Expression profiling in ovarian cancer reveals coordinated regulation of BRCA1/2 and homologous recombination genes

  Ci a ion: Cus ódio, N.; Sa isaa , R.; Ca alho, C.; Bak-Go don, P.; Ribei o, M.I.; Ta a es, J.; Nunes, P.B.; Peixo o, A.; Pin o, C.; Escudei o, C.; e al. Exp ession P o iling in O a ian Cance Re eals Coo dina ed Regula ion o BRCA1/2 and Homologous Recombina ion Genes. Biomedicines 2022,10, 199. h ps://doi.o g/ 10.3390/biomedicines10020199 Academic Edi o : Luísa Romão Recei ed: 16 No embe 2021 Accep ed: 13 Janua y 2022 Published: 18 Janua y 2022 Publishe ’s No e: MDPI s ays neu al wi h ega d o ju isdic ional claims in published maps and ins i u ional a il- ia ions. Copy igh : © 2022 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). biomedicines A icle Exp ession P o iling in O a ian Cance Re eals Coo dina ed Regula ion o BRCA1/2 and Homologous Recombina ion Genes Noélia Cus ódio 1,*,† , Rosina Sa isaa 1,† , Célia Ca alho 1, Ped o Bak-Go don 1,‡ , Ma ia I. Ribei o 1, Joana Ta a es 2, Paula B. Nunes 3,4, Ana Peixo o 5, Ca la Pin o 5, Ca la Escudei o 5, Manuel R. Teixei a 5 and Ma ia Ca mo-Fonseca 1 1Ins i u o de Medicina Molecula João Lobo An unes, Faculdade de Medicina, Uni e sidade de Lisboa, 1649-028 Lisboa, Po ugal; [email p o ec ed] (R.S.); [email p o ec ed] (C.C.); go donped [email p o ec ed] (P.B.-G.); [email p o ec ed] (M.I.R.); [email p o ec ed] (M.C.-F.) 2Se iço de Ana omia Pa ológica, Hospi al de San a Ma ia, Cen o Hospi ala Uni e si á io Lisboa No e, 1649-028 Lisboa, Po ugal; [email p o ec ed] 3Hospi al CUF Descobe as, 1998-018 Lisboa, Po ugal; [email p o ec ed] 4Faculdade de Medicina, Uni e sidade de Lisboa, 1649-028 Lisboa, Po ugal 5Se iço de Gené ica, Ins i u o Po uguês de Oncologia do Po o F ancisco Gen il, 4200-072 Po o, Po ugal; [email p o ec ed] (A.P.); [email p o ec ed] (C.P.); [email p o ec ed] (C.E.); [email p o ec ed] (M.R.T.) *Co espondence: [email p o ec ed]; Tel.: +35-121-799-9411 † These au ho s con ibu ed equally o his wo k. ‡ Cu en add ess: Depa men o Biological Sciences, Columbia Uni e si y, New Yo k, NY 10027, USA. Abs ac : P edic i e bioma ke s a e c ucial in cla i ying he bes s a egy o use poly(ADP- ibose) polyme ase inhibi o s (PARPi) o he g ea es bene i o o a ian cance pa ien s. PARPi a e speci ically le hal o cance cells ha canno epai DNA damage by homologous ecombina ion (HR), and HR de iciency is equen ly associa ed wi h BRCA1/2 mu a ions. Gene ic es s o BRCA1/2 mu a ions a e cu en ly used in he clinic, bu esul s can be inconclusi e due o he high p e alence o a e DNA sequence a ian s o unknown signi icance. Mos es s also ail o de ec epigene ic modi ica ions and mu a ions loca ed deep wi hin in ons ha may al e he mRNA. The aim o his s udy was o in es iga e whe he quan i a ion o BRCA1/2 mRNAs in o a ian cance can p o ide in o ma ion beyond he DNA es s. Using he nCoun e assay om NanoS ing Technologies, we analyzed RNA isola ed om 38 o a ian cance specimens and 11 no mal allopian ube samples. We ound ha BRCA1/2 exp ession was highly a iable among umo s. We u he obse ed ha umo s wi h lowe le els o BRCA1/2 mRNA showed down egula ed exp ession o 12 addi ional HR genes. Analysis o 299 o a ian cance samples om The Cance Genome A las (TCGA) con i med he coo dina ed exp ession o BRCA1/2 and HR genes. To acili a e he ou ine analysis o BRCA1/2 mRNA in he clinical se ing, we de eloped a a ge ed d ople digi al PCR app oach ha can be used wi h FFPE samples. In conclusion, his s udy unde sco es he po en ial clinical bene i o measu ing mRNA le els in umo s when BRCA1/2 DNA es s a e nega i e o inconclusi e. Keywo ds: o a ian cance ; BRCA1/2 mRNA; homologous ecombina ion; nCoun e assay; ddPCR 1. In oduc ion O a ian cance is he deadlies o m o gynecological cance [ 1 , 2 ]. The a e age li e ime isk o de eloping o a ian cance is 1.3%, he equi alen o 1 in 78 women [ 1 ]. Al hough o a ian cance accoun s o 2.5% o all malignancies in women, i is