Rett Syndrome ResearchRett Syndrome Research

The Sydney ExperienceThe Sydney Experience

John ChristodoulouJohn Christodoulou

NSW Centre for Rett Syndrome ResearchNSW Centre for Rett Syndrome Research

Western Sydney Genetics Program, Children’s Hospital at WestmeadWestern Sydney Genetics Program, Children’s Hospital at Westmead

Disciplines of Paediatrics & Child Health and Medical Genetics, Disciplines of Paediatrics & Child Health and Medical Genetics,

University of SydneyUniversity of Sydney

Presentation OutlinePresentation Outline

• Predicting disease severity by knowing Predicting disease severity by knowing thethe MECP2 MECP2 gene mutationgene mutation

• CDKL5CDKL5 – a second Rett syndrome gene – a second Rett syndrome gene

• hunt for MeCP2 targetshunt for MeCP2 targets

MECP2 MECP2 Mutation Studies:Mutation Studies:

phenotype-genotype correlationsphenotype-genotype correlations

Rett SyndromeRett Syndrome• first described by first described by Andreas Rett Andreas Rett in 1966in 1966

• almost exclusively affects femalesalmost exclusively affects females

• progressive loss ofprogressive loss of– intellectual functioningintellectual functioning

– fine and gross motor skillsfine and gross motor skills

• stereotypic hand movementsstereotypic hand movements

• 1:8,000 females by 15 yrs1:8,000 females by 15 yrs

• rarely familial recurrencesrarely familial recurrences

• most cases caused by mutations in most cases caused by mutations in MECP2MECP2

Clinical DiagnosisClinical Diagnosis• specific developmental profile based on a consistent specific developmental profile based on a consistent

constellation of clinical features constellation of clinical features (diagnosis is provisional < 3 yrs)(diagnosis is provisional < 3 yrs)

• diagnostic criteria developed and recently reviseddiagnostic criteria developed and recently revised

• classical and variant RTT phenotypesclassical and variant RTT phenotypes

Preserved Preserved SpeechSpeech

Forme Forme FrusteFruste

ClassicalClassicalCongenital Congenital RTTRTT

Late and Late and slow onset slow onset RTTRTT

Modified from Hagberg et al Eur J Paediatr Neurol 2002 (6) 293 - 297Modified from Hagberg et al Eur J Paediatr Neurol 2002 (6) 293 - 297

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

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

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

““Rett syndrome is caused by mutations in Rett syndrome is caused by mutations in X-linked X-linked MECP2MECP2, encoding methyl-CpG , encoding methyl-CpG

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

MECP2 MECP2 Mutations IdentifiedMutations Identified

> 270 different mutations to date> 270 different mutations to date > 3000 individuals> 3000 individuals

RettBASE: http://mecp2.chw.edu.auRettBASE: http://mecp2.chw.edu.au

Nucleotide change

Amino acid change

Cases reported

percentage

c.316C>T p.R106W 95 3.07

c.397C>T p.R133C 134 4.33

c.473C>T p.T158M 276 8.92

c.502C>T p.R168X 254 8.21c.763C>T p.R255X 222 7.17

c.808C>T p.R270X 198 6.40c.880C>T p.R294X 169 5.46

c.916C>T p.R306C 152 4.91

20 – 100 bp

deletions

various

truncations

203 6.5555%

• 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

• both databases have been funded by IRSA (and now both databases have been funded by IRSA (and now the IRSF)the IRSF)

• development of clinical and mutation databases development of clinical and mutation databases

(J Child Neurol, 2003; Hum Mut, 2003)(J Child Neurol, 2003; Hum Mut, 2003)

Our Our MECP2MECP2 Mutation Studies Mutation Studies

• MECP2MECP2 mutation screening of a clinically well- mutation 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 mutationstruncation mutations clinically more severe than missense mutations

– NLS & TRD mutations clinically more severe than MBD mutationsNLS & TRD mutations clinically more severe than MBD mutations

– higher proportion with skewing of X-inactivation Vs normal controlshigher proportion with skewing of X-inactivation Vs normal controls

