Variation at 2q35 (PNKD and TMBIM1) influences colorectal cancer risk and identifies a pleiotropic effect with inflammatory bowel disease

ASSOCIATION STUDIES ARTICLE Va ia ion a 2q35 (PNKD and TMBIM1) in luences colo ec al cance isk and iden i ies a pleio opic e ec wi h in lamma o y bowel disease Giulia O lando 1,† , Philip J. Law 1,† , Kimmo Palin 2,3,† , Sa i Tuupanen 2,3 , Alexand a Gyl e 2,3,‡ , Ul ika A. H€ anninen 2,3 , Ta iana Cajuso 2,3 , Tomas Tanskanen 2,3 , Johanna Kondelin 2,3 , Ee i Kaasinen 2,3 , An i-Pekka Sa in 4 , Jaakko Kap io 4,5 , Johan G. E iksson 6,7 , Ha i Rissanen 5 , Paul Knek 5 , Ee o Pukkala 8,9 , Pekka Jousilah i 5 , Veikko Salomaa 5 , Samuli Ripa i 4 , Aa no Palo ie 4,10,11,12 , Heikki J€ a inen 13 , Lau a Renkonen-Sinisalo 14 , Anna Lepis o¨ 14 , Jan Bo¨hm 15 , Jukka-Pekka Mecklin 16 , Nada A. Al-Tassan 17 , Clai e Palles 18 , Lynn Ma in 18 , Ella Ba clay 18 , Albe Tenesa 19,20 , Susan Fa ing on 19 , Ma ia N. Timo ee a 19 , B ian F. Meye 17 , Salma M. Wakil 17 , Ha y Campbell 21 , Ch is ophe G. Smi h 22 , Shelley Idziaszczyk 22 , Timo hy S. Maughan 23 , Richa d Kaplan 24 , Rachel Ke 25 , Da id Ke 26 , Daniel D. Buchanan 27,28 , Aung Ko Win 28 , John Hoppe 28 , Ma k Jenkins 28 , No alane M. Lindo 29 , Polly A. Newcomb 30 , S e e Gallinge 31 , Da id Con i 32 , F ed Schumache 32 , G aham Casey 32 , Jussi Taipale 2,3,33 , Je emy P. Cheadle 22 , Malcolm G. Dunlop 19 , Ian P. Tomlinson 18 , Lau i A. Aal onen 2,3 and Richa d S. Houls on 1, * 1 Di ision o Gene ics and Epidemiology, The Ins i u e o Cance Resea ch, London SW7 3RP, UK, 2 Genome-Scale Biology Resea ch P og am, Resea ch P og ams Uni , 3 Depa men o Medical and Clinical Gene ics, Medicum and 4 Ins i u e o Molecula Medicine Finland, Uni e si y o Helsinki, Helsinki 00014, Finland, 5 Na ional Ins i u e o Heal h and Wel a e, Helsinki 00271, Finland, 6 Folkh€ alsan Resea ch Cen e, Helsinki 00250, Finland, 7 Uni o Gene al P ac ice and P ima y Heal h Ca e, Uni e si y o Helsinki and Helsinki Uni e si y Hospi al, Helsinki 00014, Finland, 8 Finnish Cance Regis y, Ins i u e o S a is ical and Epidemiological Cance Resea ch, Helsinki 00130, Finland, 9 School o Heal h Sciences, Uni e si y o Tampe e, Tampe e 33014, Finland, † These au ho s con ibu ed equally o his wo k. ‡ P esen add ess: Human Longe i y Inc., La Jolla, CA 92121, USA. Recei ed: Oc obe 16, 2015. Re ised: Feb ua y 5, 2016. Accep ed: Ma ch 14, 2016 V CThe Au ho 2016. Published by Ox o d Uni e si y P ess. This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (h p://c ea i ecommons.o g/licenses/by/4.0/), which pe mi s un es ic ed euse, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. 2349 Human Molecula Gene ics, 2016, Vol. 25, No. 11 2349–2359 doi: 10.1093/hmg/ddw087 Ad ance Access Publica ion Da e: 22 Ma ch 2016 Associa ion S udies A icle a Tampe e Uni e si y Lib a y. Depa men o Heal h Sciences on Oc obe 31, 2016h p://hmg.ox o djou nals.o g/Downloaded om 10 Analy ic and T ansla ional Gene ics Uni , Depa men o Medicine, Massachuse s Gene al Hospi al, Bos on, MA 02114, USA, 11 P og am in Medical and Popula ion Gene ics, The B oad Ins i u e o MIT and Ha a d, Camb idge, MA 02142, USA, 12 Depa men o Neu ology, Massachuse s Gene al Hospi al, Bos on, MA 02114, USA, 13 Depa men o Su ge y, Helsinki Uni e si y Cen al Hospi al, Hospi al Dis ic o Helsinki and Uusimaa, Helsinki 00029, Finland, 14 Depa men o Su ge y, Abdominal Cen e , Helsinki Uni e si y Hospi al, Helsinki 00029, Finland, 15 Depa men o Pa hology, Cen al Finland Cen al Hospi al, Jy € askyl€ a 40620, Finland, 16 Depa men o Su ge y, Jy € askyl€ a Cen al Hospi al, Uni e si y o Eas e n Finland, Jy € askyl€ a 40620, Finland, 17 Depa men o Gene ics, King Faisal Specialis Hospi al and Resea ch Cen e , Riyadh 12713, Saudi A abia, 18 Wellcome T us Cen e o Human Gene ics and NIHR Comp ehensi e Biomedical Resea ch Cen e, Ox o d OX3 7BN, UK, 19 Colon Cance Gene ics G oup, Uni e si y o Edinbu gh and MRC Human Gene ics Uni , Wes e n Gene al Hospi al, Edinbu gh EH4 2XU, UK, 20 The Roslin Ins i u e, Uni e si y o Edinbu gh, Eas e Bush, Roslin EH25 9RG, UK, 21 Cen e o Popula ion Heal h Sciences, Uni e si y o Edinbu gh, Edinbu gh EH8 9AG, UK, 22 Ins i u e o Cance and Gene ics, School o Medicine, Ca di Uni e si y, Ca di CF14 4XN, UK, 23 CRUK/MRC Ox o d Ins i u e o Radia ion Oncology, Uni e si y o Ox o d, Ox o d OX3 7DQ, UK, 24 MRC Clinical T ials Uni , A ia ion House, London WC2B 6NH, UK, 25 Depa men o Oncology, Ox o d Cance Cen e, Chu chill Hospi al and 26 Nu ield Depa men o Clinical Labo a o y Sciences, John Radcli e Hospi al, Uni e si y o Ox o d, Ox o d OX3 7LE, UK, 27 Colo ec al Oncogenomics G oup, Gene ic Epidemiology Labo a o y, Depa men o Pa hology and 28 Cen e o Epidemiology and Bios a is ics, The Uni e si y o Melbou ne, Melbou ne, Vic. 3010, Aus alia, 29 Depa men o Heal h Sciences Resea ch, Mayo Clinic, Sco sdale, AZ 85259, USA, 30 Cance P e en ion P og am, F ed Hu chinson Cance Resea ch Cen e , Sea le, WA 98109, USA, 31 Lunen eld- Tanenbaum Resea ch Ins i u e, Moun Sinai Hospi al, To on o, ON M5G 1X5, Canada, 32 Depa men o P e en i e