esponsible o 5% o emale cance dea hs [ 2 ]. This high a ali y a e is usually a ibu ed o equen la e diagnosis, limi ed he apeu ic op ions, and a lack o e ec i e bioma ke s o p edic ing ea men esponse [3]. Biomedicines 2022,10, 199. h ps://doi.o g/10.3390/biomedicines10020199 h ps://www.mdpi.com/jou nal/biomedicines Biomedicines 2022,10, 199 2 o 17 The s anda d ea men o newly diagnosed o a ian cance is cy o educ i e su ge y ollowed by pla inum-based chemo he apy [ 4 ]. Howe e , app oxima ely 70% o pa ien s ha e a elapse wi hin he subsequen 3 yea s [ 3 ]. Recu en o a ian cance is ypically incu able, wi h mos pa ien s ecei ing mul iple addi ional lines o ea men . O e he pas 10 yea s, a new class o small molecule d ugs ha inhibi poly(adenosine diphospha e- ibose) polyme ases (PARPs) has a ac ed much a en ion o he ea men o o a ian cance [ 5 ]. The PARP amily comp ises se e al p o eins in ol ed in a a ie y o cellula p ocesses, including he s ess esponse, ch oma in emodeling, DNA epai , and apop osis [ 6 ]. One o he bes -cha ac e ized a ge s o PARP inhibi o s (PARPi) is PARP1, a p o ein ha plays a majo ole in he de ec ion and epai o single-s and DNA b eaks [ 6 , 7 ]. PARP1 is also likely in ol ed in o he DNA epai pa hways, such as nucleo ide excision epai , non-homologous end joining, homologous ecombina ion, and DNA misma ch epai [ 6 , 7 ]. Mechanis ically, PARP1 is a DNA damage senso and a signal ansduce ha binds o DNA b eaks and hen syn hesizes poly(ADP- ibose) chains on a ge p o eins (PARyla ion), including PARP1 i sel (au oPARyla ion). Au o-PARyla ion u he ac i a es PARP1 and enables he PARyla ion o his ones and o he ch oma in-associa ed p o eins, which leads o he ec ui men o addi ional DNA epai molecules o he si e o damage [ 7 ]. In 2005, wo s udies e ealed ha umo cells lacking BRCA1 o BRCA2 p o eins a e selec i ely ulne able o PARPi [ 8 , 9 ]. Inhibi ion o PARP1 alone is no le hal o no mal cells, bu in he absence o BRCA1/2, i becomes cy o oxic o umo cells [ 8 , 9 ]. The p oposed mechanism o ac ion is ha PARP1 inhibi ion p e en s PARyla ion and locks PARP1 on damaged DNA, s alling he p og ess o eplica ion o ks. In di iding cells ea ed wi h PARPi, s alled eplica ion o ks can be escued by homologous ecombina ion (HR), a p ocess ha enables high- ideli y, empla e-dependen epai o DNA damage [ 10 ]. Tumo cells ha a e de ec i e in HR due o loss o BRCA1 o BRCA2 use al e na i e e o -p one pa hways ha lead o agmen a ion o he genome and ul ima ely esul in cell dea h [ 5 ]. In addi ion o BRCA1 and BRCA2, se e al o he p o eins a e in ol ed in he HR p ocess [ 11 ], and umo s wi h BRCA-independen HR de iciency a e also sensi i e o PARPi [ 6 ]. Al- hough he i s app o ed PARPi (olapa ib) ini ially a ge ed pa ien s wi h o a ian cance ha ca ied ge mline mu a ions in he BRCA1 o BRCA2 genes, a la e s udy demons a ed esponses wi h olapa ib in 41% and 24% o ge mline BRCA mu a ion ca ie s and non- ca ie s, espec i ely [ 12 ]. Among he non-ca ie s, a sub-se had soma ic BRCA mu a ions and a ew o he s had mu a ions in o he HR genes, such as RAD50, RAD51C/D, CDK12, o PALB2 [ 13 ]. These clinical s udies we e conduc ed in pa ien s p e iously ea ed wi h pla inum sal s, which a e DNA damaging agen s ha cause DNA c osslink epai in pa by HR, and e ealed a clea associa ion be ween pla inum sensi i i y and esponse o PARPi [ 14 ]. Thus, u he ials shi ed owa ds using PARPi in pa ien s who esponded o pla inum-based chemo he apy. Cu en ly, PARPi a e app o ed o he ea men o all pla inum-sensi i e pa ien s, ega dless o BRCA1/2 s a us. Howe e , mo e bene i is seen in pa ien s wi h ei he ge mline o soma ic BRCA1/2 mu a ions [5]. Inhe i ed suscep ibili y o o a ian cance is ound in app oxima ely 15% o pa ien s, he as majo i y o which ha e ge mline he e ozygous mu a ions in he BRCA1 o BRCA2 genes [ 15 ]. Loss o he wild- ype BRCA allele can be de ec ed in cance s ha de elop in he e ozygous ca ie s, bu whe he his second hi is equi ed o