The Genetics of Rett SyndromeThe Genetics of Rett Syndrome

John ChristodoulouJohn Christodoulou

Head, NSW Rett Syndrome Research UnitHead, NSW Rett Syndrome Research Unit

Western Sydney Genetics Program, Children’s Hospital at Western Sydney Genetics Program, Children’s Hospital at WestmeadWestmead

Discipline of Paediatrics & Child Health, University of SydneyDiscipline of Paediatrics & Child Health, University of Sydney

Clinical DiagnosisClinical Diagnosis

•• specific developmental profile based on a specific developmental profile based on a consistent constellation of clinical featuresconsistent constellation of clinical features

•• diagnostic criteria developeddiagnostic criteria developed

•• classical and variant RTT phenotypesclassical and variant RTT phenotypes–– atypical Rett syndromeatypical Rett syndrome–– “speech preserved” variant“speech preserved” variant–– congenital onset variantcongenital onset variant–– male Rett syndrome equivalentmale Rett syndrome equivalent

Genetics of Rett SyndromeGenetics of Rett SyndromeX: autosome translocations:X: autosome translocations:

-- t (X; 22) t (X; 22) -- Xp11.22Xp11.22-- t (X; 3)t (X; 3) -- Xp21.3Xp21.3

Deletions:Deletions:-- del (3) (3p25.1 del (3) (3p25.1 -- p25.2)p25.2)-- del (13) (13q12.1 del (13) (13q12.1 -- q21.2)q21.2)

mtDNA mutation screening:mtDNA mutation screening:-- 16S rRNA 16S rRNA -- A2706GA2706G (1 patient & mother)(1 patient & mother)

Exclusion mapping in familial cases:Exclusion mapping in familial cases:(incl. Brazilian family with 3 affected sisters)(incl. Brazilian family with 3 affected sisters)

-- gene likely to be in Xq28 or gene likely to be in Xq28 or XpterXpter

6 mutations identified in 21 sporadic classical cases6 mutations identified in 21 sporadic classical cases-- 4 4 de novode novo missense mutations in methylmissense mutations in methyl--binding domain (MBD)binding domain (MBD)

-- 1 1 de novode novo frameframe--shift mutation in transcription repression domain (TRD)shift mutation in transcription repression domain (TRD)

-- 1 1 de novode novo nonsense mutation in TRDnonsense mutation in TRD

““Rett syndrome is caused by mutations in Rett syndrome is caused by mutations in XX--linked linked MECP2MECP2, encoding , encoding

methylmethyl--CpG binding protein 2”CpG binding protein 2”(Amir et al, Nature Genet 1999: 23; 185 (Amir et al, Nature Genet 1999: 23; 185 -- 188)188)

MECP2 MECP2 Mutations IdentifiedMutations Identified

> 200 to date> 200 to dateRettBASE: http://mecp2.chw.edu.auRettBASE: http://mecp2.chw.edu.au

Large Deletions in RTT PatientsLarge Deletions in RTT Patients

teltel cencen11 22 33 ExonExon 44

MBDMBD TRDTRD 3’UTR3’UTR

11

~15 kb~15 kb

4.609 kb4.609 kb

~47 kb~47 kb

~37 kb~37 kb

~40 kb~40 kb

~65 kb~65 kb

MethylatedMethylatedDNADNA

MeCP2MeCP2

AcAc

AcAc AcAc

AcAc

chromatin accessible & activechromatin accessible & active

Gene Silencing by Gene Silencing by Chromatin CondensationChromatin Condensation

chromatin condensed & inactivechromatin condensed & inactive

MeCP2MeCP2mSin3amSin3a

SWI/SNFSWI/SNFHDACHDAC

MeCP2 binds to methylMeCP2 binds to methyl--CpGsCpGs

recruitment of mSin3a & recruitment of mSin3a & histone deacetylase (HDAC)histone deacetylase (HDAC)

HDACHDAC

mSin3amSin3a

Factors Contributing to Factors Contributing to Phenotypic VariabilityPhenotypic Variability

•• type of mutationtype of mutation–– truncation mutations worse than missense mutationstruncation mutations worse than missense mutations

•• location of mutationlocation of mutation–– MBD mutations worse than TRD mutationsMBD mutations worse than TRD mutations

•• skewing of Xskewing of X--inactivationinactivation–– favourable or unfavourable effect depending on which favourable or unfavourable effect depending on which

X is preferentially inactivatedX is preferentially inactivated

•• other epigenetic factors?other epigenetic factors?

