This article isprotected bycopyright. Allrights reserved. Development (R21HD074278,A.R.)andtheMWRIPostdoctoral Fellowship(M.W-T). comments. ThisworkwassupportedbytheNational Institute of ChildHealth andHuman scientific editor atthe Magee-Womens Research We thankthefamilies forparticipatinginth Acknowledgements Conflict of interest: Email: [email protected] Phone: 412-641-6693;Fax: 412-641-2255 University of PittsburghSchoolofMedicine, Assistant Professor,Department ofObstet Svetlana A.Yatsenko,MD. *Corresponding Author: 5 Center, Memphis,TN 4 3 School ofMedicine,Pittsburgh,PA 2 Womens Hospital ofUPMC,Pittsburgh, PA 1 carriers affectedbyX-linked High-resolution microarray analysis unravels

Accepted ArticleDepartment of Human Genetics,Graduate School Department ofOphthalmology,Hamilton EyeInstitu Department ofPathology,UniversityP Department ofObstetrics,GynecologyandRe Pittsburgh CytogeneticsLabor Archana Kishore

article as doi: 10.1111 /cge.12638 differencesbetween version lead to this may been through the copyediting, typesetting, pagination andproofreading process, which This article hasbeen accepted for publication andundergone full peer review but has not Cercone Svetlana A.Yatsenko 2 , Alexander N.Yatsenko 2 , BarbaraJ.Jennings Theauthorsdeclareno 1,2,3* blueconemonochromacy atory, CenterforMedical ,

Heather A. Bakos 2,3 4 , AleksandarRajkovic , UrvashiSurti rics, GynecologyandReproductiveSciences ittsburgh SchoolofMedicine,Pittsburgh, PA conflict ofinterest. UPMC, 300HalketStreet,Pittsburgh, PA15213

productive Sciences,UniversityofPittsburgh and the Versionof Record. Please cite this is study.We aregrateful toBruceCampbell, complexXq28aberrationsinpatientsand Institute (MWRI), fortheediting anduseful of PublicHealth,Universityof Pittsburgh 1 , KathleenVitullo te, UniversityofTennesseeHealthScience 1,2,3,5 Genetics andGenomics, Magee- , MichelleWood-Trageser 1,2,3,5 , Alessandro Iannaccone 1 , MarinaKedrov

2 , Stephen 4 , 4 .

This article isprotected bycopyright. Allrights reserved. Acceptedlinked disease Key Words: previously unrecognizedX-linkeddiseases. aCGH analysisandwhichcanimprove molecular advantages indetectionofsmall aberrationsthat application ofhigh-resolutionX regulatory locuscontrol a novelfoundermutation thatconsisted ofacomplex3-kbdeletionthatembracedthe characterized bysequencing ofthebreakpoint junc Articlecarriers fromthreeunrelatedfamilies. Pathogeni targeted CGHmicroarray wasappliedtotestfo Furthermore, carriertestinginfemales islim PCR-based technologies;however,alterations and issusceptibletodeletions,duplications, . Theconeopsingenecl X-linked conditioncausedbyalo with aspecificgeneticcond The human Xchromosome contains~1,600genes, ABSTRACT blue conemonochromacy;Xq28deletion; region andinsertionof ition, mainly affectingmales. uster iscomposed of2-9para chromosomemicroarray inclini ss-of-function ofboththe and mutations.Current di ited orunavailable.High-resolutionXchromosome- remain undetermined in 10%ofpatients. r rearrangements inmales withBCMandfemale- an additionalaberrant arebeyondtheresolutionof c alterationswererevealedinallprobands, diagnosis oftheknownconditionsandunravel tions andqPCR.Intwo about 15%ofwhichhavebeenassociated ;Xchromosome; aCGH;X-

Blue conemonochromacy (BCM)isan logs with99.8% OPN1LW cal diagnosisbringssignificant and agnostic tests employ OPN1MW OPN1MW families, weidentified sequence homology clinicallyavailable .The