oncogenesis is unclea [ 16 ]. Soma ic mu a ions in BRCA1 o BRCA2 genes a e also occasionally p esen in spo adic (non-he edi a y) o a ian cance s [15]. De eloping p edic i e bioma ke s o PARPi is o c i ical impo ance o deli e ing p ecision ea men o pa ien s. Cu en ly, DNA es s o de ec bo h ge mline and soma ic mu a ions in BRCA1 and BRCA2 genes a e o en used in he clinic. Howe e , he u ili y o such assays is limi ed by he high p e alence o a e DNA sequence a ian s o un- known signi icance (VUS). Indeed, oughly equal numbe s o VUS and pa hogenic/likely pa hogenic a ian s in BRCA1/2 a e lis ed in he Na ional Ins i u es o Heal h (NIH) epos- i o y ClinVa (h ps://www.ncbi.nlm.nih.go /clin a , las accessed on 25 May 2020). Mo eo e , mos cu en ly used DNA assays ail o de ec epigene ic modi ica ions (such as Biomedicines 2022,10, 199 3 o 17 p omo e hype me hyla ion), as well as mu a ions loca ed deep wi hin in ons ha may al e he mRNA [ 17 ].He e, we hypo hesized ha measu ing he le el o BRCA1 and BRCA2 mRNA in o a ian cance issue may p o ide addi ional in o ma ion when DNA es s a e nega i e o inconclusi e. Down egula ion o BRCA mRNA was p e iously shown o cause dys unc ional exp ession o BRCA p o eins, esul ing in HR de iciency and sensi i i y o PARPi [ 18 ]. Mechanisms leading o educed mRNA le els include epigene ic silencing and ameshi mu a ions. In addi ion o classical ameshi mu a ions (i.e., inse ions, dele- ions, o duplica ions o exonic nucleo ides ha change he eading ame o he mRNA), splicing mu a ions o en induce a ameshi as a consequence o exon skipping, use o unna u al (c yp ic) splice si es, o in on e en ion [ 17 ]. Changing he eading ame o ansla ion o en in oduces a p ema u e e mina ion codon (PTC) in o he mRNA, and a cellula su eillance sys em e med nonsense-media ed mRNA decay (NMD) deg ades he as majo i y o mRNAs wi h PTCs [ 19 ]. Because NMD leads o he down egula ion o exp ession and loss o unc ion o he a ec ed genes, i con ibu es o many gene ic diseases including cance . An example o his is he inac i a ion o umo supp esso genes ha bo ing PTCs h ough NMD [20]. The ex en o which BRCA1 and BRCA2 mRNA le els a e egula ed in o a ian cance has emained poo ly unde s ood. In his s udy, we iden i ied a sub-se o umo s lacking any de ec ed BRCA1/2 mu a ion ha exp ess low le els o BRCA1/2 mRNA simila o umo s wi h known NMD-elici ing PTCs. No ably, we ound ha he exp ession o 12 o he HR genes is also down egula ed in umo s wi h low le els o BRCA1/2 mRNA. These esul s unde sco e he po en ial clinical bene i o measu ing mRNA le els in umo s when BRCA1/2 DNA es s a e nega i e o inconclusi e. 2. Ma e ials and Me hods 2.1. Biological Samples We analyzed a chi ed o malin- ixed and pa a in-embedded (FFPE) issue biopsies, including o a ian cance and no mal allopian ube samples. Samples we e collec ed om he a chi es o he Hospi al San a Ma ia/Cen o Hospi ala Uni e si á io Lisboa No e, E.P.E., Lisbon, Po ugal, and Ins i u o Po uguês de Oncologia, Po o, Po ugal. An expe ienced pa hologis assessed he quali y o each sample. In he case o umo samples, he diagnosis was con i med, and he issue was mac o dissec ed o ensu e ha only umo egions we e u he analyzed. The s udy was app o ed by he e hical commi ee o he Lisbon Academic Medical Cen e , Lisbon, Po ugal (app o al numbe : 170/18). 2.2. RNA isola ion and Quan i ica ion RNA was ex ac ed om 5–8 cu ls o issue (10 µ m hick) sec ioned om each sample. RNA was pu i ied using he Pu eLink TM FFPE RNA Isola ion Ki (, In i ogen, Ca lsbad, CA, USA), as desc ibed by he manu ac u e . The amoun and pu i y o o al RNA eco - e ed we e de e mined wi h he Nanod op TM 2000 spec opho ome e (The mo Scien i ic, Wal ham, MA, USA). T aces o con aminan DNA we e emo ed by DNase I diges ion. Finally, RNA concen a ion and size we e analyzed in he Agilen 4200 TapeS a ion sys em using an Agilen RNA Sc eenTape assay (Agilen , San a Cla a, CA, USA). 