FANCM mutation c.5791C>T is a risk factor for triple-negative breast cancer in the Finnish population

EPIDEMIOLOGY FANCM mu a ion c.5791C>T is a isk ac o o iple-nega i e b eas cance in he Finnish popula ion Johanna I. Kiiski 1 •Anna Te asma ¨ki 2,3 •Liisa M. Pel a i 1 •So ia Khan 1 • Tuomo Man e e 2,3 •Ka i Pylka ¨s 2,3 •A o Manne maa 4,5 •Ma ia Tengs o ¨m 6,7 • Ande s K is 8 •A ˚ke Bo g 8 •Veli-Ma i Kosma 4,5 •Anne Kallioniemi 9 • Johanna Schleu ke 10,11 •Ral Bu ¨ zow 1,12 •Ca l Blomq is 13 •K is iina Ai oma ¨ki 14 • Robe Winq is 2,3 •Heli Ne anlinna 15 Recei ed: 21 Feb ua y 2017 / Accep ed: 7 July 2017 / Published online: 12 July 2017 ÓThe Au ho (s) 2017. This a icle is an open access publica ion Abs ac Pu pose The FANCM c.5101C[T nonsense mu a ion was p e iously ound o associa e wi h b eas cance in he Finnish popula ion, especially among iple-nega i e cases. He e, we s udied he p e alence o h ee o he FANCM a ian s: c.5791C[T, which has been epo ed o p edis- pose o amilial b eas cance , and he c.4025_4026delCT and c.5293dupA a ian s ecen ly iden i ied in Finnish cance pa ien s. Me hods We geno yped he FANCM c.5791C[T mu a ion in 4806 in asi e b eas cance pa ien s, including BRCA1/2 mu a ion nega i e amilial cases and unselec ed cases, and in 2734 heal hy popula ion con ols om ou di e en geog aphical a eas o Finland. The associa ion o he mu a ion wi h b eas cance isk among pa ien subg oups was s a is ically e alua ed. We u he analyzed he com- bined isk associa ed wi h c.5101C[T and c.5791C[T mu a ions. We also geno yped 526 unselec ed o a ian &Heli Ne anlinna [email p o ec ed] Johanna I. Kiiski [email p o ec ed] Anna Te asma ¨ki [email p o ec ed] Liisa M. Pel a i [email p o ec ed] So ia Khan [email p o ec ed] Tuomo Man e e [email p o ec ed] Ka i Pylka ¨s [email p o ec ed] A o Manne maa [email p o ec ed] Ma ia Tengs o ¨m [email p o ec ed] Ande s K is [email p o ec ed] A ˚ke Bo g [email p o ec ed] Veli-Ma i Kosma [email p o ec ed] Anne Kallioniemi [email p o ec ed] Johanna Schleu ke [email p o ec ed] Ral Bu ¨ zow [email p o ec ed] Ca l Blomq is [email p o ec ed] K is iina Ai oma ¨ki [email p o ec ed] Robe Winq is [email p o ec ed] 1 Depa men o Obs e ics and Gynecology, Uni e si y o Helsinki and Helsinki Uni e si y Hospi al, Helsinki, Finland 2 Labo a o y o Cance Gene ics and Tumo Biology, Cance and T ansla ional Medicine Resea ch Uni , Biocen e Oulu, Uni e si y o Oulu, Oulu, Finland 3 Labo a o y o Cance Gene ics and Tumo Biology, No he n Finland Labo a o y Cen e, No dLab, Oulu, Finland 4 School o Medicine, Ins i u e o Clinical Medicine, Pa hology and Fo ensic Medicine, and Cance Cen e o Eas e n Finland, Uni e si y o Eas e n Finland, Kuopio, Finland 5 Imaging Cen e , Clinical Pa hology, Kuopio Uni e si y Hospi al, Kuopio, Finland 123 B eas Cance Res T ea (2017) 166:217–226 DOI 10.1007/s10549-017-4388-0 cance pa ien s o he c.5791C[T mu a ion and 862 amilial b eas cance pa ien s o he c.4025_4026delCT and c.5293dupA a ian s. Resul s The equency o he FANCM c.5791C[T mu a- ion was highe among b eas cance cases han in con ols (OR 1.94, 95% CI 0.87–4.32, P=0.11), wi h a s a is i- cally signi ican associa ion wi h iple-nega i e b eas cance (OR 5.14, 95% CI 1.65–16.0, P=0.005). The combined analysis o c.5101C[T and c.5791C[T ca ie s con i med a s ong associa ion wi h b eas cance (OR 1.86, 95% CI 1.32–2.49, P=0.0002), especially among he iple-nega i e pa ien s (OR 3.08, 95% CI 1.77–5.35, P=0.00007). Fo he o he a ian s, only one addi ional c.4025_4026delCT ca ie and no c.5293dupA ca ie s we e obse ed. Conclusions These esul s suppo he ole o FANCM as a b eas cance suscep ibili y gene, pa icula ly o iple- nega i e b eas cance . Keywo ds FANCM B eas cance T iple-nega i e b eas cance Familial b eas cance DNA epai Abb e ia ions FA Fanconi anemia ICL In e s and c osslink OR Odds a io CI Con idence in e al TNBC T iple-nega i e b eas cance ER Es ogen ecep o PR P oges e one ecep o HNPCC He edi a y non-polyposis colo ec al cance In oduc ion Fanconi Anemia complemen a ion g oup M (FANCM) is a mul i unc ional p o ein, in e ac ing wi h se e al pa ne s o ac i a e he Fanconi anemia (FA) epai pa hway. The pa