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Ge mline NPAT inac i a ing a ian s as cause o he edi a y colo ec al cance 1
Sho i le: NPAT mu a ions in colo ec al cance p edisposi ion 2
Ma iona Te adas1,2*, S ephanie A. Schube 3,*, Julen Viana-E as i1, Dina Ruano3, Gemma 3
Aiza1,2, Maa je Nielsen4, Paula Ma ciel1, Ca li M. Tops4, Genís Pa a5, Hans Mo eau3, Da id 4
To en s6,7, Monique E. an Lee dam8,9, Gab iel Capellá1,2, Noel F.C.C. de Mi anda3, Lau a 5
Valle1,2,**, Tom an Wezel3,10**. 6
1. He edi a y Cance P og amme, Ca alan Ins i u e o Oncology; Oncobell P og amme,7
IDIBELL, Hospi ale de Llob ega , Ba celona, Spain.8
2. Cen o de In es igación Biomédica en Red de Cánce (CIBERONC), Mad id, Spain.9
3. Depa men o Pa hology, Leiden Uni e si y Medical Cen e, Leiden, The Ne he lands.10
4. Depa men o Clinical Gene ics, Leiden Uni e si y Medical Cen e, Leiden, The11
Ne he lands12
5. CNAG-CRG, Cen e o Genomic Regula ion (CRG), The Ba celona Ins i u e o Science13
and Technology, Ba celona, Spain14
6. Li e Sciences Depa men , Ba celona Supe compu ing Cen e (BSC), Ba celona, Spain15
7. ICREA, Ba celona, Spain16
8. Depa men o Gas oen e ology and Hepa ology, Leiden Uni e si y Medical Cen e,17
Leiden, The Ne he lands18
9. Depa men o Gas oin es inal Oncology, Ne he lands Cance Ins i u e, Ams e dam, The19
Ne he lands.20
10. Depa men o Pa hology, Ne he lands Cance Ins i u e, Ams e dam, The Ne he lands.21
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*These au ho s con ibu ed equally o his s udy.23
** Senio and co esponding au ho s. 24
his is an AAM, he inal e sion can be oud a :
Te adas, M., Schube , S.A., Viana-E as i, J. e al. Ge mline NPAT inac i a ing a ian s as cause o he edi a y colo ec al cance . Eu J Hum
Gene (2024). h ps://doi.o g/10.1038/s41431-024-01625-8
© 2024 Sp inge Na u e Limi ed
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Co espondence o:
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Lau a Valle, PhD. He edi a y Cance P og am, Ca alan Ins i u e o Oncology, IDIBELL. A .
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G an Via 199-203, 08908 Hospi ale de Llob ega , Ba celona, Spain. Email: [email p o ec ed]
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Tom an Wezel, PhD. Depa men o Pa hology, Leiden Uni e si y Medical Cen e, Leiden
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Uni e si y, Albinusd ee 2, 2333 ZA Leiden, The Ne he lands. Email: [email p o ec ed]
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ABSTRACT
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Two independen exome sequencing ini ia i es aimed o iden i y new genes in ol ed in he
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p edisposi ion o nonpolyposis colo ec al cance led o he iden i ica ion o he e ozygous loss-o -
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unc ion a ian s in NPAT, a gene ha encodes a cyclin E/CDK2 e ec o equi ed o S phase
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en y and a coac i a o o his one ansc ip ion, in wo amilies wi h mul iple membe s a ec ed
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wi h colo ec al cance . En ichmen o loss-o - unc ion and p edic ed dele e ious NPAT a ian s
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was iden i ied in amilial/ea ly-onse colo ec al cance pa ien s compa ed o non-cance gnomAD
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indi iduals, u he suppo ing he associa ion wi h he disease. P e ious s udies in D osophila
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models showed ha NPAT ab oga ion esul s in ch omosomal ins abili y, inc ease o double s and
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b eaks, and induc ion o umou o ma ion. In line wi h hese esul s, colo ec al cance s wi h NPAT
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soma ic a ian s and no DNA epai de ec s ha e signi ican ly highe aneuploidy le els han
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NPAT-wild ype colo ec al cance s. In conclusion, ou indings sugges ha cons i u ional
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inac i a ing NPAT a ian s p edispose o misma ch epai -p o icien nonpolyposis colo ec al
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cance .
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Keywo ds: Colo ec al cance p edisposi ion; He edi a y cance ; Cance gene ics; Nonpolyposis
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colo ec al cance .