cis - This article isprotected bycopyright. Allrights reserved. genomic contributing tohuman imbalances di of diagnosticandcarrierte Acceptedgenomic regionamong human populationspose multip opsin genes,thepresenceofhighlyhomologous type opsingenes,orextendintotheclus efficiency ofgenetherapy.TheXq28deletions carriera andfemale-carriers, affected males defects remain unknown(2,6),precluding accurate PCR-based moleculartesting,while~10%ofpa deletions orpointmutations inactivatingboth located ~4kbupstream of the genepromoterwithalocuscontrolregion(LCR), aunique expressed (2). Redorgreenexpression incone Articleformation of region tonon-allelichomologous The closephysicallocationandhigh(99.8%) and , andinabilitytodiscriminate colo recessive disorder characterized bymarkedly photopigment genescausesblueconemonoc INTRODUCTION OPN1MW Microarray platforms arenow In thenormal human retina,onlythemost A lossoffunctionboththe OPN1LW/OPN1MW- genesarearrangedina5 OPN1LW sting infamilies withBCM. recombination, whichresultsin hybrid genes(4-6). (7).Inabout90%ofmales affectedbyBCM,Xq28 OPN1LW used extensivelyfordiagnosis andresearchtodetect ′ -3 ′ orientationwithinXq28,fo nd prenataltesting,poten rs. Inhumans, uptoninecopiesofthe hromacy OMIM#303700),arareX-linked, (BCM; reduced vision,severephotophobia, congenital sease andpopulationdi sequence homology predisposethisgenomic OPN1LW ter (4,6).Thecomplex st photoreceptors isaccomplished byinteractionof may remove theLCR,inactivatingbothwild- (red) sequences,andvariabil tients havenegativeresultsandthemolecular proximaltwogenesintheclusterare information ondiseaseprogression in

and le challengesandlimit theeffectiveness and OPN1MW OPN1MW cis -regulatory DNAsequence deletions, duplications,andthe (green)coneopsin versity (8-9).High- tial approaches,and ructure oftheLCRand havebeenidentifiedby rming thecluster(1-3). rming ity withintheopsin

OPN1LW

This article isprotected bycopyright. Allrights reserved. versus male reference(Coriell,NA12891), 10male carriers (Family-1 III-4,Family-2 IV-11 andIV AcceptedArray-CGH studies samples withXchromosome imbalances andimplemented forclinicaldiagnostictesting. or 10probes/gene)coverageofallX-linkedge covering theXchromosome withanaverage (Agilent, SantaClara,CA).TheX-HRmicroa X-HRmicroarraydesign Pittsburgh IRB guidelines. microarray clinicaltestingunrelatedtoBCMc protocols. Controlsamples from 268wome Informedfor testingbyX-HRmicroarray. ArticleSubjects ANDMETHODS MATERIALS microarray analysisinthediagnosisof comparative genomic hybridization(aCGH)a alterations inthreeBCM families, usinghigh insufficient todetect smaller Xchromosome imbalances. We identified novel molecular the resolutionofclinicalw disorders (9).Singlegenealterationsaremore lik resolution microarrays provide The X-HRmicroarray studieswereperforme We developedacustom oligonucleotidemi Three unrelatedmen (P1-P3;Fig.1)withaclin hole-genome microarray platforms,

distinct benefitsinstudying BCMandotherX-linkedconditions. consents were obtained using the UTHSC IRB consents wereobtainedusingtheUTHSCIRB n and42de-identifiedmales whounderwent -resolution Xchromosome-targeted (X-HR) array 1 probe/kb,andanenhanced(1probe/200-500bp nd reporttheadvantages rray consistsof166,000 ondition wererecruitedund nes. Thismicroarray hasbeenvalidatedon -13). DNAsamples 42males from hybridized ely tobepresentinaffectedmales; however, croarray usinga4x180KCGHplatform s hybridizedagainstAgilent male reference, d onmale patientsP1-P3andfemale- ical diagnosisofBCM males withX-linked affected rangingfrom25-200kb,isoften andlimitations of oligonucleotide probes, er theUniversity of (10) werereferred