2.3. Gene Exp ession P o iling wi h he nCoun e Sys em We used he nCoun e ® FLEX Analysis Sys em and he Van age 3D ™ RNA Panel o DNA Damage and Repai (NanoS ing Technologies, Sea le, WA, USA). The mRNA hyb idiza ion, de ec ion, and scanning we e pe o med a he NanoS ing nCoun e Co e Facili y a he Se iço de Gené ica—Ins i u o Po uguês de Oncologia do Po o F ancisco Gen il, E.P.E., Po o, Po ugal. B ie ly, pu i ied RNA (200 ng) was hyb idized o e nigh a 67 ◦ C. The ull olume o he hyb idiza ion eac ions was immedia ely in oduced in he P ep S a ion, which is a liquid handling obo ha pe o ms he pu i ica ion o he hyb idized complexes and hei immobiliza ion on o he su ace o a ca idge. The ba codes cap u ed o each sample we e hen iden i ied and coun ed by he Digi al Analyze . Biomedicines 2022,10, 199 4 o 17 The esul ing da a we e impo ed in o and analyzed by he nSol e 4.0 Analysis So wa e Sys em. Quali y con ol pa ame e s ecommended by NanoS ing we e applied using he nSol e de aul se ings. Samples ha ailed he quali y con ol me ics we e excluded om u he analysis. Fo da a no maliza ion, aw coun s we e he i s backg ound adjus ed using he in e nal nega i e con ols ollowed by a wi hin-sample no maliza ion using he in e nal posi i e con ols. Finally, da a we e no malized ac oss samples (i.e., co ec ed o inpu ) using he mean RNA coun s om e e ence (housekeeping) genes. This esul ed in a o al o 49 samples, 11 om no mal allopian ubes and 38 om o a ian cance issue. 2.4. BRCA1 and BRCA2 mRNA Analysis by D ople Digi al PCR The le el o BRCA1 and BRCA2 mRNA in o a ian cance samples p e iously analyzed wi h he nCoun e assay was quan i ied by d ople digi al PCR (ddPCR) using he Bio-Rad QX200 sys em (Bio-Rad Labo a o ies, He cules, CA, USA), acco ding o he manu ac u e ’s ins uc ions and he digi al MIQE guidelines [ 21 , 22 ]. Fi s , pu i ied RNA samples we e ea ed wi h DNAse I (Roche, Mannheim, Ge many) 0.5 U/ µ L, a 37 ◦ C o 30 min, in he p esence o RiboSa e (Bioline, London, UK) 1 U/ µ L. A e pu i ica ion wi h an equal olume o phenol:chlo o o m:isoamyl alcohol (25:24:1) in he p esence o 0.3M Sodium ace a e pH 5.2 and 50 µ g/mL GlycoBlue ™ cop ecipi an (The moFishe Scien i ic, Wal ham, MA, USA), RNA was p ecipi a ed wi h 2 olumes 100% e hanol, dissol ed in 0.1 mL o RNAse ee wa e and quan i ied in NanoD op 2000 spec opho ome e . Then, RNA was e o ansc ibed and ampli ied using he One-S ep RT-ddPCR Ad anced Ki o P obes (Bio- Rad, He cules, CA, USA,) and P imeTime S d ® qPCR P obe Assays om IDT (In eg a ed DNA Technologies, Co al ille, IA, USA). P ime s and Taqman luo escen p obes we e designed wi h IDT so wa e ools a ailable online and a e desc ibed in Supplemen a y Table S1. All genes we e analyzed wi h 6-FAM/ZEN/IBFQ labeled p obes, excep e e ence gene GUSB o which we used a HEX/ZEN/IBFQ labeled p obe. Duplex expe imen s we e pe o med a ge ing ei he BRCA1, BRCA2, POLR1E o NUBP1 (FAM) and GUSB (HEX) simul aneously. The ddPCR wo k low s a ed by pa i ioning he RT-PCR eac ion mix ( o al olume 20 µ L) con aining o wa d and e e se p ime s (900 nM inal concen a ion), p obes (250 nM inal concen a ion each), and sample RNA in o aqueous d ople s in oil. Nega i e con ols included samples wi h no e e se ansc ip ase in he RT-PCR mas e mix. The d ople s we e hen subjec ed o a he mocycling p o ocol o e o ansc ip ion and ampli ica ion (50 ◦ C o 1 h; 95 ◦ C o 10 min; 46 cycles o 95 ◦ C o 35 s and 60 ◦ C o 1 min; and 98 ◦ C o 10 min). The PCR p oduc s we e kep a 4 ◦ C o up o 1 day be o e eading he luo escen con en o he d ople s in he QX200 D ople Reade (Bio-Rad, He cules, CA, USA). Da a we e analyzed wi h he Quan aSo so wa e (Ve sion 1.7.4.0917, Bio-Rad, He cules, CA, USA). The ac ion o posi i e and nega i e d ople s was de e mined based on he luo escen signal, and he esul s we e i ed o a Poisson dis ibu ion o es ima e absolu e concen a ions (copies/µL) o each mRNA. To ensu e ha simila amoun s o e e ence mRNA we e loaded pe sample, wo assays we e sequen ially pe o med o each sample. In he i s assay, 30 ng o o al RNA was loaded and he numbe o copies pe d ople (CPD) o GUSB mRNA was quan i ied. The amoun o o al RNA loaded in he second assay con ained GUSB mRNA a a concen a ion o 0.05 CPD (50 copies/µL). Fo compa ison, we used he same ddPCR assays o es RNA ex ac ed om non- ixed, eshly isola ed pe iphe al blood mononuclea cells (PBMCs). 