Variant Rett Syndrome in a Girl with a Pericentric X-Chromosome Inversion Leading to Epigenetic Changes and Overexpression of the MECP2 Gene

ContentslistsavailableatScienceDirect

International

Journal

of

Developmental

Neuroscience

jo u r n al h om ep age :w w w . e l s e v i e r . c o m / l o c a t e / i j d e v n e u

Variant

Rett

syndrome

in

a

girl

with

a

pericentric

X-chromosome

inversion

leading

to

epigenetic

changes

and

overexpression

of

the

MECP2

gene

José

Pedro

Vieira

_,

_Fátima

_Lopes

_,

_Anabela

_{Silva-Fernandes}

_,

_Maria

_Vânia

_Sousa

_,

Sofia

Moura

_,

_Susana

_Sousa

_,

_Bruno

_M.

_Costa

_,

_Mafalda

_Barbosa

_,

_Bauke

_Ylstra

_,

Teresa

Temudo

_,

_Teresa

_Lourenc¸

_o

_,

_Patrícia

_Maciel

a_{NeurologyDepartment,HospitalDonaEstefânia,CentroHospitalardeLisboaCentral,Portugal}

b_{LifeandHealthSciencesResearchInstitute(ICVS),SchoolofHealthSciences,UniversityofMinho,Braga,Portugal} c_{ICVS/3B’s–PTGovernmentAssociateLaboratory,Braga/Guimarães,Portugal}

d_{DepartmentofGeneticsandGenomicSciences,TheMindichChildHealth&DevelopmentInstitute,TheSeaverAutismCenterforResearchandTreatment,}

IcahnSchoolofMedicineatMountSinai,NY,USA

e_{InstitutoGulbenkiandeCiência,Oeiras,Portugal}

f_{DepartmentofPathology,VUUniversityMedicalCenter,Amsterdam,TheNetherlands} g_{NeuropediatricsDepartment,CentroHospitalardoPorto,Portugal}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received18May2015

Receivedinrevisedform3July2015 Accepted15July2015

Availableonline10August2015 Keywords: Epigenetics Epilepsy Intellectualdisability Neurodevelopment

a

b

s

t

r

a

c

t

RettsyndromeisaneurodevelopmentaldisordercausedbymutationsintheMECP2gene.We investi-gatedthegeneticbasisofdiseaseinafemalepatientwithaRett-likeclinical.Karyotypeanalysisrevealed apericentricinversionintheXchromosome−46,X,inv(X)(p22.1q28),withbreakpointsinthecytobands wheretheMECP2andCDKL5genesarelocated.FISHanalysisrevealedthattheMECP2geneisnot dislo-catedbytheinversion.However,andinspiteofabalancedpatternofXinactivation,thispatientdisplayed hypomethylationandanoverexpressionoftheMECP2geneatthemRNAlevelinthelymphocytes(mean foldchange:2.55±0.38)incomparisontoagroupofcontrolindividuals;theexpressionoftheCDKL5 genewassimilartothatofcontrols(meanfoldchange:0.98±0.10).Nogainsorlossesweredetectedin thebreakpointregionsencompassingknownorsuspectedtranscriptionregulatoryelements.We pro-posethatthede-regulationofMECP2expressioninthispatientmaybeduetoalterationsinlong-range genomicinteractionscausedbytheinversionandhypothesizethatthistypeofepigeneticde-regulation oftheMECP2maybepresentinotherRTT-likepatients.

©2015ElsevierLtd.Allrightsreserved.

1. Introduction

Rettsyndrome(RTT)isaneurodevelopmentaldisorder charac-terizedbyanearlyregressionfollowedbystagnationingrowth anddevelopment,leadingtomentalretardation,stereotypiesand motor apraxia that may be associated with epilepsy and with dysautonomicfeatures,includingdisturbedbreathing,sleepand gastrointestinalmotility(Hagbergetal.,1983;Rett,1966).RTTis causedbymutationsintheMECP2gene,encodingamethyl-CpG

∗ Correspondingauthorat:LifeandHealthSciencesResearchInstitute(ICVS), School of Health Sciences, University of Minho, 4710-057 Braga, Portugal. Tele/Fax:+351253604824/20.