This article isprotected bycopyright. Allrights reserved. 153,459,120), consistent withadeletion insufficient coveragewithintheopsin Accepteddisability, butdidnotdetectal gain inthe4p16.1-pterregion(S (Fig. 2B).Inthebrother(IV-3;Fig.1) DNA. Identical,butheterozygousdeletion(log showed againsuggestiveofanextraopsingene (minimum) 6.126kb(maximum). to the chrX:153,406,875-153,409,337segment, indicati breakpoint withinthechrX 153,406,875;hg19) encompassinganat-least-1.5-kbse Microarray studiesinBCMfamilies Article RESULTS DNA (supplemental methods). Green Supermix (Bio-RadLaboratories,Hercules Quantitative real-time PCR(qPCR) IV-3). Seesupplemental data. CGH+SNP microarray (ISCAdesign,Agilent)was frequency ofopsingenecopynumber in were analyzedbyX-HR,servingas hybridizedagainstfemaleand 268females reference(Promega, G152)notaffectedbyBCM In P2,X-HRanalysis revealed aloss(log In P1,X-HRmicroarray detect To determine the total number of reda :153,403,612-153,405,331 interval,andthedi terations inXq28.Visualexaminat upplemental Fig.S2A),whichpr anegativecontrol,andwere This deletionincludestheLCR genecluster(Supplemental Fig. S2B). , awhole-genome identifieda microarray ed ahemizygous loss(chrX:153,405,331– of atleasttwooutthr a normal population. Awhole-genome 180K nd greenopsingenes,qPCRusingiQ-SYBR 2 (1/2)=–1.0) wasdetectedinthepatient’smother copy inthepatientasco 2 (2/3)=–1.58) inXq28(chrX:153,416,333- , CA)wasperformed onP2andthereference performed onthebrotherofP1(Family-1 ng adeletionrangingfrom 1.544kb gment (Fig.2A),withtheproximal alsousedtocalculatethe ee copiesoftheopsingenes ion oftheaCGHplotrevealed obably explainshisintellectual (Fig. 2E).Inaddition,X-HR stal breakpointwithin mpared tothereference de novo

9.3-Mb This article isprotected bycopyright. Allrights reserved. within two 1, andapartof ( deletion embracingtheentireLCR,insertionof upstream ofthe amplify thegenomic male andfemale referenceDNA. deletion detectedbyaCGH. Neitherjunctionwa 2.7 kb)intheaffectedmalesandcarriermo brother (F1IV3)andmother (F1III4),andP Long-range PCRwasperformed on designed toamplify thebreakpointjunctiona p1R1, mapped totheproximal andthedistal Breakpoint junctionanalysis absent inthe268female and42male controlsamples. close relationshipbetweenthesetwopatients.De variants alongtheXchromosome betweenP1a cluster region,identicaltothos Two daughters(Family-2 IV-11and or functionofthethirdopsinge and thedistalbrea (Fig. 2C),withtheproxima

OPN1MW Accepted Article Direct sequencingofthepatient-specific We furtheranalyzedbreakpoints in individua In P3,X-HRmicroarray detectedalossinvol ), andatandemduplicationof1,345-bpse Alu elements, OPN1MW OPN1LW kpoint downstreamof AluSx gene.Thisaberration,identical intron1(Fig.3C,D).The deletion l breakpointinchrX:153,414,302-153,416,333( e ofpatientP1(Fig.2A).Comp and ne inthecluster.Seesupplem AluSx1 DNA from a control(wt)female andmale,DNA from a P1,hisaffected IV-13; Fig.1),showedthesame OPN1MW , orientedinthesame di sequencesflankingtheLCRdeletion,were 3. PCRyieldedtwo abnormal fragments (~4kband nd a6,234-bpwild-typefragment (TableS1). ther (Fig.3A,B),cons anextracopyofthegreenpigment gene nd P3showednosimilarity, thusexcludinga s observedusingthesamePCRprimersto products revealedacomplex rearrangement letions detectedintheaffectedmales were exon6(Fig. 2E),probablyalteringstructure ving theLCRandagaininopsingene ls from Families 1and3.Primers p1F1and quence encompassing thepromoter, exon inP1andP3,includeda2,991-bp -insertion breakpointswere located ental dataforqPCRconfirmation. arison ofthebenigncopynumber rection, with~79%homology Xq28alteration(Fig.2D). istent withtheXq28 OPN1LW

intron1) This article isprotected bycopyright. Allrights reserved. by recombination betweenflankingsegmental duplica cone opsingenecluster(1,5-7). taking intoconsideration thecomplexity of the Acceptedpopulation ofunaffectedfemale andmale 180K CGH+SNParraytostudymales andcarrier can detectX chromosome imbalances withatle of priorknowledgetheregioninterest(8-9). duplications, andamplifications, ataveryhighre techniques haveenableddetec this method isofverylimited utility. Incont precise breakpointsofadeletion as itrequirespriortargetsequen diagnosis, PCR amplification ofrepetitiveandre Articledue totheirhigherresolutioncompared tocytogene DISCUSSION same haplotype; therefore,thecomplex rearra sequences. Comparison intheunique oftheSNPs using thep1F1andp1R1primers. Theduplicat segment comprisesexon1;therefore,twomuta OPN1MW (Supplemental(AGAGGTTGT/CAGTGAGC) Fig.S3). between them(Fig.3D).Thejunctionoccurredina16-bpmicrohomology segment It isnot surprising that previous PCR-basedtesting wasunsuccessful inthese families, Historically, X-linkedrecessive geneisalteredbyaduplicationof

