Germline EMSY sequence alterations in hereditary breast cancer and ovarian cancer families

RESEARCH ARTICLE Open Access Ge mline EMSY sequence al e a ions in he edi a y b eas cance and o a ian cance amilies Ki si M. Mää ä 1,2† , Riikka Nu minen 1,2† , Minna Kanku i-Tammileh o 3 , Anne Kallioniemi 1 , Sa u-Leena Laasanen 4,5 and Johanna Schleu ke 1,2,6,7* Abs ac Backg ound: BRCA1 and BRCA2 mu a ions explain app oxima ely one- i h o he inhe i ed suscep ibili y in high- isk Finnish he edi a y b eas and o a ian cance (HBOC) amilies. EMSY is loca ed in he b eas cance - associa ed ch omosomal egion 11q13. The EMSY gene encodes a BRCA2-in e ac ing p o ein ha has been implica ed in DNA damage epai and genomic ins abili y. We analysed he ole o ge mline EMSY a ia ion in b eas /o a ian cance p edisposi ion. The p esen s udy desc ibes he i s EMSY sc eening in pa ien s wi h high amilial isk o his disease. Me hods: Index indi iduals om 71 high- isk, BRCA1/2-nega i e HBOC amilies we e sc eened o ge mline EMSY sequence al e a ions in p o ein coding egions and exon-in on bounda ies using Sange sequencing and TaqMan assays. The iden i ied a ian s we e u he sc eened in 36 Finnish HBOC pa ien s and 904 con ols. Mo eo e , one no el in onic dele ion was sc eened in a coho o 404 b eas cance pa ien s unselec ed o amily his o y. Haplo ype block s uc u e and he associa ion o haplo ypes wi h b eas /o a ian cance we e analysed using Haplo iew. The unc ionali y o he iden i ied a ian s was p edic ed using Haplo eg, RegulomeDB, Human Splicing Finde , and Pa hogenic-o -No -Pipeline 2. Resul s: Al oge he , 12 ge mline EMSY a ian s we e obse ed. Two al e a ions we e loca ed in he coding egion, i e al e a ions we e in onic, and i e al e a ions we e loca ed in he 3'un ansla ed egion (UTR). Va ian equencies did no signi ican ly di e be ween cases and con ols. The no el a ian , c.2709 + 122delT, was de ec ed in 1 ou o 107 (0.9%) b eas cance pa ien s, and he ca ie showed a bila e al o m o he disease. The dele ion was absen in 897 con ols (OR = 25.28; P= 0.1) and in 404 b eas cance pa ien s unselec ed o amily his o y. No haplo ype was iden i ied o inc ease he isk o b eas /o a ian cance . Func ional analyses sugges ed ha a ian s, pa icula ly in he 3'UTR, we e loca ed wi hin egula o y elemen s. The no el dele ion was p edic ed o a ec splicing egula o y elemen s. Conclusions: These esul s sugges ha he iden i ied EMSY a ian s a e likely neu al a he popula ion le el. Howe e , hese a ian s may con ibu e o b eas /o a ian cance isk in single amilies. Addi ional analyses a e wa an ed o a e no el in onic dele ions and he 3'UTR a ian s p edic ed o ha e unc ional oles. Keywo ds: B eas cance , O a ian cance , Ge mline, EMSY * Co espondence: [email p o ec ed] † Equal con ibu o s 1 Ins i u e o Biosciences and Medical Technology - BioMediTech, Uni e si y o Tampe e, Lääkä inka u 1, FI-33520 Tampe e, Finland 2 Fimlab Labo a o ies, Tampe e Uni e si y Hospi al, Bioka u 4, FI-33520 Tampe e, Finland Full lis o au ho in o ma ion is a ailable a he end o he a icle © The Au ho (s). 2017 Open Access This a icle is dis ibu ed unde he e ms o he C ea i e Commons A ibu ion 4.0 In e na ional License (h p://c ea i ecommons.o g/licenses/by/4.0/), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided you gi e app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o he C ea i e Commons license, and indica e i changes we e made. The C ea i e Commons Public Domain Dedica ion wai e (h p://c ea i ecommons.o g/publicdomain/ze o/1.0/) applies o he da a made a ailable in his a icle, unless o he wise s a ed. Mää ä e al. BMC Cance (2017) 17:496 DOI 10.1186/s12885-017-3488-x Backg ound B eas cance (BC) is he mos common cance among women in Wes e n coun ies. In Finland, 4694 new BC cases we e diagnosed in 2012 (Finnish Cance Regis y). O a ian cance (OC) is he mos le hal gynaecologic ma- lignancy in de eloped coun ies [1]. In 2012, 466 new OC cases we e