Case-control analysis of truncating mutations in DNA damage response genes connects TEX15 and FANCD2 with hereditary breast cancer susceptibility

1 Scien i ic RepoR s | 7: 681 | DOI:10.1038/s41598-017-00766-9 www.na u e.com/scien i ic epo s Case-con ol analysis o unca ing mu a ions in DNA damage esponse genes connec s TEX15 and FANCD2 wi h he edi a y b eas cance suscep ibili y Tuomo Man e e 1, Anna Te asmäki1, Anna Nu mi2, Ka in Rapakko3,4, Saila Kauppila5, Jiangbo Tang6, Johanna Schleu ke 7,8, Anne Kallioniemi9, Jaana M. Ha ikainen10,11,12, A o Manne maa10,11,12, Pen i Nieminen 13, Rii a Hanhisalo3, Sini Leh o2, Maija Su an o2, Me i G ip14, A ja Jukkola-Vuo inen15, Ma ia Tengs öm11,16, Päi i Au inen11,16, Ande s K is 17, Åke Bo g17, Ca l Blomq is 18,19, K is iina Ai omäki20, Roge A. G eenbe g6, Robe Winq is 1, Heli Ne anlinna 2 & Ka i Pylkäs1 Se e al known b eas cance suscep ibili y genes encode p o eins in ol ed in DNA damage esponse (DDR) and a e cha ac e ized by a e loss-o - unc ion mu a ions. Howe e , hese explain less han hal o he amilial cases. To iden i y no el suscep ibili y ac o s, 39 a e unca ing mu a ions, iden i ied in 189 No he n Finnish he edi a y b eas cance pa ien s in pa allel sequencing o 796 DDR genes, we e s udied o disease associa ion. Mu a ion sc eening was pe o med o No he n Finnish b eas cance cases (n = 578–1565) and con ols (n = 337–1228). Mu a ions showing po en ial cance associa ion we e analyzed in addi ional Finnish coho s. c.7253dupT in TEX15, encoding a DDR ac o impo an in meiosis, associa ed wi h he edi a y b eas cance (p = 0.018) and likely ep esen s a No he n Finnish ounde mu a ion. A dele e ious c.2715 + 1G > A mu a ion in he Fanconi anemia gene, FANCD2, was o e wo imes mo e common in he combined Finnish he edi a y coho compa ed o con ols. A dele ion (c.640_644del5) in RNF168, causa i e o ecessi e RIDDLE synd ome, had high p e alence 1Labo a o y o Cance Gene ics and Tumo Biology, Cance and T ansla ional Medicine Resea ch Uni and Biocen e Oulu, No he n Finland Labo a o y Cen e No dlab Oulu, Uni e si y o Oulu, Oulu, Finland. 2Depa men o Obs e ics and Gynecology, Uni e si y o Helsinki and Helsinki Uni e si y Hospi al, Helsinki, Finland. 3Labo a o y o Gene ics, No he n Finland Labo a o y Cen e No dLab Oulu, Oulu, Finland. 4Cance Gene ic Uni , Se ice and Cen al Labo a o y o Haema ology, CHUV, Lausanne Uni e si y Hospi al, Lausanne, Swi ze land. 5Depa men o Pa hology, Oulu Uni e si y Hospi al and Uni e si y o Oulu, Oulu, Finland. 6Depa men s o Cance Biology and Pa hology, Ab amson Family Cance Resea ch Ins i u e, Basse Resea ch Cen e o BRCA, Pe elman School o Medicine, Uni e si y o Pennsyl ania, Philadelphia, PA, USA. 7Medical Biochemis y and Gene ics Ins i u e o Biomedicine, Uni e si y o Tu ku, Tu ku, Finland. 8Mic obiology and Gene ics, Depa men o Medical Gene ics, Tu ku Uni e si y Hospi al, Tu ku, Finland. 9BioMediTech and FimLab Labo a o ies, Uni e si y o Tampe e, Tampe e, Finland. 10School o Medicine, Ins i u e o Clinical Medicine, Pa hology and Fo ensic Medicine, Uni e si y o Eas e n Finland, Kuopio, Finland. 11Cance Cen e o Eas e n Finland, Uni e si y o Eas e n Finland, Kuopio, Finland. 12Imaging Cen e , Depa men o Clinical Pa hology, Kuopio Uni e si y Hospi al, Kuopio, Finland. 13Medical In o ma ics and S a is ics Resea ch G oup, Uni e si y o Oulu, Oulu, Finland. 14Depa men o Su ge y, Oulu Uni e si y Hospi al and Uni e si y o Oulu, Oulu, Finland. 15Depa men o Oncology, Oulu Uni e si y Hospi al and Uni e si y o Oulu, Oulu, Finland. 16Cance Cen e , Kuopio Uni e si y Hospi al, Kuopio, Finland. 17Depa men o Oncology and Pa hology, Depa men o Clinical Sciences Lund, Lund Uni e si y, Medicon Village, Lund, Sweden. 18Depa men o Oncology, Helsinki Uni e si y Hospi al, Helsinki, Finland. 19Depa men o Oncology, Uni e si y o Ö eb o, Ö eb o, Sweden. 20Depa men o Clinical Gene ics, Uni e si y o Helsinki and Helsinki Uni e si y Hospi al, Helsinki, Finland. Tuomo Man e e and Anna Te asmäki con ibu ed equally o his wo k. Co espondence and eques s o ma e ials should be add essed o R.W. (email: obe .[email p o ec ed]) o K.P. (email: ka i.pylkas@ oulu. i) Recei ed: 24 Janua y 2017 Accep ed: 13 Ma ch 2017 Published: xx xx xxxx OPEN www.na u e.com/scien i ic epo s/ 2 Scien i ic RepoR s | 7: 681 | DOI:10.1038/s41598-017-00766-9 in majo i y o he analyzed coho s, bu did no associa e wi h b eas cance . In conclusion, unca ing a ian s in TEX15 and FANCD2 a e po en ial b eas cance isk ac o s, wa an ing u he in es iga ions in o he popula ions. Fu he mo e, high equency o RNF168 c.640_644del5 indica es he need o i s es ing in Finnish pa ien s wi h RIDDLE synd ome symp oms. Inabili y o espond p ope ly o DNA damage leads o gene ic ins abili y, which is one o he main o ces d i ing he onse and p og ession o umo igenesis1. Acco dingly, a signi ican ac ion o inhe i ed cance p edisposi ion synd omes esul s om ge mline mu a ions in DNA epai and cell cycle checkpoin con ol genes2. Pa icula