tion ofindividualgenomic va Interstitial chromosomal rearrange ce information andcannottestforduplications.Furthermore,the are difficulttodetermine; thus disorderswerestudiedbyPCR-basedmolecular methods controls (Supplemental Figs.S4-S7). rast totargetedapproaches,microarray-based the1,345bpsequence.TheduplicatedDNA ngement probablyrepresen ion junctionoccurredbetweennon-homologous nt PCRfragments were rearrangement andthepeculiarstructureof ast 10-fold higherresolution thantheclinical arranged DNAsegments ishighlyproblematic solution inboth males andfemales regardless females affectedbyBCMaswella We appliedahigh-resolutionmicroarray that sequence revealedthatP1andP3exhibitthe tic techniques. Despiteitswide use inclinical tions (11);however,anal The structureofanextra(inserted) riations, suchasdeletions, , testingforfemale carriersby ments arefrequentlymediated obtained inP1andP3 ts afoundermutation. ysis ofrepetitive

This article isprotected bycopyright. Allrights reserved. in sporadicreoccurrence ofsimilar mutations the presence of consistent with afoundermutation; however, geno Acceptedinduced replication(MMBIR)mechanism (12). microhomology segments oftwo this alsoresultedin~3-kbdeletionof green opsingenecopytook placeatthefirst,most copy. Analysisofthejunctionfragments inP1and displacement orreplacemen the functionalcopiesonXchromosome (1-4). normal colorvision,additional hybridred/green-cone specific arrangements ofgenecodingand Articlevariants. Normal functionofred-andgreen-p opsin geneclusterareobserved cone opsinphotopigment geneontheXchromosome (3). production ofhybridred/green-cone as togeneticdiversityamong recombination orgeneconversion sequence homology(11-12).Suchregionsare“h structurally andfunctionallyrelatedgenes(p sequences isitselfchallenging.Dupl Interestingly, deletion-insertionjunction Up to10%ofmales haveredorgreenco Indeed, intragenicrecombinationbetweennor Alu repetitive sequences withextended segmentsofmicrohomology may result t ofthenormal functional healthy individuals(5,11-12). in 10-15%ofmen andareconsid Alu rearrangements thatcanleadto icated regionsofthegenome genes (3-4).About70%ofmale elements, suggestiveofa the regulatory region, resulting in BCM. the regulatoryregion,resultinginBCM. cis- aralogs) andpseudogeneswithahighdegreeof . Bothofourpatientsalso havea1.3-kb igment opsingenesstronglydependsonthe regulatory sequences inthecluster.Inmales with Complex aberrationinbothP1andP3is breakpoints werelocatedwithinthe16-bp lor visiondeficiencyastheresultof ot spots”fornonhomologous andintragenic proximal, positioninthecluster. Inaddition, mic instability intheopsin cluster regionand P3 showedthattheinsertionofanadditional mal functionalgenecopiesleadstothe OPN1LW genes mustbepositionedmore distallyto

Gains incopynumberwithinthecone

microhomology-mediated break- or ered tobebenigncopynumber pathogenicalterationsaswell usually representaclusterof OPN1MW s carrythreecopiesofthe genebyahybrid

This article isprotected bycopyright. Allrights reserved. Accepted previously unrecognizedX-linkeddiseases. with mutation analysiscanimprove molecula resolution microarray testinginto theroutine clinical diagnosis inconjunction for thediscoveryofunderlyingmolecular defect commonly problematic. Thishighlig Carrier testinginfemales dependsonthemol carriers may presentwithsome ofretinal degree Articlespecific molecular defectwithclinicalmanifest considered tobeastationary disorder; however in paralogousgeneclustersandtherefore de usually containprobesfor segmental duplicons,we duplications in thestudied families. Incontrast to population. insertion-deletion rearrangement, oritmay simultaneously. Alternatively,th duplication, anditispossiblethattheinser The high-densityX-HRmicroarray wasinvalu e exon1duplicationmay havearisenindependentlyfrom the hts theimportance ofestablis tion-deletion-duplication rearrangement occurred exist asahybridgenepolymorphism withina tected apathogenicde ecular findings intheaffectedmales andis r diagnosisforknownconditionsandunravel , therecent studies showed thecorrelationofa ations anddiseaseprogression.Moreover,female s forX-linkedconditions. degeneration orprogressi otherclinical microarray platforms thatdonot were abletoanalyzecopynumber variations able inidentifyingnoveldeletionsand hing morereliableapproaches letion inP2.BCMis ve visionloss(13-14). Incorporation ofhigh-