• detailed evaluations of specific mutations detailed evaluations of specific mutations (J Med Genet, (J Med Genet, 2003; J Med Genet 2004; Brain Dev, 2005; Eur J Hum Genet, 2005; J Med 2003; J Med Genet 2004; Brain Dev, 2005; Eur J Hum Genet, 2005; J Med Genet, 2007)Genet, 2007)

– p.R133C mutation is milder; p.R270X most severep.R133C mutation is milder; p.R270X most severe

– 58% show unusual behaviours in the first 6 months of life58% show unusual behaviours in the first 6 months of life

– X-inactivation modulates disease severity of p.T158M & p.R168XX-inactivation modulates disease severity of p.T158M & p.R168X

Our Our MECP2MECP2 Mutation Studies Mutation Studies

• evaluation of clinical aspects ofevaluation of clinical aspects of RTT RTT (J Pediatr, (J Pediatr, 2005; J Pediatr, 2006; J Child Neurol, 2006; Eur J Pediatr Neurol, 2005; J Pediatr, 2006; J Child Neurol, 2006; Eur J Pediatr Neurol, 2008)2008)

– 78% survival by 25 yrs78% survival by 25 yrs

– 25% have seizures by 2 yr, 50% by 4 yr, 79% by 10 yr25% have seizures by 2 yr, 50% by 4 yr, 79% by 10 yr

– later onset of seizures with p.R294X vs p.R255Xlater onset of seizures with p.R294X vs p.R255X

– seizure rate highest 7 – 12 yr (lower with p.R294X, p.R255X, seizure rate highest 7 – 12 yr (lower with p.R294X, p.R255X, C-term)C-term)

– 75% have scoliosis by 13 yr (less likely if have p.R294X)75% have scoliosis by 13 yr (less likely if have p.R294X)

– 4 times more likely to have a fracture4 times more likely to have a fracture

Please participatePlease participate

• if you are not a current participant and you would like to take if you are not a current participant and you would like to take part in InterRett and the work practices pilot study just email part in InterRett and the work practices pilot study just email us….us….

• rett@ichr.uwa.edu.aurett@ichr.uwa.edu.au

• or visit our website or visit our website http://http://interrett.ichr.uwa.edu.auinterrett.ichr.uwa.edu.au

• encourage your doctor or the laboratory that did the testing to encourage your doctor or the laboratory that did the testing to submit their informationsubmit their information

• gladysh@chw.edu.augladysh@chw.edu.au

• or visit our website or visit our website http://http://mecp2.chw.edu.aumecp2.chw.edu.au

CDKL5CDKL5::

a new Rett Syndrome genea new Rett Syndrome gene

Clinical Summary Clinical Summary

Family 1Family 1

II:1II:1- clinically normal mother- clinically 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 weeks- infantile spasms from 9 weeks

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

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

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

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

Further Genetic Studies of Family 1Further Genetic Studies of Family 1

atypical Rett syndromeatypical Rett syndrome autism/MRautism/MR neonatal onset seizuresneonatal onset seizures and profound MRand profound MR

DXS1193

DXS1060

DXS1135DXS8051

DXS987DXS1683DXS1226DXS1202

DXS993DXS991

ARDXS986

DXS1196DXS990

DXS1191DXS1106DXS8055DXS1001DXS1047DXS1227DXS8043DXS8091DXS1185

DXS1123DXS8103DXS1684DXS1177

DXS15DXS8087DXS1073DXS1108

I:2

II:2

III:3III:1 III:4

? ?

18.56cM18.56cM(16.4Mb)(16.4Mb)

MECP2MECP2

24.8cM24.8cM(19.7Mb)(19.7Mb)

21.36cM21.36cM(10.3 Mb)(10.3 Mb)

ARXARX

STK9STK9

II:1

III:5

I:1

?