Medicine, Uni e si y o Sou he n Cali o nia, Los Angeles, CA 90033, USA and 33 Depa men o Biosciences and Nu i ion, SciLi e Cen e , Ka olinska Ins i u e, S ockholm, SE 141 83, Sweden *To whom co espondence should be add essed. Tel: þ44 (0)208722 4175; Fax: þ44 (0)7224365; Email: [email p o ec ed] Abs ac To iden i y new isk loci o colo ec al cance (CRC), we conduc ed a me a-analysis o se en genome-wide associa ion s udies (GWAS) wi h independen eplica ion, o alling 13 656 CRC cases and 21 667 con ols o Eu opean ances y. The combined analysis iden i ied a new isk associa ion o CRC a 2q35 ma ked by s992157 (P¼3.15 10 8 , odds a io ¼1.10, 95% con idence in e al ¼1.06–1.13), which is in onic o PNKD (pa oxysmal non-kinesigenic dyskinesia) and TMBIM1 ( ansmemb ane BAX inhibi o mo i con aining 1). In iguingly his suscep ibili y single-nucleo ide polymo phism (SNP) is in s ong linkage disequilib ium ( 2 ¼0.90, D0¼0.96) wi h he p e iously disco e ed GWAS SNP s2382817 o in lamma o y bowel disease (IBD). Following on om his obse a ion we examined o pleio opy, o sha ed gene ic suscep ibili y, be ween CRC and he 200 es ablished IBD isk loci, iden i ying an addi ional 11 signi ican associa ions ( alse disco e y a e [FDR])<0.05). Ou indings p o ide u he insigh in o he biological basis o inhe i ed gene ic suscep ibili y o CRC, and iden i y isk ac o s ha may in luence he de elopmen o bo h CRC and IBD. In oduc ion Colo ec al cance (CRC), a leading cause o cance - ela ed dea h wo ldwide, has a he i able basis (1,2). Recen genome-wide associ- a ion s udies (GWAS) ha e success ully iden i ied a numbe o common single-nucleo ide polymo phisms (SNPs) in luencing CRC isk he eby indica ing he asse ion ha pa o he he i able isk is polygenic (3–7). These s udies ha e also p o ided insigh s in o he biology o CRC, highligh ing he impo ance o bone mo phoge- ne ic p o ein signalling pa hway genes (BMP2, BMP4, GREM1 and SMAD7) (4,5), candida e genes (CDH1), as well as genes no p e i- ously implica ed in CRC (POLD3, TERC, CDKN1A, VIT1A and SHROOM2) (6,7). I is well es ablished ha in lamma o y bowel dis- ease (IBD), which p ima ily p esen s as C ohn’s disease o ulce a- i e coli is, is associa ed wi h an inc eased CRC isk (8–11). Despi e IBD being s ongly he i able (12), li le e idence o sha ed gene ic suscep ibili y o di e en ial e ec s o gene ic a ia ion on IBD and CRC isk has been epo ed, al hough he p esump ion is ha he di ec ion o e ec will be consis en be ween bo h diseases. A ailu e o unco e pleio opy may be e lec i e o a lack o powe o CRC GWAS conduc ed hus a . Indeed s a is ical modelling o GWAS da a shows ha al hough 19% o he he i a- bili y o CRC can be asc ibed o common a ia ion, only 10% o his is explained by cu en ly iden i ied isk SNPs (13). To empowe he iden i ica ion o new CRC suscep ibili y SNPs in pe sons o Eu opean ances y, we conduc ed a genome-wide me a-analysis o a p e iously un epo ed GWAS wi h six pub- lished da ase s in addi ion o independen eplica ion o alling 13 810 cases and 21 754 con ols. We epo he iden i ica ion o a new CRC isk associa ion which also impac s on IBD isk. Ex ending ou analysis o 2350 |Human Molecula Gene ics, 2016, Vol. 25, No. 11 a Tampe e Uni e si y Lib a y. Depa men o Heal h Sciences on Oc obe 31, 2016h p://hmg.ox o djou nals.o g/Downloaded om es ablished IBD loci, we p o ide e idence o sha ed gene ic sus- cep ibili y be ween CRC and IBD a 11 addi ional loci. Resul s P ima y GWAS In he p ima y scan ( e med he FIN GWAS), 1172 CRC cases as- ce ained h ough he Finnish CRC collec ion and Finnish Cance Regis y we e analysed wi h con ol da a on 8266 indi iduals om he FINRISK, Heal h2000, Finnish Twin Coho and Helsinki Bi h Coho S udy coho s. A e applying s ic quali y con ol c i e ia, 283906 au osomal SNPs we e a ailable o associa ion wi h CRC isk. A quan ile–quan ile (Q–Q) plo o obse ed e sus expec ed 2 - es s a is ics showed li le e idence o an in la ion o es s a is ics, he eby excluding he possibili y o subs an i e hidden popula ion subs uc u e, c yp ic ela edness among sub- jec s o di e en ial geno ype calling (in la ion ac o k¼1.07). Me a-analysis We pe o med a me a-analysis o ou p ima y scan da a wi h six o he non-o e lapping GWAS o Eu opean ances y (CCFR1, CCFR2, COIN, UK1, Sco land1 and VQ58), which ha e been p e i- ously epo ed (14). To maximize he p ospec s o iden i ying no el isk a ian s, we impu ed he da a wi h a me ged e e - ence panel using Sequencing Ini ia i e Suomi (SISu) ( o he FIN da a) o UK10K ( o he UK da a) in addi ion o 1000 Genomes P ojec da a. A e quali y con ol p ocedu es, o e 10 million a ian s, including o e 1 million inse ion–dele ions, we e ana- lysed in 8749 cases and 18 245 con ols. Associa ions o he 37 p e iously es ablished Eu opean CRC isk SNPs showed a di ec ion o e ec consis en wi h p e i- ously epo ed s udies, wi h 10 o hese SNPs ha ing P<5.0 10 8 in his me a-analysis (Supplemen a y Ma e ial, Table S1). Excluding hese known isk SNPs, oge he wi h hose co ela ed wi h 2 >0.8, om he me a-analysis wo no el e- gions o linkage disequilib ium (LD), ma ked by s992157 and s2383207, showed he s