This article isprotected bycopyright. Allrights reserved. 5. 4. 3. 2. 1. REFERENCES 7. 6. 11. Accepted10. 9. 8. Article

green opsingeneinblueconem Reyniers E,VanThienenMN,MeireFetal. 633-651. Neitz J,M.Thegeneticsofnormal a 86: 983–987. color visiongenesamong 134men ofEuropean Drummond-Borg M,DeebSS,MotulskyAG.MolecularpatternsofXchromosome-linked pigment arraydetermines colour-visi Hayashi T,MotulskyAG, DeebSS.Positionof Science 1988:240:1669-1672. Vollrath D,NathansJ,DavisRW. Tandem arra green visualpigment gene Wang Y,MackeJP,MerbsSLetal.Alocus red andgreenconeopsins.Am JHumGenet2010:87:26-39. Gardner JC,WebbTR,KanugaN Lupski JR.Genomic rearrangements andspor efficacy outcome measures forclinical Luo X,CideciyanAV,IannacconeAetal. mutation detectioninthedystrophi Hegde MR,ChinEL,MulleJG,OkouDT, of constitutional disorders. NatGenet2007:39:S48–S54. Lee C,IafrateAJ,Brothman AR s. Neuron1992:9:429-440. onochromacy. Genomics 1995:29:323-328. . Copynumber variati et al.X-linkedconedystrophycausedbymutation ofthe n gene.Hum Mutat2008:29:1091-1099. on phenotype.NatGenet1999:22:90-93. trials.PloS One2015:10:e0125700. nd defectivecolorvision.VisionRes2011:51: Warren ST,ZwickME.Microarray-based Blue ConeMonochromacy: visualfunctionand Geneconversionbetw control regionadjacent tothe human redand adic disease.Nat Ge y ofhuman visualpi a'green-red' hybridgeneinthevisual ancestry. Proc NatlAcadSciUSA1989: ons andclinicalcy net 2007:39:S43–S47. gment genesatXq28. een redanddefective togenetic diagnosis

This article isprotected bycopyright. Allrights reserved. 12. 13. 14. Accepted Article

Hastings PJ,IraG,LupskiJR.AMicr Model fortheOriginofHuman CopyNumb mutations andassociatedphenotype Gardner JC,MichaelidesM,HolderGEet region (LCR) resultsindisruptionofthe Carroll J,RossiEA,PorterJetal.Deletion s. MolVis2009:15:876-884. ohomology-Mediated Break cone mosaic. VisionRes2010:50:1989-1999. al. Blueconemonochromacy: causative of theX-linkedopsinge er Variation.PLoS Genet2009:5:e1000327. -Induced Replication ne arraylocuscontrol

- confirmed deletioninXq28.Previous molecular hearsay. ID–brother(Family 1,IV-3)oftheprob of theprobandP2.Thematernal grandfatherofth respectively. Hx-family historyofvisionpr individuals; thearrows designate theproba and aresummarized inthesupplemental data. (BCM).Retinalfindi Fig. 1.Five-generationpedigrees. This article isprotected bycopyright. Allrights reserved. Accepted Article Figure legends Black squaresrepresent withblue affectedmales cone ngs ofthesethreesubjectswe nds P1, P2,andP3inFamilies 1,2,and3, oblems relativesonthegrandmother’s among side Slashes throughsymbols indicatedeceased e probandP3(half-filledsquare)–affectedby and P1withintellectual disability andBCM.* studies atanotherins re reportedpreviously(10) titution performed onall

This article isprotected bycopyright. Allrights reserved. Accepted Article (MPLA) assaydidnot affected individuals(P1,P2,andP3)bymultip detect Xq28deletions. le ligation-dependent probeamplification