hway includes 19 associa ed p o eins, which o m mul ip o ein complexes o epai damaged DNA, especially s alled eplica ion o ks induced by in e s and c oss-link- ing (ICL) agen s [1,2]. FA i sel is a a e, ecessi ely inhe i ed gene ic diso de causing congeni al de ec s, hema ologic abno mali ies, and cance p edisposi ion [1]. Despi e he nomencla u e, FANCM does no appea o ha e a ole in he FA disease, as he ini ial case, which led o he FANCM associa ion wi h FA, also ca ied biallelic FANCA mu a ions. Fu he mo e, homozygous loss-o - unc ion mu a ions in FANCM ha e been iden i ied in indi iduals wi hou any FA symp oms [3,4]. Howe e , FANCM has an undeniable ole in he FA pa hway, as i ec ui s o he DNA damage esponse p o eins o he si es o DNA lesions [4]. Se e al FA genes (FANCD1/BRCA2, FANCN/PALB2,FANCJ/BRIP1,FANCO/RAD51C, and FANCS/BRCA1)[5–11] a e high- o mode a e- isk b eas o o a ian cance suscep ibili y genes and p e ious s udies ha e connec ed also he e ozygous FANCM mu a ions wi h b eas cance p edisposi ion [12–14]. We p e iously iden i ied he FANCM c.5101C[T non- sense mu a ion ( s147021911, p.Gln1701*) in exon 20 by exome sequencing o ge mline DNA samples om 24 BRCA1/2-nega i e b eas cance pa ien s and u he geno- yped i in a la ge se ies o Finnish b eas cance pa ien s and heal hy popula ion con ols [12]. The mu a ion was ound o be associa ed wi h b eas cance wi h an odds a io (OR) o 1.86 [95% con idence in e al (CI) 1.26–2.75, P=0.0018], especially among iple-nega i e b eas cance [TNBC; es ogen (ER) and p oges e one (PR) ecep o and HER2 nega i e] cases (OR 3.56, 95% CI 1.81–6.98, P=0.0002). Acco ding o he ExAC [15] da abase his mu a ion is mo e common in Finland (ca ie equency *1.8%) han in o he Eu opean popula ions (ca ie equency *0.3%). Ano he , ye mo e a e FANCM mu a ion, c.5791C[T ( s144567652, p.A g1931*) in exon 22, has been iden i ied wi h exome sequencing o b eas cance pa ien s [16]. Fu he analysis o amilial b eas cance cases and heal hy con ols om se e al popula ions showed an associa ion be ween he mu a ion and amilial b eas cance (OR 3.93, 95% CI 1.28–12.11, P=0.017) [13]. A ecen s udy in a Ge man popula ion p oduced simila esul s, whe e he loss-o - unc ion mu a ions in he FANCM gene we e associa ed wi h bo h amilial b eas cance and TNBC [14]. 6 School o Medicine, Ins i u e o Clinical Medicine, Oncology, Kuopio, Finland 7 Cance Cen e , Kuopio Uni e si y Hospi al, Kuopio, Finland 8 Di ision o Oncology and Pa hology Depa men o Clinical Sciences Lund, Lund Uni e si y, Medicon Village, Lund, Sweden 9 BioMediTech Ins i u e and Facul y o Medicine and Li e Sciences, Uni e si y o Tampe e, and Fimlab Labo a o ies, Tampe e, Finland 10 Depa men o Medical Biochemis y and Gene ics, Ins i u e o Biomedicine, Uni e si y o Tu ku, Tu ku, Finland 11 Depa men o Medical Gene ics, Tyks Mic obiology and Gene ics, Tu ku Uni e si y Hospi al, Tu ku, Finland 12 Depa men o Pa hology, Uni e si y o Helsinki and Helsinki Uni e si y Hospi al, Helsinki, Finland 13 Depa men o Oncology, Uni e si y o Helsinki and Helsinki Uni e si y Hospi al, Helsinki, Finland 14 Depa men o Clinical Gene ics, Uni e si y o Helsinki and Helsinki Uni e si y Hospi al, Helsinki, Finland 15 Depa men o Obs e ics and Gynecology, Helsinki Uni e si y Cen al Hospi al (HUS), PO Box 700, 00029 Helsinki, Finland 218 B eas Cance Res T ea (2017) 166:217–226 123 c.5791C[T mu a ion has also been iden i ied in wo colo ec al cance pa ien s [17], and since FANCM is unc ionally connec ed wi h he misma ch epai genes MSH2/MSH6, i has p e iously been conside ed as a po en ial candida e gene o he edi a y non-polyposis col- o ec al cance (HNPCC) [17,18]. Howe e , a combined analysis o la ge da ase s did no e eal s a is ically sig- ni ican associa ion be ween FANCM mu a ions and he disease [19]. He e, we ha e in es iga ed he associa ion o he FANCM c.5791C[T mu a ion wi h b eas cance isk in amilial and unselec ed b eas cance cases among 4806 in asi e b eas cance pa ien s and 2734 heal hy popula- ion con ols om ou di e en geog aphical a eas o Finland. We u he e alua ed he b eas cance isk by subg oups o pa ien s as well as o a ian cance isk among 526 o