Genetic Relationship between Schizophrenia and Nicotine Dependence

1 Scien i ic RepoR s | 6:25671 | DOI: 10.1038/s ep25671 www.na u e.com/scien i ic epo s Gene ic Rela ionship be ween Schizoph enia and Nico ine Dependence Jingchun Chen1,*, Sil iu-Alin Bacanu2,3,*, Hui Yu4, Zhongming Zhao4, Peilin Jia4, Kenne h S. Kendle 2,3, Hen y R. K anzle 5, Joel Gele n e 6, Lindsay Fa e 7, Camelia Minica8, Rene Pool8, Yu i Milaneschi9, Do e I. Boomsma8, B enda W. J. H. Penninx8, Rachel F. Tyndale10, Jenni e J. Wa e11, Jacqueline M. Vink12,13, Jaakko Kap io14,15,16, Ma cus Muna ò11,17 , Xiangning Chen1,18, Co inine me a-analysis g oup & FTND me a-analysis g oup I is well known ha mos schizoph enia pa ien s smoke ciga e es. The e a e di e en hypo heses pos ula ing he unde lying mechanisms o his como bidi y. We used summa y s a is ics om la ge me a-analyses o plasma co inine concen a ion (COT), Fage s öm es o nico ine dependence (FTND) and schizoph enia o examine he gene ic ela ionship be ween hese ai s. We ound ha schizoph enia isk sco es calcula ed a P- alue h esholds o 5 × 10−3 and la ge p edic ed FTND and ciga e es smoked pe day (CPD), sugges ing ha genes mos signi ican ly associa ed wi h schizoph enia we e no associa ed wi h FTND/CPD, consis en wi h he sel -medica ion hypo hesis. The COT isk sco es p edic ed schizoph enia diagnosis a P- alues o 5 × 10−3 and smalle , implying ha genes mos signi ican ly associa ed wi h COT we e associa ed wi h schizoph enia. These esul s implica ed ha schizoph enia and FTND/CPD/COT sha ed some gene ic liabili y. Based on his sha ed liabili y, we iden i ied mul iple long non-coding RNAs and RNA binding p o ein genes (DA376252, BX089737, LOC101927273, LINC01029, LOC101928622, HY157071, DA902558, RBFOX1 and TINCR), p o ein modi ica ion genes (MANBA, UBE2D3, and RANGAP1) and ene gy p oduc ion genes (XYLB, MTRF1 and ENOX1) ha we e associa ed wi h bo h condi ions. Fu he analyses e ealed ha hese sha ed genes we e en iched in calcium signaling, long- e m po en ia ion and neu oac i e ligand- ecep o in e ac ion pa hways ha played a c i ical ole in cogni i e unc ions and neu onal plas ici y. 1Ne ada Ins i u e o Pe sonalized Medicine, Uni e si y o Ne ada a Las Vegas, 4505 S. Ma yland Pa kway, Las Vegas, NV 89154, USA. 2Vi ginia Ins i u e o Psychia ic and Beha io al Gene ics, Vi ginia Commonweal h Uni e si y, 800 E. Leigh S ee , Richmond, VA 23298, USA. 3Depa men o Psychia y Vi ginia, Commonweal h Uni e si y, 800 E. Leigh S ee , Richmond, VA 23298, USA. 4Depa men s o Biomedical In o ma ics and Psychia y, Vande bil Uni e si y School o Medicine, Nash ille, TN 37203, USA. 5Depa men o Psychia y, Uni e si y o Pennsyl ania Pe elman School o Medicine and VISN4 MIRECC, Philadelphia VA Medical Cen e , Philadelphia, PA, USA. 6Di ision o Human Gene ics, Depa men o Psychia y, Yale Uni e si y School o Medicine, and VA CT Heal hca e Cen e , New Ha en, CT 06516, USA. 7Depa men o Medicine (Biomedical Gene ics), Bos on Uni e si y School o Medicine, Bos on, MA, USA. 8Depa men o Biological Psychology, VU Uni e si y, Ams e dam, Ne he lands. 9Depa men o Psychia y and EMGO Ins i u e o Heal h and Ca e Resea ch, VU Uni e si y Medical Cen e , Ams e dam, Ne he lands. 10Depa men o Pha macology and Toxicology, Uni e si y o To on o, To on o, Canada. 11MRC In eg a i e Epidemiology Uni , Uni e si y o B is ol, B is ol, UK. 12Depa men o Biological Psychology, V ije Uni e si ei , Ams e dam, The Ne he lands. 13Beha iou al Science Ins i u e, Radboud Uni e si y, Nijmegen, The Ne he lands. 14Uni e si y o Helsinki, Depa men o Public Heal h, P.O.Box 41 (Manne heimin ie 172), 00014 Helsinki, Finland. 15Na ional Ins i u e o Heal h and Wel a e, Depa men o Men al Heal h and Subs ance Abuse Se ices, P.O. Box 30 (Manne heimin ie 166), 00300 Helsinki, Finland. 16Uni e si y o Helsinki, Ins i u e o Molecula Medicine, P.O. Box 20 (Tukholmanka u 8), 00014 Helsinki, Finland. 