ly in b eas cance , he majo i y o he known suscep ibili y genes encode p o eins wi h in eg al oles in DNA dam- age esponse (DDR), including he wo majo ones BRCA1 and BRCA2, and a numbe o o he s, such as PALB2, ATM and CHEK23–5. E en in he e a o whole exome sequencing, mos o he genes wi h p oposed ole in b eas cance p edisposi ion ha e been iden i ied among hose unc ioning in DNA epai ela ed pa hways, including RECQL, FANCM and ERCC36–9. All hese genes a e cha ac e ized by se e al, a e loss-o - unc ion mu a ions ac ing as mode a e o high- isk p edisposing alleles o b eas cance , hus p o iding suppo o he “common disease- a e a ian ” hypo hesis. Howe e , a la ge p opo ion o he gene ic ac o s p edisposing o b eas cance s ill emains unknown, as cu en ly known mode a e o high- isk genes explain less han hal o he amilial and abou 5% o he o al b eas cance incidence10, 11. In o de o iden i y no el b eas cance p edisposing alleles, we pe o med a case-con ol associa ion analysis o 39 a e p o ein- unca ing a ian s p e iously iden i ied by a ge ed nex -gene a ion sequencing (NGS) o DDR genes in 189 No he n Finnish b eas cance pa ien s wi h indica ions o he edi a y disease suscep ibili y12. The use o ounde popula ions, such as he Finns, p o ides an ad an age o pe o ming a e a ian s udies as hese a ian s migh be en iched in he popula ion, hus ep esen ing a signi ican pa o all p edisposing mu a- ions in he genes unde in es iga ion. This has p o en o be he case o example o he PALB23 and RAD5013 genes. Based on he cu en esul s, we p opose a ole in b eas cance p edisposi ion o TEX15 and FANCD2. O hese, FANCD2 has a cen al ole in he Fanconi anemia (FA) DNA epai pa hway, which has p e iously been s ongly linked o b eas cance p edisposi ion14, whe eas TEX15 ep esen s a no el suscep ibili y gene among he DDR ac o s. Func ions o he encoded p o ein (Tes is exp essed 15) a e s ill la gely unknown, al hough i has been shown o be in ol ed in DNA double-s and b eak epai du ing meiosis15. Cu en esul s also e eal se e al mu a ions in genes known o be ele an o o he inhe i ed synd omes linked o DNA epai de iciencies, hus p o iding in eg al in o ma ion o he ield o clinical gene ics. These al e a ions include a ounde mu a ion in RNF168: he encoded p o ein has p e iously been linked o BRCA1 ec ui men o he DNA damage oci and iden i ied as he causa i e gene o he ecessi ely inhe i ed RIDDLE synd ome16. Resul s Case-con ol analysis o he iden i ica ion o b eas cance associa ed alleles. Fil e ing and al- ida ion s eps p esen ed in Fig.1 we e aken in o de o selec po en ially disease associa ed p o ein unca ing o Figu e 1. Wo k low o he s udy. Ta ge ed sequencing o 796 DDR genes was pe o med in 189 No he n Finnish b eas cance pa ien s wi h indica ion o he edi a y disease suscep ibili y. Analysis ocused on a e mu a ions likely esul ing in unca ed p o ein p oduc s. A e il e ing s eps, 39 mu a ions we e geno yped in he No he n Finnish case and con ol coho s. Ex ended geno yping o ou mu a ions we e pe o med in Finnish b eas cance and con ol coho s om Helsinkia, Tampe eb and Kuopioc egions. 1Supplemen a y TableS2, 2MCPH1 c.904_916del epo ed in Man e e e al.12. ExAC: Exome Agg ega ion Conso ium da abase, BC: b eas cance , IGV: In eg a i e Genomics Viewe , MAF: mino allele equency. www.na u e.com/scien i ic epo s/ 3 Scien i ic RepoR s | 7: 681 | DOI:10.1038/s41598-017-00766-9 splice si e mu a ions o he case-con ol associa ion analysis. In o al, 39 di e en al e a ions me he il e ing c i- e ia (21 nonsense, 9 ameshi and 9 canonical splice si e mu a ions) and we e s udied u he by he case-con- ol app oach in he No he n Finnish coho s (Supplemen a y TableS1). Mu a ions in en genes (CHD1L, CYP19A1, DCLRE1A, ERCC2, EXO1, GNL3, IGHMBP2, NAT10, PTPRH and TOP3A) u ned ou o be single ons and mu a ions in MSH3, NINL and ZRANB3 we e p esen only in wo pa ien s while absen om con ols, hus lacking he powe o s a is ical compa isons. The seg ega ion o hese mu a ions in he ca ie amilies could no be s udied due o he lack o addi ional DNA samples om sui able amily membe s, lea ing hei impac on b eas cance isk unknown. In o al, ou o he ecu en mu a ions showed signi ican (TEX15 c.7253dupT and FANCD2 c.2715 + 1G > A) o bo de line associa ion (TEX15 c.8325G > A and RNF168 c.640_644del5) wi h he edi a y b eas cance in he No he n Finnish coho . These mu a ions we e u he geno yped in expanded se o Finnish b eas cance cases and con ols o igina ing om Helsinki, Kuopio and Tampe e (Table1, and me a-analyses in Table2). O no e, wo o he s udied mu a ions we e also simul aneously iden i ied in Helsinki: FANCD2 c.2715 + 1G > A in clinical panel sequencing o a pa ien wi h ea ly onse iple-nega i e b eas cance and RNF168 c.640_644del5 in exome sequencing o he edi a y b eas cance pa ien s. TEX15 c.7253dupT mu a ion is s able a mRNA le el and associa es wi h b eas cance . Two di e en p o ein unca ing mu a ions in he TEX15 gene showed po en ial associa ion wi h b eas cance in he