onges associa ion wi h CRC a P<1.0 10 6 (Supplemen a y Ma e ial, Table S2). To eplica e hese associa ions, we geno yped s992157 and s2383207 in an addi ional 5061 CRC cases and 3509 con ols, wi h only s992157 showing e idence o an associa ion wi h CRC (P¼0.023). In he combined analysis, he associa ion was signi ican a he genome-wide h eshold (P¼3.15 10 8 ; Fig. 1). The e was no a ia ion due o he e ogenei y (I 2 ¼0, P he ¼0.79). s992157 is loca ed a 2q35, and is in onic o wo genes: pa ox- ysmal non-kinesigenic dyskinesia (PNKD) on he o wa d s and and ansmemb ane BAX inhibi o mo i con aining 1 (TMBIM1) on he e e se s and (Fig. 2). Rela ionship be ween geno ype and CRC pheno ype Using da a on mic osa elli e ins abili y (MSI) s a us om he FIN (n¼1146), COIN (n¼1239) and NSCCG eplica ion (n¼1282) se ies, oge he wi h in o ma ion on KRAS and BRAF mu a ion s a us in umou s in COIN, we explo ed he possibili y ha he associa ion a s992157 is es ic ed o a speci ic molecula sub ype o CRC (Supplemen a y Ma e ial, Table S3). The e was no e idence o an associa ion be ween hese SNPs and any o he a iables a e ad- jus ing o mul iple es ing (i.e. P>0.05). Addi ionally, we ob- se ed no consis en associa ion be ween age, sex o umou si e using da a om he UK1, Sco land1, VQ58, COIN and NSCCG se- ies (Supplemen a y Ma e ial, Table S3). IBD SNPs in luence CRC Ano he associa ion a 2q35 de ined by s2382817 has p e i- ously been shown o in luence IBD isk (CRC me a P¼1.02  10 5 ), which is also in onic o PNKD and TMBIM1, and is in s ong LD wi h s992157 ( 2 ¼0.90, D0¼0.96). Pa adoxically, he isk o s2382817 in IBD is in e se o he CRC associa ion. Gi en he compelling e idence o an associa ion be ween IBD and CRC, we sough e idence o addi ional sha ed suscep i- bili y be ween he wo diseases. Speci ically, we examined he isk o CRC in ou me a-analysis a 200 loci ha ha e been shown in p e ious GWAS o a ec IBD isk (15,16) (Supplemen a y Ma e ial, Table S4). A Q–Q plo o he obse ed CRC associa ion P- alues agains he expec ed P- alues o each o he 200 IBD isk SNPs showed signi ican o e -dispe - sion (k¼1.33, Fig. 3). This obse a ion is compa ible wi h a ge- ne ic ela ionship be ween CRC and IBD. To accoun o mul iple es ing, we imposed an FDR-adjus ed P- alue o 0.05 as being s a is ically signi ican . A his h eshold, in addi ion o s2382817, 11 IBD isk SNPs we e associa ed wi h CRC isk (Table 1), o which i e we e posi i ely associa ed wi h CRC isk, whe eas he o he se en displayed an in e se ela ion- ship. A numbe o hese SNPs anno a e genes wi h documen ed oles ha a e ele an o CRC de elopmen , such as Wn -signal- ling [WNT4, (17)], umou supp ession [MAPKAPK5,FOXO1 (18,19)] and cellula ans o ma ion [CDC42,CEBPB (20,21)] (Table 1). We examined o an associa ion be ween he geno ype o hese 12 SNPs and he molecula sub ype o CRC, and ound no e idence o a ela ionship (Supplemen a y Ma e ial, Table S3). Func ional e ec p edic ion analysis The genomic egion con aining s992157 is he si e o ac i e s uc- u e and has egula o y mo i s o bo h enhance and p omo o unc ion in mul iple cell ypes (Fig. 2). Mo eo e ChIP-seq da a iden i y o e 122 ansc ip ion ac o s binding o he egion, in- cluding CRC- ela ed ansc ip ion ac o s such as MYC, HNF4A and TCF7L2 (Supplemen a y Ma e ial, Table S5). We also pe - o med an exp ession quan i a i e ai loci (eQTL) analysis and ound no signi ican ela ionship be ween he s992157 geno ype and PNKD and TMBIM1 exp ession in colo ec al adenoca cinoma cells (Supplemen a y Ma e ial, Table S6). The isk geno ype was howe e associa ed wi h al e ed gene exp ession in o he issues, including lymphoblas oid cells (FDR P- alue<0.05, Figu e 1. Fo es plo o he odds a ios o he associa ion be ween s992157 and CRC. S udies we e weigh ed acco ding o he in e se o he a iance o he log o he OR. Ho izon al lines: 95% con idence in e als (95% CI).Box: OR poin es i- ma e; i s a ea is p opo ional o he weigh o he s udy. Diamond: o e all sum- ma y es ima e, wi h con idence in e al gi en by i s wid h. Ve ical line: null alue (OR ¼1.0). 2351Human Molecula Gene ics, 2016, Vol. 25, No. 11 | a Tampe e Uni e si y Lib a y. Depa men o Heal h Sciences on Oc obe 31, 2016h p://hmg.ox o djou nals.o g/Downloaded om Supplemen a y Ma e ial, Table S6). This appa en di e ence in eQTLs may be e lec i e o he di e ences in epigene ic p o iles a 2q35 be ween CRC and lymphoblas oid cells (Fig. 2). To u he in es iga e he ela ionship be ween CRC and IBD isk we pe o med eQTL analysis on he 12 IBD SNPs associa ed wi h CRC isk in he colo ec al adenoca cinoma da a, and ound wo signi ican ela ionships be ween s174537 and he exp es- sion o a y acid desa u ase 2 (FADS2, FDR P- alue ¼3.28 10 6 ) and be ween s516246 and ucosyl ans e ase 2 (FUT2, FDR P- alue ¼2.08 10 17 ) (Supplemen a y Ma e ial, Table S6). Addi ional e idence o hese eQTLs was ound in o he issues in he Geu adis, Blood and GTEx da abases (Supplemen a y Ma e ial, Table S6). Simila ly o s992157, as epo ed abo e, s2382817 is an eQTL o PNKD and TMBIM1 in bo h lymphoblas- oid and whole blood issues. Following on om his we in es iga ed he p esence o sha ed