17UK Cen e o Tobacco and Alcohol S udies, School o Expe imen al Psychology, Uni e si y o B is ol, B is ol, UK. 18Depa men o Psychology, Uni e si y o Ne ada, Las Vegas, 4505 S. Ma yland Pa kway, Las Vegas, NV 89154, USA. *These au ho s con ibu ed equally o his wo k. Co espondence and eques s o ma e ials should be add essed o X.C. (email: Xiangning.chen@unl .edu) Recei ed: 21 Janua y 2016 Accep ed: 20 Ap il 2016 Published: 10 May 2016 OPEN www.na u e.com/scien i ic epo s/ 2 Scien i ic RepoR s | 6:25671 | DOI: 10.1038/s ep25671 La ge scale genome wide associa ion s udies (GWASs) ha e iden i ied isk genes o many complex human dis- eases and ai s (h p://www.genome.go /gwas udies/), including psychia ic diso de s such as schizoph enia and nico ine dependence (ND)1–3. These GWASs also show ha many human diseases and ai s a e polygenic in na u e and he con ibu ion o indi idual genes is limi ed4–6. Many o hese s udies ha e been deposi ed in he da abase o Geno ypes and Pheno ypes (dbGaP, h p://www.ncbi.nlm.nih.go /gap) and a e a ailable o second- a y analyses. These da ase s p o ide an oppo uni y o examine he gene ic ela ionship be ween co ela ed ai s, and o disco e and iden i y isk genes sha ed ac oss hese ai s. Pleio opy is a phenomenon in which a single locus a ec s mul iple ai s7,8. I accoun s o a leas a pa o he gene ic mechanism o many co ela ed human beha io s and diseases. Pleio opy can ake wo o ms: ei he a single p ocess, leading o a cascade o downs eam e ec s (some imes desc ibed as “media ed pleio opy”), o a single locus in luencing mul iple ai s (some imes desc ibed as “biological pleio opy”)9. Schizoph enia is highly como bid wi h ciga e e smoking10. Howe e , he unde lying biology o his como bidi y is no well unde s ood11. Se e al hypo heses ha e been p oposed. The sel -medica ion hypo hesis pos ula es ha schizoph enia pa ien s smoke o educe symp oms and an ipsycho ics-induced side e ec s and o imp o e hei a en ion and wo king memo y12. Al e na i ely, schizoph enia and ND could ha e sha ed some gene ic liabili y (i.e., biological pleio - opy)13, which is suppo ed by ecen s udies o indi idual genes14–21. A hi d possibili y is ha smoking may be causal o schizoph enia (i.e., media ed pleio opy)22. To explo e he gene ic ela ionship be ween schizoph enia and ND, we ob ained he GWAS summa y s a is ics om he Psychia ic Genomics Conso ium (PGC) schizo- ph enia analyses and ND ela ed ai s om ou unpublished s udies, and conduc ed polygenic analyses. Unde he hypo hesis o biological pleio opy, we expec ha gene ic isk sco es o schizoph enia and ND ela ed ai s p edic each o he ; whe eas sel -medica ion would an icipa e unidi ec ional (schizoph enia o ND ai s) p edic- ion. In his a icle, we epo he indings om hese analyses. Resul s Nico ine dependence and schizoph enia sha e gene ic liabili y. In hese analyses, we calcula ed gene ic isk sco es o schizoph enia (supplemen a y Figu e 1A) and es ed whe he he isk sco es p edic ed FTND and CPD. The esul s we e summa ized in Table1. Schizoph enia isk sco es p edic ed FTND sco e and CPD a he h esholds o P ≤ 5 × 10−3, 5 × 10−2 and 5 × 10−1. The co ela ion coe icien s a hese h esholds we e all posi i e, sugges ing ha a schizoph enia diagnosis was posi i ely associa ed wi h ciga e e smoking, consis en wi h he well-known como bidi y be ween schizoph enia and ND. Howe e , schizoph enia isk sco es explained only a e y small ac ion o he FTND and CPD ai s. FTND and COT isk sco es (supplemen a y Figu e 1B,C) we e calcula ed o he subjec s o he phase I PGC schizoph enia GWAS samples23 using he summa y s a is ics om he FTND (n = 17,781) and COT (n = 4,548) GWAS me a-analyses. We hen e alua ed whe he he gene ic isk sco es o COT and FTND could p edic he schizoph enia diagnosis using logis ic eg ession. The esul s we e p esen ed in Table2. The COT isk sco es calcula ed a he P- alues o 5 × 10−5, 5 × 10−4 and 5 × 10−3 p edic ed schizoph enia diagnosis, bu FTND isk sco es ailed o do so. Fo he P- alues h esholds a which he COT isk sco es p edic ed schizoph enia diagnosis, he be a coe icien s we e also posi i e, again, con i ming he posi i e pheno ypic co ela ion be ween ND and schizoph enia. Iden i ica ion o sha ed a ian s be ween ND and schizoph enia. Ou ecip ocal polygenic anal- yses sugges ed ha he e we e some sha ed gene ic liabili y be ween schizoph enia and ND as de ined by he FTND and COT ai s. We hen p oceeded o iden i y he a ian s associa ed wi h bo h schizoph enia and ND ai s. We compu ed join P- alues o each ma ke using he summa y s a is ics om he schizoph enia and COT/FTND/TFC me a-analyses, and assigned a q- alue o each o he join P- alues using an FDR me hod24,25. Table3 lis ed he loci iden i