diagnosed, making OC he en h mos common cance among Finnish women (Finnish Cance Regis y). The gene ic p edisposi ion o bo h o hese diseases has been well ecognized. App oxima ely 5– 10% o all o b eas and o a ian cance cases e lec inhe i ed gene ic de ec s p ima ily in wo well-known high-pene ance b eas and o a ian cance genes, BRCA1 (b eas cance 1, ea ly onse ) and BRCA2 (b eas cance 2, ea ly onse ) [2–4]. Bo h o hese genes encode la ge p o eins ha play c i ical oles in he DNA epai pa hway ( e iewed in [5]). Mu a ions in BRCA1 and BRCA2 p edispose o he edi a y b eas and o a ian can- ce (HBOC) synd ome cha ac e ized by mul iple amily membe s a ec ed wi h b eas o o a ian cance o bo h, ea ly onse o BC, bila e al o m o cance , and he ap- pea ance o o he cance s in amily membe s, including p os a e, panc ea ic and male BC [6]. Among Finnish HBOC amilies, BRCA1/2 mu a ions a e explain ap- p oxima ely 20% [7, 8]. Addi ionally, a low p opo ion o addi ional HBOC p edisposi ion in he Finnish amilies e lec s de ec s in o he DNA epai pa hway genes, including CHEK2 (Checkpoin kinase 2), PALB2 (Pa ne and localize o BRCA2), RAD51C (RAD51 (S. ce e isiae) Homologue C), and Ab axas [9–12]. Ne e heless, addi ional HBOC p edisposing gene ic ac o s emains unknown, and one po en ial app oach is o sc een he candida e genes o p o eins ha in e ac wi h ei he BRCA1 o BRCA2 in he DNA epai pa hway. One o he in e ac ing p o eins o BRCA2 is EMSY (C11o 30) [13]. EMSY is loca ed a ch omosomal egion 11q13 (11q13.5), which is associa ed wi h BC, pa icu- la ly he ho mone ecep o -posi i e o m o he disease [14]. EMSY encodes a p o ein wi h an e olu iona y con- se ed EMSY N- e minal domain ha is unique in he human genome [13]. The EMSY N- e minal domain has a s uc u e simila o he DNA-binding mo i ound in ansc ip ion ac o s [15]. EMSY has been implica ed in DNA epai and an- sc ip ional egula ion. The in e ac ion wi h BRCA2 co e- la es EMSY o DNA epai wi h he obse a ion ha EMSY localizes a DNA damage si es [13]. In addi ion, he o e -exp ession o a unca ed o m o EMSY esul s in a ch omosome ins abili y pheno ype in human mamma y epi helial cells, simila o ha o cells showing a loss o BRCA2 unc ion [16]. The binding o EMSY o he BRCA2 exon 3-encoding ansc ip ional ac i a ion do- main ep esses he unc ion o his domain [13]. In addi ion, EMSY in e ac s wi h he ch oma in emodelling p o eins He e och oma in p o ein 1 β(HP1 β)and BS69 [13]. EMSY has been implica ed in he egula- ion o nuclea ecep o -media ed ansc ip ion [17], he ansc ip ion o in e e on-s imula ed genes wi h BRCA2 [18], and he ansc ip ion o an ime as a ic mic oRNA miR-31 [19]. EMSY is ampli ied in b eas umou s [13, 20–23], and his ampli ica ion is associa ed wi h he poo ou come o BC [13, 20–22]. EMSY copy numbe changes ha e also been obse ed in male b eas umou s, bu he copy numbe gains a e compa a i ely mo e equen in emale b eas umou s [24]. In addi ion o b eas cance , EMSY is o e exp essed in high-g ade o a ian cance [13, 25, 26] and panc ea ic cance [27]. P e iously, we examined he associa ion o EMSY single-nucleo ide polymo phisms (SNPs) in ela ion o p os a e cance p edisposi ion and iden i ied a a e in onic SNP ha inc eases he isk o agg essi e p os a e cance [28]. In he p esen s udy, we iden i ied ge mline sequence al e a ions in he EMSY gene, encoding a BRCA2- in e ac ing p o ein pa ne , which could con ibu e o HBOC suscep ibili y by dis up ing c i ical unc ions in he DNA epai pa hway. Thus, we sequenced he EMSY coding egion and exon-in on bounda ies in a coho o 71 Finnish BRCA1/2-nega i e HBOC pa ien s p e- sc eened o mu a ions in se en known b eas cance genes and copy-numbe al e a ions a he genome-wide scale [29, 30] and u he analysed he iden i ied a ian s in addi ional HBOC pa ien s and heal hy con ols. To ou knowledge, he p esen s udy is he i s o analyse ge mline EMSY al e a ions in suscep ibili y o HBOC in high- isk amilies. Me hods Pa ien s and con ols Ge