Brian Gabrielli 1. QIMR Berghofer Medical Research Institute, Herston, Australia. This article is protected Allrights bycopyright. reserved. 10.1111/pcmr.12487 of Record.versiondifferences and article Please between the cite this Version this as doi: through the copyediting, typesetting, pagination for publication accepted This articlehasbeen QIMRBerghofer Medical ResearchInstitute, Walker, Dr. Graeme *Corresponding author: Running title:Mapping congen a Keywords: Congenital nevi,qtl,mice. * These authors contributed equally to the project. Australia 4. Dermatology Research Centre, UQSchool of Medici 3. Centre for Diabetes Research, Harry Perkins Institute of Medical Research, Perth, Australia. Research Institute, Brisbane, Australia 2 Chitsazan Arash Manuscript Category: Signaling& Cell biology (SCB) Article type :ShortCommunication Date:04-May-2016 Accepted Revised Date:01-May-2016 :03-Feb-2016 Date Received . TheUniversity ofQueensland Diamantina Institute, The University ofQueensland (UQ), Translational

Accepted ArticleA mutationinthe

2 , Peter HSoyer 1, 2 1, ,

Blake Ferguson development mediatedbyNRAS 4 , Grant Morahan Cdon 1 ital nevus modifier ital nevusmodifier , Ramesh Ram gene potentiates congenital nevus nevus potentiates congenital gene 3* , Graeme J. Walker and undergone full peer review buthasnot been review undergone fullpeer and 3 , Pamela Mukhopadhyay and proofreading process, which may lead to

ne, TranslationalResearch Institute,Brisbane, 1* . 1 ,

Q61K Herlina Y.Handoko Herlina 1 , multiple are involved). We examined varia examined We multiple genes involved). are where (those diseases complex genes for ofmodifier identification enables that aresource (CC), escaping hair theconfinesfollicles. co of We transgenes congenital develop lesi nevus-like This article is protected Allrights bycopyright. reserved. becomes trapped inthe dermis during development. Mice bearing the of age the acquisition from arise presumed to before Congenital nevidevelop Summary. Email: [email protected] +61738453508 Tel: +61738453715,Fax: QLD 4006,Australia. 300 HerstonRd,Herston, Accepted t for childhood melanomais significantly increased due tole itching, andoverheating neviareoftenassociat Largecongenital al., 2013). giant lesions cove lesions ontheheadandneck,or assmallcommonpl manifest can Congenital nevi lesions. for these measures andcontrol treatments genes that control the development ofthese lesions willbe a firststep in the development ofnew define innatevariation development ofcongenita to association studieshavebeenperformed lo can be they since removed easily not Theyare c of causeavariety Giant congenitalnevican Significance. mainly inkeratinocytes. expressed ofan context the in nevus development congenital exacerbates gene that fora candidate 9.Thebest Articlemapped a large effect quantitative trait locus (QTL) controlling cell nevus density tomurine NRAS that dramatically influences the dens the influences dramatically that mutation is l nevi.For thefirsttime, asystems weutilized genetics to approach birth, and sometimes cover large areas of the body. They are They are thebody. of areas coverlarge and sometimes birth, ss subcutaneousfat.The risk Cdon , a positive regulator of (Shh)thatis , apositive regulator ofsonichedgehog mbined thesembined mice Collaborative withthe Cross helpusunderstandthesusceptibility tothe linical problems, and can convert to melanoma. tomelanoma. linical problems,andcanconvert ons by post natal day 10, from melanocytes ons by10, frommelanocytes postnatalday ne mutation in an embryonic melanocyte that that melanocyte embryonic mutationinan ne cated deep withinthe Nogenome dermis. wide tion innevuscelldensity ace “birthmarks” anywhere on the body, large large thebody, on anywhere “birthmarks” ace ring substantialof the torso(Krengel areas et ed with increased skin fragility, dryness and and skinfragility, dryness ed withincreased hose with giant congenital nevi (Krengel et nevi(Krengel hose withgiant congenital ity of congenital nevi. Understanding ity nevi.Understanding ofcongenital of otherwise extremely rare rare extremely of otherwise Cdk4 in 66CCstrainsand R24C ::Tyr-NRAS Q61K

