1. Rett Syndrome Research The Sydney Experience John Christodoulou NSW Centre for Rett Syndrome Research Western Sydney Genetics Program, Children’s Hospital at Westmead Disciplines of Paediatrics & Child Health and Medical Genetics, University of Sydney

2. Presentation Outline Predicting disease severity by knowing the MECP2 gene mutationPredicting disease severity by knowing the MECP2 gene mutation CDKL5 – a second Rett syndrome geneCDKL5 – a second Rett syndrome gene hunt for MeCP2 targetshunt for MeCP2 targets

3. MECP2 Mutation Studies: phenotype-genotype correlations

4. Rett Syndrome first described by Andreas Rett in 1966first described by Andreas Rett in 1966 almost exclusively affects femalesalmost exclusively affects females progressive loss ofprogressive loss of – intellectual functioning – fine 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 MECP2most cases caused by mutations in MECP2

5. Clinical Diagnosis specific developmental profile based on a consistent constellation of clinical features (diagnosis is provisional < 3 yrs)specific developmental profile based on a consistent constellation of clinical features (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 Speech Forme Fruste Classical Congenital RTT Late and slow onset RTT

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

7. 6 mutations identified in 21 sporadic classical cases - 4 de novo missense mutations in methyl-binding domain (MBD) - 4 de novo missense mutations in methyl-binding domain (MBD) - 1 de novo frame-shift mutation in transcription repression domain (TRD) - 1 de novo frame-shift mutation in transcription repression domain (TRD) - 1 de novo nonsense mutation in TRD - 1 de novo nonsense mutation in TRD “Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG binding protein 2” (Amir et al, Nature Genet 1999: 23; 185 - 188)

9. MECP2 Mutations Identified > 270 different mutations to date> 3000 individuals RettBASE: http://mecp2.chw.edu.au Nucleotide change Amino acid change Cases reported percentage c.316C>Tp.R106W953.07 c.397C>Tp.R133C1344.33 c.473C>Tp.T158M2768.92 c.502C>Tp.R168X2548.21 c.763C>Tp.R255X2227.17 c.808C>Tp.R270X1986.40 c.880C>Tp.R294X1695.46 c.916C>Tp.R306C1524.91 20 – 100 bp deletions various truncations 2036.55 55%

10. 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 –families –clinicians data are stored and compiled to produce an output databasedata are stored and compiled to produce an output database –this will be a searchable form in the future both databases have been funded by IRSA (and now the IRSF)both databases have been funded by IRSA (and now the IRSF) development of clinical and mutation databasesdevelopment of clinical and mutation databases (J Child Neurol, 2003; Hum Mut, 2003) (J Child Neurol, 2003; Hum Mut, 2003)

11. Our MECP2 Mutation Studies MECP2 mutation screening of a clinically well- characterised cohort of RTT patients (Am J Med Genet, 2003)MECP2 mutation screening of a clinically well- characterised cohort of RTT patients (Am J Med Genet, 2003) –pathogenic mutations in 74% of 234 patients –(80% classical RTT patients, 70% atypical RTT patients) –truncation mutations clinically more severe than missense mutations –NLS & TRD mutations clinically more severe than MBD mutations –higher proportion with skewing of X-inactivation Vs normal controls detailed evaluations of specific mutations (J Med Genet, 2003; J Med Genet 2004; Brain Dev, 2005; Eur J Hum Genet, 2005; J Med Genet, 2007)detailed evaluations of specific mutations (J Med Genet, 2003; J Med Genet 2004; Brain Dev, 2005; Eur J Hum Genet, 2005; J Med Genet, 2007) –p.R133C mutation is milder; p.R270X most severe –58% show unusual behaviours in the first 6 months of life –X-inactivation modulates disease severity of p.T158M & p.R168X

12. Our MECP2 Mutation Studies evaluation of clinical aspects of RTT (J Pediatr, 2005; J Pediatr, 2006; J Child Neurol, 2006; Eur J Pediatr Neurol, 2008)evaluation of clinical aspects of RTT (J Pediatr, 2005; J Pediatr, 2006; J Child Neurol, 2006; Eur J Pediatr Neurol, 2008) –78% survival by 25 yrs –25% have seizures by 2 yr, 50% by 4 yr, 79% by 10 yr –later onset of seizures with p.R294X vs p.R255X –seizure rate highest 7 – 12 yr (lower with p.R294X, p.R255X, C-term) –75% have scoliosis by 13 yr (less likely if have p.R294X) –4 times more likely to have a fracture

13. Please participate if you are not a current participant and you would like to take part in InterRett and the work practices pilot study just email us….if you are not a current participant and you would like to take part in InterRett and the work practices pilot study just email us…. rett@ichr.uwa.edu.aurett@ichr.uwa.edu.aurett@ichr.uwa.edu.au or visit our website http://interrett.ichr.uwa.edu.auor visit our website http://interrett.ichr.uwa.edu.auhttp://interrett.ichr.uwa.edu.auhttp://interrett.ichr.uwa.edu.au encourage your doctor or the laboratory that did the testing to submit their informationencourage your doctor or the laboratory that did the testing to submit their information gladysh@chw.edu.augladysh@chw.edu.augladysh@chw.edu.au or visit our website http://mecp2.chw.edu.auor visit our website http://mecp2.chw.edu.auhttp://mecp2.chw.edu.auhttp://mecp2.chw.edu.au