gene ic pa hways be ween CRC and IBD using he LENS pa hway ool (22), which allows explo a ion o in e ac ions be- ween he gene p oduc s in p oximi y o he GWAS SNPs. Ac oss he 594 CRC p o eins and 1574 IBD p o eins, a ne wo k o 542 o e lapping p o eins was iden i ied. Figu e 4 shows he common ne wo k and in e ac ions be ween key p o eins. O in- e es was he di ec ion o associa ion be ween he CRC SNPs wi h IBD isk. Pa hways wi h e idence o en ichmen (i.e. P<0.001) wi h a consis en e ec be ween CRC and IBD we e in- ol ed in immune and in lamma o y esponse, such as co-s im- ula ion by he CD28 amily, Fc epsilon ecep o signalling and downs eam B-cell ecep o signalling. In con as , he p o ein ne wo ks de ined by ecip ocal SNPs associa ion o CRC and IBD we e en iched o in e leukin and calmodulin signalling. Figu e 2. Regional plo o associa ion esul s and ecombina ion a es o he 2q35 locus. In he panel, log 10 P alues (y-axis) o he SNPs a e shown acco ding o hei ch omosomal posi ions (x-axis). The op SNP is shown as a la ge iangle and is labelled by i s sID. The colou in ensi y o each symbol e lec s he ex en o LD wi h he op SNP: whi e ( 2 ¼0) h ough o da k ed ( 2 ¼1.0), wi h 2 es ima ed om he 1000 Genomes Phase 1 da a. Gene ic ecombina ion a es (cM/Mb) a e shown wi h a ligh blue line. Physical posi ions a e based on NCBI build 37 o he human genome. Also shown a e he ela i e posi ions o genes and ansc ip s mapping o each e- gion o associa ion. The lowe panel shows he ch oma in s a e segmen a ion ack (Ch omHMM) in HCT116 CRC and GM12878 lymphoblas oid cell lines. 2352 |Human Molecula Gene ics, 2016, Vol. 25, No. 11 a Tampe e Uni e si y Lib a y. Depa men o Heal h Sciences on Oc obe 31, 2016h p://hmg.ox o djou nals.o g/Downloaded om Pa hways ha we e en iched in bo h, albei in ol ing di e en p o eins, included hose ela ed o he adap i e immune e- sponse, cy okine signalling and in e e on signalling (Supplemen a y Ma e ial, Table S7). Discussion In his me a-analysis we combined se en independen GWAS, and ha e iden i ied a isk locus o CRC isk a 2q35 ma ked by s992157. As his SNP is in onic o bo h PNKD and TMBIM1, and hese a e he only ansc ip s wi hin he egion o high LD, i is a plausible ha he gene ic basis o he 2q35 associa ion o CRC is h ough unc ional e ec s on one o hese genes a p io i. This is coupled wi h he ac ha s992157 localizes o a genomic e- gion wi h egula o y unc ion and he eQTL da a showing allele- speci ic cis- egula o y ela ionship be ween SNP geno ype and PNKD and TMBIM1 exp ession. Al hough specula i e, he long iso o m o PNKD appea s o unc ion in a pa hway o de oxi y al- pha-ke oaldehyde using glu a hione as a co ac o (23). As glu a- hione is essen ial o main aining cellula edox s a us, educed glu a hione le els in cells h ough dys unc ional PNKD may lead o inc easing oxida i e s ess le els, which ha e been linked o in lamma ion (24). TMBIM1 has been epo ed o ha e a ole in egula ing he le el o Fas ligand (25,26), which medi- a es bo h apop osis and in lamma ion (27). The e o e, bo h gene p oduc s indi ec ly con ibu e o he egula ion o in lamma- ion, a physiological p ocess linked wi h he onse o IBD and CRC. Ano he SNP in he 2q35 locus ( s2382817), which is in s ong LD wi h s992157, has p e iously been shown o in luence IBD isk (15). In addi ion, con empo aneous wi h ou analysis, a e- cen s udy (28) has also ound e idence, albei no GWAS signi i- can , o a ela ionship be ween 2q35 a ia ion and CRC isk (P¼7.0 10 5 ), addi ionally inding an in e se ela ionship wi h isk o IBD. The iden i ied SNP, s11676348, is co ela ed wi h bo h s992157 and s2382817 (LD me ics, 2 and D0¼0.32, 0.65 and 0.33, 0.71, espec i ely). The opposing e ec s o he s2382817-C allele wi h inc eased isk o CRC bu dec eased isk o IBD may ini ially appea pa adoxical, gi en he inc eased isk o CRC associa ed wi h IBD. The isk o CRC in IBD inc eases wi h longe du a ion, ex en o coli is and he deg ee o in lam- ma ion (11). The in lamma o y esponse has been linked o in- c eased oxida i e s ess, and his oxida i e s a e s imula es an ioxidan de ences ha p omo e he su i al pa hways in cance cells, a ou ing umou p oli e a ion (29). None heless, hese SNPs may indica e sha ed pa hways in which he e a e opposing ela ionships be ween ca cinogenesis and in lamma ion. Mo i a ed by he obse a ion ha he 2q35 locus in luences IBD isk, we sough addi ional e idence o a common gene ic basis o bo h diseases by e alua ing he CRC isk a p e iously es ablished IBD loci (15,16). While no o mally signi ican glob- ally, he e was an o e - ep esen a ion o associa ion signals o CRC de ined by he IBD isk SNPs. Th ough his analysis we iden i ied po en ial isk a ian s o CRC mapped o egions in he p oximi y o genes encoding WNT4 and CDC42, p e iously shown o be in ol ed in he isk o CRC (14); MAPKAPK5, a mem- be o he MAPK amily epo ed o egula e MYC p o ein le els (18); and he ansc ip ion ac o CEBPB, ound o be highly ex- p essed in samples de i ed om CRC pa ien s (21). Mo eo e , ou eQTL analysis on IBD SNPs showed al e ed exp ession o FADS2 and FUT2 genes in CRC issues. Bo h he genes ha e p e- iously been