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INTRODUCTION
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Lynch synd ome is he mos common inhe i ed nonpolyposis colo ec al cance (CRC) synd ome,
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es ima ed o occu in 1 o e e y 280 o 400 indi iduals (1-3). I is caused by ge mline pa hogenic
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a ian s o epimu a ions in he DNA misma ch epai (MMR) genes MLH1, MSH2, MSH6, PMS2
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and i is cha ac e ised by he p esence o umou MMR de iciency (dMMR) (4). In con as , he
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gene ic basis o amilial and/o ea ly-onse nonpolyposis MMR-p o icien (pMMR) CRC emains
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la gely unexplained. Ge mline pa hogenic a ian s in RPS20 ha e been unequi ocally linked o
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an inc eased isk o pMMR nonpolyposis CRC (5). howe e , hey explain a negligible numbe o
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unsol ed amilies (6).
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He e, NPAT, iden i ied h ough wo independen s udies, eme ges as a new candida e gene o
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pMMR CRC p edisposi ion. Mul iple pieces o e idence suppo i s associa ion wi h he disease,
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and we p opose a po en ial ca cinogenic mechanism o NPAT, esul ing in high ch omosomal
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ins abili y.
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MATERIALS AND METHODS
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This s udy includes wo independen coho s o gene ically unexplained, pMMR amilial and/o
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ea ly-onse nonpolyposis CRC pa ien s, which include 265 Spanish and 106 Du ch pa ien s.
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GnomAD .2.1.1 non-cance indi iduals we e conside ed as con ol popula ion (7). Addi ional
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de ails on he cases and con ols s udied, and a desc ip ion o he me hods used, which include
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exome sequencing and a ian p io i iza ion s a egies, NPAT mu a ional sc eening in alida ion
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coho s, soma ic second hi assessmen s and s a is ical analyses, a e included in Supplemen a y
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Ma e ial and Me hods.
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RESULTS AND DISCUSSION
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Ge mline he e ozygous NPAT a ian s in amilial and/o ea ly-onse CRC
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In wo pMMR CRC amilies o di e en geog aphic o igins (Spain and he Ne he lands) (Figu e
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1), wo independen exome sequencing analyses we e conduc ed. No al e a ions o a ian s o
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unknown signi icance in known he edi a y cance genes we e de ec ed. He e ozygous loss-o -
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unc ion a ian s in NPAT (NM_002519.2: c.781G>T;p.(Glu261*) and c.3452C>A;p.(Se 1151*)
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[p o ein-coding sequence leng h: 1,427 amino acids] we e iden i ied in Family A and Family B
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espec i ely. Nei he o hese a ian s a e epo ed in public popula ion da ase s (gnomAD .2.1.1,
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.3.1.2), Collabo a i e Spanish Va ian Se e (h p://cs s.babelomics.o g/), o Genome o he
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Ne he lands (GoNL); accessed Feb ua y 2024), excep o one he e ozygous c.3452C>A
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indi idual in 380,952 gnomAD .4.0.0 non-Finnish Eu opeans. The a ian and gene p io i isa ion
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s a egies ha led o he selec ion o NPAT as candida e gene a e de ailed in Supplemen a y
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Ma e ial and Me hods.
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Six CRC-a ec ed amily membe s we e he e ozygous o he co esponding NPAT a ian
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(Figu e 1). In hese pa ien s, ( i s ) CRC was diagnosed a a mean age o 64 ( ange: 48-82), and
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no ex acolonic umou s we e epo ed. In Family A, only one o h ee CRC-a ec ed membe s
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was a ailable o analysis. No biological ma e ial om he CRC-a ec ed o sp ing, diagnosed
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wi h CRC ages 59 and 32 could be ob ained. In Family B, all i e CRC-a ec ed membe s we e
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he e ozygous o obliga ed he e ozygous o NPAT c.3452C>A;p.(Se 1151*), as well as 10
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ela i es wi hou a CRC diagnosis (age ange: 50-77). Due o he s ong CRC amily his o y, hese
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10 indi iduals had unde gone pe iodic colonoscopy su eillance. In se en, p emalignan lesions
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we e iden i ied and emo ed (mean age: 64.5), which may ha e p e en ed CRC occu ence. The
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emaining h ee had no colon umou pheno ypes a ages 50, 51 and 55. Eigh addi ional amily
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membe s, all cance - ee (age ange: 68-79), had wild ype NPAT geno ypes.
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To u he in es iga e he po en ial associa ion o NPAT wi h CRC p edisposi ion, we compa ed
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he equency o loss-o - unc ion and/o p edic ed dele e ious alleles in NPAT in pa ien s s.