21:79

18:82 23:77 27:73

DXS1214DXS1068

X Chromosome

markers

p22.33 3.18 4 240

p22.31 21.36 10 286p22.2 25.41 13 535

p22.12 34.06 22 794

p11.4 67.09 39 396p11.21 89.67 53457q11.2 91.02 64 000

q21.2 99.35 83 765q21.33 107.15 89 981

q22.3 118.84 107 202q23 123.36 111 780q24 137.73 116 822

q27.1 159.45 137 660q27.2 173.13 140 850

q28 181.83 145 181q28 181.94 145 212q28 181.99 145 318

q28 184.43 146 809q28 188.95 156 199

q28 190.32 158 154

q28 192.52 159 695

p22.31 15.52 10 150

p22.12 31.98 20 857

p22.11 40.32 25 130

p11.4 58.17 37 196p21.1 46.16 29 946

q13.3 98.22 76 343

q22.3 117.19 100 213

q25 147.38 125 921

q28 180.85 144 434

q28 183.59 146 798

q28 189.48 156 959

q28 190.5 158 414

Chromosomal location

Map location

(cM)

Map location

(kb)

11223212112212222221122112?2?2122

11223212112212222221122112?2?2122

122213?11111112?1111211111?1?1111

112121221233121111123321213333122

112121211111111111112111111111111

112232121122122222211221122222122

1

222122121

121111112

21213

3

122

12/11113111131/31123211231/3111111/311/3111

1?222122121?121111112?21213?3?122

112121211111111111112111111111111

112232121122122222211221122222122

III:2

MECP2 MECP2 - C426T (F142F)- C426T (F142F)

STK9/CDKL5STK9/CDKL5 Mutation Screening Mutation Screening

Family 1Family 1II: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

(p.L75X)(p.L75X)

Summary of currently known Summary of currently known CDKL5CDKL5 mutations mutations

66 55 44 33

1a1a11 1b1b 101022

ATGATG

1111 121233 44 55 66 77 88 99 1313 141415151616 1717 1818 1919 2020 2121

TelTel CenCen

CDKL5 gene

XLRS1 gene

IVS7-2A>GIVS7-2A>G

c.455G>Tc.455G>T

c.2635-2636delCTc.2635-2636delCT

c.838_847del10c.838_847del10

IVS11-2A>GIVS11-2A>G

IVS13-1G>AIVS13-1G>AIVS6-1G>TIVS6-1G>TIVS6-1G>CIVS6-1G>C

c.525A>Tc.525A>T

c.539C>Tc.539C>T

c.2343delGc.2343delG

IVS16+1G>CIVS16+1G>C

IVS16+1G>AIVS16+1G>A

c.2362_2366del5c.2362_2366del5del678_691del678_691ins683_673ins683_673

c.680T>Cc.680T>C

c.183delTc.183delTc.215T>Ac.215T>A

c.163-166delGAAAc.163-166delGAAAc.175C>Tc.175C>T

Further Mutation Screening Further Mutation Screening of CDKL5of CDKL5

• particular clinical phenotypeparticular clinical phenotype– Hanefeld variant –onset of severe seizures < 6 monthsHanefeld variant –onset of severe seizures < 6 months

• Archer et al ( J Med Genet 2006: 43; 729-734)Archer et al ( J Med Genet 2006: 43; 729-734)– 7 of 42 (17%) ♀ with seizures commencing < 6 months of age7 of 42 (17%) ♀ with seizures commencing < 6 months of age

– all with poor developmental progressall with poor developmental progress

– severe seizures mostly of myoclonic or infantile spasm typesevere seizures mostly of myoclonic or infantile spasm type

– few clinical signs suggestive of RTTfew clinical signs suggestive of RTT

– males rarely show males rarely show CDKL5CDKL5 mutations mutations

• our studies – 272 patients screened for mutations in the our studies – 272 patients screened for mutations in the CDKL5CDKL5 gene, incl. 89 RTT, 60 ISSX, 58 autism, 7 XLMR, gene, incl. 89 RTT, 60 ISSX, 58 autism, 7 XLMR, 58 others58 others– only 1 de novo missense mutation - c.586C>T (p.S196L)only 1 de novo missense mutation - c.586C>T (p.S196L)

Dapi CDKL5- Polyclonal

-Tubulin MergeUntransfected (HeLa)

CDKL5CDKL5 + Tubulin + Tubulin

• functional significance?functional significance?