““NonNon--Rett” Clinical PhenotypesRett” Clinical Phenotypes

•• XX--linked mental retardation:linked mental retardation:–– severe nonsevere non--specific XLMR specific XLMR –– mild nonmild non--specific Xspecific X--linked mental retardationlinked mental retardation–– XLMR with progressive spasticityXLMR with progressive spasticity–– PPMPPM--X; psychosis, pyramidal signs, macroX; psychosis, pyramidal signs, macro--orchidismorchidism

•• severe neonatal encephalopathy:severe neonatal encephalopathy:–– esp. if unexplained central hypoventilation, severe seizures & esp. if unexplained central hypoventilation, severe seizures &

abnormal toneabnormal tone

•• AngelmanAngelman--like syndrome: like syndrome: (no (no abnabn involving chromosome 15)involving chromosome 15)~8% (10/125) had ~8% (10/125) had MECP2MECP2 mutationsmutations•• most (but not all) retrospectively found to have regressedmost (but not all) retrospectively found to have regressed

Who Should have Who Should have MECP2MECP2Mutation Screening?Mutation Screening?

•• all patients with a clinical diagnosis of RTTall patients with a clinical diagnosis of RTT–– followfollow--up specific mutation testing in first degree female relativesup specific mutation testing in first degree female relatives–– prenatal testing where requestedprenatal testing where requested

•• male sibs of RTT who show MR &/or neurological male sibs of RTT who show MR &/or neurological abnabn

•• Angelman syndrome with no abnormality of Angelman syndrome with no abnormality of chrchr 1515–– especially if there is an evolving regressive clinical pictureespecially if there is an evolving regressive clinical picture

Definitely:Definitely:

Who Should have Who Should have MECP2MECP2Mutation Screening?Mutation Screening?

•• XLMR, XLMR, FraX(AFraX(A) negative?) negative?

•• MR + autism???MR + autism???

•• Isolated MR???Isolated MR???

Maybe:Maybe:

yield seems very low so faryield seems very low so far(decision on an individual basis)(decision on an individual basis)

SummarySummaryOur Our MECP2MECP2 Studies to DateStudies to Date

•• 75% have missense, nonsense, small frame75% have missense, nonsense, small frame--shiftsshifts

•• 15% have large deletions15% have large deletions

•• exon 1 mutations rareexon 1 mutations rare

•• promoter sequence variations of uncertain promoter sequence variations of uncertain significancesignificance

•• some phenotypesome phenotype--genotype correlationsgenotype correlations

•• 5 5 –– 10% 10% -- no apparent no apparent MECP2MECP2 mutationmutation

Family with no Family with no MECP2MECP2 mutationmutation

II:1II:1-- clinically normal motherclinically normal mother

II:1II:1 II:2II:2

III:5III:5III:1III:1 III:2III:2 III:3III:3 III:4III:4

III:1III:1-- atypical (milder RTT)atypical (milder RTT)-- infantile spasms from 9 weeksinfantile spasms from 9 weeks

-- III:2III:2-- autism & mild MRautism & mild MR-- never had seizuresnever had seizures

III:3III:3-- infantile spasms in the newborn periodinfantile spasms in the newborn period-- poor head controlpoor head control-- severe psychomotor retardationsevere psychomotor retardation-- died age 16 yrs (vegetative, frequent myoclonic jerks)died age 16 yrs (vegetative, frequent myoclonic jerks)

III:4III:4-- clinically normal brotherclinically normal brother

III:5III:5-- clinically normal sisterclinically normal sister

CDKL5CDKL5 Mutation ScreeningMutation ScreeningII:1II:1 II:2II:2

III:5III:5III:3III:3III:1III:1 III:2III:2

183T183T183T183T

183T/183T/183delT183delT 183T/183T/183delT183delT 183delT183delT 183T/183T183T/183T

c.183delTc.183delT

(L75X)(L75X)

c.163_166delGAAAc.163_166delGAAA c.2635_2636delCTc.2635_2636delCT

Summary of currently known Summary of currently known CDKL5CDKL5mutationsmutations

t(X;6)t(X;6)(severe ISSX)(severe ISSX) t(X;7)t(X;7)

(severe ISSX)(severe ISSX) 136kb deletion136kb deletion((retinoschisisretinoschisisand epilepsy)and epilepsy)IVS13IVS13--1G>A1G>Ac.183delTc.183delT

c.746A>T(R175S)c.746A>T(R175S)c.676G>T(C152F)c.676G>T(C152F)

1a1a11 1b1b 101022

ATGATG

66 55 44 33

1111 121233 44 55 66 77 88 99 1313 14141515 1616 1717 1818 1919 2020 2121

TelTel CenCen

STK9 gene

XLRS1 gene

IVS16+1G>CIVS16+1G>CIVS7IVS7--2A>G2A>G

c.215T>A (I72N)c.215T>A (I72N)

CDKL5CDKL5(aka STK9)(aka STK9)

•• novel, conserved serine/threonine kinase novel, conserved serine/threonine kinase -- function function unknown, substrate unknownunknown, substrate unknown

•• large gene of 23 exons with 2 alternative transcription large gene of 23 exons with 2 alternative transcription start sitesstart sites

•• CDKL5 protein localisation CDKL5 protein localisation -- cytoplasm/nucleus?cytoplasm/nucleus?