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con ols. We e alua ed NPAT mu a ional s a us in 464 Spanish and 105 Du ch addi ional
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amilial/ea ly-onse pMMR nonpolyposis CRC p obands (1 CRC-a ec ed indi idual/ amily)
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om he same coho s as Families A and B, and analysed he publicly a ailable da a om 1,006
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un ela ed CRC pa ien s diagnosed be o e age 60 (da a accessed ia h ps://can a .ic .ac.uk/) (8,
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9). We included 59,095 gnomAD ( .2.1.1) Eu opean non-Finnish, non-cance indi iduals as
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con ols. Loss-o - unc ion and p edic ed damaging a ian s iden i ied in cases and con ols a e
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lis ed in Table 1. In addi ion o he wo unca ing a ian s iden i ied in Families A and B, wo
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addi ional NPAT loss-o - unc ion a ian s (c.1755del;p.Glu586Asn s*30, and
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c.2635G>T;p.Gly879*) and one missense p edic ed pa hogenic a ian (c.2549T>C;p.Ile850Th ;
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REVEL: 0.544) we e iden i ied among he 1,577 amilial and/o ea ly-onse CRC pa ien s
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e alua ed. Based on he h ee coho s s udied, 0.25% (4/1,577) o amilial/ea ly onse CRC
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pa ien s ca ied a he e ozygous NPAT loss-o - unc ion a ian , o 0.32% (5/1,577) when
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conside ing bo h loss-o - unc ion and p edic ed dele e ious (REVEL >0.5) a ian s. Allele
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equencies o NPAT loss-o - unc ion a ian s in amilial/ea ly-onse CRC pa ien s (4/3,154;
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MAF=0.13%) and con ols (18/118,190; MAF=0.015%) esul ed signi ican ly di e en , wi h an
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odds a io (OR) o 8.3 (95% CI: 12.0 - 25.3; p=0.0023; Fishe ’s exac es ). The di e ences we e
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also obse ed when bo h loss-o - unc ion and p edic ed damaging NPAT a ian s we e conside ed
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(5/3,154 (MAF=0.16%) s. 26/118190 (MAF=0.022%); OR=7.2; 95% CI: 2.2-19.1; p=0.0011)
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(Table 1). The esul s o his en ichmen es may be subjec o bias due o he u iliza ion o
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di e en sequencing app oaches in cases and con ols, and among di e en coho s o cases. A
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alse posi i e esul due o he modes sample size o he case g oup and he a i y o ge mline
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NPAT damaging a ian s migh ha e also occu ed. A alida ion s udy wi h la ge coho s is
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needed o con i m he e ec s obse ed.
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Based on gnomAD .4.0.0, he a io o obse ed s. expec ed loss-o - unc ion a ian s in NPAT
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is low (36 obse ed s. 100.2 expec ed = 0.36; 90% CI: 0.27-0.47), wi h a p obabili y o loss o
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unc ion in ole ance (pLI) o 0.99. This sugges s ha NPAT is ela i ely in ole an o p o ein
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unca ion, like o he cons ain genes ha when mu a ed cause Mendelian diseases, including
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au osomal dominan he edi a y cance synd omes (10). To da e, no o he Mendelian synd ome has
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been associa ed wi h pa hogenic NPAT a ian s (h ps://www.omim.o g/; Feb ua y 2024).
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In e es ingly, NPAT sha es i s p omo e wi h he cance p edisposi ion gene ATM (bidi ec ional
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520-bp p omo e ). Howe e , an e ec on ATM exp ession o NPAT loss-o - unc ion a ian s,
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which do no a ec he p omo e DNA sequence, seems unlikely.
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NPAT soma ic s a us: e idence suppo ing haploinsu iciency
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The loss-o - unc ion na u e o he a ian s sugges s a umou supp esso na u e o NPAT. No
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soma ic second hi (LOH o mu a ion) was iden i ied in he CRC de eloped a age 76 by he
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p oband o Family A, howe e , soma ic me hyla ion o he NPAT p omo e was no analysed and
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no o he umou samples om Family A o B we e a ailable o analysis.
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We con i med he exp ession o he (no mal) majo NPAT ansc ip in no mal colon mucosa
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(CoT Ex .2; h ps://ba cu aseq.o g/co ex/) and e alua ed he p esence and equency o soma ic
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mu a ions in NPAT among he di e en TCGA umou ypes (sou ce: www.cBioPo al.o g). The
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mos equen ly NPAT-mu a ed cance s we e endome ial ca cinoma (NPAT al e ed in 9.45% o
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529 umou s), s omach adenoca cinoma (6.36% o 440), cu aneous melanoma (5.63% o 444),
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and CRC (4.55% o 594) (Figu e 2A), being colo ec al, endome ial, gas ic cance s he mos
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ep esen a i e umou s in known he edi a y CRC synd omes (e.g. Lynch synd ome).