2.5. S a is ical Analysis 2.5.1. Gene al Analysis and plo ing we e pe o med wi h R Co e Team 2019 ( e sion 3.6.1, R Foun- da ion o S a is ical Compu ing, Vienna, Aus ia). Empi ical p- alues we e calcula ed as P=n+1 m+1 , whe e nis he numbe o s imulan s ha show a alue as high as o highe han he ue alue, and m is he o al numbe o simulan s [ 23 ], (Spea man’s co ela ion coe i- Biomedicines 2022,10, 199 5 o 17 cien s we e con e ed o z’- alues and back using he unc ions Fishe Z() and Fishe ZIn () in he DescTools package . 0.99.36. 2.5.2. Analysis o NanoS ing Da a Genes we e classi ied in o pa hways based on he labels supplied wi h he NanoS ing panel. The Homologous Recombina ion and Fanconi Anemia pa hways we e g ouped, and he ele an genes a e e e ed o as Homologous Recombina ion (HR) genes h oughou he ex . Twel e o he genes in he panel (ERCC1, ERCC2, ERCC3, ERCC4, ERCC5, ERCC6, ERCC8, EXO1, FAN1, FANCA, FANCB, FANCC) we e excluded om he analysis due o a p oblem wi h a pa icula lo o TagSe s, which passed ini ial quali y con ol bu was subsequen ly ound o lead o abno mally low NanoS ing coun s. No e ha a ew o he samples we e included in wo sepa a e NanoS ing uns. In hese cases, he alues om he wo uns we e a e aged in all analyses. The hea map was p oduced wi h he hea map.2() unc ion om he gplo s package ( e sion 3.1.1., h ps://CRAN.R-p ojec .o g/package= gplo s, accessed on 16 No embe 2021), using he comple e linkage clus e ing me hod wi h 1 –Spea man0sρ as he dis ance unc ion. P io o gene a ing he hea map, da a we e scaled so ha he mean o each gene was 0 and he s anda d de ia ion was 1 so as o no malize o di e ences in NanoS ing p obe e iciency. 2.6. The Cance Genome A las (TCGA) Da a Analysis TCGA o a ian se ous cys adenoca cinoma mRNAseq RSEM alues we e e ie ed om h p:// i eb owse.o g/ (las accessed: 22 No embe 2019). The da a we e il e ed o emo e genes wi h e y low ead coun s using he il e ByExp () unc ion om he edgeR package 3.28.1 [ 24 ]. The edgeR calcNo mFac o s() unc ion was used o calcula e lib a y size no maliza ion ac o s, which we e hen used when con e ing he da a in o log 2 Coun s Pe Million (log2CPM) using oom(). All edgeR unc ions we e un wi h de aul pa ame e se ings. In o al, 164 o he 168 genes ha we e used om he NanoS ing panel we e a ailable in TCGA da a (a e ead coun il e ing). 3. Resul s 3.1. S udy Coho Ou s udy coho comp ised 42 FFPE issue samples om pa ien s diagnosed wi h o a ian cance , he majo i y o which we e classi ied as high-g ade se ous ca cinomas (Supplemen a y Table S2). All umo issues had been p e iously sc eened o he p esence o mu a ions in he BRCA1 and BRCA2 genes, and he esul s a e indica ed in Supplemen- a y Table S2. Because mos o a ian cance s a e p obably de i ed om he allopian ube epi helium [ 25 , 26 ], we included 18 samples o no mal allopian ubes su gically esec ed om indi iduals wi h no cance diagnosis in ou s udy. 3.2. Quan i ica ion o BRCA1 and BRCA2 mRNAs wi h he nCoun e Assay We used he nCoun e assay om NanoS ing Technologies because his is conside ed an ideal me hod o exp ession p o iling in FFPE issues. The assay coun s indi idual mRNA molecules wi h a sensi i i y simila o eal- ime PCR bu wi hou he bias in oduced by enzyma ic eac ions [ 27 ]. This is pa icula ly ele an when using RNA isola ed om FFPE specimens because ixa ion wi h o maldehyde induces he o ma ion o me hylene c osslinks ha limi success ul e e se ansc ip ion and PCR ampli ica ion [ 28 ]. Mo eo e , RNA becomes p og essi ely mo e agmen ed in FFPE specimens, pa icula ly when blocks a e s o ed a oom empe a u