a ian cance pa ien s. Also, he ecen ly iden i ied c.4025_4026delCT (p.Se 1342*) and c.5293dupA (p.Th 1765Asn s*3) a ian s in he FANCM gene we e s udied among 862 amilial b eas cance pa ien s om he Helsinki a ea. Mo eo e , o u he s udy he isk associa ed wi h unca ing C- e minal FANCM mu a ions, we analyzed he cu en esul s o he c.5791C[T mu a ion and isk es i- ma es o he c.5101C[T mu a ion, including he p e iously published esul s om Helsinki and Tampe e a ea [12] and geno yping esul s om Oulu and Kuopio. Me hods Subjec s Helsinki b eas cance se ies The FANCM c.5791C[T mu a ion was geno yped in 2391 b eas cance cases (including unselec ed and amilial pa ien s) and 1258 heal hy emale popula ion con ols om he Helsinki a ea. Unselec ed b eas cance pa ien samples we e collec ed a he Helsinki Uni e si y Cen al Hospi al in wo phases. Du ing 1997–1998 and 2000, 884 samples we e collec ed a he Depa men o Oncology. These samples include 79% o all consecu i e, newly diagnosed b eas cance cases du ing he collec ion pe iods [20,21]. Du ing 2001–2004, 986 samples, including 87% o all consecu i e, newly diagnosed b eas cance cases we e collec ed a he Depa men o Su ge y [21]. Only in asi e cases we e included in his s udy. The amilial cases we e collec ed a he Helsinki Uni e si y Cen al Hospi al Depa men s o Oncology and Clinical Gene ics [22,23]. O hese, 523 pa ien s had a s ong amily his o y wi h a leas h ee b eas o o a ian cance s among i s - o second-deg ee ela i es (including he p oband), and 555 pa ien s had a leas one i s -deg ee ela i e a ec ed wi h b eas o o a ian cance . All he amilial coho pa ien s wi h a leas h ee b eas o o a ian cance s among i s - o second-deg ee ela i es we e es ed nega i e o BRCA1/2 mu a ions and he pa ien s wi h one a ec ed ela i e we e es ed nega i e o he Finnish BRCA1/2 ounde mu a ions as p e iously desc ibed [24–26]. Eigh hund ed and six y wo amilial cases we e geno yped o he c.4025_4026delCT and c.5293dupA a ian s and 1078 amilial cases we e geno yped o he FANCM c.5791C[T mu a ion. The samples a e genomic DNA isola ed om pe iphe al blood. The pa ien genealogies we e con i med om he popula ion egis ies o hospi al eco ds and cance diag- noses om he hospi al eco ds o he Finnish Cance Regis y. Ho mone ecep o s a us was collec ed om pa hology epo s as desc ibed ea lie [27]. The 1258 geno yped popula ion con ols we e heal hy emale blood dono s om he Helsinki a ea. The unselec ed o a ian cance coho was collec ed a he Helsinki Uni e si y Cen al Hospi al Depa men o Obs e ics and Gynecology in 1998 as p e iously desc ibed [28]. Blood samples we e collec ed om in asi e epi helial o a ian ca cinoma ea ed pa ien s du ing ou ine ollow- up isi s o he clinic. Addi ional samples we e also col- lec ed be ween 1998 and 2006. Ou o he 526 independen samples included in he analysis in his s udy, 408 we e genomic DNA isola ed om blood and 118 samples we e umo -DNA. O he genomic samples, 171 we e se ous, 64 mucinous, and 43 endome ioid sub ype. 129 samples we e o he o a ian cance sub ypes, o one sample he sub ype is no known. Ou o he umo samples, 104 we e se ous and wo we e o he sub ypes, 12 samples we e o unknown sub ype. Tampe e b eas cance se ies The unselec ed b eas cance pa ien sample se om he Tampe e a ea was collec ed a he Tampe e Uni e si y Hospi al as p e iously desc ibed [20,22]. Addi ional 336 inciden cases we e collec ed in 1996–2004 a he Tam- pe e Uni e si y Hospi al. Two hund ed and o y nine cases had also amilial backg ound. Ho mone ecep o s a us in o ma ion was ob ained om pa ien pa hology epo s. Only in asi e cases we e included in his s udy. All samples a e genomic DNA isola ed om pe iphe al blood. The con ol coho o he Tampe e da ase consis s o 808 heal hy emale blood dono s om he Tampe e a ea. B eas Cance Res T ea (2017) 166:217–226 219 123 Kuopio b eas cance se ies 430 pa ien s wi h in asi e b eas cance om he Kuopio a ea we e geno yped o he FANCM c.5791C[T and c.5101C[T mu a ions. These pa ien s belong o p ospec- i e popula ion-based case–con ol s udy, The Kuopio B eas Cance P ojec (KBCP), which was conduc ed in be ween 1990 and 1996. Samples we e collec ed om women