an embryonic melanocyte precursor. precursor. melanocyte an embryonic ar they hypodermis, and sometimes deepdermis s occasionally or before Congenital develop nevi risk. similar patientsdevelope in melanoma reported pediatric generated progeny forboththe generated heterozygous Cdk4 To investigategenetic innate that variation regulates congenital development, nevus weused signaling Wnt w al. 1998,2000),orincreased ofstemcellorhepatocyte changes inexpression in results that engineering genetic with altered canbedramatically lesions ofthese and size Dohmenetal.2009;Shakhova nevi (e.g. mutations developdermal Many transgen important innevusdevelopment. whereby variation inepigenetic ormelanocyt inna informationabout islittle there However, congen giant present in~95%of By breeding mouse coatcolour theCC proof ofconcept, Ina sequence isknown. inherite blocks thehaplotype knowledge of for locus atany sequence genome involved). The genes fo discovery of the making itidealfor et species (Churchill inthe variation present genetic of ~90% captures and founder strains diverse eight from The CCisderived al. 2012). mousestrains,the Collabora genetically-defined This article is protected Allrights bycopyright. reserved. In studyal. 2006). prospective oflarg anearly Accepted days etal.2014),atbetween reported (Chai Article R24C/R24C ::Tyr-NRAS Cdk4 (Ram et al. 2014). etal.2014). (Ram R24C/R24C Q61K d melanoma in fourteen fourteen d melanomain melanocytic proliferations remini melanocytic proliferations ::Tyr-NRAS mice 2010,2015)in (Ferguson etal., conjunction withacohortof 2.3%, while Vourc’h-Jourdain et Vourc’h-Jourdain 2.3%, while ital melanocytic nevi (Kinsleret ital melanocytic NRAS Q61K/+ Q61K males withfemalesmany from CCstrains we ithin melanocytes (Pawlikowski etal.2013). melanocytes (Pawlikowski ithin al., 2004, Collaborative Cross Consortium2012), 2004,CollaborativeCross al., d from the founder(s), for which the genome whichthe for from thefounder(s), d 2012; Pawlikowskietal.2015). Thedevelopment mutation is the most common somatic mutation, e thought to arise from anoncogenic mutation in r complex traits (those where many genes are genes are wheremany (those traits complex r Cdk4 tive Cross (CC) (Morahan etal.2008; etWelsh tive Cross(CC)(Morahan ic mice carrying melanocyte-specific oncogenic oncogenic melanocyte-specific carrying mice ic e-microenvironment interactions may interactions e-microenvironment also be e congenitalnevus e 0 and 5 after birth (P0-P5) melanocytes are melanocytesare 0 and5afterbirth(P0-P5) growth factor from keratinocytes (Kunisada et et (Kunisada keratinocytes factorfrom growth oon after birth. Located in the superficial and and in thesuperficial Located birth. oon after could rapidly map the variants responsible for responsiblefor could rapidly mapthe variants selection-based studies, about a 2% absolute abouta2% studies, selection-based te genetic differences between individuals, between genetic differences te each strain is easily inferred based upon based inferred strainiseasily each and NRAS scent of human congenital or blue orblue scent ofhumancongenital al. (2013) found that 51 of 2578 2578 foundthat51of al. (2013) mutations. Aswe previously al. 2013; Charbel et al, 2014). al. 2013;Charbeletal,2014). patients,Haleetal.(2005) onset of stage 2/3 melanomas with the nevus cel nevus withthe melanomas 2/3 onset ofstage these from emanated they have suggests that 2or3me early stage of thesedermalnevi,and nevus cell density ( nevus celldensity betweenth count nevus we compared scored between 1 (least dense) and 10 (most dense). (mostdense). and10 (least dense) 1 scored between was dermis in cells ofnevus density and the cross, each of >3 mice from skin collected We congregation of any of there wasnosign the 12 strains for which we had data forboth we haddata the 12strainsforwhich depende countwas whether nevus amela had developed (generally was sacrificed strain isshowninThe medianscoreper