ied by he join analyses wi h q- alues ≤ 0.05. F om he join analyses be ween schiz- oph enia and COT, 11 loci eached genome-wide signi icance o associa ion wi h bo h COT and schizoph enia, o which 2 loci had no known genes nea by and 6 we e spliced ESTs o long non-coding RNAs. In he analyses be ween schizoph enia and FTND, 10 loci we e iden i ied, and 3 o hem we e ESTs o non-coding RNAs. The Th eshold (P) Be a S.E. T_ alue P (>|T|) R2 FTND 5 × 10−52.41E-04 0.000269 0.90 0.3708 0 5 × 10−43.30E-04 0.000235 1.41 0.1599 1.00E-04 5 × 10−33.91E-04 0.000194 2.01 0.0440 3.00E-04 5 × 10−23.53E-04 0.000153 2.31 0.0211 4.00E-04 5 × 10−13.22E-04 0.000118 2.73 0.0063 7.00E-04 CPD 5 × 10−51.03E-04 8.77E-05 1.17 0.2424 0 5 × 10−41.37E-04 7.64E-05 1.79 0.0736 1.00E-04 5 × 10−31.71E-04 6.32E-05 2.71 0.0067 5.00E-04 5 × 10−21.56E-04 4.99E-05 3.13 0.0018 6.00E-04 5 × 10−11.29E-04 3.84E-05 3.37 0.0008 8.00E-04 Table 1. Schizoph enia isk sco e p edic ion o FTND and CPD. www.na u e.com/scien i ic epo s/ 3 Scien i ic RepoR s | 6:25671 | DOI: 10.1038/s ep25671 join analyses be ween schizoph enia and TFC yielded 15 signi ican loci. The CHRNA5-CHRNA3-CHRNB4 locus was he only one iden i ied by all h ee smoking ai s. In addi ion o some genes known o be associa ed wi h schizoph enia (HLA-B and MAD1L1), we also iden i ied no el non-coding RNAs and RNA binding p o ein genes (DA376252, BX089737, LOC101927273, LINC01029, LOC101928622, HY157071, DA902558, RBFOX1 and TINCR), pos - ansla ion modi ica ion genes (MANBA, UBE2D3 and RANGAP1) and ene gy p oduc ion genes (XYLB, MTRF1 and ENOX1). Pa hway en ichmen and ne wo k in e ac ion analyses. We u he explo ed he pa hways sha ed by schizoph enia and ND by selec ing all ma ke s wi h q- alues less han 0.16 om he join analyses be ween schiz- oph enia and smoking ai s. A e mapping he ma ke s o genes, he genes showing po en ial associa ion wi h schizoph enia and COT/FTND/TFC we e pooled o sea ch o pa hways en iched in bo h condi ions. In hese analyses, we selec ed only he genes iden i ied by a leas 2 o he 3 smoking ai s, yielding a o al o 146 genes. A e il e ing ou he human leukocy e an igen genes (HLA-B, HLA-C, HLA-DOA, HLA-DQA1, HLA-DQB1, HLA-DRB1, and HLA-G) due o hei s ong linkage disequilib ium26, we used he emaining 139 genes in pa h- way analyses. Ou analyses iden i ied 16 unique pa hways ha we e sha ed be ween schizoph enia and ND (Table4). The mos no iceable pa hways we e Calcium Signaling, Long-Te m Po en ia ion, Neu oac i e Ligand-Recep o In e ac ion, Phospha idylinosi ol Signaling, Cell Adhesion Molecules, and Regula ion o Ac in Cy oskele on pa hways. Some o hese pa hways (Calcium Signaling, Long-Te m Po en ia ion, Cell Adhesion Molecules, and Regula ion o Ac in Cy oskele on) had been epo ed o be in ol ed in schizoph enia27–32, o he s (Cell Adhesion Molecules and Neu oac i e Ligand-Recep o In e ac ion) had been implica ed in ND33,34. We ound ha hese pa hways we e en iched in he genes associa ed wi h bo h ND and schizoph enia. Addi ionally, pa hways in ol ed in ca diomyopa hy, GnRH signaling, gas ic acid sec e ion and Alzheime ’s disease we e also ound o be sha ed be ween schizoph enia and ND. In he pa hway ne wo k in e ac ion analyses, we ound a ne wo k o c oss alk be ween pa hways (Fig.1), wi h he Long-Te m Po en ia ion loca ed a he cen e o hese in e ac ions. Discussion I is well known in psychia ic clinics ha a la ge p opo ion o schizoph enia pa ien s smoke ciga e es and smoke hea ily13. The dominan hypo hesis o explain he como bidi y is sel -medica ion12, i.e., ha schizoph e- nia pa ien s smoke o amelio a e impai men s in cogni i e unc ion and supp ess psycho ic symp oms. Ano he hypo hesis con ends ha schizoph enia and ND sha e some gene ic liabili y, and he high p e alence a e o ciga e e smoking in schizoph enia pa ien s is a mani es a ion ha is pa ially due o he common liabili y13. A hi d possibili y is ha smoking may be a isk ac o o he de elopmen o schizoph enia, gi en ha smoking ini ia ion ypically p eda es he onse o schizoph enia22. These h ee hypo heses a e no mu ually exclusi e, and all h ee may con ibu e o he obse ed co-occu ence o schizoph enia and smoking. P e ious s udies examining his issue ha e la gely ocused on indi idual unc ions/symp oms o genes using ela i ely small sample sizes. He e we