en e ing he Kuopio Uni e si y Hospi al due o b eas symp oms and who we e e en ually diagnosed as ha ing b eas cance . All samples a e genomic DNA iso- la ed om pe iphe al blood. In o ma ion abou ho monal ecep o s a us was ob ained om hospi al egis ies [29,30]. The con ol coho consis s o 158 heal hy blood dono s om he Kuopio a ea. Oulu b eas cance se ies The FANCM c.5791C[T and c.5101C[T mu a ions we e sc eened among 1323 b eas cance cases (1147 unselec ed, 153 amilial, and 56 young b eas cance pa ien s) and 510 heal hy emale con ols om he Oulu a ea. The unselec ed b eas cance samples we e collec ed om pa ien s ope - a ed a he Oulu Uni e si y Hospi al du ing 2000–2014, and hey we e unselec ed o age a disease onse and a amily his o y o cance . Only in asi e cases we e included in his s udy. The amilial b eas cance 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, and he young coho consis ed o b eas cance pa ien s unselec ed o amily his o y o cance bu wi h ea ly disease onse (B40 yea s), which sugges s a plausible he edi a y p edisposi ion ega dless o he amily his o y [31–33]. Only BRCA1/2 mu a ion neg- a i e amilial and young cases we e included in his s udy. Ho mone ecep o s a us was collec ed om pa hology epo s as desc ibed ea lie [34]. The con ol coho con- sis ed o 510 heal hy emale blood dono s om he Oulu a ea. All samples we e genomic DNA isola ed om pe iphe al blood. This s udy was pe o med wi h in o med consen om all he pa ien s and pe mission om he e hics commi ees o Uni e si y o Helsinki, Tampe e Uni e si y Hospi al, Oulu Uni e si y Hospi al, Uni e si y o Eas e n Finland, and Kuopio Uni e si y Hospi al Boa d on Resea ch E hics. Iden i ica ion o he mu a ions The FANCM c.5791C[T mu a ion has been ound o associa e wi h amilial b eas cance and TNBC [13,14]. I has also been epo ed in he Finnish popula ion as an en iched loss-o - unc ion mu a ion [3] and iden i ied in gene panel sequencing o Finnish amilial b eas cance pa ien s [35]. The c.4025_4026delCT a ian was iden i ied a Lund Uni e si y in one pa ien om 100 Finnish high- isk b eas cance cases wi h panel sequencing, in which also wo ca ie s o he p e iously published c.5101C[T mu a ion we e iden i ied. Su eselec XT Cus om 3-5.9 Mb lib a y ki (Agilen Technology) was used o cap u e DNA agmen s om he a ge genes. Sequencing was pe o med on he Illumina HiSeq 2500 wi h 2 994 o 2 9101 bp pai ed- end eads. The c.5293dupA a ian was iden i ied by exome sequencing o genomic DNA samples om 44 Finnish cance pa ien s wi h amilial his o y o b eas cance . Exome sequencing was execu ed a he Genome Quebec Inno a ion Cen e. To cap u e he exomic egions, Roche Nimblegen SeqCap EZ Exome 3 ki was used. The sequencing was pe o med on Illumina HiSeq 2000 sequence wi h 100 bp pai ed-end eads. The c.5101C[T mu a ion was iden i ied by exome sequencing as p e iously desc ibed [12]. Geno yping Geno yping o he FANCM c.5791C[T mu a ion o he Helsinki and Tampe e sample se s was pe o med wi h Sequenom MassARRAY sys em using iPLEX Gold assays (Sequenom) a FIMM (Uni e si y o Helsinki). Va ian s c.4025_4026delCT and c.5293dupA we e geno yped wi h TaqMan eal- ime PCR. 7500 Fas Real Time sys em was u ilized by using TaqMan SNP Geno yping Cus om assays and TaqMan Geno yping Mas e Mix (Applied Biosys- ems). Geno ype calling was pe o med wi h 7500 Real- Time PCR Sys em and 7500 so wa e ( e sion 2.06, Applied Biosys ems). Geno yping o he FANCM c.5101C[T mu a ion was pe o med as p e iously desc i- bed [12]. Posi i e con ols we e used in all analyses and all mu a ions we e con i med wi h Sange sequencing. I is o be no ed ha one pa ien om he Helsinki da ase (coun ed as one in he analysis) ca ies bo h c.5101C[T and c.5791C[T mu a ions. Un o una ely, we we e no able o de e mine whe he he mu a ions a e in cis o in ans; howe e , i hey we e in cis, his geno ype would be ex emely a e. FANCM c.5791C[T and c.5101C[T sc eening o Oulu and Kuopio sample se s was pe o med using High Reso- lu ion Mel analysis (CFX96, Bio-Rad) wi h Type-i HRM eagen s (Qiagen). Posi i e con ol DNA was included in all analyses, and samples wi h posi i e-like o di e ing mel ing cu es we e alida ed by Sange sequencing (ABI3130xl, Applied Biosys ems). 