No he n Finnish coho . TEX15 encodes a p o ein ha has been sugges ed o ha e a speci ic ole in DNA double-s and b eak epai , as i is necessa y o o ma ion o DMC1 and RAD51 oci on meio ic ch omosomes15. TEX15 has ini ially been classi ied in o he g oup o cance / es is (CT) an igen encoding genes ac i a ed in a - ious cance s, and i s exp ession has been conside ed o be mo e o less s ic ly es ic ed o es is among ma u e o gans17, 18. Howe e , besides es is, low le el TEX15 exp ession has been epo ed also in o he no mal issues (including u e us, b ain and smoo h muscle), whe eas in cance samples TEX15 exp ession has been ecu en ly obse ed in b eas , lung and bladde cance , and cu aneous melanoma17, 19, 20. We con i med ha TEX15 was exp essed in he es ed pa ien -de i ed LCLs and also in b eas epi helial cance cells (MCF7). The TEX15 ameshi mu a ion (c.7253dupT, Leu2418Phe sTe 6, s760604179) was obse ed in h ee cases om he No he n Finnish he edi a y coho (3/247, 1.2%), whe eas only one ca ie was iden i ied in con ols (1/1190, 0.1%, p = 0.018, odds a io [OR] = 14.6 and 95% con idence in e al [CI] = 1.5–141.1) (Table1). All h ee cases we e diagnosed wi h b eas cance a young age (39, 36 and 38) and did no ha e mu a ions in p e- iously sc eened cance p edisposi ion genes3, 21, 22. Cu iously, wo o hem had a a he diagnosed wi h p os a e cance : one was con i med as a TEX15 c.7253dupT ca ie , he o he was no a ailable o es ing (Supplemen a y Fig.S1a). The mo he o he hi d index case was diagnosed wi h b eas cance a he age o 40 yea s (ca ie s a us unknown). Th ee mo e ca ie s (diagnosed a he age o 37, 58 and 47, espec i ely) we e iden i ied in he unselec ed coho (Supplemen a y Fig.S1a). Only one sample om amily membe s was a ailable o mu a ion es ing: he mo he o he index ( amily 6), diagnosed wi h bone ma ow cance a he age o 81, was con i med as a ca ie . Da a om pa hology epo s was a ailable o all six ca ie s. Tumo s we e mainly ER/PR posi i e, and in i e ca ie s hese we e o duc al o igin (Supplemen a y TableS2). No addi ional TEX15 c.7253dupT ca ie s we e iden i ied in ei he he cases o he con ols o igina ing om Helsinki o Tampe e (bo h Sou he n Finland), indica ing ha he mu a ion is geog aphically es ic ed o No he n Finland. Ano he TEX15 unca ing mu a ion (c.8325G > A, T p2775Te , s146619272) was iden i ied in 7/247 (2.8%) o he No he n Finnish he edi a y cases and i showed a bo de line associa ion wi h b eas cance (p = 0.063, OR = 2.7 and 95% CI = 1.1–6.8). The en ichmen was es ic ed o he he edi a y coho , whe eas he p e a- lence o he mu a ion in he unselec ed cases was no di e en om he con ols (Table1). Fu he sc eening o TEX15 c.8325G > A in he addi ional Helsinki and Tampe e coho s e ealed se e al ca ie s in bo h he edi a y cases and con ols wi h simila equencies, indica ing ha TEX15 c.8325G > A migh no be a b eas cance isk ac o o ha he isk associa ed wi h i is low (Table1 and Table2). To esol e his appa en disc epancy in b eas cance associa ion be ween he wo p o ein unca ing mu a- ions in he same gene, we es ed hei e ec a mRNA le el. Cu iously, TEX15 c.7253dupT and c.8325G > A beha ed di e en ly: he b eas cance associa ed TEX15 c.7253dupT mRNA was s able, whe eas he ansc ip om he c.8325G > A allele was e icien ly a ge ed by nonsense-media ed decay (Fig.2). This indica es ha he e ec o c. 8325G > A is a he dosage le el o TEX15, whe eas c.7253dupT could dis u b a leas some cellula unc ions o TEX15, po en ially in dominan –nega i e manne . Al hough we we e able o s udy he e ec o he TEX15 c.7253dupT mu a ion a mRNA le el, he p esence o he unca ed TEX15 p o ein p oduc could no be con i med due o lack o a sui able an ibody. As de ec s in some DDR genes ha e been associa ed wi h inc eased genomic ins abili y12, 13, 23, we es ed he p e alence o ch omosomal abe a ions in he TEX15 c.7253dupT ca ie s. They exhibi ed an inc ease in he equency o spon aneous ch omosomal ea angemen s when compa ed o con ols, howe e , he di e ence showing only a bo de line signi icance (p = 0.067) (Supplemen a y TableS3). All obse ed ch omosomal abe a- ions we e conside ed andom, as no p e e ence o speci ic b eak si e o e idence o clonali y was obse ed, hus sugges ing a plausible e ec on genomic ins abili y o he he e ozygous TEX15 c.7253dupT mu a ion. Fanconi anemia allele in FANCD2 shows en ichmen in b eas cance cases. A splice si e mu a ion (c.2715 + 1G > A, E906L sX4, s201811817) in he Fanconi anemia (FA) gene, FANCD2, showed en ichmen in he No he n Finnish he edi a y coho (3/247, 1.2%) when compa ed o con ols (p = 0.036, OR = 7.5 and 95% CI = 1.3–45.3) (Table1). This mu a ion induces he use o a c yp ic splice-dono si e downs eam he exon (c.2715 + 28_29) esul ing in inclusion o a 27 bp sequence o he in on in o mRNA, which ansla es in o a p ema u e s op codon a e h ee inse ed amino acids. cDNA-speci ic sequencing o he mu a ion ca ie LCL mRNA con i med he exp ession and a leas pa ial s abili y o he mu a ed allele (Supplemen a y