Figure score. thedensity for marker we butessentially “nevus cells”, (i.e. dark pigmentation coveringa large portionofthe also dermis), try we toassess dendritic nevus dendritic For cells.the lightly heavily number (brown) pigmented of pigmented strains ligh For (1-10). scores the of ofeach examples greatly givencan vary section,soanes a across relative densitycell ofnevus nuclei across the Note section. that this, being a biological variable, cells rightacrosstheskinsec density ofnevus for the score arbitrary an instead, skin, mouse each in ofnevi number the of ameasure not surface over theirdorsal nevi many thousandsof This article is protected Allrights bycopyright. reserved. Accepted ArticleNRAS from melanocytes thatfrom the follicle break ( away hair follicles( hair dermis, around into P21thesehavedeveloped epidermis, andby not normally persist at the epidermal-dermal AtP10there junction). are melanocytes more inthe migratingfolliclethe humans, hair (unlike mouse to in interfollicular downwards melanocytes do Q61K , melanocyte numbers were much lower at numbers weremuchlower , melanocyte Figure 1D,E Figure 1A ) isgenetically determined. We scored based on the area covered by dense pigmentation as a asa pigmentation dense by covered thearea basedon scored nt upon whether ornot amouse nt uponwhether e two groups and found no correlation ( andfoundnocorrelation groups e two melanocytes in the form ofnevi( intheform melanocytes ). Hence the nevi begin to develop at P10, apparently atP10,apparently todevelop begin thenevi Hence ). spontaneous andneonatal spontaneous nevi (Wurm et al. 2010). We compared the age of age the compared 2010).We etal. (Wurm nevi tly pigmented low nevus count strains we assessed assessed strainswe count lownevus pigmented tly tion analyzed. For albino strains we estimated the the weestimated strains For albino analyzed. tion lanomas whoselocation in lanomas S4. Normal skin was collected when each mouse mouse wheneach wascollected skin Normal S4. l density score for all mice in our study ( study inour all mice for score density l timate hastobemade.Figures S2andS3show noma of ~10mm in diameter). We determined determined noma of~10mmindiameter).We (Chai et al. 2014). Note that nevus density is thatnevusdensityNote is (Chai etal.2014). nests ofcellswithin the superficial (nevi) Cdk4 all timepoints, and R24C/R24C Figure 1B previously reported the presence reported thepresence previously ::Tyr-NRAS had received neonatal UVR.In had received ). In littermates ). notcarrying UVR-induced melanoma melanoma UVR-induced Figure S1 Figure between P10andp42 the superficial dermis dermis superficial the Q61K/+ Figure 1C micedevelop ). ). Hence ). Hence Figure Figure wide linkage. We identified a major effect QTL on mouse chromosome 9 (-1-Log chromosome onmouse QTL effect amajor identified linkage. We wide discovery false useda We chi-squared). (ANOVA reconstruc the model over detected linkageat lowerlevels ofsignificanceto ( density in strain per testedWe 66 CC strains todiscover quantitative trait loci (QTLs) that modify nevus median Rank Correlationtestwith haplotypes is described in Ram haplotypes isdescribedin log strains (see DNAvariants cataloguing the bestcandidatesby 70genes).42.8 Mb,containing began We to about interval=12.3-17.4 Mb, containing 13 interval=12.3-17.4 Mb, (Mb), interval=28.2-36.2 megabases This article is protected Allrights bycopyright. reserved. 1F linked interval ( candidates are listed in Figure listedin are candidates product isinvolvedintRNAmeta is mutation missense specific a NOD- carrying 9interval thechromosome gene in other 2D).The UTR, orintrons(Figure mutations, while11hadNOD-specific ( genes just2 revealed variants NOD-specific Mining termedNOD). founder(hereafter NOD/LtJ carries the causal allele, and of the 70 andofthe allele, causal the carries (Houetal.,2015) behavior regulates neuronal missensevariant, NOD-specific “susceptibility” chromosome candidate 11only one alleleinheriteda On carries NOD. is from genes ( 13 11carries chromosome on The interval biology.

