In o ma ion Supplemen a y in o ma ion accompanies his pape a h p://www.na u e.com/s ep Compe ing inancial in e es s: The au ho s decla e no compe ing inancial in e es s. How o ci e his a icle: Loley, C. e al. No Associa ion o Co ona y A e y Disease wi h X-Ch omosomal Va ian s in Comp ehensi e In e na ional Me a-Analysis. Sci. Rep. 6, 35278; doi: 10.1038/s ep35278 (2016). This wo k is licensed unde a C ea i e Commons A ibu ion 4.0 In e na ional License. The images o o he hi d pa y ma e ial in his a icle a e included in he a icle’s C ea i e Commons license, unless indica ed o he wise in he c edi line; i he ma e ial is no included unde he C ea i e Commons license, use s will need o ob ain pe mission om he license holde o ep oduce he ma e ial. To iew a copy o his license, isi h p://c ea i ecommons.o g/licenses/by/4.0/ © The Au ho (s) 2016