Rett Syndrome Research The Sydney Experience

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Rett Syndrome ResearchThe Sydney Experience
John Christodoulou NSW Centre for Rett Syndrome
Research Western Sydney Genetics Program,
Childrens Hospital at Westmead Disciplines of
Paediatrics Child Health and Medical Genetics,
University of Sydney
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Presentation Outline

• Predicting disease severity by knowing the MECP2
  gene mutation
• CDKL5 a second Rett syndrome gene
• hunt for MeCP2 targets

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MECP2 Mutation Studiesphenotype-genotype
correlations
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Rett Syndrome

• almost exclusively affects females
• progressive loss of
• intellectual functioning
• fine and gross motor skills
• stereotypic hand movements
• 18,000 females by 15 yrs
• rarely familial recurrences
• most cases caused by mutations in MECP2

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Clinical Diagnosis

• specific developmental profile based on a
  consistent constellation of clinical features
  (diagnosis is provisional lt 3 yrs)
• diagnostic criteria developed and recently
  revised
• classical and variant RTT phenotypes

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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
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MECP2 Mutations Identified
RettBASE http//mecp2.chw.edu.au
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gt 270 different mutations to date gt 3000
individuals
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• 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)

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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

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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

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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_at_ichr.uwa.edu.au
• or visit our website http//interrett.ichr.uwa.e
  du.au

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CDKL5a new Rett Syndrome gene
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Clinical Summary Family 1
III1 - atypical (milder RTT) - infantile spasms
from 9 weeks - III2 - autism mild MR -
never had seizures III3 - infantile spasms in
the newborn period - poor head control - severe
psychomotor retardation - died age 16 yrs
(unresponsive, frequent myoclonic jerks) III4 -
clinically normal brother III5 - clinically
normal sister
II1 - clinically normal mother
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Further Genetic Studies of Family 1
MECP2 - C426T (F142F)
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STK9/CDKL5 Mutation Screening Family 1
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Summary of currently known CDKL5 mutations
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5
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3
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Further Mutation Screening of CDKL5

• particular clinical phenotype
• Hanefeld variant onset of severe seizures lt 6
  months
• Archer et al ( J Med Genet 2006 43 729-734)
• 7 of 42 (17) ? with seizures commencing lt 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.586CgtT
  (p.S196L)

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Untransfected (HeLa)
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CDKL5 Tubulin

• functional significance?
• direct or indirect interaction?
• neuronal physiology decreased dendritic
  branching in Rett Syndrome patients

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Identification of Specific MeCP2 Downstream
Targets
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Neuropathology of Rett

• adult Rett brain 900 grams (the same size as a
  non RTT 1 year old)
• regional volumetric loss

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Bind labelled probe mRNA simultaneously to spots
Label two mRNA populations (red and green)
Uniform Expression
Increased Expression
Decreased Expression
Created by Dan Catchpoole, CHW, 2001
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Gene Expression Studies

• using mRNA from RTT patient brain samples
• - compare regional expression
  patterns
• - studies using frontal and occipital cerebral
  cortex
• microarray analyses

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Abnormal expression in Rett frontal cortex
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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
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Proteomic Study on Mecp2 Mouse 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
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3 PH

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250 15
Overlaid Image
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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!
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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

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Collaborators
Childrens Hospital at Westmead Group Current
team Past team Roksana Armani Linda
Weaving Bruce Bennetts Alexandra Bezler Desiree
Cloosterman Andrew Grimm Carolyn
Ellaway Joanne Gibson Gladys Ho
Simon Hardwick Rania Kairouz-Wabhe
Hooshang Lahooti Vidya Vasudevan
Abid Mohamedali Sarah Williamson
Rose White
Childrens 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

• Womens Childrens 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
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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
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Gene Silencing by Chromatin Condensation
Methylated DNA
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Large Deletions in our RTT Patients
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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

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Unanswered Questions - CDKL5

• Does CDKL5 phosphorylate MeCP2 (and other
  proteins)?

Is CDKL5 the link??
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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?

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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) gt1.5 fold change, 5/7 biological replicates

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