1 / 41

Rett Syndrome Research The Sydney Experience

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. Presentation Outline.

psikes
Télécharger la présentation

Rett Syndrome Research The Sydney Experience

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Rett Syndrome ResearchThe 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 mutation • CDKL5 – a second Rett syndrome gene • hunt for MeCP2 targets

  3. MECP2 Mutation Studies:phenotype-genotype correlations

  4. first described by Andreas Rett in 1966 Rett Syndrome • almost exclusively affects females • progressive loss of • intellectual functioning • fine and gross motor skills • stereotypic hand movements • 1:8,000 females by 15 yrs • rarely familial recurrences • most cases caused by mutations in MECP2

  5. Classical Late and slow onset RTT Preserved Speech Forme Fruste Congenital RTT Clinical Diagnosis • specific developmental profile based on a consistent constellation of clinical features (diagnosis is provisional < 3 yrs) • diagnostic criteria developed and recently revised • classical and variant RTT phenotypes

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

  7. “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) 6 mutations identified in 21 sporadic classical cases - 4 de novo missense mutations in methyl-binding domain (MBD) - 1 de novo frame-shift mutation in transcription repression domain (TRD) - 1 de novo nonsense mutation in TRD

  8. MECP2 Mutations Identified RettBASE: http://mecp2.chw.edu.au 55% > 270 different mutations to date > 3000 individuals

  9. international study to examine clinical features of RTT • data are collected from 2 sources • families • clinicians • data 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) • development of clinical and mutation databases (J Child Neurol, 2003; Hum Mut, 2003)

  10. Our MECP2 Mutation Studies • 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) • 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

  11. 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) • 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

  12. encourage your doctor or the laboratory that did the testing to submit their information • gladysh@chw.edu.au • or visit our website http://mecp2.chw.edu.au 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…. • rett@ichr.uwa.edu.au • or visit our website http://interrett.ichr.uwa.edu.au

  13. CDKL5:a new Rett Syndrome gene

  14. III:4 III:5 III:3 III:1 III:2 II:1 II:2 Clinical Summary Family 1 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 II:1 - clinically normal mother

  15. Further Genetic Studies of Family 1 I:2 I:1 1 ? 2 2 2 1 2 2 1 2 1 ? 1 2 1 1 1 1 1 1 2 ? 2 1 2 1 3 ? 3 ? 1 2 2 II:2 II:1 1 1 2 2 3 2 1 2 1 1 2 2 1 2 2 2 2 2 2 1 1 2 2 1 1 2 ? 2 ? 2 1 2 2 1 2 2 2 1 2 2 1 2 1 1 2 1 1 1 1 1 1 2 2 1 2 1 3 3 1 2 2 1 2/1 1 1 1 3 1 1 1 1 3 1/3 1 1 2 3 2 1 1 2 3 1/3 1 1 1 1 1 1/3 1 1/3 1 1 1 X Chromosome markers Map location (kb) Map location (cM) ? ? Chromosomal location ? III:2 III:1 III:3 III:4 III:5 21.36cM (10.3 Mb) p22.33 3.18 4 240 1 1 2 1 2 1 2 2 1 2 3 3 1 2 1 1 1 1 1 2 3 3 2 1 2 1 3 3 3 3 1 2 2 DXS1060 1 1 2 2 3 2 1 2 1 1 2 2 1 2 2 2 2 2 2 1 1 2 2 1 1 2 ? 2 ? 2 1 2 2 1 2 2 2 1 3 ? 1 1 1 1 1 1 1 2 ? 1 1 1 1 2 1 1 1 1 1 ? 1 ? 1 1 1 1 1 1 2 1 2 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 2 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 3 2 1 2 1 1 2 2 1 2 2 2 2 2 2 1 1 2 2 1 1 2 2 2 2 2 1 2 2 1 1 2 2 3 2 1 2 1 1 2 2 1 2 2 2 2 2 2 1 1 2 2 1 1 2 2 2 2 2 1 2 2 p22.31 15.52 10 150 DXS8051 p22.31 21.36 10 286 DXS1135 STK9 p22.2 25.41 13 535 DXS987 24.8cM (19.7Mb) p22.12 31.98 20 857 DXS1683 p22.12 34.06 22 794 DXS1226 ARX p22.11 40.32 25 130 DXS1202 p21.1 46.16 29 946 DXS1214 p11.4 58.17 37 196 DXS1068 p11.4 67.09 39 396 DXS993 p11.21 89.67 53457 DXS991 q11.2 91.02 64 000 AR q13.3 98.22 76 343 DXS986 18.56cM (16.4Mb) q21.2 99.35 83 765 DXS1196 q21.33 107.15 89 981 DXS990 q22.3 117.19 100 213 DXS1191 q22.3 118.84 107 202 DXS1106 q23 123.36 111 780 DXS8055 q24 137.73 116 822 DXS1001 autism/MR q25 147.38 125 921 DXS1047 q27.1 159.45 137 660 DXS1227 q27.2 173.13 140 850 DXS8043 q28 180.85 144 434 DXS8091 neonatal onset seizures and profound MR q28 181.83 145 181 DXS1185 DXS1193 q28 181.94 145 212 q28 181.99 145 318 DXS1123 q28 183.59 146 798 DXS8103 q28 184.43 146 809 DXS1684 q28 188.95 156 199 DXS1177 21:79 q28 189.48 156 959 DXS15 MECP2 q28 190.32 158 154 DXS8087 q28 190.5 158 414 DXS1073 q28 192.52 159 695 DXS1108 18:82 23:77 27:73 atypical Rett syndrome MECP2 - C426T (F142F)