Accepted Article correlation(correlati foundanegative ), butwe 10 (P) values). For instance, examination ofth examination (P) values).For instance, Figure 2B Figure 2C Figure Cdk4 for the coefficients together with the corresponding ANOVA test – test ANOVA corresponding the with together coefficients for the ted haplotypes matrix to produce ge toproduce matrix ted haplotypes an in-house R script). an in-houseRscript). ) onchromosome9shows thatthecausalvariantisderivedfrom R24C 1d. Most of these have noknown have Mostofthese 1d. et al.(2014).For mapping weused Pus3 ::Tyr-NRAS Cobl bolism but not skin or melanocyte biology. Other less likely lesslikely Other bolism butnotskinormelanocyte biology. (Cordon-Bleu). It encodes a thatbindsIt actinand aprotein (Cordon-Bleu). encodes genes), and chromosome 19 (-1 -Log chromosome19(-1 genes), and single nucleotide polymorphisms (SNPs) intheir5’or3’ polymorphisms (SNPs) nucleotide single (encoding Pseudouridylate Synthase 3), whose protein whose protein 3), Pseudouridylate Synthase (encoding a region containing 23genes) ( genes in the 4 Mb interval, only one, genes the4Mbinterval, only in Q61K . On chromosome 19 the NOD haplotype again again NOD haplotype On chromosome 19the . mice. The construction of the CC founder CCfounder ofthe construction mice.The on co-efficient = -0.18, p= 0.00048, Spearman 0.00048,Spearman =-0.18,p= on co-efficient e founder haplotype coefficients through the the through coefficients e founderhaplotype Cdon rate of p=0.0001 to define significant genome significant todefine ofp=0.0001 rate ascertaingenes within the which intervals are the Sanger Mouse Genomes database for for database MouseGenomes theSanger that vary between susceptible orresistant that vary susceptible between and Figure S5A,B Figure nome-wide distribution of P values ofPvalues distribution nome-wide Pus3 ) carrying ) carrying non-synonymous a logistic regressionmatrix alogistic role in skin or melanocyte skinormelanocyte role in Figure S5 Figure ). Here also the nevus alsothe Here ). Figure 2A 10 (P) interval=38.5- (P) Tctn3 , (-1-Log ). We also). We (Tectonic 10 10 (P) (P) ( interacting withTyrosinase ( particularly Shh(sonichedgehog). expressed inthe follicular inner rootsheath ( acceptabl were notabletoobtain may bemostly withinfollicul networks, Shhwasthestrongest gene associated significantgenes. genesinte Themost between to database providesameans of FunctionalNetworks et al.2007).The (Stecca GLI1 signaling inhuman hair follicle matrix c viacross-talkbetween melanoma progression et (Allen (SHH) hedgehog sonic activator of and interactions ofthe mutant This article is protected Allrights bycopyright. reserved. theChromosome9peakThe bestcandidateunder is 9locuswasunder altogether, thechromosome separately ( genomewide linka a pigmented weran strains, scoringBecause celldensit ourmethodof nevus here. located is formation nevus congenital modifying locus effect alarge suggesting genome, thelinkage tochro nevus,however for congenital betw cooperation generally involves suggesting betweenthecausal somecooperation chromosome 9also the carry susceptibility haplotype onchromosomesand 19,( 11 to regulate melanocyte that behavior. carry Many susceptibility strains the haplotype on FamilyMember 3) carries a NOD-specific missens escape the melanocytes mice,may follicle hair nevus-prone of inthe from it disruptingbe via The Immunoglobulinfive andthreefibronectin (Ig) missense mutation;none oftheother AcceptedKrt14 Article Cdon ) toidentify keratinocyte-specificIn networks. the P456S Figure S6 Figure mutation is in the Ig domain and could modify the glycosylation, phosphorylationglycosylation, modify could Ig the and mutationis domain inthe ). Despite the relatively samples smaller sizes than with all strains analyzed Tyr ar keratinocytes. Using a commercial anti-CDON antibody we Using anti-CDON acommercial ar keratinocytes. assess tissue-specificco-expressi protein. Cdon is aneuralcell e staining