ook a sys ema ic app oach, and examined he en i e genome using la ge GWAS da ase s and mul iple ai s. We obse ed di e en pa e ns be ween he ecip ocal polygenic analyses (compa ing Tables1 and 2). When we used he gene ic isk sco es o schizoph enia o p edic ND ai s, he associa ion was e iden a P- alues ≥ 5 × 10−3, wi h he associa ion s eng h inc eased as he P- alue h eshold became la ge (Table1). Gi en ha he PGC schizoph enia GWAS did no con ol o smoking s a us and quan- i y, and he e was a la ge di e ence o smoking p e alence be ween schizoph enia pa ien s and con ols (on a e age, 65% o mo e schizoph enia pa ien s smoke, and abou 20% people smoke in he gene al popula ion), we would expec ha he PGC schizoph enia GWAS iden i y op candida es o ND ela ed ai s. Bu wha we ound was no he case. These op anked candida es (i.e. hose wi h P- alues ≤ 5 × 10−5) om he PGC schizoph enia me a-analysis1 we e no p edic ND ela ed ai s. A likely explana ion o hese esul s is ha genes mos s ongly associa ed wi h schizoph enia do no di ec ly con ibu e o he smoking beha io s in schizoph enia pa ien s. In o he wo ds, he eason why schizoph enia pa ien s smoke is ha hey wan o imp o e hei cogni i e unc ions Th eshold (P) Be a S.E. Wald Z P (>|Z|) R2 Co inine 5 × 10−50.0459 0.0169 2.72 0.0066 0.0006 5 × 10−40.0583 0.0169 3.45 0.0006 0.0010 5 × 10−30.0435 0.0169 2.58 0.0100 0.0005 5 × 10−20.0035 0.0170 0.20 0.8384 3.4E-06 5 × 10−1− 0.0226 0.0172 − 1.32 0.1886 0.0001 FTND 5 × 10−50.0220 0.0242 0.91 0.3641 0.0001 5 × 10−40.0227 0.0507 0.45 0.6537 2.5E-05 5 × 10−30.0331 0.0617 0.54 0.5913 3.5E-05 5 × 10−20.0033 0.0323 0.10 0.9174 1.3E-06 5 × 10−10.0281 0.0292 0.96 0.3362 0.0001 Table 2. Gene ic isk sco e o nico ine dependence p edic ion o schizoph enia diagnosis*. *R2 is he Nagelke ke’s R2 ob ained by he R package msb. www.na u e.com/scien i ic epo s/ 4 Scien i ic RepoR s | 6:25671 | DOI: 10.1038/s ep25671 and o supp ess psycho ic symp oms, no because ha hey a e addic ed o nico ine as egula smoke s in he gene al popula ion do. These esul s a e consis en wi h he sel -medica ion hypo hesis. In con as , when we used COT isk sco es o p edic schizoph enia diagnosis, we ound ha smalle P- alues p oduced s onge signals (Table2), indica ing ha genes mos s ongly associa ed wi h ND we e associa ed wi h schizoph enia. The esul s imply ha ei he ND and schizoph enia sha e some gene ic liabili y, o ND is a isk ac o o schizoph enia. These i he p edic ions o he sha ed liabili y hypo hesis and ha smoking is a causal isk o schizoph enia. O no e, hese wo explana ions a e no mu ually exclusi e. Bu wi hou da a on smoking o he pa ien s we a e unable o es he la e possibili y (e.g., by s a i ying ou sample on smoking s a us). Assuming biological pleio opy o be he unde lying mechanism, we de ised a es o disco e he a ian s sha ed be ween ND and schizoph enia. Using his app oach, we iden i ied mul iple genes associa ed wi h bo h condi ions (Table3). O hese genes, he CHRNA5-CHRNA3-CHRNB4 clus e had been ound o be associa ed wi h CPD2,3 and o he ND ela ed ai s, and i was epo ed o be associa ed wi h schizoph enia in he la es schizoph enia GWAS me a-analysis om PGC1. Se e al o he genes had been epo ed o be associa ed wi h schizoph enia (HLA-B and MAD1L1)28 and epilepsy (KCNT1, PRICKLE2 and RBFOX1)35–37, sugges ing ha hey migh play a ole in smoking beha io s as well. Ou analyses also iden i ied some no el genes sha ed be ween schizoph enia and ND, including a g oup o long non-coding RNAs and RNA binding p o ein genes (DA376252, BX089737, LOC101927273, LINC01029, LOC101928622, HY157071, DA902558, RBFOX1 and TINCR), a g oup o pos - ansla ion modi ica ion genes (MANBA, UBE2D3, and RANGAP1) and a g oup o ene gy p oduc ion SNP Ch Loca ion (bp) Z (ND) Z (SCZ) P- alue Q- alue Gene symbol # ma ke s COT s798015 1 117,320,907 − 3.71 − 3.69 5.08E-08 0.0020 DA376252 3 s9850756 3 18,905,739 − 3.71 3.62 8.72E-08 0.0030 DA733783 1 s709071 3 191,426,311 − 3.46 3.51 2.85E-07 0.0081 – 1 s12640124 4 118,775,172 3.61 − 3.50 2.12E-07 0.0063 BX089737 3 s2442720 6 31,320,277 − 3.49 4.62 2.26E-07 0.0067 HLA-B 1 s11779524 8 8,618,613 3.85 3.81 1.89E-08 0.0008 CF594265 4 s56235824 9 11,039,320 3.44 3.55 3.38E-07 0.0090 – 1 s11788261 9 138,611,139 3.80 3.51 1.95E-07 0.0060 KCNT1 2 s8042374 15 78,908,032 7.69 7.04 