Fig.S2). The dele e ious e ec o he mu a ion o he p o ein unc ion is suppo ed by i s occu ence in wo FA pa ien s a www.na u e.com/scien i ic epo s/ 4 Scien i ic RepoR s | 7: 681 | DOI:10.1038/s41598-017-00766-9 compound he e ozygous s a e24. All h ee c.2715 + 1G > A ca ie s om he he edi a y coho we e diagnosed wi h b eas cance a ela i ely young age (39, 40 and 42). In addi ion o se e al b eas cance cases in hei am- ilies, wo index cases had also a close ela i e (1s o 2nd deg ee) diagnosed wi h blood cance (a he age o 1 and 17 yea s, espec i ely) (Supplemen a y Fig.S1b). Howe e , he seg ega ion o he FANCD2 mu a ion wi h Mu a ion S udy coho Coho NWT (%) Mu (%) OR 95% CI pa TEX15 Oulu He edi a y BC 247 244 (98.8) 3 (1.2) 14.6 1.5–141.1 0.018 c.7253dupT BRCA1/2 neg. 228 225 (98.7) 3 (1.3) 15.9 1.6–153.1 0.014 Leu2418Phe sTe 6 Unselec ed BC 1317 1314 (99.8) 3 (0.2) 2.7 0.3–26.1 0.627 s760604179 All BC 1564 1558 (99.6) 6 (0.4) 4.6 0.6–38.1 0.149 Con ols 1190 1189 (99.9) 1 (0.1) Tampe e He edi a y BC 87 87 (100) 0 (ND) NA NA NA Con ols 93 93 (100) 0 (ND) Helsinki He edi a y BC 581 581 (100) 0 (ND) NA NA NA Con ols 640 640 (100) 0 (ND) TEX15 Oulu He edi a y BC 247 240 (97.2) 7b(2.8) 2.7 1.1–6.8 0.063 c.8325G > A BRCA1/2 neg. 228 222 (97.4) 6 (2.6) 2.5 0.9–6.6 0.104 T p2775Te Unselec ed BC 1318 1308 (99.2) 10 (0.8) 0.7 0.3–1.6 0.411 s146619272 All BC 1565 1548 (98.9) 17b(1.1) 1.0 0.5–2.1 1.000 Con ols 1203 1190 (98.9) 13 (1.1) Tampe e He edi a y BC 87 86 (98.9) 1 (1.1) NA NA 0.481 Con ols 94 94 (100) 0 (ND) Helsinki He edi a y BC 986 977 (99.1) 9c(0.9) 1.0 0.4–2.5 1.000 Con ols 1088 1078 (99.1) 10 (0.9) FANCD2 Oulu He edi a y BC 247 244 (98.8) 3b(1.2) 7.5 1.3–45.3 0.036 c.2715 + 1G > A BRCA1/2 neg. 228 226 (99.1) 2 (0.9) 5.4 0.8–38.7 0.118 E906L sX4 Unselec ed BC 1152 1150 (99.8) 2 (0.2) 1.1 0.2–7.6 1.000 s201811817 All BC 1399 1394 (99.6) 5b(0.4) 2.2 0.4–11.3 0.459 Con ols 1228 1226 (99.8) 2 (0.2) Tampe e He edi a y BC 87 87 (100) 0 (ND) NA NA 1.000 Unselec ed BC 646 644 (99.7) 2 (0.3) 1.2 0.2–8.5 1.000 All BC 733 731 (99.7) 2 (0.3) 1.0 0.1–7.4 1.000 Con ols 767 765 (99.7) 2 (0.3) Kuopio Unselec ed BC 668 668 (100) 0 (ND) NA NA NA Con ols 156 156 (100) 0 (ND) Helsinki He edi a y BC 1175 1171 (99.7) 4 (0.3) 2.2 0.4–11.9 0.436 Unselec ed BC 1727 1723 (99.8) 4d(0.2) 1.5 0.3–8.1 1.000 All BC 2513 2506 (99.7) 7 (0.3) 1.8 0.4–8.6 0.727 Con ols 1272 1270 (99.8) 2 (0.2) RNF168 Oulu He edi a y BC 247 243 (98.4) 4b(1.6) 3.2 0.9–11.5 0.077 c.640_644del5 BRCA1/2 neg. 228 225 (98.7) 3 (1.3) 2.6 0.7–10.6 0.165 Lys214Te s Unselec ed BC 1194 1185 (99.2) 9 (0.8) 1.5 0.5–4.2 0.606 s777601326 All BC 1441 1428 (99.1) 13b(0.9) 1.8 0.7–4.7 0.257 Con ols 1185 1179 (99.5) 6 (0.5) Tampe e He edi a y BC 87 87 (100) 0 (ND) NA NA 0.576 Unselec ed BC 445 444 (99.8) 1 (0.2) 0.2 0.02–1.4 0.073 All BC 532 531 (99.8) 1 (0.2) 0.1 0.01–1.1 0.048 Con ols 413 409 (99.0) 4 (1.0) Kuopio Unselec ed BC 652 635 (97.4) 17 (2.6) 0.6 0.3–1.2 0.157 Con ols 288 275 (95.5) 13 (4.5) Helsinki He edi a y BC 1170 1163 (99.4) 7 (0.6) 0.7 0.3–1.8 0.486 Con ols 1272 1261 (99.1) 11 (0.9) Table 1. F equency o TEX15, FANCD2 and RNF168 mu a ions in he s udied Finnish case-con ol coho s. ap- alue o χ2 es o Fishe ’s exac es , bone BRCA1/2 ca ie , cone homozygo e, d416 o he he edi a y pa ien s belong also o he unselec ed coho , includes one ca ie . BC: b eas cance , BRCA1/2 neg: includes only he he edi a y cases nega i e o pa hogenic BRCA1/2 mu a ions, CI: con idence in e al, Mu : mu a ion ca ie , NA: no analyzed, ND: no de ec ed, OR: odds a io, WT: wild ype. www.na u e.com/scien i ic epo s/ 5 Scien i ic RepoR s | 7: 681 | DOI:10.1038/s41598-017-00766-9 he disease emained inconclusi e due o he lack o addi ional DNA samples om he amily membe s. O no e, one index case was a double mu an o FANCD2 c.2715 + 1G > A and BRCA1 c.4097-2 A > G ( s80358019), and cu iously he e we e also se e al heal hy ca ie s o bo h mu a ions in his amily. Fu he s udies on addi ional Finnish b eas cance coho s (bo h unselec ed and he edi a y) iden i ied mo e FANCD2 c.2715 + 1G > A ca i- e s (Tables1 and 2), bu he equency emained low, which is ypical o mu a ions causa i e o FA25. In Helsinki, he FANCD2 mu a ion was iden i ied in ou b eas cance cases in he geno yped he edi a y coho (diagnosed a he age o 41, 44, 49 and 68, espec i ely). Two o he index cases we e diagnosed also wi h basal cell ca cinoma. Howe e , addi ional DNA samples we e a ailable only in one amily, in which he mu a ion did no seg ega e wi h b eas cance (Supplemen a y Fig.S1c). Da a om pa hology epo s was a ailable o ele en ca ie s, he umo s being mainly duc al (9/11). Cu iously, h ee o he umo s (27%) we e iple-nega i e (ER-/PR-/HER2-, Mu a ionaCoho bOR 95% CI p- aluec Oulu Mu / WT T e Mu / WT Kuo Mu / WT Hki Mu / WT TEX15 All BC 1.0 0.6–1.8 0.959 16/1530 1/86 NA 9/977 c.8325G > A He edi a y BC 1.6 0.8–3.1 0.208 6/222 1/86 NA 9/977 s146619272 Con ols 13/1190 0/94 NA 10/1078 FANCD2 All BC 1.6 0.6–4.1 0.375 4/1376 2/731 0/668 