• direct or indirect interaction?direct or indirect interaction?

• neuronal physiology – decreased dendritic branching neuronal physiology – decreased dendritic branching

in Rett Syndrome patients in Rett Syndrome patients

normalnormal RettRett autismautism

Zoghbi, Science 2003

Identification of Specific MeCP2 Identification of Specific MeCP2

Downstream TargetsDownstream Targets

Neuropathology of RettNeuropathology of Rett• adult Rett brain ~ 900 grams (the adult Rett brain ~ 900 grams (the

same size as a non RTT 1 year old)same size as a non RTT 1 year old)

• regional volumetric lossregional volumetric loss

Control RettControl Rett

• small densely packed neurons with decreased dendritic small densely packed neurons with decreased dendritic branching branching (cerebral cortex, basal ganglia, hippocampus)(cerebral cortex, basal ganglia, hippocampus)

• occipital cortex escapes neuropathologyoccipital cortex escapes neuropathology

normalnormal RettRettautismautism

Zoghbi, Science 2003

-------AGCT--------------AGCT-------

-------AGCT--------------AGCT-------

-------AGCT--------------AGCT-------

-------AGCT--------------AGCT-------

--------------aaaaUCGA------UCGA------

--------------aaaaUCGA------UCGA------

--------------aaaaUCGA------UCGA------

-------AGCT--------------AGCT-------

-------AGCT--------------AGCT-------

-------AGCT--------------AGCT-------

-------AGCT--------------AGCT-------

--------------aaaaUCGA------UCGA------

--------------aaaaUCGA------UCGA------

--------------aaaaUCGA------UCGA------

Increased Increased ExpressionExpression

DecreasedDecreased ExpressionExpression

-------AGCT--------------AGCT-------

-------AGCT--------------AGCT-------

-------AGCT--------------AGCT-------

-------AGCT--------------AGCT-------

--------------aaaaUCGA------UCGA------

--------------aaaaUCGA------UCGA------

UniformUniform ExpressionExpression

Label two mRNA Label two mRNA populations (populations (redred and and greengreen))

Bind labelled probe Bind labelled probe mRNAmRNA ssiimmuullttaanneeoouussllyy toto spotsspots

Created by Dan Catchpoole, CHW, 2001Created by Dan Catchpoole, CHW, 2001

Gene Expression StudiesGene Expression Studies• using mRNA from RTT patient brain samplesusing mRNA from RTT patient brain samples - compare regional expression patterns- compare regional expression patterns

- studies using frontal and occipital cerebral cortex- studies using frontal and occipital cerebral cortex

• microarray analysesmicroarray analysesExpression ProfilesExpression Profiles

increasedincreased

uniformuniform

reducedreduced

Gene NameGene Name FunctionFunction Median Median Fold Fold

ChangeChangeRTT FC:RTT OC/ RTT FC:RTT OC/ RTT FC: CON FCRTT FC: CON FC

(RNAi results)(RNAi results)

cytochrome c cytochrome c oxidase subunit Ioxidase subunit I

involved in energy involved in energy productionproduction

2.4/ 1.9 2.4/ 1.9

(4x)(4x)

clusterinclusterin control of cell survivalcontrol of cell survival 1.7/ 1.51.7/ 1.5

(3x)(3x)

guanine nucleotide guanine nucleotide binding protein binding protein

communication between communication between cellscells

2.5/ 1.82.5/ 1.8

(2x)(2x)

dynamin 1dynamin 1 communication between communication between brain cells brain cells

1.7/ 1.51.7/ 1.5

(3x)(3x)

Abnormal expression in Abnormal expression in Rett frontal cortexRett frontal cortex

Biology of Rett SyndromeBiology of Rett Syndrome

• functional abnormalities of energy production?functional abnormalities of energy production?– previous functional and structural studiesprevious functional and structural studies

• altered control of cell survival?altered control of cell survival?– increased sensitivity to agents that promote cell deathincreased sensitivity to agents that promote cell death