3.48E-24 1.68E-17 CHRNA5/CHRNA3/CHRNB4 270 s6650723 18 53,524,269 3.76 3.51 2.07E-07 0.0062 DA696352/LOC101927273 5 s7228837 18 75,817,606 4.23 3.65 6.64E-08 0.0024 LINC01029 19 FTND s56335113 1 30,427,639 3.80 5.85 2.02E-08 0.0021 – 10 s36025078 2 155,883,716 − 3.90 − 3.59 1.13E-07 0.0061 – 1 s188499496 3 38,450,183 − 3.93 − 3.62 8.82E-08 0.0050 XYLB 2 s11917643 3 64,171,754 3.62 − 4.17 8.41E-08 0.0048 PRICKLE2 6 s76923559 4 34,053,926 − 3.93 − 3.79 2.35E-08 0.0023 LOC101928622 3 s147093127 5 152,086,293 3.67 4.01 5.70E-08 0.0037 LINC01470 1 s9322751 6 104,015,759 − 3.51 − 3.54 2.03E-07 0.0089 HY157071 6 s4994764 7 1,928,662 − 3.90 − 4.32 9.17E-09 0.0012 MAD1L1 18 s3910267 11 130,810,282 − 3.84 5.71 1.50E-08 0.0018 SNX19 21 s147144681 15 78,900,908 − 7.62 − 5.55 7.85E-16 5.66E-09 CHRNA5/CHRNA3/CHRNB4 106 TFC s4658015 1 196,053,435 3.51 3.36 5.91E-07 0.0094 – 1 s1069267 3 38,435,023 − 3.63 − 4.43 8.02E-08 0.0015 XYLB 3 s11722779 4 103,827,488 4.22 − 4.30 6.00E-10 0.0001 MANBA/UBE2D3/SLC9B2 315 s2717737 8 18,459,343 − 3.36 3.73 6.05E-07 0.0096 PSD3 1 s13264022 8 21,293,046 3.50 4.01 2.16E-07 0.0038 DA902558 15 s7959287 12 103,601,638 3.84 4.49 1.54E-08 0.0003 – 5 s9594516 13 41,849,360 3.64 3.77 7.33E-08 0.0014 MTRF1 1 s112531467 13 43,865,047 − 3.66 3.52 1.86E-07 0.0033 ENOX1 1 s181676509 14 104,242,531 − 3.67 − 3.98 5.73E-08 0.0011 C14ORF2 15 s147144681 15 78,900,908 − 5.50 − 5.55 1.49E-15 1.08E-08 CHRNA5/CHRNA3/CHRNB4 72 s17665477 16 6,700,889 − 3.69 3.80 5.16E-08 0.0010 RBFOX1 11 s12462853 19 5,538,936 3.45 − 3.39 4.82E-07 0.0078 TINCR 2 s62202174 20 20,461,125 − 3.38 − 4.24 5.26E-07 0.0085 RALGAPA2 1 s5758274 22 41,664,539 3.64 − 4.45 7.43E-08 0.0014 RANGAP1 1 s8135804 22 42,334,660 3.40 3.57 4.54E-07 0.0074 CENPM 12 Table 3. Join es ing o associa ion wi h schizoph enia and smoking ai s. www.na u e.com/scien i ic epo s/ 5 Scien i ic RepoR s | 6:25671 | DOI: 10.1038/s ep25671 genes (XYLB, MTRF1 and ENOX1). Long non-coding RNAs we e sugges ed o play a ole in schizoph enia38,39, he iden i ica ion o mul iple long non-coding RNAs was in iguing. Pheno ype como bidi y is common in complex diseases and ai s7,8. Pleio opy, o sha ed gene ic liabili y, may be an unde lying mechanism o hese como bidi ies. Unde his condi ion, di e en app oaches ha e been de eloped o iden i y genes sha ed by he como bid condi ions40,41, and hese app oaches seem mo e powe ul han s anda d GWAS8,42. Ano he ad an age o hese me hods is ha hey can use he la ge numbe o GWAS da ase s p oduced by a single pheno ype/ ai analyses. The app oach we used o iden i y hese sha ed loci is conse a i e. In ou analyses, we excluded all ma ke s eaching genome-wide signi icance om bo h schizoph e- nia and smoking ai s and equi ed a balanced con ibu ion om bo h ai s. Unde his condi ion, i a ma ke eached genome wide signi icance o schizoph enia bu had a modes associa ion wi h ND ai s (say P- alues be ween 10−4 o 5 × 10−6), i was excluded om ou join es ing. Simila ly, some ma ke s would be excluded i hey eached genome wide signi icance in ND ai s. Because he GWASs used ha e di e en sample sizes, and Pa hway Genes ound in pa hway # gene in pa hway # gene obse ed # gene expec ed Obse ed/ expec ed a io Raw P- alue Adjus ed P- alue Calcium signaling pa hway ATP2B2, CACNA1C, CACNA1I, CHRM3, ITPR1, ITPR2 177 6 0.57 10.59 2.41E-05 0.0004 Long- e m po en ia ion CACNA1C, ITPR1, ITPR2, PPP1CB 70 4 0.22 17.86 7.81E-05 0.0007 Neu oac i e ligand- ecep o in e ac ion CHRM3, CHRNA3, CHRNA5, CHRNB4, NR3C1, THRB 272 6 0.87 6.89 0.0003 0.0011 Sali a y sec e ion ATP2B2, CHRM3, ITPR1, ITPR2 89 4 0.28 14.05 0.0002 0.0011 Panc ea ic sec e ion ATP2B2, CHRM3, ITPR1, ITPR2 101 4 0.32 12.38 0.0003 0.0011 Vascula smoo h muscle con ac ion CACNA1C, ITPR1, ITPR2, PPP1CB 116 4 0.37 10.78 0.0005 0.0013 Oocy e meiosis ITPR1, ITPR2, PPP1CB, SPDYA 112 4 0.36 11.16 0.0005 0.0013 Phospha idylinosi ol signaling sys em INPP5K, ITPR1, ITPR2 78 3 0.25 12.02 0.0020 0.0036 Gas ic acid sec e ion CHRM3, ITPR1, ITPR2 74 3 0.24 12.67 0.0018 0.0036 A hy hmogenic igh en icula ca diomyopa hy (ARVC) CACNA1C, ITGAV, SGCD 74 3 0.24 12.67 0.0018 0.0036 Hype ophic ca diomyopa hy (HCM) CACNA1C, ITGAV, SGCD 83 3 0.27 11.30 0.0024 0.0039 Dila ed ca diomyopa hy CACNA1C, ITGAV, SGCD 90 3 0.29 10.42 0.0031 0.0046 GnRH signaling pa hway