E-mailaddress:pmaciel@ecsaude.uminho.pt(P.Maciel).

1 _{Bothauthorscontributedequallytothiswork.}

bindingproteinthoughttobeinvolvedintranscriptional regula-tion(Amiretal.,1999;Hoffbuhretal.,2001;Philippeetal.,2006). Mutationsinthisgenearepresentinmorethan90%ofpatientsthat fulfillthediagnosticcriteria,andwithlesserfrequencyinpatients withso-calledvariantformsofthedisease,thatdonotcomplywith allthesecriteria(Amiretal.,1999;Hoffbuhretal.,2001;Philippe etal.,2006).

Themostfrequent mutationsin MECP2arepoint mutations, smallinsertionsordeletions.Thegenotypeofpatientswithoutone ofthesemutationshasalsobeeninvestigatedandsomecaseswith grossrearrangementsinvolvingtheMECP2genewerefound, usu-allylargedeletions.Inboys,MECP2duplicationistherarecauseof asevereencephalopathywithrecurrentinfections,severemental retardation,microcephalyandepilepsy(Ramockietal.,2009;Van Eschetal.,2005).MECP2duplicationhasalsobeendescribedina

http://dx.doi.org/10.1016/j.ijdevneu.2015.07.010

girlwithapreserved-speechvariantofRTTandinanotherwith nonspecificmentalretardation(Arianietal.,2004;Makrythanasis etal.,2010).RecentlyBijlsmaetal.reviewedMECP2duplication inelevenfemales;fivewerefromtheirstudyand sixhadbeen previouslyreported.Intellectualdisabilitywasaprominent fea-turewhilehandstereotypies,epilepsy,dysmorphismandrecurrent infectionswerelessfrequent(Bijlsmaetal.,2012).Overexpression ofnormalMeCP2hasalsobeenshowntocauseaprogressive neuro-logicaldisorderinamousemodel,showingthatthenervoussystem requiresaveryprecisedosageofthisproteinforproperfunctioning (Collinsetal.,2004).

Here, we describea patientwith a RTT-likeclinical presen-tationin whom wefound nomutationsin theMECP2gene by directsequencing,norchangesinexondosage.Karyotype analy-sis,however,revealedapericentricinversionintheXchromosome 46,X,inv(X)(p22.1q28).Thisinversionisaccompaniedbyepigenetic changesintheformofdecreasedDNAmethylationlevelswithin the5regulatoryregionofMECP2gene,andledtoasignificantly increasedexpressionofthegene, inspiteof itsnormal dosage, throughamechanismthatmayinvolverelativelydistantregions oftheXchromosome.

2. Materialsandmethods

2.1. Patientassessment

Theprobandwasascertainedandassessedthroughthe Hospi-talDonaEstefânia(CentroHospitalardeLisboaCentral)andMECP2 studywasrequestedattheMolecularDiagnosticServiceatLifeand HealthSciencesResearchInstitute(ICVS)–SchoolofHealth Sci-ences,UniversityofMinho,Braga,Portugal.Informedconsentwas obtainedfromthechild’sparentsforbloodsamplingandgenetic analyses.GenomicDNAwasextractedfromperipheralbloodusing thePuregeneDNAisolationkit(Gentra,Minneapolis,MN).

2.2. MECP2mutationdetection

Thecodingregionand exon-intronboundaries oftheMECP2 genewereamplifiedbyPCRandsequencedonanautomated DNA-sequencer (ABI-3130GeneticAnalyzer,Applied Biosystems, Life technologies).