mouseskin,butinhuman of skin CDON ismostly ) toidentify and Keratin melanocyte-specific 14 networks, We stratifiedthehair We een multiplelowriskgeneticva een founder strains havethischange( founder strains ontrols normal proliferation of human melanocytes the most significant peak in both cases. both cases. mostsignificantthe peakin the GLI and RAS-MEK/AKT pathways. SHH- GLI andRAS-MEK/AKT the Figure 2E,F Figure type III domains (Sanchez-Arrones etal.2012). IIItype (Sanchez-Arrones domains al. 2011). SHH-GLI signaling is crucial for crucial is signaling al. 2011).SHH-GLI , suggesting, thattheactivity of Cdon viaShh racting with Cdon were withCdonwere racting ge analysis on albino and pigmented strains strains and pigmented onalbino ge analysis TissuesinMouse(http://fntm.princeton.edu) e variant. Neither Cobl nor Tctn3 are reported reported are nor Tctn3 Cobl Neither e variant. y foralbinoand isinsomeways different variants. Susceptibility tocommondiseases mosome 9isby strongesthitinthe the far Cdon folliclegene networ , the NOD allele of which carries a , theNODallele a ofwhichcarries Krt14 ). If). Cdonderegulation facilitates on andfunctional adhesion molecule that is an that molecule adhesion and riants. This may be similar Thismaybe similar riants. Cdon Figure 2D in thehairfollicle, k looking forgenes (butnotthe Figure S5C Figure ). Cdonhas interactions Tyr ), ), ) In a mouse model carrying thehuman Ina mousemodelcarrying lesion formationhastoourknow al. 2003).Thisnoncell- basal epidermis(Elliset pigmented lesionsonhind-limbs, dueto skin have hyperpigmented (Powell etal.2015).Further, miceove transgenic ofinterest. noconflict The authorsreport Interest of Conflict nevi. development ofcongenital astohowinnatege provide thefirstclues CC strains,allowing morede mi DO al. 2012). et (Churchill system outbred (DO)

densitya revealed highly significantchromosome locus 9, with on formation ofthenevi,notwhetherthey proceed gene we Further, the neonatal UVRexposure. the insomestrainshardlyprogeny developing Nevus ce take theCCapproach. nevi.Mice ofdermal mousemodels characterized c human phenocopy these micedonotperfectly melanocytesescapingfollicle. the atnevi day ~post-natal from hair formed 10,probably While This article is protected Allrights bycopyright. reserved. and cadherin localization, inzebr adhesion, since cell-cell Acceptedtesting furtherCCstrains withthe the Fine of mapping 11and19. nevus density. modifier driving Other high Article Cdon tailed mapping within theintervals.Thesenovelfindings within tailed mapping may knockout suppresses melanocyte migr knockout suppresses ledge not been followed up. followed notbeen ledge increased melanocytes near the epidermis, and develop discrete discrete anddevelop epidermis, nearthe melanocytes increased afish Cdon promotes migration by regulating N- regulating by migration crest neural promotes afish Cdon made up of masses ofhighly made upofmasses ll density varied greatly with genetic background, withCC withgenetic greatly ll density varied Cdk4 NRAS R24C Q61K ::Tyr-NRAS lesions. Nevuscelldensity lesions. to melanoma. Genetic linka netic variation may control thegrowthnetic variationmaycontrol and minor, butsignificantloci were located on autonomous influence of the Shh on pigmented Shhonpigmented influenceofthe autonomous cDNA, lesions very re cDNA,lesionsvery are the only mammals in which we could have couldhave whichwe mammalsin theonly are rexpressing Shhunderakeratin rexpressing genepromoter genomic intervals can now be performed by by now beperformed genomic intervalscan have localized seems toonly influence the ongenital nevus, they are one of the best areoneofthebest ongenital nevus,they ce carry many more recombinations than the thanthe recombinations more many ce carry Q61K mice and/or using the diversity mice and/orusing thediversity pigmented melanocytes near the the melanocytes near pigmented ation to the ventral surface ation totheventralsurface Cdon miniscent ofcongenital was not influenced by was notinfluencedby ge analysis ofnevus ge analysis a strong candidate candidate astrong congenital melanocy Associ al. (2005). Hale, E.K.,et Oncogene progression. tumor stages of all MelanomasusceptibilityFerguson,B., etal.