mline EMSY sequence al e a ions we e sc eened in index pa ien s om 71 high- isk Finnish HBOC amilies. S udy ma e ial was collec ed om he Tampe e Uni e si y Hospi al Gene ics Ou pa ien Clinic be ween Janua y 1997 and May 2008. Pa ien s belonging o a coho o 82 high- isk HBOC indi iduals p e iously well cha ac e ized and sc eened o ge mline al e a ions in BRCA1, BRCA2, CHEK2, PALB2, BRIP1, RAD50, and CDH1 genes and ge mline copy numbe al e a ions a he genome-wide scale [29, 30]. F om he p e iously desc ibed coho , we included only he 71 a ec ed index indi iduals in he p esen s udy, including 57 emales wi h BC, 8 emales wi h bila e al BC, 1 emale wi h OC, and 5 emales wi h bo h BC and OC. O iginally, all pa ien s we e de e mined as nega i e o 28 Finnish BRCA1/2 ounde -mu a ions based on minisequencing and p o ein unca ion es s (PTTs) o BRCA1 exon 11 and BRCA2 exons 10 and 11. Addi ionally, he exons and exon-in on bounda ies o BRCA1 and BRCA2 ha e p e iously been analysed using Mää ä e al. BMC Cance (2017) 17:496 Page 2 o 8 Sange sequencing and Mul iplex Liga ion-dependen P obe ampli ica ion (MLPA) o exclude o he mu a ions [29]. All o he iden i ied EMSY sequence al e a ions we e sc eened om he DNA samples o 36 addi ional index pa ien s om HBOC amilies collec ed om he Tu ku egion and om anonymous heal hy emale blood dono s (n= 380–904) ( e e ed as con ols) ob- ained om he Finnish Red C oss. Addi ionally, a a e no el c.2709 + 122delT a ian was u he sc eened in a coho o 404 BC cases unselec ed o amily his o y om he Tampe e egion [31]. All pa- ien s we e in o med o he analyses and p o ided w i en consen o he use o exis ing DNA samples in he p esen s udy. The E hical Commi ees o Tampe e and Tu ku Uni e si y Hospi als and he Na ional Au ho i y o Medicolegal A ai s app o ed his esea ch p ojec . Sample p epa a ion and mu a ion sc eening Genomic DNA om con ol samples was ex ac ed om pe iphe al leukocy es using he Pu egene Ki acco ding o he manu ac u e ’sins uc ions(Gen aSys ems, Inc.,Minneapolis,MN,USA).Mu a ionsc eeningwas achie ed h ough Sange sequencing. Re e ence se- quence NM_020193.3 was ob ained om he USCS genome b owse [32, 33]. The genome build GRCh37 (hg19) was used. Whole coding egions and exon-in on bounda ies we e analysed. EMSY p ime sequences and PCR condi ions a e a ailable upon eques . Sequencing was pe o med using he Big Dye Te mina o .3.1 Cycle Sequencing Ki and he ABIPRISM 3130xl Gene ic Analyse (Applied Biosys ems, Fos e Ci y, CA, USA). The sequences we e analysed using Sequenche .5.1 so wa e (Gene Codes Co po a ion, Ann A bo , MI, USA). The Re SNP numbe o he iden i ied a ian s was ob ained om he NCBI Single Nucleo ide Polymo phism da abase (dbSNP) [34]. Two a ian s, s1044265 and s2513513, we e analysed om con ols using TaqMan SNP Geno yp- ing Assays acco ding o he manu ac u e ’s ins uc ions wi h he ABI P ism® 7900HT ins umen and SDS2.2.2 so wa e (Applied Biosys ems). The geno yping call a es o he SNPs we e ≥0.98. S a is ical and bioin o ma ics analyses Obse ed a ian s we e es ed o Ha dy-Weinbe g equi- lib ium in he con ols. The associa ion o a ian s wi h b eas /o a ian cance was examined using Fishe ’s exac es . P alues we e wo-sided, and P< 0.05 was consid- e ed s a is ically signi ican . The associa ion o he mino allele was examined. Odds a ios (OR) and con idence in e als (CI) we e calcula ed using PLINK 1.07 [35]. I he a ian was no obse ed in cases o con ols, hen he OR was calcula ed using G aphPad P ism e sion 5.02 o Windows (G aphPad So wa e, San Diego, CA, USA) by adding 0.5 o each alue o ob ain nume ic alues. Linkage disequilib ia (LD) be ween a ian s, haplo- ype blocks and he associa ion o haplo ypes wi h b eas / o a ian cance we e analysed using Haplo iew 4.2 [36]. Haplo ype blocks we e de ined using 107 cases and 380 con ols ia Gab iel’s me hod, which includes by de aul SNPs wi h mino allele equencies (MAFs) > 0.05 [37]. The e ec o he amino-acid changing a ian , c.2861 T > G (Leu954A g), was p edic ed using he Pa hogenic-o -No - Pipeline (PON-P2) p og amme[38].The unc ionali yo heobse edSNPswi h sIDswasanalysedusing HaploReg 2 [39], including conse ed egion, open ch o- ma in, egula o y ch oma in s a e om he Encyclopaedia o DNA Elemen s (ENCODE) [40], p o ein binding, and al- e ed mo i s. The ou al e na i e posi ions o he dele ion c.2709 + 122delT (ch 11 76,253,530–76,253,533) we e analysed o unc ional elemen s using RegulomeDB [41], which includes da a o p edic ed and known egula o y elemen s, such as egions o DNA hype - sensi i i y, binding si es o ansc ip ion ac o s, and egions wi h enhance ac i i y. The e ec o he dele ion c.2709 + 122delT on splicing was analysed using Human Splicing Finde (HSF) 2.4.1 [42]. The al e na i e dele ion loca ions we e analysed using HSF o de ec po en ial splice si es (HSF ma ices), po en ial b anch poin s, enhance mo i s (ESE Finde ) and hnRNP mo i s (expe imen al) o SR p o eins. Resul s In he p esen s udy, we iden i ied 12 di e en ge mline EMSY sequence al e a ions in index pa ien s om 71 HBOC amilies, and he al e a ions we e u he sc eened in a coho o 36 HBOC pa ien s and heal hy con ols. Va ian equencies a e p esen ed in Table 1. Two o he iden i ied al e a ions we e loca ed in he p o ein-coding egion, i e al e a ions we e in onic, and i e a ian s we e loca ed in he 3′UTR. Wi h he excep ion o one al- e a ion, c.2709 + 122delT, all o he iden i ied a ian s a e epo ed in he NCBI Single Nucleo ide Polymo phism (SNP) da abase wi h s-numbe s (Table 1). All SNPs we e in Ha dy Weinbe g equilib ium in con ols. The allele equencies did no signi ican ly di e be ween cases and con ols (Table 1). One o he SNPs, s2508740, was iallelic. In e es ingly, he T allele, ob- se ed in one con ol sample, has no p e iously been e- po ed in he SNP da abase. Howe e , his con ol sample was no included in he associa ion es ing o he SNP. One o he wo a ian s obse ed in he p o ein-coding e- gion s184345272 esul ed in an amino acid subs i u ion o a hyd ophobic leucine wi h a posi i ely cha ged a gin- ine a posi ion 954. The he e ozygous s184345272 a ian was iden i ied in wo pa ien s (2 o 106, 1.9%) and h ee con ols (3 o 376, 0.8%) (OR = 2.40; P= 0.3). PON-P2 p edic s he unknown e ec s o subs i u ions. Mää ä e al. BMC Cance (2017) 17:496 Page 3 o 8 The mos in e es ing inding was he a e no el se- quence al e a ion, c.2709 + 122delT (Fig. 1). The p ecise loca ion o he dele ed T could no be de e mined based on he sequence because o a s e ch o ou T bases in he e e ence sequence (ch 11 76,253,530–76,253,533). This dele ion has been named acco ding o he Human Genome Va ia ion Socie y (HGVS) nomencla u e (h p:// a nomen.hg s.o g, .15.11). The he e ozygous dele ion was iden i ied in one pa ien (1 o 107, 0.9%) wi h bila e al BC diagnosed a 39 and 42 yea s o age. Howe e , none o he con ols (0 o 897, 0%) had his dele ion (OR = 25.28; P= 0.1) (Table 1), and i was no obse ed in a coho o 404 BC cases unselec ed o amily his o y (da a no shown). The dele ion ca ie pa ien died o BC a age 52. The clinical ea u es o he pa ien included in asi e duc al ype g ade 1 umou in he le b eas and in asi e duc al ype g ade 2 umou in he igh b eas . Bo h he umou s had oes ogen and p oges e one ecep o posi i e and human epide mal g ow h ac o ecep o 2 nega i e s a uses. The pa ien ’smo he hadBCdi- agnosed a age 51, and he a he had panc ea ic can- ce diagnosed a age 64. The pa ien had one heal hy b o he . Addi ionally, he dele ion ca ie pa ien had ou o he he e ozygous EMSY a ian s, s42445443, s2508740, s2513513, and s1044265. Based on haplo ype block analysis, se en obse ed a - ian s ( s4245443, s2508740, s11363199, s3753051, s2513513, s187735484 and s1044265) o med a haplo- ype block (Fig. 2). A o al o six haplo ypes we e ob- se ed. The haplo ype AAATAAA was mo e common in con ols (2.6%) han cases (0.5%) wi h a bo de line Table 1 Iden i ied EMSY sequence al e a ions Nucleo