epo ed o ha e a ole in he de elopmen o IBD (30,31) p o iding u he e idence o possible sha ed genes. Fu he s udies a e equi ed o delinea e he gene ic basis and implica e pe u ba ion o a speci ic gene as he unc ional basis Figu e 3. Quan ile–quan ile (Q–Q) plo o obse ed and expec ed CRC associa ion P- alues o 200 IBD isk SNPs (15, 16). Table 1. Table o he IBD SNPs wi h FDR-co ec ed P- alue <0.05 in he CRC GWAS sID Ch Posi ion Tag genes CRC isk allele IBD isk allele CRC RAF CRC P- alue CRC FDR co ec ed CRC OR CRC 95% CI s12568930 1 22702231 WNT4, CDC42 T T 0.85 6.58 10 05 3.29 10 03 1.12 (1.06; 1.18) s7554511 1 200877562 GPR25, C1o 106 A C 0.29 6.95 10 04 0.02 1.08 (1.03; 1.13) s7608910 2 61204856 PUS10, REL A G 0.63 7.28 10 04 0.02 1.07 (1.03; 1.12) s17229285 2 199523122 PLCL1, SATB2 C C 0.49 2.46 10 03 0.04 1.06 (1.02; 1.1) s2382817 2 219151218 TMBIM1, PNKD C A 0.62 1.02 10 05 1.02 10 03 1.09 (1.05; 1.14) s4722672 7 27231762 HOXA13, HOXA11 C C 0.20 2.46 10 03 0.04 1.08 (1.03; 1.13) s174537 11 61552680 MYRF, TMEM258 G T 0.67 2.63 10 03 0.04 1.06 (1.02; 1.11) s653178 12 112007756 ATXN2, MAPKAPK5 T C 0.54 2.23 10 05 1.49 10 03 1.09 (1.05; 1.13) s17085007 13 27531267 GPR12, UPS12 C C 0.19 5.81 10 04 0.02 1.09 (1.04; 1.15) s941823 13 41013977 MRPS31, FOXO1 T C 0.27 2.47 10 03 0.04 1.07 (1.02; 1.12) s516246 19 49206172 FUT2, MAMSTR T T 0.54 4.71 10 04 0.02 1.07 (1.03; 1.11) s913678 20 48955424 CEBPB, PTPN1 C T 0.34 7.30 10 06 1.02 10 03 1.10 (1.05; 1.14) 2353Human Molecula Gene ics, 2016, Vol. 25, No. 11 | a Tampe e Uni e si y Lib a y. Depa men o Heal h Sciences on Oc obe 31, 2016h p://hmg.ox o djou nals.o g/Downloaded om o he associa ions. Collec i ely hese da a a e consis en wi h a deg ee o commonali y in gene ically de ined pa hways in he de elopmen be ween CRC and IBD, albei ha many o he as- socia ions ha e opposi e e ec s. Conside ing he low p e alence o IBD in Eu opean popu- la ions (<0.5%) (32), oge he wi h he obse a ion ha o he SNPs ha a e s ongly associa ed wi h isk o IBD we e no associa ed wi h CRC, i is unlikely ha sampling has biased ou indings. Mo eo e i he associa ion be ween hese IBD SNPs and CRC was simply media ed by i s associa ion wi h IBD pe se, we would ha e expec ed di ec ionali y o he asso- cia ion o be iden ical bu his was no he case o many o he SNPs. In summa y, we ha e iden i ied a new isk associa ion o CRC which also in luences IBD isk. Ou associa ion signals o CRC de ined by o he es ablished IBD isk SNPs also se e o highligh he impo ance o sha ed gene pa hways in he de el- opmen o CRC and IBD. Deciphe ing he unc ional and biologi- cal basis o hese SNPs associa ions has he po en ial o ansla e in o a be e unde s anding o he biological basis o how IBD ansi ions o CRC. Finally ou analysis se es o illus- a e ha in e - ela ionships be ween diseases do no necessa - ily equa e o consis en allelic a chi ec u e in isk, hus adding an ex a laye o complexi y o in e p e a ion. Ma e ials and Me hods E hics Collec ion o blood samples and clinico-pa hological in o ma- ion om subjec s was unde aken wi h in o med consen and e hical e iew boa d app o al a all si es in acco dance wi h he ene s o he Decla a ion o Helsinki. P ima y GWAS The Finnish GWAS (FIN) was based on 1172 CRC cases and 8266 cance ee con ols asce ained h ough Finnish Hospi als (33) and h ough he Finnish Cance Regis y. Cases we e geno yped using Illumina HumanOmni 2.5M8 1 acco ding o he manu ac- u e ’s ecommenda ions. Fo con ols, we made use o Illumina HumanHap 670k and 610k a ay da a on indi iduals om he FINRISK (34), Heal h 2000 (35), Finnish Twin Coho (36) and Helsinki Bi h Coho S udies (37). Indi iduals we e ex- cluded wi h: <90% success ully geno yped SNPs, disco dan sex in o ma ion, duplica ion o c yp ic ela edness (iden i y by de- scen >0.2). We excluded SNPs om he analysis wi h: call a e <95%, (mino allele equency [MAF]) <0.01 and depa u e om Ha dy–Weinbe g equilib ium in con ols a P<10 6 . The adequacy o he case–con ol ma ching and he possibili y o di e en ial geno yping o cases and con ols we e assessed us- ing quan ile–quan ile (Q–Q) plo s o es s a is ics. Published GWAS o me a-analysis We made use o six p e iously published GWAS: UK1 (CORGI s udy) (7) comp ised 940 cases wi h colo ec al neoplasia and 965 con ols; Sco land1 (COGS s udy) (7) included 1012 CRC cases and 1012 con ols; VQ58 comp ised 1800 CRC cases om he UK-based VICTOR and QUASAR2 adju an chemo he apy clini- cal ials (38) and 2690 popula ion con ol geno ypes om he Wellcome T us Case Con ol Conso ium 2 (WTCCC2) 1958 bi h coho (39); CCFR1 comp ised 1290 amilial CRC cases and 1055 con ols om he Colon Cance Family Regis y (CCFR) (40); CCFR2 included a u he 796 cases om he CCFR and 2236 con- ols om he Cance Gene ic Ma ke s o Suscep ibili y (CGEMS) s udies o b eas and p os a e cance (41,42); and he COIN Figu e 4. Hi e plo o common p o ein–p o ein in e ac ions be ween CRC and IBD de ined by isk SNPs. Each a c ep esen s an in e ac ion be ween wo p o eins, and he dis ance om he cen e o he plo co esponds o a g ea e numbe o p o ein–p o ein in e ac ions (highe deg ee o he node). The le a m ep esen s p o eins ha we e only iden i ied using he CRC SNPs, he igh a m ep esen s p o eins ha we e only iden i ied using he IBD SNPs, and he cen al a m ep esen s he com- mon p o eins, highligh ing he p e iously associa ed ag genes. 