(2015). 771-80 andmela naevus inmurine Arf/p53 pathways et Muller,H.,Handoko,H.Y., Ferguson,B., Konrad 263 em suppresses Hedgehog ofSonic Overexpression (2003). Ellis T,SmythI, Bowles Adolphe Riley C,Rothnagel Wicking E, J, C,Wainwright JA, BJ. This article is protected Allrights bycopyright. reserved. mice. Cancer Cell Braf Oncogenic Dhomen, N.,etal.(2009). congenital melanocy Charbel, NRASmutational. isthe (2014). sole C.,et recurrent somatic mutation inlarge Pigment Cell MelanomaRes. andestimation ne count probability of ofnevus B, T,Soyer P,Walk Chai E,Ferguson Prow Dev.Cell pathway function. CDO,andBOCinSHH GAS1, roles Overlapping (2011). etal. Allen, B.L., References. Council ofQueensland. This workwassupportedby Research Melanoma Acknowledgements Acceptedmouse genetic referenc Collaborative CrossConsortium. population. Mamm.Genome Churchill GA,GattiDM,Munger SC,Svenson Article , 203-15. 15 tic naevi--results fromtheNYU- tic naevi--results tic nevi. J. Invest. J. Dermatol. tic nevi. , 294-303. e population. Genetics 23 27 , 713-8. , 713-8. ation of melanoma and neurocutan ation ofmelanoma , 317-9. (2012). The genome architectur genome (2012). The 34 induces melanocyte senescence and melanomain senescence melanocyte induces , 2879-86. , 2879-86. 190 noma genesis.Pigmen noma KL. (2012). The Diversity Outbred mouse TheDiversity KL. (2012). er G. (2014). Three-dimensional modelling for modelling for Three-dimensional G.(2014). er and collective requirement for the coreceptors coreceptors forthe requirement collective and vus-melanoma progression ina murinemodel.vus-melanoma progression 134 , 389-401. bryonic hair follicle morphogenesis. Dev Biol. Dev bryonic folliclemorphogenesis. hair Alliance, Washington DC, and the Cancer Alliance,Washington DC,andtheCancer al. (2010). Differential roles ofthepRband al.(2010).Differential as a complex trait: gene a complex as , 1067-74. LCMN registry. Br J Dermatol. BrLCMN registry. J 20 , 775–787. e of the Collaborative Cross e ofthe eous melanocytosis with large eous melanocytosis with large t Cell MelanomaRes. t Cell tic variation controls tic variationcontrols 152 , 512-7. , 512-7. 23 , This article is protected Allrights bycopyright. reserved. Dermatol. ModelDrivenby inaMurine Nevus Syndrome signaling etal.(2013).Wnt Pawlikowski, J.S., S0012-1606 migration(2015). Cdonpromotesneuralcrest by Powell, D.R.,Williams, Hernandez-Lagunas,J.S., Development promotes survival,proliferation,differentia of expression Kunisada T,etal.(1998).Transgene growth andinvivo.PLoSmelanoma One. invitro antagonist reduces selectiveSMOOTHENED and apotent A.,etal.(2013).NVP-LDE225, Jalili, Is byNucleator Cobl Controlled TheActin Izadi, B. (2015). M.M.,andQualmann, S., Haag, M.,Nemitz, N.,Kessels, Hou, W., Pawlikowski, J.S., etal.InhibPawlikowski, J.S., (2015).Acute USA. rapid identification trait genes. ofcomplex Genome Mamm. L.,Morahan, G.,Balmer, Monley, D.(2008).Estab melanocyte development, leading todermal melanocytosis. Mech Dev. expression Keratinocyte Kunisada T,etal.(2000). AcadDermatol. Am categoriza recommendations forthe S.W., A., Dusza, S.,Scope, Krengel, Dermatol. Br J review. systematic T.(2006).Me A.,Schäfer, Krengel, S.,Hauschild, mutationsin postzygotic by are caused Kinsler, Multiple V.A.,et congenital al. (2013). melanocytic nevi and neurocutaneous melanosis AcceptedGenome C.E.,etal.(2012).Statusand acce Welsh, Article 110 23 , 16009-14. 135 , 706-12. , 706-12. , 30097-X. , 30097-X. 125 , 2093-101. , 2093-101. , 2915-23. , 2915-23. 