ide change Amino acid change Posi ion s numbe Geno ype dis ibu ion n(%) a P- alue OR; 95% CI Cases Con ols c.1108 + 40A > G −76,183,924 s4245443 16/49/42 (15.0/45.8/39.3) 54/182/137 (14.5/48.8/36.7) 0.8 0.96; 0.70–1.31 c.1514-4G > A −76,227,182 s2508740 15/50/42 (14.0/46.7/39.3) 44/178/149 (11.9/48.0/40.1) 0.7 1.07; 0.78–1.46 c.1685-14C > T −76,234,185 s11600501 103/4/0 (96.3/3.7/0) 366/11/0 (97.1/2.9/0) 0.8 1.29; 0.41–4.08 c.1995 + 47delA −76,237,726 s11363199 49/44/14 (45.8/41.1/13.1) 168/168/41 (44.6/44.6/10.8) 0.9 1.02; 0.74–1.41 c.2709 + 122delT −76,253,530– 76,253,533 −106/1/0 (99.1/0.9/0) 897/0/0 (100/0/0) 0.1 25.28; 1.02–625.0 c.2861 T > G p.Leu954A g 76,255,454 s184345272 104/2/0 (98.1/1.9/0) 376/3/0 (99.2/0.8/0) 0.3 2.40; 0.40–14.44 c.3648 T > C p.Th 1216Th 76,257,215 s3753051 49/44/14 (45.8/41.1/13.1) 166/165/41/ (44.6/44.4/11.0) 0.9 1.02; 0.74–1.41 c.*343A > G −76,261,533 s2513513 16/45/46 (15.0/42.1/43.0) 55/186/139 (14.5/48.9/36.6) 0.5 0.88; 0.64–1.21 c.*631C > G −76,261,821 s148932730 105/2/0 (98.1/1.9/0) 370/5/0 (98.7/1.3/0) 0.7 1.41; 0.27–7.30 c.*744A > C −76,261,934 s187735484 98/9/0 (91.6/8.4/0) 331/45/0 (88.0/12.0/0) 0.4 0.69; 0.33–1.44 c.*753G > C −76,261,943 s72932407 99/8/0 (92.5/7.5/0) 351/27/0 (92.9/7.1/0) 0.8 1.05; 0.47–2.34 c.*938A > G b −76,262,128 s1044265 38/51/18 (35.5/47.7/16.8) 351/417/128 (39.2/46.5/14.3) 0.4 1.14; 0.85–1.52 CI con idence in e al, OR odds a io a Geno ype dis ibu ion deno es homozygo es/he e ozygo es/homozygo es in he o de indica ed in he nucleo ide change column. In he case o dele ion, geno ype dis ibu ion deno es no dele ion/he e ozygous dele ion/homozygous dele ion b Due o he Ha dy-Weinbe g disequilib ium in 380 con ols, he a ian was geno yped in a la ge numbe o con ols *3'UTR a ian , i.e. he a ian is downs eam (3') o he ansla ion e mina ion si e (h p:// a nomen.hg s.o g/bg-ma e ial/numbe ing/) a b Fig. 1 The no el ge mline sequence al e a ion c.2709 + 122delT in EMSY.aCon ol sample sequence wi hou he dele ion. bHe e ozygous dele ion in a BC pa ien sample. The p ecise posi ion o he dele ed T is unce ain ( he egion whe e he T has been dele ed is unde lined in black in bo h aand b) Mää ä e al. BMC Cance (2017) 17:496 Page 4 o 8 signi icance di e ence (P= 0.05). The SNP s2508740 was in high LD ( 2 ≥0.8) wi h s3753051, s11363199, s2513513, and s4245443 (Fig. 2). In addi ion, high LD was obse ed o SNPs s4245443 and s2513513 and SNPs s11363199 and s3753051 (Fig. 2). The unc ionali y o he iden i ied SNPs wi h s IDs (all bu c.2709 + 122delT) was analysed in silico using Hap- loReg .2 [39] (Table 2). Some di e ences we e obse ed in conse ed egions be ween GERP and SiPhy ap- p oaches, bu he conse ed egions p ima ily o e lapped wi h he SNPs loca ed in exonic and 3′UTR egions. The 3′UTR SNPs we e loca ed wi hin open ch oma in in se e al cell lines and coincided wi h he ch oma in s a es o s ong enhance s. In addi ion, wo in onic a ian s ( s11600501 and s11363199) we e loca ed wi hin ch o- ma in s a es o weak enhance s. Nine o he 11 analysed a ian s we e p edic ed o a ec egula o y mo i s. None o he a ian s we e loca ed wi hin p o ein binding si es. The unc ionali y o he c.2709 + 122delT a ian was examined using he Regulome DB [41], and he ou al e na i e loca ions o he dele ion coincided wi h he same unc ional elemen s, including he open ch oma in and ansc ip ion ac o binding si e o GATA6 (Addi ional ile 1: Table S1). In addi ion, he ou -base loca ion coincides wi h h ee egula ion mo i posi ions. The e ec o c.2709 + 122delT on splicing egula o y elemen s was analysed using Human Splicing Finde [42] (Addi ional ile 1: Table S2), which indica es bo h he in oduc ion and dele ion o an accep o splice si e as a esul o he dele ion o ei he one o he al e na i e nucleo ides. The dele ion o he hi d o ou h nucleo- ide in oduced and abolished a po en ial b anch poin . No enhance mo i s o SR p o eins o silence mo i s o hnRNP we e a ec ed by his dele