CACNA1C, ITPR1, ITPR2 101 3 0.32 9.28 0.0042 0.0058 Cell adhesion molecules (CAMs) ITGAV, NCAM2, PTPRM 133 3 0.43 7.05 0.0091 0.0117 Alzheime ’s disease CACNA1C, ITPR1, ITPR2 167 3 0.53 5.61 0.0167 0.0188 Regula ion o ac in cy oskele on CHRM3, ITGAV, PPP1CB 213 3 0.68 4.40 0.0313 0.0331 Table 4. Pa hways en iched in schizoph enia and smoking ai s. Figu e 1. Pa hway c oss alk ne wo k. The size o he node is p opo ional o he P- alues o pa hway en ichmen es . The hickness o he edge is p opo ional o he P- alues o pa hway c oss alk. www.na u e.com/scien i ic epo s/ 6 Scien i ic RepoR s | 6:25671 | DOI: 10.1038/s ep25671 he e o e a ied in hei s a is ical powe , i is ine i able ha we would miss some ma ke s om he mo e powe - ul GWAS when we equi ed balanced summa y s a is ics in he join es ing. Ou pa hway analyses iden i ied mul iple pa hways sha ed by schizoph enia and ND. The mos signi ican pa hways we e Calcium Signaling, Long-Te m Po en ia ion and Neu oac i e Ligand-Recep o In e ac ion. These pa hways a e in ol ed in neu o ansmi e ansduc ion and communica ion be ween neu ons, and hey a e essen ial o cogni i e unc ions. These pa hways ha e been shown o be in ol ed in schizoph enia28,43,44 and ND45,46. The Cell Adhesion Molecules and Regula ion o Ac in Cy oskele on pa hways ha e also been epo ed in schizoph enia31,47–49 and ND45,50,51. Thus, ou esul s a e consis en wi h hese s udies. I is wo h no ing ha he ca diomyopa hy pa hways we e iden i ied in ou analyses and ha , in a p e ious s udy, we ound ha CMYA5 was associa ed wi h schizoph enia52. Ano he gene, NDUFV2, causa i e o hype ophic ca diomyopa hy53,54, he gene ic o m o ca diomyopa hy, was also ound o be associa ed wi h schizoph enia55–57. Pa hway c oss alk analyses showed ha many o hese pa hways in e ac wi h each o he and oge he hey o m an in e linked ne wo k wi h he Long-Te m Po en ia ion pa hway a he cen e o hese in e ac ions. In animal s udies, nico- ine al e s long- e m po en ia ion58–60 and lea ning and memo y61. In humans, smoking may alle ia e cogni i e impai men 62, and bo h nico ine wi hd awal and schizoph enia a e associa ed wi h cogni i e impai men s63,64. Thus, compensa ing cogni i e impai men s may be a common mo i a ional ac o be ween egula smoke s and schizoph enia pa ien s. In summa y, ou esul s suppo ed he sel -medica ion hypo hesis. We also ound e idence ha schizoph enia and ND sha e some gene ic liabili y and hese esul s did no con adic he hypo hesis ha smoking was a causal isk ac o o schizoph enia. Assuming sha ed liabili y and a balanced con ibu ion, we iden i ied no el candi- da e genes associa ed wi h bo h schizoph enia and ND. Analyses o he sha ed genes e ealed mul iple pa hways and an in e ac ing ne wo k cen e ed on long- e m po en ia ion. These esul s p o ided some new insigh s o ou unde s anding o smoking beha io s in bo h schizoph enia pa ien s and he gene al popula ion. Me hods Pheno ypes and GWAS da ase s. Fo schizoph enia, we ob ained he summa y s a is ics om he PGC GWAS o schizoph enia1. This s udy used 52 independen samples, o hem 46 we e case con ol samples o Eu opean ances y, 3 we e Asian case con ol samples and 3 we e Eu opean amily samples. Since he sam- ples we e collec ed om di e en coun ies, bo h he c i e ia o Diagnos ic and S a is ical Manual o Men al Diso de s (DSM) and In e na ional Classi ica ion o Diseases (ICD) we e used in he diagnosis o he pa ien s. Please see o iginal pape 1 o de ails. We selec ed o use he summa y s a is ics o he 46 Eu opean case con ol samples (32,405 cases and 46,839 con ols). Fo ND- ela ed ai s, we used he summa y s a is ics o ou co inine s udy65 and 2 unpublished da ase s (manusc ip s in p epa a ion). One da a used he sum sco es o he Fage s öm Tes o Nico ine Dependence (FTND)66 as a ai , which is a commonly used pheno ype o ND based on sel - e- po ed smoking beha io s. The second da a used a single i em o he FTND ques ionnai e, “How soon a e you wake up do you smoke you i s ciga e e”, o ime o smoke he i s ciga e e (TFC) as a ai . This ques ion can be seen as a measu e o nico ine wi hd awal since he hal -li e o nico ine in he human body is abou 2 hou s67. Smoke s o en expe ience nico ine wi hd awal in he mo ning a e no smoking o e nigh . The hi d da a65 used he plasma co inine concen a ion (COT) as a ai . Co inine