2354 |Human Molecula Gene ics, 2016, Vol. 25, No. 11 a Tampe e Uni e si y Lib a y. Depa men o Heal h Sciences on Oc obe 31, 2016h p://hmg.ox o djou nals.o g/Downloaded om GWAS (14) was based on 2244 CRC cases asce ained h ough wo independen Medical Resea ch Council clinical ials o ad- anced/me as a ic CRC (COIN and COIN-B) (43) and con ols comp ised 2162 indi iduals om he UK Blood Se ice Con ol G oup geno yped as pa o he WTCCC2 (39). The VQ58, UK1 and Sco land1 GWAS se ies we e geno yped using Illumina Hap300, Hap240S, Hap370, Hap550 o Omni2.5M a ays. 1958BC geno yping was pe o med as pa o he WTCCC2 s udy on Hap1.2M-Duo Cus om a ays. The CCFR sam- ples we e geno yped using Illumina Hap1M, Hap1M-Duo o Omni-exp ess a ays. CGEMS samples we e geno yped using Illumina Hap300 and Hap240 o Hap550 a ays. The COIN cases we e geno yped using A yme ix Axiom A ays and he Blood Se ice con ols we e geno yped using A yme ix 6.0 a ays. A e applying he same quali y con ol as ha pe o med o FIN, da a on 8749 CRC cases and 18 245 con ols we e a ailable o he me a-analysis. The adequacy o he case–con ol ma ching and possibili y o di e en ial geno yping o cases and con ols we e assessed using Q–Q plo s o es s a is ics. k GC alues (44) o he UK1, Sco land1, VQ58, CCFR1, CCFR2 and COIN s udies we e 1.02, 1.01, 1.01, 1.02, 1.03 and 1.05, espec i ely. Any e hnic ou lie s o indi iduals iden i ied as ela ed we e excluded. Replica ion se ies In o al, 5061 CRC cases om he Na ional S udy o Colo ec al Cance Gene ics (NSCCG) (45) we e geno yped. Con ols (n¼3509) we e om NSCCG and he Gene ic Lung Cance P edisposi ion S udy (46). None o he con ols had a known his- o y o malignancy a asce ainmen . All subjec s we e B i ish esiden s wi h sel - epo ed Eu opean e hnici y and he e we e no ob ious demog aphic di e ences be ween cases and con- ols. DNA was ex ac ed om EDTA- enous blood samples us- ing con en ional me hodologies and PicoG een quan i ied (In i ogen Co po a ion, Ca lsbad, CA, USA). Geno yping o wo SNPs was conduc ed using KASPa compe i i e allele-speci ic PCR chemis y (LGC, Hoddesdon, UK; p ime sequences and condi ions a ailable on eques ). To moni o quali y con ol, du- plica e samples we e included in assays, and conco dance be- ween duplica e samples was >99%. Impu a ion and me a-analysis Analyses we e unde aken using R ( 3.02) (47) and PLINK ( 1.9) (48) so wa e. Phasing o GWAS SNP geno ypes was pe o med us- ing SHAPEIT ( 2. 644 and 2. 790 o FIN) (49). P edic ion o he un yped SNPs was ca ied ou using IMPUTE ( 2.3.1) (50). The FIN da ase used a me ged e e ence panel based on da a om he 1000 Genomes P ojec (Phase 1 3) (51) oge he wi h an addi ional popula ion ma ched e e ence panel o 3882 SISu haplo ypes. The UK samples used a me ged e e ence panel using da a om he 1000 Genomes P ojec and UK10K (Ap il 2014 elease). The ideli y o impu a ion, as assessed by he conco dance be ween impu ed and sequenced SNPs, was examined in a subse o 200 UK cases (14). The associa ion be ween each SNP and he isk o CRC was assessed by a equen is associa ion es unde an addi i e model, using SNPTEST ( 2.5.1) (52), u ilizing he geno ype p oba- bili ies om IMPUTE whe e an SNP was no di ec ly yped. Popula ion s a i ica ion was con olled in he FIN samples using sex and six p incipal componen s. Associa ion me a-analyses only included ma ke s wi h in o sco es >0.8, impu ed call a es/ SNP >0.9 and MAFs >0.005. Me a-analyses we e ca ied ou using META ( 1.6) (53). We calcula ed Coch an’s Qs a is ic o es o he e ogenei y and he I 2 s a is ic o quan i y he p opo ion o he o al a ia ion ha was caused by he e ogenei y (54). I 2 al- ues 75% a e conside ed cha ac e is ic o la ge he e ogenei y (54). Cha ac e iza ion o cance pheno ype Associa ions by sex, age and clinico-pa hological pheno ypes we e examined by logis ic eg ession. MSI s a us was de e mined using BAT25 and BAT26 ma ke s, and samples showing5no el alleles when compa ed wi h no mal DNA a ei he o bo h ma ke s we e assigned as MSI-H (co esponding o MSI-high) (55). Tumou s we e sc eened o KRAS codons 12, 13 and 61 and BRAF codon 600 mu a ions by py osequencing (43). Addi ionally, KRAS (all h ee codons) and BRAF (codons 594 and 600) we e sc eened o mu a ions by MALDI-TOF mass a ay (Sequenom, San Diego, CA, USA) (56). Di e ences be ween he a ious si es o he umou (colonic [ICD-9:153], ec al [ICD-9:154.1] and ec o sigmoid junc- ion [ICD9:154.0]) we e also analysed. Func ional p edic ion To explo e epigene ic p o iles o genomic loca ion associa ed wi h CRC, we used ENCODE his one modi ica ion da a, HaploReg and RegulomeDB (57,58) o examine whe he any o he SNPs o hei p oxies (i.e. 2 >0.8 in he 1000 Genomes EUR e e ence panel) anno a e ansc ip ion ac o binding o enhance ele- men s. Addi ionally, we made use o ChIP-seq da a on he LoVo CRC cell line (59). We used Ch omHMM o in eg a e DNase, H3K4me3, H3K4me1, H3K27ac, Pol2 and CTCF s a es om he CRC cell line HCT116 using a mul i a ia e Hidden Ma ko Model (60). Ch omHMM acks o lymphoblas oid cells we e ob ained om ENCODE (61). We assessed sequence conse a ion using: Phas Cons (62) (>0.3 indica i e o conse a ion) and Genomic E olu iona y Ra e P o iling (63) (>2 indica i e o conse a ion). SNAP plo s we e c ea ed using he isPIG ool (64). eQTL analysis To examine o a ela ionship be ween SNP geno ype and mRNA exp ession in CRC, we analysed Tumo Cance Genome A las (TCGA) RNA-seq exp ession and A yme ix 6.0 SNP da a (dbGaP accession numbe : phs000178. 