e [29]. In ou s udy, RNA in eg i y (RIN) alues anged om 1.8 o 5.0 and concen a ions om 19.7 o 353.4 ng/ µ L (Supplemen a y Figu e S1A and Supplemen a y Table S2). One o he quali y con ol equi emen s o he nCoun e gene exp ession assay is ha a leas 50% o he pu i ied RNA molecules a e longe han 300 nucleo ides (Supplemen a y Figu e S1B). As shown in Supplemen a y Figu e S1C, he RNA ex ac ed om some samples did no mee his quali y equi emen and was he e o e disca ded om u he analysis. To Biomedicines 2022,10, 199 6 o 17 assess ep oducibili y, RNA pu i ied om no mal allopian ubes and umo samples was epea edly un wi h di e en eagen and p obe ba ches and he esul s showed simila RNA coun s (Supplemen a y Figu e S2). To p o ile he exp ession o BRCA1 and BRCA2 genes, we used he nCoun e Van age ™ RNA Panel o DNA Damage and Repai ha hyb idizes o mRNA om a o al o 192 genes, including 12 housekeeping genes and 180 unique genes ela ed o DNA damage and epai pa hways (Supplemen a y Table S3). In no mal allopian ubes, we es ima ed means o BRCA1 and BRCA2 mRNA le els co esponding o 185 and 86 NanoS ing coun s, espec i ely, wi h s anda d de ia ions o 45 and 29 (Figu e 1A). In umo s, he mean le el o BRCA1 and BRCA2 mRNA co esponded o 280 and 338 NanoS ing coun s, espec i ely, wi h s anda d de ia ions o 159 and 192. Thus, in con as o he li le a ia ion in mRNA le els o no mal allopian ubes, BRCA1 and BRCA2 exp ession was highly a iable among umo s. Some umo s had alues compa able o no mal issue while o he s we e up o 10- old highe han he median o no mal samples (Figu e 1A–C). The median exp ession was signi ican ly highe in umo s han in no mal issue o bo h BRCA1 ( allopian ube median: 195.23; umo median: 256.515; p~0.010; wo- ailed Welch’s - es on anks) and BRCA2 ( allopian ube median: 74.13; umo median: 305.87; p~5.59 × 10 −12 ; wo- ailed Welch’s - es on anks). Biomedicines 2022, 10, x FOR PEER REVIEW 7 o 19 Figu e 1. Exp ession le el o BRCA1 and BRCA2 genes in 11 no mal allopian ubes and 38 umo samples analyzed wi h he NanoS ing nCoun e assay. Tumo samples showed bo h highe median exp ession le els as well as g ea e a iabil- i y. (A) NanoS ing coun s o BRCA1 and BRCA2, in bo h allopian ube and umo samples. The ho izon al ba ma ks he median. (B,C) Indi idual mRNA coun s o each umo (T) a e shown o de ed om lowes o highes BRCA1 (B) and BRCA2 (C) exp ession le els. Tumo s wi h iden i ied mu a ions a e highligh ed ( ed, ameshi mu a ions; yellow, Po - uguese BRCA2 ounde mu a ion; ligh blue, missense mu a ions). Fo he 11 no mal allopian ube (FT) samples, he mean and s anda d de ia ion a e depic ed. Nex , we ocused on umo s ha bo ing mu a ions in he BRCA1/2 genes (Figu e 1B,C). Ten umo s had ameshi mu a ions loca ed in ei he BRCA1 exon 11 o BRCA2 exons 10 and 11 (Supplemen a y Figu e S3). These mu a ions gene a e PTCs and a e p e- dic ed o elici NMD acco ding o ecen ly alida ed ules [30]. Indeed, he PTCs we e mo e han 50–54 bp ups eam o he las exon-exon junc ion, bo h BRCA1 and BRCA2 genes comp ised mo e han wo exons, and he PTCs we e mo e han 200 bp downs eam Figu e 1. Con . Biomedicines 2022,10, 199 7 o 17 Biomedicines 2022, 10, x FOR PEER REVIEW 7 o 19 Figu e 1. Exp ession le el o BRCA1 and BRCA2 genes in 11 no mal allopian ubes and 38 umo samples analyzed wi h he NanoS ing nCoun e assay. Tumo samples showed bo h highe median exp ession le els as well as g ea e a iabil- i y. (A) NanoS ing coun s o BRCA1 and BRCA2, in bo h allopian ube and umo samples. The ho izon al ba ma ks he median. (B,C) Indi idual mRNA coun s o each umo (T) a e shown o de ed om lowes o highes BRCA1 (B) and BRCA2 (C) exp ession le els. Tumo s wi h iden i ied mu a ions a e highligh ed ( ed, ameshi mu a ions; yellow, Po - uguese BRCA2 ounde mu a ion; ligh blue, missense mu a ions). Fo he 11 no mal allopian ube (FT) samples, he mean and s anda d de ia ion a e depic ed. Nex , we ocused on umo s ha bo ing mu a ions in he BRCA1/2 genes (Figu