• abnormalities of communication between brain cells?abnormalities of communication between brain cells?– MeCP2 also found in synaptic regions, & shows punctate MeCP2 also found in synaptic regions, & shows punctate

cytoplasmic staining in COS-7 cells, WBC, fibroblasts and cytoplasmic staining in COS-7 cells, WBC, fibroblasts and PC12 cellsPC12 cells

plan to study these in more detail using our cell plan to study these in more detail using our cell culture model and mouse models at our disposalculture model and mouse models at our disposal

11stst Dimension Dimension Isoelectric Focusing Isoelectric Focusing EquilibrationEquilibration

22ndnd Dimension Dimension SDS- SDS-Polyacrylamide Gel Polyacrylamide Gel ElectrophoresisElectrophoresis

image Gelimage GelUsing the Typhoon MULTI LASER Using the Typhoon MULTI LASER ScannerScanner

Image AnalysisImage Analysis

Proteomic Study on Proteomic Study on Mecp2Mecp2

Mouse ModelMouse ModelDIGE (Differential Imaging GEL Electrophoresis)DIGE (Differential Imaging GEL Electrophoresis)

Overlaid ImageOverlaid Image

33 PHPH 10 10

250250

1515

MS/MS analysisMS/MS analysis

Compare with Compare with Mw/pI from gelMw/pI from gel

Verify with Online Verify with Online toolstools

Tryptic digestTryptic digest

Gel extractionGel extraction

00 1000 1000 1500 1500 2000 2000 2500 2500 3000 3000

Mass SpectrometryMass Spectrometry

1115.531115.531116.591116.591193.621193.621209.561209.561218.631218.631248.551248.551349.621349.62------

1466.71466.71480.651480.651506.731506.731576.791576.791592.761592.761705.921705.921723.91723.9------

Peak List = Peak List = Mass fingerprintMass fingerprintDatabase searchDatabase searchProtein identified!Protein identified!

Identification of the target Identification of the target spotspot

ConclusionsConclusions

• the biological processes involved in RTT may in part the biological processes involved in RTT may in part be a consequence of abnormalities of:be a consequence of abnormalities of:

– energy productionenergy production

– cell survivalcell survival

– communication between brain cellscommunication between brain cells

• study of CDKL5/MeCP2 interactions will yield further study of CDKL5/MeCP2 interactions will yield further insights into RTT biologyinsights into RTT biology

• combination of clinical, combination of clinical, in vitroin vitro and animal model and animal model research is needed to answer questions relating to research is needed to answer questions relating to the biology of RTTthe biology of RTT

• the clinical - laboratory interface is critical to the clinical - laboratory interface is critical to translating research into clinical practicetranslating research into clinical practice

CollaboratorsCollaboratorsChildren’s Hospital at Westmead GroupChildren’s Hospital at Westmead Group

Current teamCurrent team Past teamPast teamRoksana ArmaniRoksana Armani Linda WeavingLinda WeavingBruce BennettsBruce Bennetts Alexandra BezlerAlexandra Bezler

Desiree Cloosterman Andrew GrimmDesiree Cloosterman Andrew Grimm Carolyn EllawayCarolyn Ellaway Joanne Gibson Joanne GibsonGladys HoGladys Ho Simon Hardwick Simon HardwickRania Kairouz-WabheRania Kairouz-Wabhe Hooshang LahootiHooshang Lahooti Vidya VasudevanVidya Vasudevan Abid Mohamedali Abid MohamedaliSarah Williamson Rose WhiteSarah Williamson Rose White

Children’s Medical Research InstituteChildren’s Medical Research Institute Patrick TamPatrick Tam

Gregory PelkaGregory Pelka Abid Mohamedali Abid Mohamedali

Phil RobinsonPhil Robinson

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

Westmead Millennium InstituteWestmead Millennium InstituteBarry Slobedman, Chris Bye & Josh SternBarry Slobedman, Chris Bye & Josh Stern

Baylor College of Medicine, HoustonBaylor College of Medicine, Houston

Huda ZoghbiHuda Zoghbi

Institute of Medical Genetics, Institute of Medical Genetics, University College of Medicine, CardiffUniversity College of Medicine, Cardiff