7/2506 c.2715 + 1G > A He edi a y BC 2.6 0.8–9.1 0.131 2/226 0/87 NA 4/1171 s201811817 Unselec ed BC 1.3 0.4–3.6 0.675 2/1150 2/644 0/668 4/1723 Con ols 2/1226 2/765 0/156 2/1270 RNF168 All BC 0.7 0.5–1.2 0.211 12/1410 1/531 17/635 7/1163 c.640_644del5 He edi a y BC 0.9 0.4–2.0 0.757 3/225 0/87 NA 7/1163 s777601326 Unselec ed BC 0.7 0.4–1.3 0.241 9/1185 1/444 17/635 NA Con ols 6/1179 4/409 13/275 11/1261 Table 2. Me a-analyses combining Oulu, Tampe e, Kuopio and Helsinki sc eening esul s. aTEX15 c.7253dupT was excluded om he me a-analysis since he mu a ion was p esen only in he No he n Finnish coho . bAll he known BRCA1/2 ca ie s we e excluded om he me a-analyses. cLogis ic eg ession model, all he analyzed coho s combined and all indi idual da ase s exploi ed. CI: con idence in e al, BC: b eas cance , Hki: Helsinki, Kuo: Kuopio, Mu : mu a ion ca ie , NA: no a ailable, OR: odds a io, T e: Tampe e, WT: wild ype. Figu e 2. Sequencing o TEX15 c.7253dupT and c.8325G > A mu a ion si es a genomic DNA and cDNA le el. www.na u e.com/scien i ic epo s/ 6 Scien i ic RepoR s | 7: 681 | DOI:10.1038/s41598-017-00766-9 Supplemen a y TableS2), a sub ype ha has p e iously been associa ed wi h de ec s in o he DDR genes26. Fo he unselec ed ca ie s om all o he analyzed coho s, he mean age a diagnosis was 57 yea s ( a ia ion 48–66 yea s). In he me a-analysis, he mu a ion had an OR o 2.6 among he BRCA1/2 mu a ion nega i e he edi a y cases when compa ed o con ols, bu he associa ion emained unde he le el o s a is ical signi icance (Table2). Mu a ions in genes causa i e o o he inhe i ed synd omes. Besides he FA gene FANCD2, a - ge ed sequencing o DDR genes e ealed se e al unca ing a ian s in genes known o esul in ecessi ely inhe - i ed synd omes wi h a iable pheno ypes, including sensi i i y o UV-ligh , impai men o eyesigh o muscle unc ion and also suscep ibili y o a ious cance s (OMIM h p://www.omim.o g/): APTX, CEP164, CYP19A1, ERCC2, ERCC6, IGHMBP2, NTHL1, RNF168 and UVSSA. The obse ed ecu en he e ozygous alleles in hese genes did no associa e wi h b eas cance , al hough he associa ion o he single on mu a ions in CYP19A1, ERCC2 and IGHMBP2 could no be excluded (Supplemen a y TableS1). Among his g oup o synd ome genes, RNF168 was pa icula ly in e es ing since i encodes an E3 ubiqui in-p o ein ligase in ol ed in he signaling pa h- way ha is equi ed o ec ui BRCA1 o he si e o DNA damage16. In addi ion, biallelic RNF168 unca ing mu a- ions ha e p e iously been epo ed in wo cases wi h ecessi ely inhe i ed RIDDLE synd ome (Radiosensi i i y, Immunode iciency, Dysmo phic ea u es and Lea ning Di icul ies)16. The cu en ly iden i ied RNF168 allele (c.640_644del5, Lys214Te s, s777601326) was obse ed in 4/247 (1.6%) o he No he n Finnish he edi a y cases, bu i was also de ec ed in 6/1185 (0.5%) o he con ols and showed only a bo de line associa ion wi h b eas cance suscep ibili y (p = 0.077, OR = 3.2 and 95% CI = 0.9–11.5) (Table1). The p e alence o he RNF168 c.640_644del5 allele was also es ed in Helsinki, Tampe e and Kuopio case-con ol coho s. Unexpec edly, i was obse ed a highe equency in con ols han in b eas cance pa ien s in all h ee se ies and showed no associa- ion wi h b eas cance in he me a-analysis (Table2). Fu he mo e, i had a s ikingly high ca ie equency in Kuopio (Eas e n Finland) no only in cases (2.6%) bu also in con ols (4.5%) (Table1), indica ing ha homozy- go es o his mu a ion should be expec ed in he Finnish popula ion. Discussion The ex ensi e case-con ol associa ion analysis pe o med he e o he a e, p esumably dele e ious alleles in genes encoding impo an playe s in he DDR pa hway iden i ied wo po en ially b eas cance p edisposing mu a ions: TEX15 c.7253dupT and FANCD2 c.2715 + 1G > A. Whe eas FANCD2 has an in eg al ole in he canonical FA pa hway, he biological unc ions o TEX15 a e only g adually eme ging. Howe e , TEX15 has al eady been shown o ope a e in homologous ecombina ion and o guide he loading o RAD51 o he si es o DNA double-s and b eaks along wi h BRCA1/2 du ing male meiosis15, 27. Re lec ing his, biallelic mu a ions in TEX15 lead o spe ma ogenic ailu e in bo h men and mice15, 28. Despi e hese es is-speci ic unc ions, we con i med he TEX15 exp ession in LCLs and MCF7 cell line. Toge he wi h he obse ed associa ion wi h b eas cance his indica es ha he ole o TEX15 ex ends beyond es is and meiosis-speci ic double-s and b eak epai . The b eas cance associa ed TEX15 c.7253dupT di e ed om he o he obse ed TEX15 mu a ion, c.8325G > A, in ha i was s able a mRNA le el whe eas he c.8325G > A mRNA was e icien ly deg aded. I is possible ha his s able allele migh dis u b some o he no mal cellula unc ions o TEX15, po en ially ela ing o cance p edisposi ion, mo e se e ely han a comple e null allele. Bo h c.7253dupT and c.8325G > A mu a ions eside in he same long exon (al oge he 1259 base pai s), p eceding he las one. The deg aded TEX15 null mu a- ion (c.8325G > A) c ea es a s op codon close o he o iginal, whe eas he