e 1B,C). Ten umo s had ameshi mu a ions loca ed in ei he BRCA1 exon 11 o BRCA2 exons 10 and 11 (Supplemen a y Figu e S3). These mu a ions gene a e PTCs and a e p e- dic ed o elici NMD acco ding o ecen ly alida ed ules [30]. Indeed, he PTCs we e mo e han 50–54 bp ups eam o he las exon-exon junc ion, bo h BRCA1 and BRCA2 genes comp ised mo e han wo exons, and he PTCs we e mo e han 200 bp downs eam Figu e 1. Exp ession le el o BRCA1 and BRCA2 genes in 11 no mal allopian ubes and 38 umo samples analyzed wi h he NanoS ing nCoun e assay. Tumo samples showed bo h highe median exp ession le els as well as g ea e a iabili y. ( A ) NanoS ing coun s o BRCA1 and BRCA2, in bo h allopian ube and umo samples. The ho izon al ba ma ks he median. ( B , C ) Indi idual mRNA coun s o each umo (T) a e shown o de ed om lowes o highes BRCA1 ( B ) and BRCA2 ( C ) exp ession le els. Tumo s wi h iden i ied mu a ions a e highligh ed ( ed, ameshi mu a ions; yellow, Po uguese BRCA2 ounde mu a ion; ligh blue, missense mu a ions). Fo he 11 no mal allopian ube (FT) samples, he mean and s anda d de ia ion a e depic ed. Nex , we ocused on umo s ha bo ing mu a ions in he BRCA1/2 genes (Figu e 1B,C). Ten umo s had ameshi mu a ions loca ed in ei he BRCA1 exon 11 o BRCA2 exons 10 and 11 (Supplemen a y Figu e S3). These mu a ions gene a e PTCs and a e p edic ed o elici NMD acco ding o ecen ly alida ed ules [ 30 ]. Indeed, he PTCs we e mo e han 50–54 bp ups eam o he las exon-exon junc ion, bo h BRCA1 and BRCA2 genes comp ised mo e han wo exons, and he PTCs we e mo e han 200 bp downs eam o he s a codon (Supplemen a y Figu e S3). Despi e in e -sample a iabili y, umo s wi h ameshi mu a ions we e among hose wi h lowe exp ession le els (Figu e 1B,C). In hese samples, he median BRCA1 exp ession was 235.63 (median o all umo s: 256.52), and he median BRCA2 exp ession was 225.71 (median o all umo s: 305.87). Th ee umo s had he c.156–157 insAlu BRCA2 mu a ion, which has only been epo ed in amilies o Po uguese ances y. This Po uguese BRCA2 ounde mu a ion is p esen in ~30% o all Po uguese amilies wi h he edi a y b eas and o a ian cance , ep esen ing 55% o all BRCA2 ge minal mu a ion ca ie s in Po ugal [ 31 ]. This mu a ion leads o ull in- ame skipping o exon 3 [31,32], which encodes amino acids 23 o 105. Possibly, his p o ein dele ion educes he in e ac ion be ween BRCA2 and PALB2, and impai s homologous ecombina ion, hus con e ing hype sensi i i y o DNA damage [ 33 ]. The le els o BRCA2 exp ession in hese umo s anged om 157.44 o 686.05, wi h a median o 438.28 (Figu e 1C). Fou umo s had missense mu a ions. In umo s wi h BRCA1 missense mu a ions, he le els o BRCA1 exp ession anged om 195.1 o 557.5, whe eas he ange o BRCA2 exp ession in umo s wi h ameshi s in BRCA2 anged om 273.93 o 477.36 (Figu e 1B,C). 3.3. Exp ession P o iling o DNA Damage and Repai Genes Ha ing shown ha he le els o BRCA1/2 mRNAs we e highly a iable be ween umo samples, we nex analyzed he exp ession o he addi ional genes p obed wi h he nCoun e Van age ™ RNA Panel o DNA Damage and Repai (Supplemen a y Table S3). We obse ed s ikingly di e en exp ession pa e ns be ween no mal issue and many o he umo samples, al hough he e was high a ia ion be ween umo s (Figu e 2A, see Supplemen a y Table S4 o he NanoS ing coun s o all he genes in all he samples). We ound 72 genes o be di e en ially exp essed be ween umo and no mal samples ( wo- ailed Welch’s - es on anks wi h Bon e oni co ec ion; genes we e conside ed o be signi ican ly di e en ially exp essed i he co ec ed p- alue was below 0.05; Figu e 2B, Biomedicines 2022,10, 199 8 o 17 Table 1). Among he 47 genes showing signi ican ly highe exp ession in umo s, 13 (~28%) belonged o he Homologous Recombina ion (HR) pa hway ((A) in Table 1). Biomedicines 2022, 10, x FOR PEER REVIEW 9 o 19 Figu e 2. Exp ession p o ile o DNA damage and epai genes in 11 no mal allopian ube and 38 umo samples analyzed wi h he NanoS ing nCoun e assay. (A) Hea