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To ind e idence suppo ing NPAT haploinsu iciency, as he po en ial absence o a second hi in
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he umou o he Family A p oband sugges ed, we u he analysed he soma ic s a us o he gene
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in he NPAT-mu a ed TCGA CRCs. Due o he mu a ional complexi y o hype - and ul a-mu a ed
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umou s, we s udied he TCGA umou s wi h nei he dMMR no mu a ions in he exonuclease
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domains o polyme ases ε (POLE) o δ (POLD1). These included one CRC wi h a s op-gain
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mu a ion, and i e CRCs wi h i e di e en soma ic missense a ian s. None o he six umou s
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had a second a ian in NPAT, and he allele equency o he a ian s ( ange: 9% - 56%) did no
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sugges loss o he wild ype NPAT allele (Suppl. Table 1), suppo ing, al hough no de ini i ely,
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haploinsu iciency o his gene.
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NPAT unc ion and i s po en ial ole in ca cinogenesis
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NPAT is a cyclin E/cyclin-dependen kinase 2 (CDK2) e ec o equi ed o p og ession h ough
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he G1 and S phases o he cell cycle and o S phase en y, and is a key componen o he his one
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locus body, in ol ed in he ansc ip ion o canonical his one genes and in p e-mRNA p ocessing
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(11, 12). No associa ion o NPAT mu a ions wi h CRC p edisposi ion has been epo ed o da e.
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Howe e , ge mline NPAT c.2437-2438del;p.(Leu814Phe s*6) was iden i ied in ou membe s o
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a amily a ec ed wi h Hodgkin lymphoma in hei 20s, and in i e cance - ee ela i es (13).
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S udies in D osophila showed ha : i) NPAT (o holog o mxc in D osophila) mu a ions esul in
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ch omosomal ins abili y caused by abno mal ch omosome seg ega ion p omo ed by diminished
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canonical his one le els (14); ii) NPAT (mxc) knockdown causes inc eased a e o DNA double
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s and b eaks (15); and iii) se e al s udies suppo he umou supp esso ole o NPAT (mxc) in
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his model o ganism (16-18). Based on he e idence obse ed in D osophila whe e NPAT/mxc
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wild ype would p e en ch omosome ins abili y, we analysed he cha ac e is ics o he six TCGA
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non-hype /ul amu a ed CRCs wi h NPAT soma ic a ian s. The analysis, pe o med h ough he
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cBioPo al websi e (h ps://www.cbiopo al.o g/), e ealed a signi ican ly highe aneuploidy le el
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(numbe o al e ed ch omosome a ms) in hese umou s han in NPAT-wild ype CRCs wi h he
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same cha ac e is ics (n=50) (Figu e 2B; TCGA umou s), suppo ing he obse a ions in
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D osophila and p o iding a po en ial explana ion o he NPAT-d i en ca cinogenic mechanism.
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Un o una ely, he e alua ion o ch omosomal ins abili y in he umou s o he pa ien s
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he e ozygous o NPAT dele e ious a ian s could no be pe o med due o una ailabili y o
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umou ma e ial.
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CONCLUSIONS
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Ou indings s ongly sugges ha inac i a ing ge mline pa hogenic a ian s in NPAT a e
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associa ed wi h an au osomal dominan p edisposi ion o pMMR CRC. A ailable e idence,
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al hough s ill sca ce, sugges s ha NPAT de iciency migh lead o high ch omosomal ins abili y
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in umou s. Unlike mos umou supp esso s, soma ic inac i a ion o he second allele does no
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seem o be equi ed, al hough addi ional e idence is needed o con i m haploinsu iciency. Fu he
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con i ma ion o he causal associa ion o NPAT wi h CRC p edisposi ion is needed be o e
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inco po a ing his gene in o gene ic es ing in clinical p ac ice.
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DATA AVAILABILITY
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Da a suppo ing he epo ed esul s may be ound in he a icle o supplemen a y ma e ial.
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Addi ional da a, analy ic me hods, and s udy ma e ials will be made a ailable o o he esea che s
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upon eques .
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REFERENCES
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1. Win AK, Jenkins MA, Dow y JG, An oniou AC, Lee A, Giles GG, e al. P e alence and
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Pene ance o Majo Genes and Polygenes o Colo ec al Cance . Cance Epidemiol
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Bioma ke s P e . 2017;26(3):404-12.
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