68 , 441-51. Calcium and Calmodulin. PLoS.Calcium andCalmodulin. Biol. 155 tion of cutaneous featuresof tion ofcutaneous , 1-8. , 1-8. codon 61 of NRAS. J Invest codon61ofNRAS.J Dermatol. ss to theCollaborative ss to ition ofMEKSuppresses ition Vonthein, R., Marghoob, AA. (2013). New New (2013). AA. R.,Marghoob, Vonthein, tion andmigration of potentiates nevogenesis. potentiates nevogenesis. Activated Signaling. NRAS and Wnt Invest. J regulating N-cadherin localization. DevBiol. localization. regulating N-cadherin lanoma risk in congenital melanocytic naevi: a melanocytic naevi: lanoma riskincongenital 8 of transgenic hepatocyte growth factor affects growth affects factor of transgenichepatocyte L., Salcedo, E., O'Brien, J.H., Artinger, K.B. Artinger,L., J.H., K.B. E.,O'Brien, Salcedo, , e69064. lishment of "The Gene Mine": a resource for lishment of"The Mine": for aresource Gene steelfactorinthebasallayer ofepidermis 19 , 390-3. , 390-3. congenital melanocytic nevi. J Cross population. Mamm.Cross 13 94 Congenital Melanocytic melanocyte precursors. precursors. melanocyte , e1002233. , , 67-78. Proc. Natl. Acad. Sci. Proc. Natl.Acad. 133 , 2229-36. (F) Heat map showing the lack of correlation ofcorrelation lack showing the map (F) Heat with very density. low one panel, lower astrainA pigmented panel: high, withvery Upper strain. panel: high a withvery cell strain with very density.a density, nevus strain (E) lower low panel, (D) et al.,2010) (Ferguson asdescribed exposure 3- (A98004M). institute animalethicsapproval scored themblind,and a third(BF) agreed toneonatal and withoutpreviousexposure bulb.(C hair panel), the (right escaped panel), or and the RAS-MEK/AKT pathways. Proc. Natl. Acad. Sci. USA. USA. Acad. Sci. Natl. pathways. Proc. and theRAS-MEK/AKT HEDGEH (2007). Melanomasrequire I. F., Ruiz, Beermann, V., Piguet, R., Correa, V.,Zbinden, M., Clement, C., B., Mas, Stecca, supplementary methods file. supplementary methods nevi. arrowsdenote points afterbirth.Yellow This article is protected Allrights bycopyright. reserved. 1 Figure legends Figure Dermatol. NRAS(Q61K) model.Exp. of murinemelanocy A blueprintforstaging Soyer, H.P.(2012). G.J., Walker, Lambie, Prow,T.W., Ferguson,Lin,B., D., L.L., E.M., Wurm, 68 Dermatol. Acad. Am. J. review. asystematic risk: andmelanoma management therapeutic nevi: Martin,L., Vourc'h-Jourdain, Barbarot, M., CellBiol. Nat. naevi andmelanoma. promotesthe Sox10 (2012). Shakhova, O.,etal. 44 Biol. Cell Biochem Int.J. communication. cell-cell in implicated Two transmembrane M.,Nieto-LopeL., Cardozo, Sanchez-Arrones, geneseffect collaborative using the cross.Genetics Ram, R.,Mehta,M.,Balmer,L., D.M.,Mora Gatti, , 698-702. Accepted , 493-8. Article . (A) Sox10-stained skin taken from skintaken Sox10-stained . (A) ( b). Yellow arrows show Sox10-positive melanocytes Sox10-positive escaping arrowsshow (left b). Yellow 14 OG-GLI signaling regulated by interactions between GLI1 by betweenGLI1 interactions regulated OG-GLI signaling 21 , 882-90. , 676-81. , 676-81. tic lesions basedontheCdk4(R24C/R24C)::Tyr-tic lesions between congenital nevus congenital between Cdk4 UVR. independently Twopeople(ACandGW) on the score. Experiments were undertaken with with were undertaken on thescore.Experiments S; aRED. (2013). Large (2013). S; aRED. z, F., and Bovolenta, P. (2012). Cdon and Boc: Boc: Cdonand P.(2012). Bovolenta, F., and z, See immunohistochemistry methodsinstaining See ) Average nevus score per strains for mice with with mice strainsfor per score nevus Average ) day-old mice were exposed to a single UVB UVB toasingle exposed were day-old mice formation andmaintenan H&E skin sections from albinostrains.Upper H&E from skinsections R24C/R24C han, G. (2014). Rapid han, G.(2014). 198 , 75-86. ::Tyr-NRAS 104 , 5895-5900. , 5895-5900. Q61K cell density and neonatal densitycell and neonatal congenital me identification ofmajor- mice at various time- various at mice ce of giant congenital lanocytic lanocytic points after birth. (B). (B) Sox10-stained skin taken from Sox10-stained points afterbirth.