map o mRNA coun s ob ained wi h he NanoS ing nCoun e assay o DNA damage and epai genes in 11 no mal allopian ube and 38 umo samples. Genes a e depic ed in ows and samples in columns. Bo h ows and columns ha e been hie a chically clus e ed based on Spea man co ela ions. No mal allopian ube (FT) samples a e labeled in g ey, whe eas umo samples (T) a e labeled in blue. No e ha he da a ha e been scaled and cen e ed in ows (se ing he ow mean o 0 and he ow s anda d de ia ion o 1) in o de o no malize o di e ences in p obe e iciencies. Tumo and allopian ube samples clus e sepa a ely, wi h ma kedly di e en exp ession pa e ns. (B) Di e en ial exp ession o DNA damage epai genes be ween umo and allopian ube samples, as de e mined Figu e 2. Exp ession p o ile o DNA damage and epai genes in 11 no mal allopian ube and 38 umo samples analyzed wi h he NanoS ing nCoun e assay. ( A ) Hea map o mRNA coun s ob ained wi h he NanoS ing nCoun e assay o DNA damage and epai genes in 11 no mal allopian ube and 38 umo samples. Genes a e depic ed in ows and samples in columns. Bo h ows Biomedicines 2022,10, 199 9 o 17 and columns ha e been hie a chically clus e ed based on Spea man co ela ions. No mal allopian ube (FT) samples a e labeled in g ey, whe eas umo samples (T) a e labeled in blue. No e ha he da a ha e been scaled and cen e ed in ows (se ing he ow mean o 0 and he ow s anda d de ia ion o 1) in o de o no malize o di e ences in p obe e iciencies. Tumo and allopian ube samples clus e sepa a ely, wi h ma kedly di e en exp ession pa e ns. ( B ) Di e en ial exp ession o DNA damage epai genes be ween umo and allopian ube samples, as de e mined h ough a wo- ailed Welch’s - es on anks wi h Bon e oni co ec ion pe o med on he NanoS ing coun s. Log2 old changes abo e 0 indica e highe exp ession in umo s, whe eas hose below 0 indica e highe exp ession in allopian ubes. Mos di e en ially exp essed genes show highe exp ession in umo s. Table 1. Genes signi ican ly di e en ially exp essed in umo s. (A) Genes Up egula ed in Tumo s. Pa hway Genes Apop osis BCL2L1, CASP8, MYD88 Base excision epai APEX2, FEN1, NEIL3, PARP1, SMUG1, UNG Base excision epai —T anslesion Syn hesis—Cell Cycle and Signaling PCNA Cell Cycle and Signaling AURKA, BUB1B, CDKN2A, KRAS, RAD21, RM12, SUMO3 Checkpoin Ac i a ion H2AFX Checkpoin Ac i a ion—Cell Cycle and Signaling CHEK1/2 Homologous Recombina ion and Fanconi Anemia BRIP1, BLM, BRCA2, FANCD2, FANCG, FANCI, GEN1, RAD51, RAD54L, UBE2T, USP1, XRCC2, XRCC3 Independen Repai Enzymes/Polyme ases DNA2, POLQ Independen Repai Enzymes/Polyme ases–Base excision epai POLD1, POLD4, POLE2 Independen Repai Enzymes/Polyme ases—Cell Cycle and Signaling MAD2L2 Independen Repai Enzymes/Polyme ases–Nucleo ide Excision Repai POLR2D, POLR2H Independen Repai Enzymes/Polyme ases–T anslesion Syn hesis HLTF Misma ch Repai MSH2 Misma ch Repai –T anslesion Syn hesis RFC4 Non-homologous End Joining—Cell Cycle and Signaling PRKDC T anslesion Syn hesis RAD18 Housekeeping SF3FA3 (B) Genes down egula ed in umo s. Pa hway Genes Apop osis AKT3, BCL2, NFKB2, PIK3R1 Base Excision Repai NEIL1, NEIL2, OGG1 Cell Cycle and Signaling ABL1, CCND2, CDKN1A, EGFR Checkpoin Ac i a ion TIPIN Homologous Recombina ion and Fanconi Anemia RAD51B, WRN Independen Repai Enzymes/Polyme ases ALKBH3, CRY1, POLK, REV1 Independen Repai Enzymes/Polyme ases–T anslesion Syn hesis POLI Misma ch Repai MSH3 Non-homologous End Joining LIG4 Nucleo ide Excision Repai XPA Nucleo ide Excision Repai —Apop osis PTEN Housekeeping COG7, NUBP1 We nex asked whe he he exp ession o BRCA1 and BRCA2 co ela es wi h he exp es- sion o he o he HR genes ha we ound up egula ed in umo s. We di ided he coho in o low and high g oups based on hei BRCA1/2 exp ession. The low g oup included umo s wi h BRCA1/2 mRNA le els equal o o below he median alue, while he high g oup included umo s wi h mRNA le els abo e he median. 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