Angus Clarke, Hayley ArcherAngus Clarke, Hayley Archer

Women’s & Children’s Hospital, AdelaideWomen’s & Children’s Hospital, Adelaide Jozef GJozef Géécz, Kathie Friend & Olivia McKenziecz, Kathie Friend & Olivia McKenzie

West Australian Institute for Medical ResearchWest Australian Institute for Medical ResearchDavid Ravine & Alka SaxenaDavid Ravine & Alka Saxena

Funding AcknowledgementsFunding AcknowledgementsNHMRCNHMRC

International Rett Syndrome AssociationInternational Rett Syndrome Association

Rett Syndrome Research FoundationRett Syndrome Research Foundation

International Rett Syndrome FoundationInternational Rett Syndrome Foundation

Rotary Club of NarellanRotary Club of Narellan

CWA of NSWCWA of NSW

Rett Syndrome Australian Research Rett Syndrome Australian Research FundFund

Tissue Resource Centre, SydneyTissue Resource Centre, SydneyHarvard Brain BankHarvard Brain Bank

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

MeCP2MeCP2mSin3amSin3aHDACHDAC

MeCP2 binds to methyl-CpGsMeCP2 binds to methyl-CpGs

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

HDACHDAC

mSin3amSin3a

Large Deletions in our RTT Large Deletions in our RTT PatientsPatients

teltel cencen

11 22 33 Exon 4Exon 4

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

CDKL5/STK9CDKL5/STK9

• novel, conserved serine/threonine kinasenovel, conserved serine/threonine kinase

• large gene of 23 exons with 2 alternative transcription large gene of 23 exons with 2 alternative transcription start sites generating two isoformsstart sites generating two isoforms

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

• wide tissue expression, including fetal and adult brainwide tissue expression, including fetal and adult brain

Unanswered Questions - CDKL5Unanswered Questions - CDKL5

• Does CDKL5 Does CDKL5 phosphorylate phosphorylate MeCP2 (and other MeCP2 (and other proteins)?proteins)?

POPO44

HDAC

mSin3amSin3a

MeCP2

HDAC

mSin3amSin3a

MeCP2

rat BDNF exon IIIrat BDNF exon III

rat BDNF exon IIIrat BDNF exon III

depolarizationdepolarization

Is CDKL5 the link??Is CDKL5 the link??

Unanswered QuestionsUnanswered Questions- CDKL5- CDKL5

• Does CDKL5 phosphorylate MeCP2 (and other proteins)?Does CDKL5 phosphorylate MeCP2 (and other proteins)?

• Do the different isoforms have different functions?Do the different isoforms have different functions?

• What is the developmental expression profile of What is the developmental expression profile of Cdkl5Cdkl5 in in mouse?mouse?

• Will mouse models for Will mouse models for Cdkl5Cdkl5 deficiency help us deficiency help us understand the biology of Rett syndrome?understand the biology of Rett syndrome?

Rett frontal cortexRett frontal cortex

Control occipital cortexControl occipital cortex

Rett occipital cortexRett occipital cortex

Control frontal cortexControl frontal cortex

Expression profilingExpression profiling

• cDNA microarrays with 19,000+ probe sequences cDNA microarrays with 19,000+ probe sequences (University Health Network, Ontario)(University Health Network, Ontario)

• 7 Rett and 7 control human frontal and occipital 7 Rett and 7 control human frontal and occipital corticescortices

• (a) Significance Analysis of Microarrays (a) Significance Analysis of Microarrays (modified t-test)(modified t-test)

• (b) >1.5 fold change, 5/7 biological replicates(b) >1.5 fold change, 5/7 biological replicates

Differentially expressed genesDifferentially expressed genes

13 UP13 UP

21 DOWN21 DOWN

14 unknown 14 unknown functionfunction

Rett frontal cortexRett frontal cortex

Control occipital cortexControl occipital cortex

Rett occipital cortexRett occipital cortex

Control frontal cortexControl frontal cortex

3434

1313

33

447 UP7 UP

6 DOWN6 DOWN

4 unknown 4 unknown functionfunction