is he majo me aboli e o nico ine, and i s hal -li e is much longe han ha o nico ine. The e o e, i s concen a ion in plasma can be conside ed an index o nico ine in ake in ecen days68,69. Because he quan i y o nico ine in ake is one o he mos impo an measu es o ND, COT may be conside ed a measu e o ND as well. In hese s udies, FTND, TFC and COT we e ea ed as quan- i a i e ai s. The sample size o FTND was 16,237, excluding he Ne he lands Twin Regis y sample because some o i s subjec s we e also used in COT GWAS. The sample sizes o TFC and COT we e 15,705 and 4,575 espec i ely. The FTND and TFC measu es we e de i ed om he same subjec s, he e o e, only FTND was used in polygenic analyses. TFC we e used only o he iden i ica ion o sha ed genes be ween schizoph enia and ND ela ed pheno ypes. The samples used in hese 3 ND ela ed GWASs we e lis ed in Supplemen a y Table S1. All subjec s used in his s udy we e o Eu opean ances y. Polygenic analyses. Schizoph enia isk sco es we e calcula ed o 9 independen smoking ela ed s udies (Table S1, n = 10,794) wi h FTND and CPD measu es using he summa y s a is ics om he PGC schizoph enia me a-analysis. The con ol subjec s om he Molecula Gene ics o Schizoph enia (MGS) we e included in he GWASs o bo h FTND and PGC schizoph enia, he e o e hey we e excluded om his analysis. Risk sco es o COT and FTND we e calcula ed o 13,326 indi iduals om he NIMH gene ics conso ium eposi o y (h ps:// www.nimhgene ics.o g/). We es ima ed he isk sco es o each ai using he algo i hms implemen ed in he PLINK so wa e70. Speci ically, he isk sco e o an indi idual was he sum o he numbe o isk alleles mul- iplied by he loga i hm o odds a io (OR, o schizoph enia) o be a coe icien ( o FTND and COT), which was hen no malized subsequen ly by he p oduc o maximal numbe o isk alleles and log(OR)s/be a coe i- cien s. Fo each ai , we calcula ed isk sco es a 5 P- alue h esholds: 5 × 10−5, 5 × 10−4, 5 × 10−3, 5 × 10−2 and 5 × 10−1. The numbe s o ma ke s used o calcula e schizoph enia isk sco es a hese h esholds we e 6,014, 94,804, 268,070, 1,021,476 and 5,370,899. The numbe s o ma ke s used o FTND and COT we e 731, 6,312, 55,378, 500,542 and 4,752,196; and 1,621, 6,357, 48,575, 473,100, and 4,737,313 espec i ely. We hen es ed whe he schizoph enia isk sco es p edic ed FTND sco es and ice e sa using logis ic (schizoph enia) and linea eg ession (FTND sco es). Since he numbe o ciga e es smoked pe day (CPD) was a ailable om he FTND da ase s, we also es ed whe he he gene ic isk sco es o schizoph enia p edic ed he CPD pheno ype. Because we did no ha e indi idual geno ypes o all da ase s used in he COT me a-analyses, we used only he COT isk sco e o p edic schizoph enia diagnosis. Sex, age and s udy we e included as co a ia es in eg ession analyses. www.na u e.com/scien i ic epo s/ 7 Scien i ic RepoR s | 6:25671 | DOI: 10.1038/s ep25671 Iden i ica ion o sha ed isk genes. While he e a e pape s looking a pleio opy om a condi ional FDR poin o iew71, we a i e o quali a i ely simila conclusions using a somewha simple app oach o amily-wise e o a e. Ou es a emp s o disco e sha ed isk genes be ween schizoph enia and ND using summa y s a is- ics om hei espec i e GWASs. To ensu e ha such a es is no o e ly in luenced by a s ong signal in jus one ai , we implemen ed a “weakes link” app oach (i.e., choosing he la ge P- alue o he pai o ai es s a he SNP unde in es iga ion)72. In mo e de ail, le Xj and Pj be he χ 2 dis ibu ed s a is ics and hei associa ed (back- g ound en ichmen adjus ed) P- alues, j = 1, … , m, o associa ion es s be ween he m ai s and a SNP. As he o e lap s a is ic o all ai s we use =R Pmax jj (o , al e na i ely, =||TXmin j j). Unde he assump ion ha he ai es s a e independen , he P- alue (also deno ed as o e lap P- alue) o a gi en o e lap s a is ic, , a a SNP is ≤= ∏≤ = PR PP () () j m j 1 . I we u he assume ha (unde he null hypo hesis - H0) none o he ai s is asso- cia ed wi h he gene ic a ian , he o e lap P- alue simpli ies o P(R ≤ ) = m (1). O he wise, P(Pj ≤ ) can be compu ed based on he dis ibu ion o he j- h ai P- alues. Fo ins ance, o wo pheno ype con igu a ion and a pu a i e h eshold o 5 × 10−8, he pa ame ic e sion o ou me hod equi es ha , o a signi ican pleio opic signal, he p- alues o bo h pheno ypes o be < 2.2 × 10−4 ( = − x510 