s op codon e en ually c ea ed by he c.7253dupT ameshi is mo e dis an . Acco ding o a ecen pape by Lindeboom e al.29, he dis ance o a new s op codon om he o iginal one and he long leng h o he exon whe e he mu a ion esides a e he key ac o s o de ining he e iciency o nonsense-media ed decay. Indeed, his could p o ide a plausible explana ion o he cu en ly obse ed si ua ion, which is ac ually analogous o ha obse ed o he Finnish PALB2 and MCPH1 ounde mu a ions. Bo h o hem eside in a long exon o he gene, a e s able a mRNA le el, and a e mo e com- mon in people a ec ed wi h b eas cance compa ed o heal hy con ols12, 23. Besides in oducing TEX15 as a pu a i e new b eas cance p edisposi ion gene, he cu en esul s can be o ele ance o indi iduals su e ing om idiopa hic spe ma ogenic ailu e. The TEX15 null allele (c.8325G > A) has a ca ie equency o app ox- ima ely 1% in he Finnish popula ion, and indi iduals homozygous o his mu a ion a e likely o su e om spe ma ogenic ailu e as in he amily desc ibed by Oku man e al.28. The p e alence o FANCD2 c.2715 + 1G > A mu a ion among he s udied coho s suppo s he p e iously es ablished link be ween FA and b eas cance : monoallelic mu a ions in BRCA2/FANCD1, BRIP1/FANCJ, PALB2/FANCN, RAD51C/FANCO and BRCA1/FANCS genes p edispose o b eas and/o o a ian cance , while hei biallelic mu a ions cause FA, cha ac e ized by genomic ins abili y, bone ma ow ailu e, de elopmen al abno mali ies and inc eased incidence o cance , pa icula ly leukemia14, 30, 31. B eas cance associa ed genes om he FA pa hway unc ion in he homologous ecombina ion epai pa o he pa hway, while mu a ions in he co e complex FA genes ha e no been associa ed wi h b eas cance 25, 32, he excep ions po en ially being FANCM8, 33 and FANCC34. Cu iously, also in he s udy by Thompson e al. 2012, one o he index cases wi h unca ing FANCC mu a ion ca ied a dele e ious BRCA2 mu a ion34, analogous o he si ua ion obse ed in he cu en s udy, whe e one FANCD2 and BRCA1 double mu an was iden i ied. Al hough high FANCD2 exp ession le els in b eas umo s ha e p e iously been linked o poo su i al o he pa ien s35, and one common FANCD2 SNP has been associa ed wi h spo adic b eas cance isk36, o ou knowledge, his s udy is he i s one epo ing FANCD2 mu a ions in he con ex o amilial b eas cance . This s udy also p o ides in eg al in o ma ion o he ield o clinical gene ics as many biallelic mu a ions in DDR genes a e known o cause se e e congeni al diso de s. Pa icula ly in e es ing is he ecu en RNF168 c.640_644del5 mu a ion, which clea ly ep esen s a ounde mu a ion in Finland, bu whe he i is causa i e o RIDDLE synd ome emains o be demons a ed. Based on he obse ed ca ie equency, and i he mu a ion is no emb yonically le hal when homozygous, i is likely ha RIDDLE synd ome pa ien s exis in Finland, hus www.na u e.com/scien i ic epo s/ 7 Scien i ic RepoR s | 7: 681 | DOI:10.1038/s41598-017-00766-9 wa an ing u he in es iga ions. Only wo pa ien s wi h his condi ion ha e so a been desc ibed wo ldwide16, 37 and as he pheno ype o e en hese pa ien s is di e en , i is possible ha he se e i y o he condi ion migh a y. Fo a e condi ions like RIDDLE synd ome, he iden i ica ion o addi ional pa ien s is impo an o he be e cha ac e iza ion and unde s anding o he disease pheno ype and e iology, and i may ul ima ely lead o be e diagnosis and po en ial ea men . O e all, his s udy shows ha many he e ozygous p o ein- unca ing mu a ions in DDR genes iden i ied in b eas cance cases a e also equen ly ound in he heal hy popula ion and hus a e no high- isk suscep ibili y ac o s o b eas cance , al hough any low-pene ance e ec s canno be excluded. Ou esul s indica e a po en ial ole in b eas cance p edisposi ion o he e ozygous unca ing mu a ions in FANCD2 and TEX15. Based on ou esul s, FANCD2 c.2715 + 1G > A migh ac as a mode a e b eas cance isk allele, adding FANCD2 o he lis o sha ed genes be ween FA and b eas cance . Acco ding o he ExAC da abase38, FANCD2 c.2715 + 1G > A occu s also ou side Finland and should be s udied u he in la ge sample se s o cla i y i s ole in b eas cance p edis- posi ion. TEX15 ep esen s ye ano he po en ial b eas cance suscep ibili y gene om he DDR pa hway. E en hough he cu en ly iden i ied TEX15 c.7253dupT mos likely ep esen s a No he n Finnish ounde mu a ion, o he unca ing mu a ions, and hei e ec a mRNA le el, wa an u he s udies in o he popula ions. Ma e ials and Me hods Ta ge ed sequencing o 796 DDR genes in No he n Finnish b eas cance pa ien s. Ini ial sequencing was p e iously pe o med o 189 No he n Finnish b eas cance pa ien s wi h indica ion o he edi- a y disease suscep ibili y [62 cases wi h young disease onse (≤40y), and 127 cases wi h amily his o y o b eas o b eas and o a ian cance ]12. Selec ion and sequencing o he 796 DDR genes (Supplemen a y TableS4), and anno a ion o he a ian s a e desc ibed in Man e e e al.12. B ie ly, he selec ed genes encoded (1) p o eins iden- i ied as being pa o DNA epai p ocesses using he GeneOn ology sea