7.p6) on 416 colo ec al adenoca cinoma samples (65). Associa ion be ween no malized RNA coun s pe -gene and SNP geno ype was quan i ied using he K uskal–Wallis end es . To look o a ela ionship be- ween SNP geno ype and exp ession le els in o he issues, we used publicly a ailable exp ession da a gene a ed om he MuTHER (66), eQTL Blood B owse (67), GTEx (68) and Geu adis/ 1000 Genomes (69) esou ces. Fo he Geu adis da a, he ela- ionship be ween SNPs and exp ession o genes loca ed wi hin 1 Mb was analysed using he Ma ix eQTL (70) package unde a linea model. When he SNPs we e no di ec ly yped, a p oxy SNP was used ( 2 0.8). In all he da ase s, eQTL esul s we e in- cluded whe e FDR P<0.05. Rela ionship be ween es ablished isk SNPs o IBD and CRC To in es iga e pleio opic (sha ed gene ic suscep ibili y) be- ween CRC and IBD, we examined he 201 SNPs iden i ied in GWAS ha ha e been shown o a ec IBD isk (15,16). One SNP ( s71559680) is an indel ha was no p esen in he CRC 2355Human Molecula Gene ics, 2016, Vol. 25, No. 11 | a Tampe e Uni e si y Lib a y. Depa men o Heal h Sciences on Oc obe 31, 2016h p://hmg.ox o djou nals.o g/Downloaded om geno yping a ays o he e e ence panels, and was hus e- mo ed om he analysis. We ob ained he lead SNPs om he IBD GWAS and ex ac ed he P- alues o he co esponding SNPs in ou CRC me a-analysis. Pa hway analysis To in es iga e he possibili y o sha ed gene ic suscep ibili y be- ween CRC and IBD, we pe o med pa hway analysis. Fi s , we selec ed he wo closes coding genes o he leading SNPs in each GWAS and hen pe o med pa hway analysis using LENS ool (22), which iden i ies gene p oduc and p o ein–p o ein in- e ac ions om HPRD (71) and BioGRID (72). En ichmen o pa h- ways was assessed using Fishe ’s exac es , compa ing he o e lap o he genes in he ne wo k wi h he genes in he pa h- way. Pa hway da a we e ob ained om REACTOME (73). Cy oscape was used o pe o m ne wo k analyses (74), and he Hi e Plo was d awn using Hi eR (academic.depauw.edu/ hanson/Hi eR/Hi eR.h ml, las accessed Ma ch 29, 2016). Supplemen a y Ma e ial Supplemen a y Ma e ial is a ailable a HMG online. Acknowledgemen s We would like o hank Ami Sud o his c i ical eading o he manusc ip . We a e g a e ul o all indi iduals who pa icipa ed in he a ious s udies. This s udy made use o geno yping da a om he 1958 Bi h Coho , kindly made a ailable by he Wellcome T us Case Con ol Conso ium 2. A ull lis o he in- es iga o s who con ibu ed o he gene a ion o he da a is a ailable a h p://www.w ccc.o g.uk/, las accessed Ma ch 29, 2016. The esul s published he e a e in whole o pa based upon da a gene a ed by The Cance Genome A las pilo p ojec es ablished by he NCI and NHGRI. In o ma ion abou TCGA and he in es iga o s and ins i u ions ha cons i u e he TCGA e- sea ch ne wo k can be ound a h p://cance genome.nih.go /, las accessed Ma ch 29, 2016. Con lic o In e es s a emen . None decla ed. Funding In Finland, his wo k was suppo ed by g an s om he Academy o Finland [Finnish Cen e o Excellence P og am 2012–2017, 250345], he Jane and Aa os E kko Founda ion, he Finnish Cance Socie y [pe sonal g an o K.P.], he Eu opean Resea ch Council [ERC; 268648], he Sig id Juselius Founda ion, SYSCOL, he No dic In o ma ion o Ac ion eScience Cen e (NIASC), he No dic Cen e o Excellence inanced by No dFo sk [p ojec 62721, pe sonal g an o K.P.] and S a e Resea ch Funding o Kuopio Uni e si y Hospi al [B1401]. We acknowledge he compu a ional esou ces p o ided by he ELIXIR node, hos ed a he CSC–IT Cen e o Science, Finland, and unded by he Academy o Finland [g an s 271642 and 263164], he Minis y o Educa ion and Cul u e, Finland. V.S. was suppo ed by he Finnish Academy [g an numbe 139635]. Sample collec- ion and geno yping in he Finnish Twin Coho has been sup- po ed by he Wellcome T us Sange Ins i u e, ENGAGE— Eu opean Ne wo k o Gene ic and Genomic Epidemiology, FP7- HEALTH-F4-2007 [g an ag eemen numbe 201413], he Na ional Ins i u e o Alcohol Abuse and Alcoholism [g an s AA- 12502 and AA-00145 o Richa d J. Rose and K02AA018755 o Danielle M. Dick] and he Academy o Finland [g an s 100499, 205585, 265240 and 263278 o J.K.]. A he Ins i u e o Cance Resea ch, his wo k was suppo ed by Cance Resea ch UK [C1298/A8362—Bobby Moo e Fund o Cance Resea ch UK]. Addi ional suppo was p o ided by he Na ional Cance Resea ch Ne wo k and he NHS ia he Biological Resea ch Cen e o he Na ional Ins i u e o Heal h Resea ch a he Royal Ma sden Hospi al NHS T us . In Edinbu gh, he wo k was suppo ed by P og amme G an und- ing om Cance Resea ch UK [C348/A12076]. In Ox o d, addi- ional unding was p o ided by he Ox o d Comp ehensi e Biomedical Resea ch Cen e [ o C.P. and I.P.M.T.] and he EU FP7 CHIBCHA g an [I.P.M.T.]