220 B eas Cance Res T ea (2017) 166:217–226 123 S a is ical analyses All s a is ical analyses we e pe o med using R ( e sion 3.02) s a is ical so wa e (h p://www. -p ojec .o g) o IBM SPSS S a is ics o Windows, e sion 22.0. Fo he isk analyses, wo-sided P- alues we e calcula ed using Pea - son ´s 2 - es o Fishe ´s exac es i he expec ed numbe o cell coun was i e o less. P 0.05 was conside ed s a- is ically signi ican . Be as and s anda d e o s o he di - e en s udies we e combined in ‘‘ me a’’-package o examine he e ogenei y be ween s udies. In he e ogenei y analysis, P 0.10 was conside ed s a is ically signi ican . In he combined analyses, all he da ase s we e pooled and he odds a ios and P- alues we e es ima ed wi h logis ic eg ession model s a i ied by s udy. Sepa a e analysis we e conduc ed o he subg oups de ined by his opa hology and amily his o y o he disease. In he combined analysis including da ase s o bo h FANCM c.5791C[T and c.5101C[T mu a ions, pa ien s wi h geno yping esul s o bo h mu a ions we e included in he analysis. Resul s Geno yping o he FANCM c.5791C>T mu a ion in he case–con ol sample se s The FANCM c.5791C[T mu a ion was iden i ied in al o- ge he 28 b eas cance pa ien s and eigh con ols among 4806 b eas cance pa ien s and 2734 popula ion con ols om ou di e en geog aphical a eas o Finland (Helsinki, Tampe e, Kuopio, and Oulu). The popula ion equency was highes in No he n and Eas e n Finland (Oulu 0.6% and Kuopio 0.6%) and lowes in Sou he n and Sou h- wes e n Finland (Helsinki 0.2% and Tampe e 0.1%). Among he BRCA1/2-nega i e amilial pa ien s om Hel- sinki and Oulu da ase s (N=1231), eigh mu a ion ca ie s we e iden i ied, and among he 526 o a ian cance pa ien s om Helsinki, wo mu a ion ca ie s we e iden i ied (Table 1). We e alua ed he b eas cance isk among all geno- yped pa ien s (all BC) in each da ase sepa a ely (Table 1). Fu he mo e, geno yped pa ien s we e di ided in o sub- g oups acco ding o amily his o y o cance , ER s a us, and iple-nega i e sub ype o s udy he isks by b eas cance pheno ypes. B eas cance isk was inc eased among all b eas cance pa ien s in he Helsinki (OR 2.11, 95% CI 0.59–7.49, P=0.24), Tampe e (OR 6.14, 95% CI 0.72–52.70, P=0.10), and Oulu da ase s (OR 1.16, 95% CI 0.31–4.30, P=1), albei wi h no s a is ically signi ican P- alues. In he Kuopio da ase , only wo mu a ions ca ie s we e iden i ied (OR 0.73, 95% CI 0.07–8.15, P=1) (Table 1). No signi ican he e ogenei y was seen be ween he s udies (P=0.9). Howe e , he FANCM c.5791C[T mu a ion associa ed signi ican ly wi h TNBC in he Helsinki da ase (OR 9.09, 95% CI 1.82–45.49, P=0.02). In he Tampe e and Oulu da ase s, wo addi ional iple-nega i e cases we e iden i ied. The analysis o o a ian cance cases om he Helsinki da ase sugges ed possibly sligh ly inc eased isk among c.5791C[T ca ie s (OR 1.60, 95% CI 0.27–9.58, P=0.64, Table 1); howe e , he e we e only wo mu a- ion ca ie s iden i ied. One pa ien has se ous o a ian cance , whe eas he o he mu a ion ca ie has been diag- nosed wi h mucinous sub ype o he disease. We u he pe o med a combined analysis o he ou s udies o e alua e he isk among all s udied b eas cance pa ien s. An ele a ed b eas cance isk o he c.5791C[T ca ie s was seen among all b eas cance pa ien s (OR 1.94, 95% CI 0.87–4.32, P=0.11), and pa icula ly in he TNBC subg oup (OR 5.14, 95% CI 1.65–16.0), wi h a s a is ically signi ican P alue (0.005) (Table 2). In addi ion, he b eas cance isk was inc eased also in he o he subg oups, e.g., ER nega i e (OR 2.34, 95% CI 0.75–7.35, P=0.14) and amilial b eas cance (OR 2.50, 95% CI 0.83–7.51, P=0.10) (Table 2), bu he esul s did no each s a is ical signi icance. Combined analyses o he FANCM c.5791C>T and c.5101C>T mu a ions We combined he esul s om he cu en analyses o he FANCM c.5791C[T mu a ion wi h he p e ious isk s udy o he FANCM c.5101C[T mu a ion in he Helsinki and Tampe e da ase s [12], and geno yping esul s om Oulu and Kuopio cases and con ols. Highly signi ican associ- a ion was seen be ween b eas cance and ca ying ei he o he mu a ions (OR 1.86, 95% CI 1.32–2.49, P=0.0002). The isk was consis