Copy-numberanalysisoftheMECP2genewasperformedby multiplexligation-dependentprobeamplification(MLPA) (MRC-Holland, Amsterdam, Netherlands) using SALSA P015 kit and quantitativePCRanalysis(qPCR).For MLPA,PCRreactionswere carried out using C1000 – Thermal Cycler (BioRad) thermo-cycler according to the manufacturer’s instructions. Products wereseparatedina3130GeneticAnalyzer(AppliedBiosystems, Foster City, CA, USA) using ROX-500 as internal pattern. The datawasanalyzed for copy-numberdifferenceswithCoffalyser software (MRC-Holland, Amsterdam, Netherlands). Primers for qPCR were designed using Primer3Plus software (http://www. bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi)andtaking intoaccountstandardrecommendationsforqPCRprimer develop-ment(Jovanovicetal.,2003).Foursetsofprimersweredesignedfor theMECP2gene(ENSG00000169057)withinthecodingregionof exonone,twoandthreeandinthe3UTRnon-codingregionofexon four.ThereferencegenesusedwereSDC4(ENSG00000124145)and ZNF80(ENSG00000174255)localizedinthe20q12-q13and3p12 regions,respectively.qPCRreactionswerecarriedoutina 7500-FASTRealTimePCRmachine(AppliedBiosystems,FosterCity,CA, USA)usingPowerSYBRGreen®(AppliedBiosystems).The speci-ficityofeach ofthereactionswasverifiedbythegenerationof ameltingcurveforeachoftheamplifiedfragments.Theprimer efficiencywascalculatedbythegenerationofastandardcurve fit-tingtheacceptednormalefficiencypercentage.Quantificationwas

performedasdescribedelsewhere(Hoebeecketal.,2005).Ct val-ues obtainedforeachtestwereanalyzedinDataAssistTM_software

(AppliedBiosystems,FosterCity,CA,USA). 2.3. Xchromosomeinactivation

TheX-inactivationassay(XCI)wascarried outbyanalysisof theHUMARA(humanandrogenreceptor)locus,whichisahighly polymorphicCAGrepeatwithaverynearHhalenzymerestriction site,accordingtothedescribedprotocol(Jovanovicetal.,2003).The productswereseparatedonanautomatedDNA-sequencer (ABI-3130GeneticAnalyzer,AppliedBiosystems,Lifetechnologies)the allelepeakheightservingasthesemi-quantitativemeasureofthe amountofPCRproductamplifiedfromeachallele.Thesamples weredefinedashavingadeviatedXCIpatterniftheypresenteda correctedalleleratiooutofthe0.33–3range.

2.4. ConventionalkaryotypingandFISH

Standard karyotpe analysis was performed using cells cul-tured from peripheral blood leukocytes, and FISH analysis was undertaken using two distinct probes covering the Xq28 region: RP4-671D9 (153286964–153386210) and RP11-617G06 (153110141–153283527)(BlueGnome,UK).

2.5. Molecularkaryotyping

TheaCGHanalysiswasperformedona humangenomeCGH Agilent 180Kcustomarraydesigned by theLowLands Consor-tium (LLC, Dr. KlasKok)in order to beused inthe analysisof childrenwithID/DD(AMADID:023363;Agilent,SantaClara,CA). DNAlabelingwasperformedusingtheENZOLabelingKitforOligo Arrays(EnzoLifeSciences,Inc.).AsreferenceDNAweused Kreat-ech´ısMegaPollReferenceDNA(KreatechDiagnostics).Arrayswere thenhybridizedusingtheAgilentSurePintG3HumanCGH Microar-ray Kit.Data wasextracted withtheAgilentFeatureExtraction (FE)Softwarev10.5usingdefaultsettingsforCGHhybridizations. Imageanalysiswasperformedusingtheacross-arraymethodology describedpreviously(Buffartetal.,2008).CGHdatawasanalyzed usingNexusCopyNumber6.0softwarewithFASST2 Segmenta-tionalgorithm.Thecustom1MAgilentarraywasdesignedusing theeArraywebtool(https://earray.chem.agilent.com/earray/).The maximalamountofprobesavailablewasselectedforthe break-pointregions(Xq22.1andXq28).DNAlabeling,hybridizationand dataextractionwereperformedinthesamewayasforaCGH180K (describedabove).CGHdatawasanalyzedusingNexusCopy Num-ber6.0softwarewiththeFASST2Segmentationalgorithm.