•• wide tissue expression, including wide tissue expression, including fetalfetal and adult brainand adult brain

•• participates in the regulation of expression of other participates in the regulation of expression of other genes (upstream of or parallel to MeCP2?)genes (upstream of or parallel to MeCP2?)

SummarySummary

MECP2 MECP2 -- major RTT genemajor RTT gene(80(80--90% classical RTT, 6090% classical RTT, 60--70% atypical RTT)70% atypical RTT)

?? mutations involving the promotermutations involving the promoter?? mutations outside mutations outside MECP2MECP2 ORF?ORF?

CDKL5CDKL5 -- new RTT/atypical RTT genenew RTT/atypical RTT gene12 patients with 12 patients with STK9STK9 mutations identifiedmutations identified

?? ISSXISSX?? autism spectrum disorderautism spectrum disorder?? AicardiAicardi syndromesyndrome?? otherother

NetrinNetrin--G1G1: a 3: a 3rdrd RTT gene?RTT gene?

•• single case report of a female with atypical RTT single case report of a female with atypical RTT and early onset seizuresand early onset seizures

•• de novode novo translocation 46XX, t(1;7) (p13.3; q31.33)translocation 46XX, t(1;7) (p13.3; q31.33)–– disrupts the disrupts the NTNG1NTNG1 (Netrin(Netrin--G1) gene on chromosome 1G1) gene on chromosome 1–– involved in axonal guidance & signalling & in NMDA involved in axonal guidance & signalling & in NMDA

receptor functioningreceptor functioning

•• but but no mutations in 115 patients with RTT no mutations in 115 patients with RTT (females (females -- 25 classic and 84 atypical; males 25 classic and 84 atypical; males -- 6) 6)

ConclusionsConclusions

•• most cases of RTT are due to mutations in the most cases of RTT are due to mutations in the XX--linked gene linked gene MECP2MECP2

•• subset of RTT patients have mutations in the subset of RTT patients have mutations in the CDKL5CDKL5 genegene–– responsible for other clinical phenotypesresponsible for other clinical phenotypes

•• role of role of NTNG1NTNG1 in RTT uncertainin RTT uncertain

•• pathogenesis of RTT remains largely unknownpathogenesis of RTT remains largely unknown

Funding AcknowledgementsFunding AcknowledgementsNHMRCNHMRC

Apex Foundation for Research into Intellectual DisabilityApex Foundation for Research into Intellectual Disability

International Rett Syndrome AssociationInternational Rett Syndrome Association

Rett Syndrome Research FoundationRett Syndrome Research Foundation

Rotary Club of NarellanRotary Club of Narellan

CWA of NSWCWA of NSW

Rett Syndrome AustralianRett Syndrome Australian

Research FundResearch Fund

CollaboratorsCollaboratorsChildren’s Hospital at Westmead GroupChildren’s Hospital at Westmead GroupCurrent teamCurrent team Past teamPast teamAngela Angela BeatonBeaton Linda WeavingLinda WeavingBruce BennettsBruce Bennetts Joanne GibsonJoanne GibsonCarolyn EllawayCarolyn Ellaway Vince RepaciVince RepaciAndrew Grimm Andrew Grimm Alexandra BezlerAlexandra BezlerHooshang LahootiHooshang Lahooti Kirsten ReuterKirsten ReuterVidya VasudevanVidya Vasudevan Lauren Lauren CurphyCurphyRose WhiteRose White AbidAbid MohamedaliMohamedaliSarah WilliamsonSarah Williamson

Children’s Medical Research InstituteChildren’s Medical Research InstitutePatrick TamPatrick Tam

Catherine WatsonCatherine WatsonGregory Pelka Gregory Pelka

Phil RobinsonPhil Robinson

TVW Telethon Research Institute, PerthTVW Telethon Research Institute, PerthHelen Leonard & her APSU teamHelen Leonard & her APSU team

Westmead Millennium InstituteWestmead Millennium InstituteBarry Barry SlobedmanSlobedman, Chris Bye & Josh Stern, Chris Bye & Josh Stern

Baylor College of Medicine, HoustonBaylor College of Medicine, HoustonHudaHuda ZoghbiZoghbi