ion. Discussion Gene ic ac o s p edisposing o b eas and o a ian cance p ima ily emain unknown in HBOC amilies nega i e o BRCA1 and BRCA2 mu a ions. He e, we sc eened he EMSY gene, encoding he BRCA2-in e ac ing p o ein, o ge mline sequence al e a ions and analysed he associa ion o he obse ed a ian s wi h b eas /o a ian cance isk. We u ilized a coho o index indi iduals om 71 high- isk BRCA1/2-nega i e HBOC amilies p e iously sc eened o ge mline al e a ions in se en known BC genes and copy numbe al e a ions a he genome-wide scale [29, 30]. Acco ding o p e ious analyses, no known p edisposing a ian s ha e been iden i ied in a majo i y (87%) o he sc eened high- isk amilies [29, 30], indica ing he exis ence o ye unknown gene a ian s con ibu ing o b eas /o a - ian cance suscep ibili y. To ou knowledge, his s udy is he i s o sc een he EMSY gene o ge mline a ia ions in ela ion o b eas /o a ian cance in high- isk amilies. We iden i ied 12 di e en a ian s in he coding e- gions and exon-in on bounda ies, bu none o hese a ian s showed a s a is ically signi ican associa ion wi h b eas /o a ian cance isk a he popula ion le el, which may e lec he limi ed sample size in he p esen s udy. Mo eo e , haplo ype analysis iden i ied one haplo ype as mo e common in con ols compa ed o cases bu wi h bo de line signi icance di e ence. Howe e , he iden i- ied a ian s, pa icula ly he a e no el dele ion, c.2709 + 122delT, could be impo an p edisposing ac- o s in indi idual pa ien s and in a ew cance amilies. In e es ingly, he dele ion ca ie pa ien was a ec ed wi h bila e al BC, which may indica e ha his dele ion con ibu es o a poo clinical ou come o he disease. The dele ion ca ie pa ien did no ha e dele e ious mu- a ions o copy numbe changes in he p e iously sc eened BC genes, BRCA1,BRCA2,CHEK2,PALB2, BRIP1,RAD50, and CDH1 [29, 30]. Un o una ely, we did no ob ain blood samples om he ela i es o he dele ion ca ie pa ien o examine he seg ega ion o he dele ion wi h he disease. Al hough his dele ion is loca ed in an in onic egion, i may ep esen a unc- ional a ian , acco ding o he esul s o he in silico unc ional analyses. Thus, u he s udies o his dele ion a e wa an ed. O no e, du ing he e iew p ocess o his manusc ip , he dele ion was published in he dbSNP and ecei ed e e ence SNP id numbe 983125332. Only one missense a ian ( s184345272) was iden i- ied in he p o ein-coding egion o EMSY, sugges ing ha mu a ions a e ei he a e o no ole a ed. The Fig. 2 The haplo ype block s uc u e. Haplo ype analysis included he geno ypes o he obse ed a ian s om 107 b eas and/o o a ian cance cases and 380 con ols. The i s al e na i e ma ke posi ion was used o c.2079 + 122delT. Linkage disequilib ium (LD) alues ( 2 × 100) a e ep esen ed in he cha in shades o black. The igu e was ob ained using Haplo iew [36] Mää ä e al. BMC Cance (2017) 17:496 Page 5 o 8 s184345272 a ian , p edic ed o ha e unknown e ec on p o ein unc ion, was 2.4 imes mo e common in cases compa ed o con ols, bu ob iously a la ge num- be o samples should be sc eened o de e mine whe he his a ian could be a low- isk allele. In e es ingly, i e o he 12 (41.6%) al e a ions occu ed in he 3′UTR and we e p edic ed o play unc ional oles. Fu he sc een- ing o hese a ian s would be in e es ing, pa icula ly o he s148932730 a ian , which was de ec ed as 1.4 imes mo e common in cases s. con ols. No unca ing mu a ions, such as ameshi o non- sense mu a ions, we e de ec ed. Because EMSY is ampli- ied o o e -exp essed in b eas and o a ian cance umou s [13, 20–23, 25, 26], p edisposing ge mline al e - a ions a e expec ed o esul in he gain-o - unc ion a he han he loss-o - unc ion o EMSY. Based on he in silico unc ional anno a ion, ei he o he al e na i es can be uled ou , since, o example, he po en ial e ec o he c.2709 + 122delT dele ion on splicing migh a ec he unc ion o EMSY. Un o una ely, umou DNA was no a ailable om he dele ion pa ien o addi ional analysis, o example, loss o he e ozygosi y (LOH). The e o e, u he unc ional s udies a e needed o cla i y hese ind- ings and cha ac e ize he e ec s o he a ian s on he unc ions o o he genes h ough gene egula ion. Common EMSY a ia ions associa ed wi h b eas and o a ian cance isks ha e p e iously been examined in B i ish popula ion-based s udies [43]. Th ee ou o he six SNPs obse ed in he B i ish s udy [43] ( s4245443, s2508740, and s11600501) we e also iden i ied in he coho in he p esen s udy. We did no obse e an asso- cia ion o hese SNPs wi h b eas /o a ian cance isk, consis en wi h p e ious esul s [43]. We p e iously examined he associa ion o EMSY SNPs wi h p os a e cance p edisposi ion and iden i ied a a e in onic SNP ha inc eases he isk o agg essi e p os a e cance ( e e ed o as agg essi e SNP) [28]. In e es ingly, he agg essi e SNP was seg ega ed wi h b eas cance in a p os a e cance amily [28]. Howe e , in he p esen s udy, we did no de ec he agg essi e SNP. Since we only sc eened one indi idual pe b eas /o a ian cance amily, we canno ule ou ha he o he a ec ed amily membe s in he examined coho could be ca ie s o he p e iously de ec ed agg essi e SNP. Table 2 Func ional anno a ion o he iden i ied EMSY SNPs wi h s IDs SNP Loca ion Conse ed egion Open ch oma in a ; Regula o y ch oma in s a e a ; P o ein binding Mo i s changed GERP SiPhy Cell ID b (Cell ID b ) s4245443 in on No No –– –Pou1 1_2, YY1_known6 s2508740 in on No Yes –– –– s11600501 in on Yes Yes –7_Weak_Enhance (HepG2) –EWSR1-FLI1, GATA_known8, TATA_disc7 s11363199 in on No No –7_Weak_Enhance (HepG2) –Foxa_known4, Pou1 1_2, Pou2 2_known2, Pou3 2_2, Sox_5 s184345272 exon Yes Yes –– –NRSF_known3 s3753051 exon Yes Yes HeLa-S3 ––CEBPB_disc2, Foxa_known2 s2513513 3′UTR Yes Yes HA-sp., HCPEpiC, 5_S ong_Enhance (Hu ec) –– HMVEC-dBl-Neo s148932730 3′UTR Yes Yes –4_S ong_Enhance (Hu ec) –DMRT2, Nanog_disc2, Sox_13, Sox_14, Sox_16, Sox_18, Sox_19, Sox_2, Sox_4, Sox_9 s187735484 3′UTR Yes No HMVEC-LBl, 4_S ong_Enhance (Hu ec) –TATA_disc7 HMVEC-dLy-Neo s72932407 3′UTR Yes No HBMEC, HMVEC-LBl, 4_S ong_Enhance (Hu ec) –MAZ, SREBP_known3 HMVEC-dLy-Neo, HPAEC, HRGEC s1044265 3′UTR Yes Yes HUVEC 5_S ong_Enhance (Hu ec) –Foxo_3, Me 2_known1, Me 2_known6, Pou2 2_known2, TCF4_known3 a The ENCODE da a [40] b HeLa-S3 ce ical ca cinoma, HA-sp. as ocy es spinal co d, HCPEpiC cho oid plexus epi helial cells, HMVEC-dBl-Neo neona al blood mic o ascula endo helial cells, de mal-de i ed, HMVEC-LBl blood mic o ascula endo helial cells, lung-de i ed, HMVEC-dLy-Neo neona al lympha ic mic o ascula endo helial cells, de mal-de i ed, HBMEC b ain mic o ascula endo helial cells, HPAEC pulmona y a e y endo helial cells, HRGEC enal glome ula endo helial cells, HUVEC umbilical ein endo helial cells, HepG2 hepa ocellula ca cinoma Mää ä e al. BMC Cance (2017) 17:496 Page 6 o 8 Conclusions In conclusion, his s udy is he i s o analyse he ole o ge mline EMSY sequence al e a ions in HBOC p edis- posi ion in Finnish amilies. None o he obse ed EMSY a ian s showed s a is ically signi ican associa ions wi h b eas /o a ian cance isk a he popula ion le el. Based on he esul s, we canno exclude he likelihood a ian s con ibu ing o cance isk in ce ain high- isk amilies, pe haps as p i a e mu a ions. The a ian s could be used o de e mine cance isk wi h o he p edisposing mu a- ions in hese amilies. Fu he analyses a e wa an ed, pa icula ly o he a e no el in onic dele ion and 3′ UTR a ian s p edic ed o ha e unc ional oles. Addi ional ile Addi ional ile 1: Table S1. Func ional anno a ion o he loca ion o he c.2709 + 122delT dele ion. Table S2. The e ec o he c.2709 + 122delT a ian on splicing egula o y elemen s. (DOCX 16 kb) Abb e ia ions BC: B eas cance ; BRCA1:B eas cance 1, ea ly onse ;BRCA2:B eas cance 2, ea ly onse ;CHEK2:Checkpoin kinase; CI: Con idence in e al; HBOC: He edi a y b eas and o a ian cance ; HGVS: Human Genome Va ia ion Socie y; HP1 β: He e och oma in p o ein 1 β; LD: Linkage disequilib ia; LOH: Loss o he e