2.6. mRNAexpressionanalysis

TotalRNAwasisolatedfromleucocytesusingTRIZOL (Invitro-gen,Calrsbad,CA,USA)accordingtothemanufacturer’sprotocol. First-strandcDNA,synthesizedusingiScriptTM_{cDNASynthesisKit}

(Bio-rad),wasamplifiedbyquantitativereverse-transcriptasePCR (qRT-PCR)usingQuantiTectSYBRGreenPCRKit(Quiagen).Human MECP2,CDKL5andHPRTprimerswereusedforrelative quantifica-tionanalysis.MECP2(Forward:5-GGCGCTCCATCATCCGTGAC-3; Reverse: 5-CTGGGGATTGATCAAATACACAT-3), CDKL5 (Forward: 5-CATTCACTGTCTGCACCTCAC-3; Reverse: 5-GTTCTCCAGGTCGGGGTGAC-3) and HPRT (Forward: 5-CTAATTATGGACAGGGACTGAACG-3; Reverse: 5 -CAGTCATAGGAATGGATCTATCA-3). The expression levels of thegeneswerenormalizedtotheHPRTgeneandrelative quantifi-cationwasusedtodeterminethefoldchangedifferencebetween MECP2, CDKL5 andHPRT, using theDDCT method,asdescribed before (Pfaffl, 2001). The comparison between the case study

(n=1)andcontrols(n=6)ispresented,withtherespectivet-test analysisfor replicate1. Thisdifference wasreproducedfor the threereplicates.

2.7. Methylationanalysis

ForepigeneticanalysistheEpiTectMethylIIPCRPrimerAssay for Human MECP2wasused where a region of theCpG island 106 (ChrX: 153362568–153363473) was assessed (CpG Island 115358)EPHS115358-1A(Quiagen®)intheindexcase,motherand 4healthyfemalecontrols.Briefly,250ngofDNAfromeach sam-plewasdigestedfor6hat37◦Cdegrees.Thesampleswerethem amplifiedbyrealtimePCRusingthekitspecificMECP2primers (EPHS115358-1A)withRT2_{qPCRSYBRGreenROXmastermixin}

a7500-FASTRealTimePCRmachine(AppliedBiosystems,Foster City,CA,USA).Ctvalues obtainedforeachsamplewereanalyzed accordinglywiththemanufacturer’sinstructionsinordertoobtain thegenemethylationstatus,asthepercentageofunmethylated (UM)andmethylated(M)DNAintheinputgenomicsequence.A t-testanalysiswasperformedfortheunmethylatedDNAin repli-cate1oftheassaywherethecasestudy(n=1)iscomparedwith thecontrols(n=4).Thisdifferencewasreproducedforthethree replicates.