14. CDKL5: a new Rett Syndrome gene

15. Clinical Summary Family 1 II:1 - clinically normal mother II:1II:2III:5 III:1III:2 III:3 III:4 III:1 - atypical (milder RTT) - infantile spasms from 9 weeks - III:2 - autism & mild MR - never had seizures III:3 - infantile spasms in the newborn period - poor head control - severe psychomotor retardation - died age 16 yrs (unresponsive, frequent myoclonic jerks) III:4 - clinically normal brother III:5 - clinically normal sister

16. Further Genetic Studies of Family 1 MECP2 - C426T (F142F)

17. STK9/CDKL5 Mutation Screening Family 1 II:1II:2III:5 III:3 III:1III:2 183T 183T 183T/183delT 183delT183T/183T c.183delT(p.L75X)

18. Summary of currently known CDKL5 mutations 6543 1a 1 1b 102ATG11123456789131415161718192021 Tel Cen CDKL5 gene XLRS1 gene IVS7-2A>Gc.455G>T c.2635-2636delCT c.2635-2636delCT c.838_847del10IVS11-2A>G IVS13-1G>A IVS6-1G>TIVS6-1G>C c.525A>T c.525A>T c.539C>T c.539C>Tc.2343delGIVS16+1G>CIVS16+1G>Ac.2362_2366del5del678_691ins683_673 c.680T>C c.183delTc.215T>Ac.163-166delGAAAc.175C>T

19. Further Mutation Screening of CDKL5 particular clinical phenotypeparticular clinical phenotype –Hanefeld 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 age –all with poor developmental progress –severe seizures mostly of myoclonic or infantile spasm type –few clinical signs suggestive of RTT –males rarely show CDKL5 mutations our studies – 272 patients screened for mutations in the CDKL5 gene, incl. 89 RTT, 60 ISSX, 58 autism, 7 XLMR, 58 othersour studies – 272 patients screened for mutations in the CDKL5 gene, incl. 89 RTT, 60 ISSX, 58 autism, 7 XLMR, 58 others –only 1 de novo missense mutation - c.586C>T (p.S196L)

20. Dapi CDKL5- Polyclonal  -Tubulin Merge Untransfected (HeLa)

21. CDKL5 + Tubulin functional significance? functional significance? direct or indirect interaction? direct or indirect interaction? neuronal physiology – decreased dendritic branching in Rett Syndrome patients neuronal physiology – decreased dendritic branching in Rett Syndrome patients normal normal Rett Rett autism autism Zoghbi, Science 2003

22. Identification of Specific MeCP2 Downstream Targets

23. Neuropathology of Rett adult Rett brain ~ 900 grams (the same size as a non RTT 1 year old)adult Rett brain ~ 900 grams (the same size as a non RTT 1 year old) regional volumetric lossregional volumetric loss Control Rett small densely packed neurons with decreased dendritic branching (cerebral cortex, basal ganglia, hippocampus)small densely packed neurons with decreased dendritic branching (cerebral cortex, basal ganglia, hippocampus) occipital cortex escapes neuropathologyoccipital cortex escapes neuropathology normalRettautism Zoghbi, Science 2003

24. -------AGCT------- -------AGCT------- -------AGCT------- -------AGCT------- ------- aa UCGA------ -------AGCT------- -------AGCT------- -------AGCT------- -------AGCT------- IncreasedExpressionDecreased Expression Expression -------AGCT------- -------AGCT------- -------AGCT------- -------AGCT------- ------- aa UCGA------ Uniform Expression Expression Label two mRNA populations (red and green) Bind labelled probe mRNA simultaneously to spots Created by Dan Catchpoole, CHW, 2001

25. Gene 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 microarray analysesmicroarray analyses Expression Profiles increaseduniformreduced

26. Gene Name Function Median Fold Change RTT FC:RTT OC/ RTT FC: CON FC (RNAi results) cytochrome c oxidase subunit I involved in energy production 2.4/ 1.9 2.4/ 1.9 (4x) (4x) clusterin control of cell survival 1.7/ 1.5 1.7/ 1.5 (3x) (3x) guanine nucleotide binding protein  communication between cells 2.5/ 1.8 2.5/ 1.8 (2x) (2x) dynamin 1 communication between brain cells 1.7/ 1.5 1.7/ 1.5 (3x) (3x) Abnormal expression in Rett frontal cortex

27. Biology of Rett Syndrome functional abnormalities of energy production?functional abnormalities of energy production? –previous functional and structural studies altered control of cell survival?altered control of cell survival? –increased 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 cytoplasmic staining in COS-7 cells, WBC, fibroblasts and PC12 cells plan to study these in more detail using our cell culture model and mouse models at our disposal