ches and STRING .9.0 (n = 612)39, 40, and (2) no el BRCA1 and PALB2 in e ac ing p o eins iden i ied in p o ein complex pu i ica ion assays pe o med wi h epi ope agged e sions o he p o eins in HeLaS3 cells (n = 184). wANNOVAR41, Su eCall (Agilen ech- nologies, San a Cla a, CA, USA) and In eg a i e Genomics Viewe (IGV)42 we e used o anno a ion and isuali- za ion o he a ian s. All a ian s selec ed o u he s udies we e con i med by Sange sequencing (ABI3130xl, Applied Biosys ems, Fos e Ci y, CA, USA). Case-con ol coho s. Oulu coho (No he n Finland). The he edi a y b eas cance coho (n = 247) consis ed o 166 amilial and 81 young b eas cance cases (includes amilial and young cases om he disco e y coho ). The amilial cases we e a ec ed index indi iduals o No he n Finnish b eas , o b eas and o a ian cance amilies. Inclusion c i e ia we e he ollowing: 1) h ee o mo e b eas and/o o a ian cance s in i s - o second-deg ee ela i es (n = 86); 2) wo cases o b eas , o b eas and o a ian cance in i s - o second-deg ee ela i es. O hese a leas one had ea ly disease onse (<35 yea s), bila e al b eas cance , o mul iple p ima y umo s including b eas o o a ian cance in he same indi idual (n = 26); o 3) wo cases o b eas cance in i s - o second-deg ee ela i es (n = 54). Young b eas cance cases we e unselec ed o a amily his o y o can- ce and diagnosed wi h b eas cance a o below he age o 40 (median 38, a ia ion 25–40 yea s). These cases we e combined wi h he amilial coho o o m a single he edi a y coho , based on he assump ion ha when a woman below he age o 40 yea s de elops b eas cance , a he edi a y p edisposi ion may be suspec ed ega dless o he amily his o y43. In he he edi a y coho ul illing he inclusion c i e ia o he cu en s udy, 19/247 (8%) o he cases ca ied known mu a ions in BRCA1 o BRCA221, Supplemen a y TableS5. These we e included in he analysis in o de o iden i y addi ional isk ac o s, as in some amilies BRCA1/2 mu a ions could no alone explain he amily bu den o he disease, and also se e al indi iduals wi h double mu a ions in in eg al DDR genes ha e p e iously been desc ibed in he li e a u e3, 12, 34. The unselec ed b eas cance coho consis ed o 1326 consecu i e cases ope a ed a he Oulu Uni e si y Hospi al du ing 2000–2014. They we e unselec ed o a amily his o y o cance and age a disease onse . Al oge he 1228 heal hy geog aphically ma ched Finnish Red C oss blood dono s (758 emales and 470 males) we e used as popula ion con ols. Helsinki coho (Sou he n Finland). The unselec ed b eas cance coho om Helsinki was collec ed consecu- i ely a Helsinki Uni e si y Hospi al Depa men o Oncology in 1997–1998 and 200044, 45 and Depa men o Su ge y in 2001–200446. The unselec ed coho consis ed o 1727 pa ien s wi h in asi e b eas cance . Al oge he 416 pa ien s om he unselec ed se ies had a amily his o y o b eas cance and we e included also in he he ed- i a y b eas cance coho . Addi ional amilial b eas cance pa ien s we e collec ed a Depa men s o Oncology and Clinical Gene ics as p e iously desc ibed46, 47. In o al, he he edi a y b eas cance coho consis ed o 1175 pa ien s. O hese, 612 pa ien s we e om amilies wi h h ee o mo e b eas o o a ian cance s among i s - o second-deg ee ela i es and 563 pa ien s had one a ec ed i s -deg ee ela i e. The amilial b eas cance pa ien s ha e been es ed nega i e a leas o BRCA1/2 ounde mu a ions. Geog aphically ma ched 1272 heal hy Finnish Red C oss blood dono s we e used as con ols. All pa ien s and con ols we e emales. Tampe e coho (Sou he n Finland). The he edi a y cases om Tampe e consis ed o 87 index cases o BRCA1/2 mu a ion nega i e he edi a y b eas , o b eas and o a ian cance amilies as desc ibed in Kuusis o e al.48. The unselec ed b eas cance cases (n = 646) we e collec ed du ing 1997–1999 a he Tampe e Uni e si y Hospi al44. 767 geog aphically ma ched emale Finnish Red C oss blood dono s we e used as con ols. Kuopio coho (Eas e n Finland). Al oge he 487 cases and 288 con ols we e a ailable om he Kuopio B eas Cance P ojec (KBCP)49. The KBCP ma e ial includes p ospec i e b eas cance cases (unselec ed o he am- ily his o y o b eas cance ) om he p o ince o No he n Sa o in Eas e n Finland, diagnosed a he Kuopio Uni e si y Hospi al be ween 1990 and 1995. The con ol indi iduals in KBCP we e selec ed om he Na ional www.na u e.com/scien i ic epo s/ 8 Scien i ic RepoR s | 7: 681 | DOI:10.1038/s41598-017-00766-9 Popula ion Regis e a he same ime in e al and we e ma ched o sex, age and long- e m a ea o esidence o he cases. Addi ional se o unselec ed b eas cance cases (n = 181) we e a ailable om he ILRS b eas can- ce coho . The ILRS cases we e diagnosed and collec ed a he Kuopio Uni e si y Hospi al du ing 2011–2014. Al oge he hese accoun ed o 668 unselec ed b eas cance cases and 288 con ols. Genomic DNA ex ac ed om pe iphe al blood was used o mu a ion sc eening om all samples. The s udy was ca ied ou wi h in o med consen om all he pa icipa ing indi iduals. The s udy was app o ed by he E hical Boa d o