3. Results

3.1. Clinicaldescription

Thepatientisthesecondchildofhealthy,non-consanguineous parentsandhasnofamilyhistoryofRettsyndrome,intellectual dis-abilityorepilepsy.Gestationwasuneventful.Delivery,at39weeks, wascomplicatedbyadifficultforcepsextraction.Apgarscoreswere 8at1minand10at5min.Birthweightwas3755g(75thcentile), length50cm(50thcentile)andheadcircumference35.5cm(75th centile). Abrain computed tomography (CT)showed a parietal skullfracture and asmallunderlyingepiduralhematoma with-outparenchymatousbrainlesions.Inthefirstdays,sheremained clinicallywell,and acontrolCTscanshowedtotalresolution of thehematoma.Thechildwasfirstseenbyuswhenshewasforty daysold,afterabriefleft-sidedclonicpartialmotorseizure.The neurologicexaminationwasnormal.Electroencephalogram(EEG) showedfocalparoxysmalactivity.CTscanandmagneticresonance imaging (MRI)were normal.Phenobarbital wasintroduced and uptofivemonthssheexperienced afew more seizureswitha similarpattern. Subsequently she remainedasymptomatic. The EEG,repeatedatfivemonths,wasnormal.Atthistime,however, psychomotordevelopmentwasclearlyinthedelayedrange.The patienthadaxialhypotoniaandherhandsinfrequentlyreachedthe midline.Atninemonthsshedidnotsitwithoutsupport,wasunable toreachoutandpickobjectsandherlowerlimbswereslightly spas-tic.Fromtwelvemonthson,RTT-likehandstereotypiesappeared andbecameprogressivelymorefrequent.Shedidnotacquireat thistimeanyexpressiveorcomprehensivelanguageanddidnot pointtoobjects.Intheperiodbetweenfourandeighteenmonths headcircumferencewentfromthe75thcentiletoslightlybelow the5thcentile.Formaldevelopmentalevaluationwasperformed usingtheRuthGriffithsscaleattheageoffour(45months), reveal-ingamentalageof15monthsandaGlobalDevelopmentalQuotient of34%.

Attheageofsixtosevenshewaswalkingwithminimal sup-port,hand movementsweremore coordinatedand intentional, sheheldapenandscratched,wasabletopointtosomefigures inabookandtosaysixtoeightsimplewords.Currently,atthe ageoften,hervocabularyisstilllimitedtoeightwords,fineand grossmotor skills remain at thesame level and RTT-likehand stereotypiesarestillpresent,althoughlessfrequentlythanbefore.

Teethgrindingisfrequent. Hyperpnea,apnea,insomnia, unpro-vokedlaughing/screamingandautisticfeatureswerenotreported bytheparents.Thereisnohistoryofrecurrentand/orsevere infec-tiousdiseases.Recently,epilepsyrelapsedwithbriefhypomotor seizuresthatwerecontrolledwithvalproateandoxcarbazepine. EEGrevealsposteriortemporalspikes.

Thechildisgenerallyhappy,smilesquiteoftenandhasgood visualinteraction.Shehasadivergentstrabismus,misalignment anddeformityoftheteeth,aslightdorsolumbarscoliosisandno dysmorphicfeatures.Herfeetareoftencoldandpale.Neurologic examinationis remarkablefor themicrocephaly,expressionless facesuggestinganextrapyramidalhypokineticdisorder,Rett-like handmovements,spastichypertoniaoflowerlimbs,dyspraxicgait andbriskreflexeswithflexorplantarresponses.

3.2. MECP2mutationanalysisandkaryotyping

We foundnovariants in thecoding region andintron-exon boundariesofthisgenebydirectSangersequencing,norchanges in exon dosage by MLPA and quantitative PCR analysis (qPCR) forthe4exons.Karyotypeanalysisincellsculturedfrom periph-eralbloodleukocytes,however,revealedapericentric inversion intheXchromosome46,X,inv(X)(p22.1q28)inthepatient,which wasnotpresentinthemotherorfather(Fig.1A).Ofnotice,the MECP2gene is located in cytoband Xq28, and the CDKL5 gene inXp22.1. We thenperformedFISH totestthehypothesis that thepericentricinversioncouldhavedisplacedtheMECP2coding sequenceoritsnearbyregulatoryregions.We usedtwoprobes, one(RP4-671D9) covering theMECP2gene and 22998bpofits 5 region,andthesecond(RP11-G17G06)covering173386bpof 3 downstream region of the MECP2 gene (see Fig. 1, panel B andC).Theanalysis,however,revealedthattheMECP2gene5, exonic,and 3 regionshadnot beenseparatedbytheinversion. TheseresultsplacethebreakpointatXq28moreproximallythan thelocationoftheRP11-617G06probe(153110141–153283527, hg19 coordinates), meaning that the breakpoint is not occur-ring within a distance of 176.883 Kb downstream the 3 UTR of the MECP2gene. In order to clarify if the inversion lead to anyloss orgain in thebreakpoints weundertook aCGH analy-sisonahumangenomeCGHAgilent180Kcustomarray(Buffart etal.,2008)andona high-resolutioncustom1MAgilentarray designed so that themaximal amountof Agilent probes avail-ablewasselectedfor thebreakpointregions(Xp22.1 andXq28 where the CDKL5 and MECP2 genes are located, respectively) andat thecitobands14q12 (wheretheFOXG1geneis located). NosignificantCNVswerefoundintheseregionsin both analy-ses.