28. 1 st Dimension Isoelectric Focusing Equilibration 2 nd Dimension SDS- Polyacrylamide Gel Electrophoresis image Gel Using the Typhoon MULTI LASER Scanner Image Analysis Proteomic Study on Mecp2 Mouse Model DIGE (Differential Imaging GEL Electrophoresis)

29. Overlaid Image 3PH 10 25015

30. MS/MS analysis Compare with Mw/pI from gel Verify with Online tools Tryptic digest Gel extraction 0 1000 1000 1500 1500 2000 2000 2500 2500 3000 3000 Mass Spectrometry 1115.531116.591193.621209.561218.631248.551349.62---1466.71480.651506.731576.791592.761705.921723.9--- Peak List = Mass fingerprint Database search Protein identified! Identification of the target spot

31. Conclusions the biological processes involved in RTT may in part be a consequence of abnormalities of:the biological processes involved in RTT may in part be a consequence of abnormalities of: –energy production –cell survival –communication between brain cells study of CDKL5/MeCP2 interactions will yield further insights into RTT biologystudy of CDKL5/MeCP2 interactions will yield further insights into RTT biology combination of clinical, in vitro and animal model research is needed to answer questions relating to the biology of RTTcombination of clinical, in vitro and animal model research is needed to answer questions relating to the biology of RTT the clinical - laboratory interface is critical to translating research into clinical practicethe clinical - laboratory interface is critical to translating research into clinical practice

32. Collaborators Children’s Hospital at Westmead Group Current team Past team Current team Past team Roksana ArmaniLinda Weaving Bruce BennettsAlexandra Bezler Bruce Bennetts Alexandra Bezler Desiree Cloosterman Andrew Grimm Carolyn Ellaway Joanne Gibson Gladys Ho Simon Hardwick Rania Kairouz-WabheHooshang Lahooti Rania Kairouz-Wabhe Hooshang Lahooti Vidya Vasudevan Abid Mohamedali Sarah Williamson Rose White Children’s Medical Research Institute Patrick Tam Gregory Pelka Gregory Pelka Abid Mohamedali Abid Mohamedali Phil Robinson TVW Telethon Research Institute, Perth Helen Leonard & her ARSD team Westmead Millennium Institute Barry Slobedman, Chris Bye & Josh Stern Baylor College of Medicine, Houston Huda Zoghbi Institute of Medical Genetics, University College of Medicine, Cardiff Angus Clarke, Hayley Archer Women’s & Children’s Hospital, Adelaide Jozef Gécz, Kathie Friend & Olivia McKenzie Jozef Gécz, Kathie Friend & Olivia McKenzie West Australian Institute for Medical Research David Ravine & Alka Saxena

33. Funding Acknowledgements NHMRC International Rett Syndrome Association Rett Syndrome Research Foundation International Rett Syndrome Foundation Rotary Club of Narellan CWA of NSW Rett Syndrome Australian Research Fund Tissue Resource Centre, Sydney Harvard Brain Bank

35. MethylatedDNAMeCP2 Ac Ac Ac Ac chromatin accessible & active Gene Silencing by Chromatin Condensation chromatin condensed & inactive MeCP2mSin3aHDAC MeCP2 binds to methyl-CpGs recruitment of mSin3a & histone deacetylase (HDAC) HDAC mSin3a

36. Large Deletions in our RTT Patients telcen 12 3 Exon 4 MBDTRD3’UTR 1 ~15 kb 4.609 kb ~47 kb ~37 kb ~40 kb ~65 kb

37. CDKL5/STK9 novel, conserved serine/threonine kinasenovel, conserved serine/threonine kinase large gene of 23 exons with 2 alternative transcription start sites generating two isoformslarge gene of 23 exons with 2 alternative transcription start 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

38. Unanswered Questions - CDKL5 Does CDKL5 phosphorylate MeCP2 (and other proteins)?Does CDKL5 phosphorylate MeCP2 (and other proteins)? PO 4 HDAC mSin3a MeCP2 HDAC mSin3a MeCP2 rat BDNF exon III depolarization Is CDKL5 the link??

39. Unanswered Questions - 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 Cdkl5 in mouse?What is the developmental expression profile of Cdkl5 in mouse? Will mouse models for Cdkl5 deficiency help us understand the biology of Rett syndrome?Will mouse models for Cdkl5 deficiency help us understand the biology of Rett syndrome?

40. Rett frontal cortex Control occipital cortex Rett occipital cortex Control frontal cortex Expression profiling cDNA microarrays with 19,000+ probe sequences (University Health Network, Ontario)cDNA microarrays with 19,000+ probe sequences (University Health Network, Ontario) 7 Rett and 7 control human frontal and occipital cortices7 Rett and 7 control human frontal and occipital cortices (a) Significance Analysis of Microarrays (modified t-test)(a) Significance Analysis of Microarrays (modified t-test) (b) >1.5 fold change, 5/7 biological replicates(b) >1.5 fold change, 5/7 biological replicates

41. Differentially expressed genes 13 UP 21 DOWN 14 unknown function Rett frontal cortex Control occipital cortex Rett occipital cortex Control frontal cortex 34 13 3 4 7 UP 6 DOWN 4 unknown function