8). This subs an ially less han 5 × 10−8 p- alue h eshold is simila in spi i o he one om Andeassen e al.71 While he o e lap p- alue (1) does elimi- na e mos o he in luence o an ex eme signal o one pheno ype, i does no elimina e i comple ely. Howe e , o a pu a i e h eshold o 5 × 10−8 in (1), unde he wo s case scena io o an ex eme signal in one pheno ype, he alse posi i e a e pe SNP is s ill a he small, i.e. 2.2 × 10−4. E en mo e, as seen in And eassen e al., he alse posi i e a e is likely o be subs an ially lowe . Mo eo e , a wo s -case-scena io 2.2 × 10−4 alse posi i e a e is adequa e o he pa hway analyses73. We used FDR24 o e alua e he app oxima e signi icance o he gene ic o e - lap (desc ibed by ela ion (1)) be ween schizoph enia and smoking pheno ypes. To selec p omising ma ke s o pa hway and ne wo k analyses we applied a h eshold o q- alue ≤ 0.16, co esponding o a ac o o 2 in Akaike In o ma ion C i e ion penal y in a likelihood a io χ 2 es wi h 1 deg ee o eedom. Pa hway and ne wo k analyses. We conduc ed pa hway en ichmen analysis o genes wi h a leas one ma ke wi h q- alues lowe han 0.16 om he join es ing o schizoph enia and COT/FTND/TFC ai s. I a ma ke was wi hin a gene egion, i was assigned o he gene; o he wise, i was mapped o i s mos p oxima e gene using he 50-kb lanking egions (bo h 5′ and 3′ sides). Genes iden i ied using SNPs associa ed wi h COT, FTND, o TFC we e me ged o he pa hway en ichmen analysis, o which we used he hype geome ic es implemen ed in he ool WebGes al (2013 upda e)74 and he canonical pa hways om he Kyo o Encyclopedia o Genes and Genomes (KEGG) da abase. We equi ed each pa hway o ha e a leas h ee genes om ou gene lis and no mo e han 300 genes om he e e ence genome. The P- alues om hype geome ic es s we e u he adjus ed by he Benjamini-Hochbe g me hod23. Only pa hways wi h adjus ed P- alues < 0.05 we e conside ed s a is ically signi ican ly en iched. We u he examined pa hways in e ac ion using he Cha ac e is ic Sub-Pa hway Ne wo k (CSPN) algo- i hm31,75 he human p o ein-p o ein in e ac ion (PPI) ne wo k76. We es ic ed he analysis speci ically o he a o emen ioned me ged gene se and hei en iched pa hways. In he inal s ep, we selec ed he signi ican pa h- way in e ac ion pai s based on pe mu a ion P- alues less han 0.05. Re e ences 1. Schizoph enia Wo king G oup o he Psychia ic Genomics Conso ium. Biological insigh s om 108 schizoph enia-associa ed gene ic loci. Na u e 511, 421–427 (2014). 2. Tho gei sson, T. E. e al. Sequence a ian s a CHRNB3-CHRNA6 and CYP2A6 a ec smoking beha io . Na . Gene . 42, 448–453 (2010). 3. Tobacco and Gene ics Conso ium. Genome-wide me a-analyses iden i y mul iple loci associa ed wi h smoking beha io . Na . 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In eg a ed ne wo k analysis pla o m o p o ein-p o ein in e ac ions. Na . Me hods 6, 75–77 (2009). Acknowledgemen s This wo k was suppo ed in pa by NIH g an s ML011177, MH101054 and DA032246. JK has been suppo ed by he Academy o Finland (g an s # 265240 and 263278). We a e g a e ul o he PGC schizoph enia analysis g oup o p o iding he summa y s a is ics o i s GWAS me a-analysis. Al hough un ela ed o his esea ch, H. K anzle has been a consul an o ad iso y boa d membe o Alke mes, Lilly, Lundbeck, O suka, P ize , and Roche. He is also a membe o he Ame ican Socie y o Clinical Psychopha macology’s Alcohol Clinical T ials Ini ia i e, which is suppo ed by AbbVie, Alke mes, E hypha m, Lilly, Lundbeck, and P ize . Au ho Con ibu ions J.C. and S.-A.B. pe o med polygenic and joined analyses, and w o e he pape ; H.Y., Z.Z. and P.J. pe o med pa hway and ne wo k analyses; K.S.K., H.R.K., J.G., L.F., C.M., R.P., Y.M., D.I.B., B.W.J.H.P., R.F.T., J.J.W., J.V., J.K. and M.M. con ibu ed o he samples used his manusc ip and commen ed on he ex ; co inine me a-analysis g oup and FTND me a-analysis g oup con ibu ed samples o his pape ; and XC concei ed, designed and w o e he pape . All au ho s ha e ead and ag eed on he con en o his 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: Chen, J. e al. Gene ic ela ionship be ween schizoph enia and nico ine dependence. Sci. Rep. 6, 25671; doi: 10.1038/s ep25671 (2016). This wo k is licensed unde a C ea i e Commons A ibu ion 4.0 In e na ional License. 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