3.3. MECP2mRNAexpressionandmethylationstatus

WetheninvestigatedbyqRT-PCRtheexpressionlevelsofthe MECP2 (located at Xq28) and CDKL5 (located at Xp22) genes. Intriguingly,thepatientdisplayedanoverexpressionoftheMECP2 geneatthemRNAlevelinthelymphocytes(meanfoldchangefor thethreereplicates:2.55±0.38),incomparisontoagroupof6 con-trolfemaleindividuals;incontrast,theexpressionoftheCDKL5 gene wassimilartothat of controls(meanfold changeforthe three replicates: 0.98±0.10) (Fig.2A). TheX-inactivationassay (XCI), carried out by analysisof the HUMARA locus(Jovanovic etal.,2003),showedthatthepatienthada balancedpatternof Xinactivation,witharatioof1/1.Tofurtherexplorethe mech-anismleadingtotheoverexpression ofMECP2,weassessedthe methylationstatusofaportionoftheCpGislandlocatedinthe 5-regulatoryregionof theMECP2usingtheEpiTect® MethylII PCRAssay(Quiagen®).Theseanalysesshoweddecreasedlevelsof

Fig.1.Cytogeneticandmolecularanalysis.(A)Karyotypeofthepatient;(B)schematicrepresentationofthepositionoftheFISHprobesrelativetotheMECP2gene;(C) SchemeoftheresultsoftheFISHanalysisofthepatient,undertakenusingtwodistinctprobestargetingtheXq28region:RP4-671D9(153286964–153386210)and RP11-617G06(153110141–153283527)(coordinatesinhg19);(D)FISHimage(arrowsindicatetheinvertedandnormalXchromosomes);(E)180Kcustomarrayoverviewofthe XchromosomeshowingabsenceofCNVs.

methylationattheassessedCpGsite(Fig.2C),apatternthatislikely associatedwiththeincreasedMECP2geneexpressionobservedin lymphocytes,andwhichwespeculatemayalsobehappeningin thenervoussystem.ThesefindingsleadustospeculatethatMECP2 overexpressionisthemostlikelydiseasecausingmechanisminthis patient.

4. Discussion

Rettsyndrome(RTT)resultsinalargefractionofpatients(more than90%)frommutationsintheMECP2geneinpatientsthatfulfill thediagnosticcriteriaand,inlesserfrequency,inpatientswith so-calledvariantformsofthedisease,that donotcomplywithall

Fig.2. MECP2andCDKL5mRNAexpressionlevelsandmethylationstatusofMECP2.(A)qRT-PCRanalysisrevealedanupregulationofMECP2mRNAlevels(meanfold changeforthethreereplicates:2.55±0.38)inthelymphocytesoftheindexcaseincomparisontoagroupofcontrolfemaleindividuals(n=4)(theHPRTgenewasusedasa housekeepinggene);(B)StatisticalanalysisforMECP2andCDKL5mRNAexpressionbetweenthecasestudy(n=1)andcontrols(n=6)ispresented,withtherespectivet-test analysisforreplicateone(p=0.000532;***p<0.001).Thisdifferencewassignificantforthethreereplicates.(C)AnalysisoftheDNAmethylationstatusofthepartofthe CpGisland106(ChrX:153362568–153363473,hg19coordinates)intheindexpatient,motherandcontrols(n=4)usingtheEpiTectMethylIIPCRPrimerAssayforHuman MECP2(meanvaluesforthethreereplicates).(D)Statisticalanalysis(t-test)forpercentageofunmethylatedDNAforreplicateonewherethecasestudy(n=1)iscompared withthecontrols(n=4)(p=0.002152;**p<0.05).Thisdifferencewassignificantforthethreereplicates.

thesecriteria(Amiretal.,1999;Hoffbuhretal.,2001;Philippeetal., 2006).