(B). (B) after birth.Seeimmunohistoch This article is protected Allrights bycopyright. reserved. S3 Figure haplotype. denotesanintronic variant. (C)Yellow box Redasterisk Log10(P) interval. Founder across chromosome11 strain coefficients S2. Figure Figure 2. = 100 bars scale All and late yellow onset, lowscore melanoma age of . melanoma. and spontaneous UVR-induced Accepted S1. Figure Figure legends. Supplementary epidermis (notshown). suprabasal the in positivity weaker much some is There arrows. yellow by indicated sheath root ha CDON inthehumananagen (E)andtelogen (F) yellow.for (E)Staining in highlighted intronicvariants, or 3’UTR, 5 carrying synonymous, missensevariants haplotype. Candidatescarrying specific carryingchromosome genes totheNOD variants Columnsat rightshow 9interval. yellowasterisk in denotes box anintronic,5’or Article interval. asterisk within the Red -1-Log10(P) over th alleles of eight founder 9.(C)Founder alleleco chromosome scores along theP-values 2log10(P) values; shows ranging Thefrom onethe x-axis toten. chromosomal y-axis positionand the showsthe al., 2014). (Ram et described in ofCC construction Genotyping, nevus on based scan Genome-wide (A) . (A) Sox10-stained skintakenfrom Sox10-stained . (A) (A) Summaryof linkagepeakonmurine Graph shows spread of nevus scores for 66CCstrains. for scores spreadofnevus shows Graph μ M emistrymethods file. methodsinsupplementary staining e chromosome where the founders are colour-coded. (D) Genes Genes (D) colour-coded. are thefounders where e chromosome were derived from the linkage haplotype data. (B) Plot of LOD Plotof data.(B) thelinkage from haplotype derived were in blue box denotes missense mutation, black asterisk in yellowin blueboxdenotesmissensemutation,black asteriskin Nevus score was treated as a multinomial analysis with values withvalues multinomialanalysis as a treated was score Nevus strain haplotypes, and linkage analysis was performed as as wasperformed analysis andlinkage strain haplotypes,

boxes denote strains that carry the NOD founder NODfounder denote strainsthatcarry the boxes Red denotes high nevus cell density and early and early density cell nevus denoteshigh Red Cdk4 3’ UTR variant. (D) Table showing genes3’ UTRvariant.(D)Tableshowing inthe in blue box denoted missense mutation, black mutation, black denotedmissense in bluebox efficients: plotofthecalculatedlog-odds ratio are marked in red with a blue box. Genes aremarkedinredwithabluebox. are shown below. (B) Genes within the -1 - -1 withinthe (B) Genes shownbelow. are onset. Black denotes data not yet not available. data denotes onset. Black ir follicle,withpositivity intheinner (red) R24C/+ chromosome 11(seelegend 2). toFigure cell in66CCstrains. density score Cdk4 ::Tyr-NRAS R24C/+

mice atvarioustime-points Q61K mice at various time- various at mice

LODchromosome 9, alongcontain plotof scores fr derived were 2log10(P) the P-values values; containing and (B), 40 pigmented CCstrains. 40pigmented and (B), S6. Figure sections. ofspecific examination S5. Figure Nevus 1-10. cell density scores This article is protected Allrights bycopyright. reserved. Accepted Article S4. Figure Examples of Examples Cdon Genome-wide scan based onnevuscelldens scanbased Genome-wide Same images as in Figure S4, except at except S4, Figure asin images Same . The x-axis shows . Thex-axis the chromosomalpositiony-axis and the showsthe Cdk4 R24C/+ cell deposits inthe not scored. subcutis fat were layer

Red arrows denote the major linkage peak on chromosome 9 chromosome 9 on peak themajorlinkage denote Red arrows ::Tyr-NRAS Q61K and colour coded founder allelecoefficients. founder and colourcoded om thelinkagehaplotyp mouse skin for each or our arbitrary nevus nevus skin mouse foreachorourarbitrary the original size for ease of closer foreaseofcloser size original the ity score in (A), 26albinoCCstrains,ity in(A), score e data. Panels below

This article is protected Allrights bycopyright. reserved. Accepted Article

This article is protected Allrights bycopyright. reserved. Accepted Article