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

  17. c.2343delG IVS16+1G>C IVS16+1G>A c.2362_2366del5 c.525A>T c.539C>T c.183delT c.215T>A c.163-166delGAAA c.175C>T del678_691 ins683_673 ATG 1a 1b 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 c.2635-2636delCT c.680T>C Cen Tel IVS13-1G>A IVS6-1G>T IVS6-1G>C c.838_847del10 IVS11-2A>G XLRS1 gene IVS7-2A>G c.455G>T CDKL5 gene Summary of currently known CDKL5 mutations 6 5 4 3

  18. Further Mutation Screening of CDKL5 • particular clinical phenotype • Hanefeld variant –onset of severe seizures < 6 months • 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 others • only 1 de novo missense mutation - c.586C>T (p.S196L)

  19. Dapi CDKL5- Polyclonal b-Tubulin Merge Untransfected (HeLa)

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

  21. Identification of Specific MeCP2 Downstream Targets

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

  23. -------aaUCGA------ -------AGCT------- -------aaUCGA------ -------aaUCGA------ -------AGCT------- -------AGCT------- -------AGCT------- -------AGCT------- -------aaUCGA------ -------aaUCGA------ -------AGCT------- -------AGCT------- -------AGCT------- -------aaUCGA------ -------AGCT------- -------aaUCGA------ -------aaUCGA------ -------AGCT------- -------AGCT------- -------AGCT------- Bind labelled probe mRNAsimultaneouslytospots Label two mRNA populations (redand green) Uniform Expression Increased Expression Decreased Expression Created by Dan Catchpoole, CHW, 2001

  24. Expression Profiles increased uniform reduced Gene Expression Studies • using mRNA from RTT patient brain samples - compare regional expression patterns - studies using frontal and occipital cerebral cortex • microarray analyses

  25. Abnormal expression in Rett frontal cortex

  26. Biology of Rett Syndrome • functional abnormalities of energy production? • previous functional and structural studies • altered control of cell survival? • increased sensitivity to agents that promote cell death • 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

  27. + - + - Proteomic Study on Mecp2Mouse Model DIGE (Differential Imaging GEL Electrophoresis) Equilibration 1st Dimension Isoelectric Focusing 2nd Dimension SDS-Polyacrylamide Gel Electrophoresis image Gel Using the Typhoon MULTI LASER Scanner Image Analysis

  28. 3 PH 10 250 15 Overlaid Image

  29. pH10 pH3 1115.53 1116.59 1193.62 1209.56 1218.63 1248.55 1349.62 --- 1466.7 1480.65 1506.73 1576.79 1592.76 1705.92 1723.9 --- Identification of the target spot Gel extraction Tryptic digest Compare with Mw/pI from gel Mass Spectrometry MS/MS analysis l V Z G A M S 0 1000 1500 2000 2500 3000 Verify with Online tools Peak List = Mass fingerprint Database search Protein identified!

  30. Conclusions • 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 biology • combination 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 practice

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

  32. 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

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

  34. MBD TRD 3’UTR 2 Exon 4 1 1 3 tel cen ~15 kb 4.609 kb ~47 kb ~37 kb ~40 kb ~65 kb Large Deletions in our RTT Patients

  35. CDKL5/STK9 • novel, conserved serine/threonine kinase • large gene of 23 exons with 2 alternative transcription start sites generating two isoforms • CDKL5 protein localisation - cytoplasm/nucleus? • wide tissue expression, including fetal and adult brain

  36. HDAC mSin3a MeCP2 rat BDNF exon III depolarization MeCP2 HDAC PO4 mSin3a rat BDNF exon III Unanswered Questions - CDKL5 • Does CDKL5 phosphorylate MeCP2 (and other proteins)? Is CDKL5 the link??

  37. Unanswered Questions- CDKL5 • Does CDKL5 phosphorylate MeCP2 (and other proteins)? • Do the different isoforms have different functions? • What is the developmental expression profile of Cdkl5 in mouse? • Will mouse models for Cdkl5 deficiency help us understand the biology of Rett syndrome?

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

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

More Related