he No he n Os obo hnia Heal h Ca e Dis ic , he E hical commi ee o Helsinki Uni e si y Cen al Hospi al, he E hical Commi ee o Tampe e Uni e si y Hospi al and he Na ional Au ho i y o Medicolegal A ai s, he E hical commi ee o he Uni e si y o Eas e n Finland and Kuopio Uni e si y Hospi al and he Minis y o Social A ai s and Heal h in Finland. The s udy and he me hods we e conduc ed in acco d- ance wi h he app o ed guidelines. Mu a ion sc eening. Case-con ol compa isons o Oulu, Kuopio and Tampe e coho s we e pe o med by High Resolu ion Mel (HRM) analysis (CFX96, Bio-Rad, He cules, CA, USA) using Type-I HRM eagen s (Qiagen, Hilden, Ge many). A posi i e con ol was included in all analyses, and samples wi h di e ing mel ing cu es we e u he con i med by Sange sequencing (ABI3130xl, Applied Biosys em, Fos e Ci y, CA, USA). The Helsinki coho s we e geno yped using Cus om TaqMan SNP Geno yping Assays and TaqMan Geno yping Mas e Mix (The moFishe Scien i ic, Wal ham, MA, USA). PCR was pe o med on 7500 Fas Real-Time PCR Sys em o 9800 Fas The mal Cycle and geno ypes we e analyzed on 7500 Fas Sys em SDS so wa e 1.3.1 o 7500 so wa e 2.0.6 (Applied Biosys ems, Wal ham, MA, USA). He e ozygous ca ie s we e included as posi i e con ols in all analyses. Ch omosomal analysis. Ch omosomal analysis o six he e ozygous TEX15 c.7253dupT ca ie s [ h ee a ec ed index cases, one male diagnosed wi h p os a e and skin cance , and wo heal hy emales (aged 25 and 58)] and nine wild ype con ols was ca ied ou on me aphases ob ained om egula sho - e m 3-day cul u es o pe iphe al blood T-lymphocy es13. The blood samples o he pa ien s selec ed o ch omosomal analysis we e collec ed a leas 5 yea s a e he ini ial b eas cance diagnosis and ea men . The con ols we e heal hy emale indi iduals. A minimum o 50 Giemsa-banded me aphases o each sample we e e alua ed by ligh mic os- copy and pho og aphed wi h an au oma ic ch omosome analyze (Cy oVision e sion 7.2, Applied Imaging). Ch omosomal abe a ions we e di ided in o i e classes as p e iously desc ibed13. mRNA analysis. To al RNA was isola ed om MCF7 b eas cance cell line and Eps ein-Ba i us ans o med lymphoblas oid cell lines (LCLs) o TEX15 c.7253dupT, TEX15 c.8325G > A and FANCD2 c.2715 + 1G > A mu a ion ca ie s and wild ype con ols using RNeasy Mini Ki (Qiagen, Hilden, Ge many) and e e se ansc ibed wi h iSc ip cDNA syn hesis Ki (Bio-Rad, He cules, CA, USA). The exp ession and ou come o di e en mu a ions a mRNA le el was s udied using cDNA speci ic sequencing in LCLs (p ime s in Supplemen a y TableS6). TEX15 exp ession in MCF7 cell line was es ed by cDNA speci ic ampli ica ion. S a is ical analysis. Ca ie equencies be ween cases and con ols we e compa ed using Fishe ’s exac o χ2 es . Mann-Whi ney U- es was used o de e mine he di e ence in he numbe o di e en ch omosomal abe a ions be ween TEX15 c.7253dupT ca ie s and con ols. The me a-analyses o he mu a ions occu ing in mo e han one s udy coho (FANCD2 c.2715 + 1G > A, TEX15 c.8325G > A and RNF168 c.640_644del5) we e pe o med using logis ic eg ession model combining all he analyzed coho s and exploi ing all indi idual da a- se s. All he analyses we e pe o med using IBM SPSS S a is ics 22.0 o Windows (SPSS Inc., Chicago, IL, USA). All p- alues we e wo-sided and alues <0.05 we e conside ed s a is ically signi ican . Re e ences 1. Khanna, K. K. & Jackson, S. P. DNA double-s and b eaks: Signaling, epai and he cance connec ion. Na . Gene . 27, 247–254 (2001). 2. Hoeijmake s, J. H. J. Genome main enance mechanisms o p e en ing cance . Na u e 411, 366–374 (2001). 3. E kko, H. e al. A ecu en mu a ion in PALB2 in Finnish cance amilies. Na u e 446, 316–319 (2007). 4. Pylkäs, K. e al. 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This wo k was suppo ed by he Academy o Finland (g an numbe s 250083, 266528, 122715 o K.P., H.N. and R.W., and Cen e o Excellence g an numbe 284605 o R.W.), he Finnish Cance Founda ion, he Sig id Juselius Founda ion, he Uni e si y o Oulu, he Uni e si y o Oulu Suppo Founda ion, he special Go e nmen al EVO unds o Oulu Uni e si y Hospi al- based esea ch ac i i ies and Helsinki Uni e si y Hospi al Resea ch unding. The unde s had no ole in s udy design, da a collec ion and analysis, decision o publish, o p epa a ion o he manusc ip . Au ho Con ibu ions K.P., R.W. and H.N. concei ed he s udy. T.M., A.T. and K.P. pe o med he NGS a ian anno a ions and p io i iza ions. A.T., T.M., A.N., S.L., M.S. pe o med he mu a ional analyses. K.P., K.R. and R.H. pe o med ch omosomal analyses. S.K., J.S., A.K., J.M.H., A.M., M.G., A.J.V., M.T., P.A., A.K., Å.B., C.B., K.A., R.W., H.N. and K.P. pa icipa ed in he collec ion o pa ien coho s and clinical da a. J.T. and R.G. con ibu ed o he gene lis . A.T., T.M., P.N. and A.N. analyzed he s a is ics. T.M., A.T. and K.P. d a ed he manusc ip , and all au ho s ead, e ised and app o ed he manusc ip . Addi ional In o ma ion Supplemen a y in o ma ion accompanies his pape a doi:10.1038/s41598-017-00766-9