Institute of Medical Genetics, Institute of Medical Genetics, University College of Medicine, CardiffUniversity College of Medicine, CardiffAngus Clarke, Hayley Archer & Julie EvansAngus Clarke, Hayley Archer & Julie Evans

Women’s & Children’s Hospital, AdelaideWomen’s & Children’s Hospital, AdelaideJozef GJozef Gééczcz, Kathie Friend & Olivia McKenzie, Kathie Friend & Olivia McKenzie

West Australian Institute for Medical ResearchWest Australian Institute for Medical ResearchDavid Ravine & David Ravine & AlkaAlka SaxenaSaxena

Male Lethality or Male Sparing?Male Lethality or Male Sparing?•• XX--linked dominant disorderslinked dominant disorders

–– increased male lethalityincreased male lethality–– increased spontaneous miscarriage rateincreased spontaneous miscarriage rate

cytosinecytosine 55--methylmethyl--cytosinecytosine uraciluracilmethyltransferasesmethyltransferases spontaneousspontaneous

deaminationdeamination

•• Rett SyndromeRett Syndrome–– 85% of single base mutations involve CpG “hotspots”85% of single base mutations involve CpG “hotspots”–– sperm highly methylated; X completely methylatedsperm highly methylated; X completely methylated

–– 3 studies reviewing parental origin of 3 studies reviewing parental origin of de novode novo mutationsmutations(Kondo, AJHG 2000; Trappe, AJHG 2001; Girard, EJHG 2001)(Kondo, AJHG 2000; Trappe, AJHG 2001; Girard, EJHG 2001)

–– 90% (54/60) 90% (54/60) -- mutation arose on the paternal Xmutation arose on the paternal X-- many but not all at many but not all at CpGsCpGs

““NonNon--Rett” Clinical PhenotypesRett” Clinical Phenotypes•• XX--linked mental retardation:linked mental retardation:

–– severe male congenital encephalopathy (Wan, AJHG 1999; Villard, severe male congenital encephalopathy (Wan, AJHG 1999; Villard, Neurology 2000)Neurology 2000)

–– severe nonsevere non--specific XLMR specific XLMR ((OrricoOrrico, FEBS , FEBS LettLett 2000)2000)–– XLMR with progressive spasticityXLMR with progressive spasticity ((MeloniMeloni, AJHG 2001), AJHG 2001)–– MR in isolated male cases (2MR in isolated male cases (2--3%?) (3%?) (CouvertCouvert, Hum Mol Gen 2001), Hum Mol Gen 2001)

•• male neonatal encephalopathy:male neonatal encephalopathy:–– no reports of mutations in isolated cases yetno reports of mutations in isolated cases yet

•• Angelman syndrome: (no Angelman syndrome: (no abnabn involving chromosome 15)involving chromosome 15)–– ((ImessaoudeneImessaoudene, JMG 2001; Watson, JMG 2001), JMG 2001; Watson, JMG 2001)

–– ~9% (11/127) had ~9% (11/127) had MECP2MECP2 mutationsmutations»» most (but not all) retrospectively found to have regressedmost (but not all) retrospectively found to have regressed

Our Our MECP2MECP2 Mutation StudiesMutation Studies

•• MECP2MECP2 mutation screening of a clinically wellmutation screening of a clinically well--characterised cohort of RTT patients characterised cohort of RTT patients (Am J Med Genet, 2003)(Am J Med Genet, 2003)

–– pathogenic mutations in 74% of 234 patientspathogenic mutations in 74% of 234 patients(80% classical RTT patients, 70% atypical RTT patients)(80% classical RTT patients, 70% atypical RTT patients)

–– truncation mutations clinically more severe than missense mutatitruncation mutations clinically more severe than missense mutationsons–– TRD mutations clinically more severe than MBD mutationsTRD mutations clinically more severe than MBD mutations–– higher proportion with skewing of Xhigher proportion with skewing of X--inactivation Vs normal controlsinactivation Vs normal controls

•• detailed evaluations of specific mutations detailed evaluations of specific mutations (J Med Genet, (J Med Genet, 2003; J Med Genet 2004)2003; J Med Genet 2004)

•• development of clinical and mutation databases development of clinical and mutation databases (J Child (J Child NeurolNeurol, 2003; Hum , 2003; Hum MutMut, 2003), 2003)

•• international study to examine clinical features of RTTinternational study to examine clinical features of RTT

•• data are collected from 2 sourcesdata are collected from 2 sources–– FamiliesFamilies–– CliniciansClinicians

•• data are stored and compiled to produce an output data are stored and compiled to produce an output databasedatabase–– this will be a searchable form in the futurethis will be a searchable form in the future

•• funded by IRSA funded by IRSA -- International Rett Syndrome International Rett Syndrome AssociationAssociation

InterRettInterRett