en ly inc eased in all subg oups o pa ien s, wi h highes isk and he mos signi ican associ- a ion seen among he iple-nega i e pa ien s (OR 3.08, 95% CI 1.77–5.35, P=0.00007) (Table 3). No he e o- genei y was seen be ween mu a ions (P=0.7). All anal- yses we e s a i ied by s udy. Geno yping o he FANCM c.4025_4026delCT and c.5293dupA a ian s The FANCM c.5293dupA and c.4025_4026delCT a ian s we e geno yped among 862 amilial b eas cance pa ien s om he Helsinki a ea. No addi ional mu a ion ca ie s we e iden i ied o c.5293dupA a ian which may ep e- sen a unique mu a ion in he amily whe e i was B eas Cance Res T ea (2017) 166:217–226 221 123 iden i ied. The mu a ion was o iginally iden i ied in exome sequencing o a u e ine cance pa ien wi h a amily his o y o b eas cance . One addi ional ca ie was iden i ied o c.4025_4026delCT, o aling wo ca ie s o his a e a ian and was no s udied u he . The pa ien s ´ages a diagnosis we e 42 and 54, espec i ely. One ca ie had ER-nega i e b eas cance , whe eas he o he had ER- posi i e disease. Bo h had amily his o y o b eas cance , bu no addi ional samples we e a ailable o geno yping he ela i es. The ExAC popula ion equency o he c.4025_4026 dele ion in Finland is 0.015%. Discussion In his cu en case–con ol s udy, we e alua ed he b eas and o a ian cance isk o he FANCM c.5791C[T mu a ion as well as o c.4025_4026delCT and c.5293dupA a ian s among Finnish popula ion. We u he examined Table 1 F equency o he FANCM c.5791C[T mu a ion in he s udied sample se s by b eas cance subg oups and in he popula ion con ols S udy coho NCC (%) CT (%) OR 95% CI P alue Helsinki Con ols 1258 1255 (99.8) 3 (0.2) – – – All BC 2391 2379 (99.5) 12 (0.5) 2.11 0.59–7.49 0.24 Unselec ed BC 1699 1690 (99.5) 9 (0.5) 2.23 0.60–8.25 0.22 Familial BC 1078 1073 (99.5) 5 (0.5) 1.95 0.46–8.18 0.48 C3 a ec ed 523 520 (99.4) 3 (0.6) 2.41 0.49–12.00 0.37 2 a ec ed 555 553 (99.6) 2 (0.4) 1.51 0.25–9.08 0.64 ER?1795 1786 (99.5) 9 (0.5) 2.11 0.57–7.80 0.25 ER-412 409 (99.3) 3 (0.7) 3.07 0.62–15.26 0.16 TNBC 141 138 (97.9) 3 (2.1) 9.09 1.82–45.49 0.02 Unselec ed OC 526 524 (99.6) 2 (0.4) 1.60 0.27–9.58 0.64 Tampe e Con ols 808 807 (99.9) 1 (0.1) – – – All BC 662 657 (99.2) 5 (0.8) 6.14 0.72–52.70 0.10 ER?494 490 (99.2) 4 (0.8) 6.59 0.73–59.11 0.07 ER-122 121 (99.2) 1 (0.8) 6.67 0.41–107.34 0.25 TNBC 68 67 (98.5) 1 (1.5) 12.04 0.74–194.74 0.15 Oulu Con ols 510 507 (99.4) 3 (0.6) – – – All BC 1323 1314 (99.3) 9 (0.7) 1.16 0.31–4.30 1 Unselec ed 1147 1141 (99.5) 6 (0.5) 0.89 0.22–3.57 0.87 Familial BC 153 150 (98.0) 3 (2) 3.38 0.68–16.92 0.14 YBR 56 56 (100) 0 (0) – – – ER?434 432 (99.5) 2 (0.5) 0.78 0.13–4.70 1 ER-109 108 (99.1) 1 (0.9) 1.56 0.16–15.19 0.54 TNBC 69 68 (98.6) 1 (1.4) 2.49 0.25–24.23 0.40 Kuopio Con ols 158 157 (99.4) 1 (0.6) – – – All BC 430 428 (99.5) 2 (0.5) 0.73 0.07–8.15 1 ER?318 317 (99.7) 1 (0.3) 0.50 0.03–7.97 1 ER-95 95 (100) 0 (0) – – – TNBC 47 47 (100) 0 (0) – – – All BC: all geno yped b eas cance cases in designa ed s udy. C3 a ec ed: amilies wi h h ee o mo e b eas o o a ian cance cases among i s - o second-deg ee ela i es. 2 a ec ed: amilies wi h wo i s - deg ee ela i es wi h b eas o o a ian cance The pa ien subg oups a e o e lapping wi h 386 amilial cases belonging also o he unselec ed coho in he Helsinki da a se and 33 amilial cases belonging o he unselec ed coho in Oulu da a se . The pa ien s may also belong o di e en subg oups by umo pheno ype BC b eas cance , OC o a ian cance , ER es ogen ecep o , TNBC iple-nega i e b eas cance , YBR young b eas cance pa ien s (age a diagnosis B40 yea s) among Oulu da a se 222 B eas Cance Res T ea (2017) 166:217–226 123 he isk associa ed wi h ca ying ei he o he FANCM mu a ions c.5101C[T and c.5791C[T. The s udy e ealed he FANCM c.5791C[T mu a ion being mo e equen among he s udied b eas cance cases han in con ols in he Finnish popula ion. I was pa icu- la ly en iched among he TNBC cases, showing a signi i- can associa ion in he combined analysis (OR 5.14, P=0.005). This obse a ion is consis en wi h p e ious s udies on FANCM and b eas cance isk, as he c.5101C[T mu a ion has also been ound o associa e wi h TNBC [12,14]. Consis en esul s