ozygosi y; MAF: Mino allele equency; MLPA: Mul iplex Liga ion-dependen P obe ampli ica ion; OC: O a ian cance ; OR: Odds a io; PALB2:Pa ne and localize o BRCA2; PTT: P o ein unca ion es ; RAD51C:RAD51 (S. ce e isiae) Homologue C; SNP: Single-nucleo ide polymo phism; UTR: Un ansla ed egion Acknowledgemen s The au ho s would like o hank Ms. Linda En o h, Tiina Wahl o s, Ph.D., and Sanna- Kaisa Ha junen, M.Sc., o hei assis ance. The au ho s would also like o hank he cance pa ien s o hei pa icipa ion in his s udy and he pe sonnel o he Tampe e Uni e si y Hospi al Gene ics Ou pa ien Clinic o assis ance ela ed o s udy ma e ial collec ion. The ENCODE da a we e ob ained om he ENCODE conso ium. Funding This wo k was inancially suppo ed by g an s om he Compe i i e S a e Resea ch Financing o he Expe Responsibili y o Tampe e Uni e si y Hospi al (#X51003), The Finnish Cance O ganisa ions, he Sig id Juselius Founda ion, and he Academy o Finland (#251074). The Funding body has no ole in he design o he s udy o in collec ion, analysis, and in e p e a ion o da a o in w i ing he manusc ip . A ailabili y o da a and ma e ials The da ase s used and/o analysed du ing he cu en s udy a e a ailable om he co esponding au ho on easonable eques . Au ho s’con ibu ions KMM pa icipa ed in pa ien collec ion, s udy design, pe o med labo a o y analyses and d a ed he manusc ip . RN con ibu ed o he s udy design, pa icipa ed in labo a o y analyses, pe o med s a is ical and bioin o ma ics analyses and d a ed he manusc ip . MKT and AK con ibu ed s udy ma e ials and c i ically e iewed he manusc ip . S-LL and JS pa icipa ed in he s udy design and coo dina ion and c i ically e iewed he manusc ip . S-LL also pa icipa ed in pa ien collec ion and was esponsible o he gene ic counselling o pa ien s. All au ho s ead and app o ed he inal manusc ip . E hics app o al and consen o pa icipa e All pa ien s we e in o med o he analyses and p o ided w i en consen o he use o exis ing DNA samples in he p esen s udy. The E hical Commi ees o Tampe e and Tu ku Uni e si y Hospi als and he Na ional Au ho i y o Medicolegal A ai s app o ed his esea ch p ojec . Consen o publica ion ‘No applicable’. Compe ing in e es s The au ho s decla e ha hey ha e no compe ing in e es s. Publishe ’sNo e Sp inge Na u e emains neu al wi h ega d o ju isdic ional claims in published maps and ins i u ional a ilia ions. Au ho de ails 1 Ins i u e o Biosciences and Medical Technology - BioMediTech, Uni e si y o Tampe e, Lääkä inka u 1, FI-33520 Tampe e, Finland. 2 Fimlab Labo a o ies, Tampe e Uni e si y Hospi al, Bioka u 4, FI-33520 Tampe e, Finland. 3 Depa men o Clinical Gene ics, Tu ku Uni e si y Hospi al, Kiinamyllynka u 4-8, FI-20521 Tu ku, Finland. 4 Depa men o Pedia ics, Gene ics Ou pa ien Clinic, and Depa men o De ma ology, Tampe e Uni e si y Hospi al, PO BOX 2000, FI-33521 Tampe e, Finland. 5 Depa men o De ma ology, Tampe e Uni e si y Hospi al, PO BOX 2000, FI-33521 Tampe e, Finland. 6 Ins i u e o Biomedicine, Uni e si y o Tu ku, Kiinamyllynka u 10, FI-20014 Tu ku, Finland. 7 Depa men o Medical Gene ics, Tu ku Uni e si y Hospi al, Kiinamyllynka u 10, FI-20521 Tu ku, Finland. Recei ed: 30 Augus 2016 Accep ed: 17 July 2017 Re e ences 1. Jemal A, B ay F, Cen e MM, Fe lay J, Wa d E, Fo man D. Global cance s a is ics. CA Cance J Clin. 2011;61(2):69–90. 2. 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Common a ia ion in EMSY and isk o b eas and o a ian cance : a case-con ol s udy using HapMap agging SNPs. BMC Cance . 2005;5:81. • We accep p e-submission inqui ies • Ou selec o ool helps you o ind he mos ele an jou nal • We p o ide ound he clock cus ome suppo • Con enien online submission • Tho ough pee e iew • Inclusion in PubMed and all majo indexing se ices • Maximum isibili y o you esea ch Submi you manusc ip a www.biomedcen al.com/submi Submi you nex manusc ip o BioMed Cen al and we will help you a e e y s ep: Mää ä e al. BMC Cance (2017) 17:496 Page 8 o 8