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