< -0.5). Gain in DNA copy number isdetected < -0.5).GaininDNAcopy the probandsP1,P2,and P3areshownbyhorizontal indicates apromoter sequence.LocusControlRe and gray font(graydashedarrowsindicatethe5 by boldfont(blackarrowsindicate adjacent greenopsingene (13.6kb)areexpressed. distance betweentheopsingenes region. Top,genomic location(chrX:153.400-153.525 daughter (Family 2,IV-11).( shaded area)ofanat-least-42.8-kbsegmen This article isprotected bycopyright. Allrights reserved. Accepted(log (blue dots).IntheprobandP1( Articlenumber (log logarithmic (log The fluorescenceintensity ratioCy5/Cy3(test/refe to genomic location,fromtheproximal (left)to On thearrayCGHplot,eachdotrepresents upstream ofthe by aredline.Below,magnifiedviewofthe mother (Family 1,III-4). Top,anidiogram of copy number alterationsintheXq28region Fig. 2.TheXq28deletionsdetectedbyX-HRmicroarray.(A) 2 OPN1MW (0/1) =- 2 above-0.5andlessthan+0.2).Reddot ∞ consistsof6exons,indicatedby ) upstreamofthe 2 OPN1LW ) valueontheY-axis.Blackdotsrepres geneandagain(blue shadedarea) E) A Aschematic representationof ), anaveragelog OPN1LW is shown.Thesingleredopsin thedirectionoftranscription) t intheprobandP2 (Family 2,III-6)and gene. an oligonucleotideDNApr ′ -3 Xq28 regionshowingaloss(pinkshadedarea) the Xchromosome. TheX for thesegments withanaveragelog ′ the male patient P1 directionofaninactivegene).Each the distallongarm (right)oftheXchromosome. vertical bars,separatedbythe gion (LCR)isshownasabluebox.Deletionsin (C) 2 Functionalcopiesofthegenesareindicated of<-2.0indicatesahemizygous deletion rence signal)foreachprobe isdisplayed asa pinkbars.InP2,theexact positionofthe s indicate a loss in DNA copy number (log s indicatealossinDNAcopynumber X-HR microarray plot Mb; hg19)ofthecluster,size,and ent probeswithnochangeinDNAcopy in thetotalnumber ofopsingenes. the human opsingenecluster gene (14.8kbinsize)andthe . Inactivecopiesaremarked by An arrayCGHplotshowing (Family 1,IV-2)and obe, arrangedaccording obe, q28 regionisindicated showing aloss(pink TEX28

(D) 2 OPN1LW >+0.2 genes.“P” his (B) his

2

box). Reverseprimer p1R1islocated with a wtfragment. Primer p1F1ismapped within indicates anon-functiona (wt) band. (Family 1,III-4).Amplification ofthemale (mut) of~2.7kband4wereobtainedfrom the Fig. 3.BreakpointjunctionsinFamilies1and 3 This article isprotected bycopyright. Allrights reserved. Accepted Article sequences inthatregion.Hatchedbari distal breakpointcould (B) S tructure oftheopsinclus not bepreciselydetermined duetotherepetitivestructureofDNA l (greenorhybrid)copy.Primers ndicates regionofuncertaintyforP2. ter inanormal wild-type(w in theexon1,whichisidentical in and female referenceDNAyieldedasingle6.2-kb theuniqueXq28sequence upstream ofLCR(blue affected male patientsandthemother ofP1 . (A) Two patient-specific p1F1 andp1R1wereusedtoamplify t) male.Grayarrowhead junction fragments OPN1LW

and

This article isprotected bycopyright. Allrights reserved. Accepted ArticleUnderlined nucleotidesindicatemicr promoter. Below,thesequencesofjuncti Duplication junctionoccursbetweenintron1 left. “Promoter-g” designates P1 andP3.Thedeletion-insertionjunction(das containing exon1. The structureofaninserted opsin geneisinsertedupstream oftheredpi Dotted rectangleindicates adeletion encompa OPN1MW. (C) A complex rearrangement inP1andP3.A (D) Direct sequence analysis of themuta OPN1MW OPN1MW ohomology atthebreakpointjunction. geneisalteredbyadup promoter. gment gene(depictedby on fragments andgenomic coordinatesaregiven. and sequencelocatedups ssing theLCR.Anadditional copyofagreen hed box/doublelinestructure)isshownonthe Alu sequencesareindicatedbybluearrows. total offouropsingenesarepresent. nt junctionfragments fromprobands lication of a1,345-bp sequence, a double-linerectangle). tream ofthegreen