Althoughourpatientdoesnotmeetthediagnosticcriteriafor classicRTT(becauseshefulfillsonlytwoofthefourmajorcriteria), herdevelopmentwasnormalinthefirstmonthsoflifeandlatera severeretardationemerged,shehasacquiredmicrocephaly, neuro-logicsignsinlowerlimbs,coldandpalefeet,scoliosis,bruxismand, importantly,apersistentandprolongedhistoryofRTT-likehand stereotypies,whichareamongthemoststrikingclinicalfeatures ofRTT.Withthiscombinationofsignsandsymptomsshealsodoes notstrictlymeetdiagnosticcriteriaforvariantRTTaspublishedin 2001(Hagbergetal.,2002)or,morerecently,in2010(Neuletal., 2010);shedoes,however,meetthecriteriaforvariantRTT accord-ingtotheearlyHagbergclassification(HagbergandSkjeldal,1994). Themaindifferencebetweenlaterandformerdiagnosticcriteria isthatthe2001and2010criteriaemphasizeregression(whichis questionableinthiscase)asakeydiagnosticfeatureofRTT. There-fore,andgiventhisphenotypicresemblance,wewereencouraged tostudytheMECP2gene.

AlthoughtheMECP2geneitselfwasnotmutatednorinvolvedin thechromosomalrearrangement,itsexpressionwasaltered,and thisderegulationmightunderlietheneurodevelopmental pheno-typeobserved.Thisphenotypediffersfromthatofmalepatients withMECP2duplications(VanEsch,2012).Heterozygousfemales withMECP2duplicationusuallyshowextremetocomplete skew-ingofX-chromosomeinactivationandareasymptomaticorhave mainlyneuropsychiatricdisorders,butseverementalretardation

hasbeenreported(Bijlsmaetal.,2012).Wewerenotableto deter-minethemechanismthroughwhichthepericentricinversionis affectingthemethylationandthetranscriptionalregulationofthe MECP2gene.aCGHanalysis,provided noevidencefordeletions orduplicationsencompassingothergenesdescribedasmutated inRTT-likephenotypesoraknownorsuspectedregulatory ele-mentthatcouldberelatedwithMECP2alteredexpression.Taking thesefindings in consideration, we hypothesizethat the struc-turalalterationoftheXchromosome,evenwithoutsignificantly largeCNVs,couldbedisruptingchromosomaldomainorganization andlong-rangegenomicinteractions(MaksimenkoandGeorgiev, 2014;Robyretal.,2011),thusinfluencingMECP2epigenetic modi-fications,includingDNAmethylation,andconsequentlymodifying itsexpression. Thistype of perturbationhasbeen described in fibroblasts ofpatientswith Downsyndrome(Letourneauet al., 2014).

Insummary,wereportthecaseofagirlwithaRett syndrome-like clinical presentation with a pericentric inversion in the X chromosome 46,X,inv(X)(p22.1q28) that doesn´ıt disrupt the MECP2 gene but is accompanied by epigenetic changes in the formofdecreasedDNAmethylationlevelswithinthe5 regula-toryregion of MECP2gene, leading toa significantlyincreased expressionof thegene.We speculatethat this de-regulationof MECP2 expression may be a novel mechanism underlying dis-ease in other RTT-like patients who test negative for MECP2 mutations,a hypothesisthat shouldbeinvestigatedin thenear future.

Acknowledgments

Wethankthepatientandfamilyfortheirparticipation,allthe membersoftheMicroarrayCoreFacility,CancerCenter Amster-damatVrijeUniversiteitMedicalCenterfortechnicalhelpwith thearraydesignandCGCGenetics/CentrodeGenéticaClínicaS.A. forkaryotypeandFISHanalysis.

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