we e seen also in he o he subg oups s udied (Table 2). We u he combined he esul s o he FANCM c.5101C[T isk analysis [12] wi h he cu en esul s o c.5791C[T o s udy he isk associa ed wi h ca ying ei he o hese C- e minal FANCM mu a ions. The isk was sig- ni ican ly inc eased in all subg oups o pa ien s, especially among iple-nega i e cases (OR 3.08, 95% CI 1.77–5.35, P=0.00007). S udying he c.5791C[T mu a ion in he o a ian cance cases showed an ele a ed isk (OR 1.60), bu he associa ion was no s a is ically signi ican . P e ious s ud- ies on FANCM mu a ions ha e shown simila esul s [12,14]; howe e , a e y ecen s udy ound signi ican associa ion be ween FANCM mu a ions and high g ade se ous o a ian cance [36]. Sc eening o he o he wo FANCM a ian s exposed only one addi ional c.4025_4026delCT ca ie and no c.5293dupA ca ie s among 862 amilial b eas cance pa ien s, and hese we e no s udied u he . Howe e , hei iden i ica ion may sugges a wide mu a ion spec um o e y a e mu a ions in he FANCM gene, po en ially ela ing o sub ype-speci ic b eas cance p edisposi ion. O all b eas cance s, abou 10–20% a e ound o be ho monally iple-nega i e and hese a e usually agg essi e wi h a poo p ognosis, as his sub ype does no espond o ho monal he apy. These umo s a e o en highe g ade and la ge size han he o he umo sub ypes [37]. Ge mline mu a ions in BRCA1 a e common in TNBC cases, bu dele e ious changes in o he homologous ecombina ion DNA epai pa hway genes ha e also been obse ed o occu ela i ely equen ly in hese pa ien s, including Table 2 Combined analysis including all he sample se s (Helsinki, Tampe e, Kuopio, and Oulu) in di e en subg oups, wi h numbe o he c.5791C[T mu a ion ca ie s and non-ca ie s S udy coho OR P95% CI N Helsinki w /mu N Tampe e w /mu N Oulu w /mu N Kuopio w /mu All BC 1.94 0.11 0.87–4.32 2379/12 657/5 1314/9 428/2 Familial BC 2.50 0.10 0.83–7.51 1073/5 – 150/3 – Unselec ed BC 1.87 0.14 0.82–4.26 1690/9 657/5 1141/6 428/2 ER?1.86 0.16 0.78–4.41 1786/9 490/4 432/2 317/1 ER-2.34 0.14 0.75–7.35 409/3 121/1 108/1 95/0 TNBC 5.14 0.005 1.65–16.0 138/3 67/1 68/1 47/0 BC b eas cance , ER es ogen ecep o , TNBC iple-nega i e b eas cance , w wild ype, mu mu a ion ca ie Table 3 Combined analysis including all he sample se s (Helsinki, Tampe e, Kuopio, and Oulu) in di e en subg oups, wi h he numbe o he FANCM c.5101C[T and c.5791C[T mu a ion ca ie s (combined) and non-ca ie s S udy coho OR P95% CI N Helsinki w /mu N Tampe e w /mu N Oulu w /mu N Kuopio w /mu N Helsinki con ols w /mu N Tampe e con ols w /mu N Oulu con ols w /mu N Kuopio con ols w /mu All BC 1.86 0.0002 1.32–2.49 2285/81 656/35 1293/ 30 419/11 1234/21 782/21 498/12 157/1 Familial BC 1.99 0.004 1.24–3.19 1020/38 – 148/5 – – – – – Unselec ed BC 1.77 0.0006 1.28–2.45 1652/58 656/35 1124/ 23 419/11 – – – – ER?1.64 0.005 1.17–2.32 1721/55 480/22 426/8 292/8 – – – – ER- 2.02 0.004 1.25–3.25 405/16 114/8 108/1 99/2 – – – – TNBC 3.08 0.00007 1.77–5.35 130/10 60/5 68/1 58/2 – – – – BC b eas cance , ER es ogen ecep o , TNBC iple-nega i e b eas cance , w wild ype, mu mu a ion ca ie B eas Cance Res T ea (2017) 166:217–226 223 123 BRCA2,PALB2,BARD1, and RAD51C [38]. The FA pa hway has also been connec ed o TNBC, as compa ison o mRNA exp ession in di e en b eas umo ypes e ealed se e al FA pa hway genes (BRCA1,FANCD2, FANCF, and PALB2) being signi ican ly less exp essed in TNBC umo s compa ed o luminal A (ER o PR posi i e) umo s [39]. This u he suppo s he cu en inding ha also FANCM deple ion is linked especially wi h TNBC, bu deepe unde s anding o his connec ion wa an s u - he in es iga ions. Howe e , as he de elopmen al mech- anisms o TNBC a e no well known, he iden i ica ion o addi ional isk ac o s o his b eas cance sub ype is aluable o unde s anding i s e iology and o he iden i- ica ion o po en ial he apeu ic a ge s. FANCM has a c ucial unc ion in he DNA damage esponse o ICLs. I ac s as a helicase/ anslocase and binds adjacen o he c osslink, inducing he ec ui men o he FA co e complex o he si e. The co e complex monoubiqui ina es he FANCI–FANCD2 complex which igge s he accumula ion o mul iple nucleases and ini i- a es he ac ual DNA epai p ocesses [2]. Deple ion o FANCM has been epo ed o ha e e ec s on bo h he FA pa hway e iciency and umo igenesis: FANCM de icien mouse emb yonic ib oblas s displayed inc eased ch omo- somal b eakage, esidual FANCD2 monoubiqui ina ion, and inc eased spon aneous sis e ch oma id exchanges, and he FANCM knockou mice had educed o e all and umo - ee su i al [40]. The unc ional e ec o FANCM c.5791C[T has p e- iously been s udied by Pe e longo e al. [13]. Ins ead o being a con en ional nonsense mu a ion, i was p oposed o c ea e a binding si e o a splicing ac o hnRNP A1, which causes exon 22 skipping in mRNA and in oduces a p e- ma u e s op codon, leading o loss o 132 amino acids om he C- e minus o he p o ein. Two domains wi h impo an oles in binding o DNA, ERCC4 and helix–hai pin–helix (HhH)2, a e loca ed in he C- e minus o FANCM [41], and loss o hem likely dis u bs FANCM ac i i y. Suppo ing his hypo hesis, when he FANCM c.5791C[T mu a ion was in oduced o mouse emb yonic ib oblas s, he mu an cells displayed dec eased DNA epai ac i i y and inc eased ch omosomal b eakage [13]. This is consis en wi h he e ec o he majo i y o he known b eas cance - associa ed gene mu a ions [42], u he suppo ing he impac o FANCM c.5791C[T on genomic ins abili y and inc eased cance isk. Al oge he , he c.5791C[T mu a ion has o da e been connec ed o amilial b eas cance [13], and also o TNBC [14]. The cu en esul s on FANCM c.5791C[T, oge he wi h hose o c.5101C[T, suppo a ole o FANCM as a mode a e- isk b eas cance suscep ibili y gene and u he emphasize i s connec ion wi h he iple-nega i e b eas umo s. Conclusions The cu en esul s p o ide u he suppo o he p e i- ously sugges ed associa ion be ween FANCM mu a ions and iple-nega i e b eas cance . Fu he s udies wi h la - ge da ase s a e needed o p ecise isk es ima ions. Also, o he a e a ian s may occu in he FANCM gene and wa an u he in es iga ions in o he popula ions. Acknowledgemen s The au ho s wish o hank all he olun ee ed pa ien s pa icipa ing in his s udy. Helsinki B eas Cance S udy hanks esea ch nu ses I ja E kkila ¨and Vi pi Palola o hei help wi h collec ing pa ien da a and samples. Fo he cance diagnos ic da a, The Finnish Cance Regis y is g a e ully acknowledged. HEBCS wish also o hank he s a a he Technology Cen e, Ins i u e o Molecula Medicine Finland (FIMM), o SNV ma ke geno yping. Oulu b eas cance s udy hanks Annika Va ¨n a ¨nen and Leena Keski alo o echnical assis ance. Funding Helsinki b eas cance s udy has been suppo ed by he Helsinki Uni e si y Cen al Hospi al Resea ch Fund, he Academy o Finland (266528), he Sig id Juselius Founda ion, he Cance Socie y o Finland, he Finnish Cul u al Founda ion o LMP, and he Bio- medicum Helsinki Founda ion o JK. Oulu b eas cance s udy has been suppo ed by he Academy o Finland (250083) o KP, and by he Academy o Finland (122715 and Cen e o Excellence 284605), he Cance Socie y o Finland, he Sig id Juselius Founda ion, he Uni e si y o Oulu, he Uni e si y o Oulu Suppo Founda ion, and he special Go e nmen al EVO unds o Oulu Uni e si y Hospi al- based esea ch ac i i ies o RW. The Kuopio b eas cance s udy was suppo ed by he special Go e nmen Funding o Kuopio Uni e si y Hospi al G an s, The Cance Socie y o Finland, and he s a egic und o he Uni e si y o Eas e n Finland. Au ho con ibu ions JIK, LMP, and HN designed he s udy. JIK and AT pe o med he molecula gene ic s udies. JIK analyzed and pooled he da a. JIK and AT ca ied ou he s a is ical analyses and d a ed he manusc ip wi h HN. LMP, SK, TM, KP, AM, MT, AK, A ˚B, V-MK, AK, JS, RB, CB, KA, and RW con ibu ed samples, da a and pa ien in o ma ion. All au ho s ead and app o ed he inal manusc ip . Compliance wi h e hical s anda ds Con lic o in e es The au ho s decla e ha hey ha e no con lic o in e es . E hical app o al All p ocedu es pe o med in s udies in ol ing human pa icipan s we e in acco dance wi h he e hical s anda ds o he ins i u ional esea ch commi ees and wi h he 1964 Helsinki Decla a ion and i s la e amendmen s o compa able e hical s anda ds. In o med consen In o med consen was ob ained om all indi id- ual pa icipan s included in he s udy. 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