. Co e in as uc u e suppo o he Wellcome T us Cen e o Human Gene ics, Ox o d was p o- ided by g an [090532/Z/09/Z]. We a e g a e ul o many col- leagues wi hin UK Clinical Gene ics Depa men s ( o CORGI) and o many collabo a o s who pa icipa ed in he VICTOR and QUASAR2 ials. We also hank colleagues om he UK Na ional Cance Resea ch Ne wo k ( o NSCCG). Suppo om he Eu opean Union [FP7/207-2013, g an 258236] and FP7 collabo a- i e p ojec SYSCOL and COST Ac ion in he UK is also acknowl- edged [BM1206]. The COIN and COIN-B ials we e unded by Cance Resea ch UK and he Medical Resea ch Council and we e conduc ed wi h he suppo o he Na ional Ins i u e o Heal h Resea ch Cance Resea ch Ne wo k. COIN and COIN-B ansla- ional s udies we e suppo ed by he Bobby Moo e Fund om Cance Resea ch UK, Teno us, he Kidani T us , Cance Resea ch Wales and he Na ional Ins i u e o Social Ca e and Heal h Resea ch Cance Gene ics Biomedical Resea ch Uni (2011–2014) [all awa ded o J.P.C.]. N.A.A., B.F.M. and S.M.W. we e unded and suppo ed by KFSHRC. The wo k o he Colon Cance Family Regis y (CCFR) was sup- po ed by om he Na ional Cance Ins i u e [g an UM1 CA167551], Na ional Ins i u es o Heal h and h ough coope a- i e ag eemen s wi h he ollowing CCFR cen es: Aus alasian Colo ec al Cance Family Regis y [U01 CA074778, U01/U24 CA097735], USC Conso ium Colo ec al Cance Family Regis y [U01/U24 CA074799], Mayo Clinic Coope a i e Familial Regis y o Colon Cance S udies [U01/U24 CA074800], On a io Familial Colo ec al Cance Regis y [U01/U24 CA074783], Sea le Colo ec al Cance Family Regis y [U01/U24 CA074794] and Uni e si y o Hawaii Colo ec al Cance Family Regis y [U01/U24 CA074806]. The CCFR Illumina GWAS was suppo ed by unding om he Na ional Cance Ins i u e, Na ional Ins i u es o Heal h [U01 CA122839 and R01 CA143237 o G.C.]. Sea le CCFR esea ch was also suppo ed by he Cance Su eillance Sys em o he F ed Hu chinson Cance Resea ch Cen e [Con ol Nos. N01-CN-67009 (1996–2003), N01-PC-35142 (2003–2010)] and he Su eillance, Epidemiology and End Resul s (SEER) P og am o he Na ional Cance Ins i u e [Con ac No. HHSN2612013000121 (2010–2017)] wi h addi ional suppo om he F ed Hu chinson Cance Resea ch Cen e . The collec ion o cance incidence da a o he S a e o Hawai’i used in his s udy was suppo ed by he Hawai‘i Depa men o Heal h as pa o he s a ewide cance epo ing p og am manda ed by Hawai‘i Re ised S a u es; he Na ional Cance Ins i u e’s Su eillance, Epidemiology and End Resul s P og am (SEER) awa ded o he Uni e si y o Hawai‘i [Con ol Nos. N01-PC-67001 (1996–2003), N01-PC-35137 (2003–2010), Con ac Nos. HHSN26120100037C (2010–2013), HHSN261201300009I (2010–cu en )]. The ideas and opinions ex- p essed he ein a e hose o he au ho (s) and endo semen by he S a e o Hawai‘i, Depa men o Heal h, he Na ional Cance Ins i u e, SEER P og am o hei Con ac o s and Subcon ac o s 2356 |Human Molecula Gene ics, 2016, Vol. 25, No. 11 a Tampe e Uni e si y Lib a y. Depa men o Heal h Sciences on Oc obe 31, 2016h p://hmg.ox o djou nals.o g/Downloaded om is no in ended no should be in e ed. The collec ion o cance incidence da a used in his s udy was suppo ed by he Cali o nia Depa men o Public Heal h as pa o he s a ewide cance e- po ing p og am manda ed by Cali o nia Heal h and Sa e y Code Sec ion 103885; he Na ional Cance Ins i u e’s Su eillance, Epidemiology and End Resul s P og am [unde con ac HHSN261201000035C] awa ded o he Uni e si y o Sou he n Cali o nia, and [con ac HHSN261201000034C] awa ded o he Public Heal h Ins i u e; and he Cen e s o Disease Con ol and P e en ion’s Na ional P og am o Cance Regis ies, unde ag ee- men [U58DP003862-01] awa ded o he Cali o nia Depa men o Public Heal h. The ideas and opinions exp essed he ein a e hose o he au ho (s) and endo semen by he S a e o Cali o nia, Depa men o Public Heal h he Na ional Cance Ins i u e, and he Cen e s o Disease Con ol and P e en ion o hei Con ac o s and Subcon ac o s is no in ended no should be in- e ed. The con en o his manusc ip does no necessa ily e- lec he iews o policies o he Na ional Cance Ins i u e o any o he collabo a ing cen es in he CCFR, no does men ion o ade names, comme cial p oduc s, o o ganiza ions imply en- do semen by he US Go e nmen o he CCFR. Funding o pay he Open Access publica ion cha ges o his a icle was p o ided by COAF: Cha i y Open Access Fund. Re e ences 1. Jiao, S., Pe e s, U., Be nd , S., B enne , H., Bu e bach, K., Caan, B.J., Ca lson, C.S., Chan, A.T., Chang-Claude, J., Chanock, S. e al. (2014) Es ima ing he he i abili y o colo- ec al cance . Hum. Mol. Gene ., 23, 3898–3905. 2. Pe e s, U., Jiao, S., Schumache , F.R., Hu e , C.M., A agaki, A.K., Ba on, J.A., Be nd , S.I., Bezieau, S., B enne , H., Bu e bach, K. e al. (2013) Iden i ica ion o gene ic suscep i- bili y loci o colo ec al umo s in a genome-wide me a- analysis. Gas oen e ology,144, 799–807. e724. 3. Whi in, N. and Houls on, R.S. 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