RETT SYNDROME

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RETT SYNDROME

• UNDERSTANDING RETT SYNDROME AND THE ROLE OF MECP2
  
• NEUROSCIENCE
• JANUARY 2009

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OUTLINE

• CLINICAL BACKGROUND
• MOLECULAR IMPLICATIONS
• PHENOTYPE-GENOTYPE RELATION
• ANIMAL MODELS
• THERAPY

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Rett syndrome is caused by mutations in X-linked
MECP2, encoding methyl-CpG-binding protein 2

• Ruthie E. Amir, Ignatia B. van den Veyver, Mimi
  Wan, Charles Q. Tran, Uta Francke Huda Y.
  Zoghbi Nature Genet 199923185

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RETT SYNDROMEA NEURODEVELOPMENTAL DISORDER OF
YOUNG FEMALES CHARACTERIZED BY

• PROFOUND COGNITIVE IMPAIRMENT
• COMMUNICATION DYSFUNCTION
• STEREOTYPIC MOVEMENTS
• PERVASIVE GROWTH FAILURE

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RETT SYNDROME CONSENSUS CRITERIA - 2001

• Normal at birth
• Apparently normal early development (may be
  delayed from birth)
• Postnatal deceleration of head growth in most
• Lack of achieved purposeful hand skills
• Psychomotor regression Emerging social
  withdrawal, communication dysfunction, loss of
  learned words, and cognitive impairment
• Stereotypic movements Hand washing/wringing/squee
  zing Hand clapping/tapping/rubbing Hand
  mouthing
• Gait dysfunction Impaired (dyspraxic) or failing
  locomotion

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RETT SYNDROME TEMPORAL PROFILE

• APPARENTLY NORMAL DEVELOPMENT
• ARREST OF DEVELOPMENTAL PROGRESS
• FRANK REGRESSION WITH POOR SOCIAL CONTACT AND
  FINGER SKILLS
• STABILIZATION BETTER SOCIAL CONTACT AND EYE
  GAZE, BUT GRADUAL SLOWING OF MOTOR FUNCTIONS

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RETT SYNDROMEWhat do we know?

• Genetic disorder - mainly in females
• Diagnosis based on meeting clinical criteria
• Variable clinical expression
• Significant longevity
• Consistent neuropathology

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RETT SYNDROMEBRAIN MORPHOLOGY

• REDUCED BRAIN WEIGHT
• REDUCED VOLUME OF SPECIFIC REGIONS
• REDUCED MELANIN PIGMENTATION
• SMALL NEURONS SIMPLIFIED DENDRITES WITH REDUCED
  SPINES
• ABSENCE OF RECOGNIZABLE DISEASE

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Spine Dysgenesis in Mental Retardation
Normal DS MR FraX
FMR1 KO mice
wt
Rett Syndrome

• Downs Syndrome (Huttenlocher 70, 74
  Marin-Padilla 72, 76 Purpura 74, 75)
  Fragile X Syndrome - and FMR1 KO mice (Wisniewski
  85 Greenough 97) Rett Syndrome (Balichenko
  94)

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OTHER NEURODEVELOPMENTAL DISORDERS

• DOWN SYNDROME
• REDUCED DENDRITIC BRANCHES AND SPINES AFTER EARLY
  INFANCY
• AUTISM
• INCREASED PACKING DENSITY
• DECREASED CELL SIZE
• ANGELMAN AND FRAGILE X SYNDROMES
• REDUCED DENDRITIC ARBORIZATIONS AND SPINES

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RETT SYNDROME AND MECP2

• RETT SYNDROME IS A CLINICAL DIAGNOSIS
• RETT SYNDROME IS NOT SYNONYMOUS WITH MECP2
  MUTATIONS
• RETT SYNDROME MAY BE SEEN WITH MECP2 MUTATIONS
• RETT SYNDROME MAY BE SEEN WITHOUT MECP2 MUTATIONS
• MECP2 MUTATIONS MAY BE SEEN WITHOUT RETT SYNDROME

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MECP2 AND RETT SYNDROME

• 90-95 of classic RTT due to mutations in MECP2
• 4 missense and 4 nonsense mutations account for
  65 c-terminal truncations and large deletions
  account for another 15-18
• Sporadic RTT majority of paternal origin
• Familial RTT (ltlt1 of total) majority due to
  large deletions
• Phenotypes extend beyond classic RTT

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Continuum of MECP2 Associated Phenotypes
Females
Males
Normal Learning Disabilities Behavioral
Phenotype Autistic OCD Hyperactive/aggressiv
e Moderate cognitive delay Forme
fruste Preserved speech variant Rett syndrome
95 with MECP2 mutation 80-85 of RTT and
variants Early onset seizure variant Congenital
variant
MECP2 Duplication syndrome /- recurrent
infections X-linked cognitive impairment
/- progressive spasticity other NDD
features/OCD aggression Rett syndrome XXY
somatic mosaicism Severe encephalopathy
Likely under-represented
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Medical Issues

• Longevity
• Growth
• Epilepsy
• GI dysfunction
• Scoliosis
• Osteopenia
• Breathing irregularities
• Sleep
• Cardiac conduction
• Sexual Maturation

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LONGEVITY IN RETT SYNDROME

• Unpublished study from Baylor College of
  Medicine survival follows that of all females
  until age 10 70 survival to age 35 versus 98
  in all females
• Recent study from Australia 78 survival to age
  25 versus 99.9 in all females - Laurvick et
  al. J Pediatr 2006148347

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RETT SYNDROME SURVIVAL BY DATE OF BIRTH
Figure 1
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MOLECULAR IMPLICATIONS
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METHYL-CpG-BINDING PROTEIN 2

• ONE OF FAMILY OF METHYL-BINDING PROTEINS
• CAPABLE OF TRANSCRIPTIONAL SILENCING OR
  REGULATION
• UBIQUITOUS IN MAMMALIAN TISSUES
• HIGHLY EXPRESSED IN MAMMALIAN BRAIN
• SPECIFIC TARGET GENES UNDEFINED
• MAY FUNCTION IN MAINTENANCE OF DEVELOPING AND
  MATURE NEURONS

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MeCP2 DISTRIBUTION IN HUMAN BRAIN DURING
DEVELOPMENT

• CAUDAL-ROSTRAL GRADIENT OF MeCP2 IN HUMAN BRAIN
• CORTICAL NEURONS LAST TO EXPRESS
• Shahbazian et al. Hum Mol Genet 200211115

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Mecp2 plays complex role in the nucleus with
differential effects on transcription
Yasui et al. PNAS 2007
Chahrour et al. Science 2008
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Mutated MeCP2
MeCP2
Methylated CpG
Chromatin
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PHENOTYPE-GENOTYPE RELATION
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Does mutation predict outcome

• R133C, R294X, and R306C mutations and c-term
  truncations are associated with lower severity
  scores, slower progression, preserved speech
• Classic RTT missense and nonsense
• Variant RTT nonsense
• Non-RTT missense and frameshift

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Medical IssuesFrequency () by Mutation Type
Mutation (n) Seizures Scoliosis Sc Surgery
R106W (20) 78 45 15
R133C (28) 50 25 0
T158M (64) 74 50 16
R168X (55) 54 35 11
R255X (60) 49 42 7
R270X (34) 53 50 12
R294X (37) 69 43 3
R306C (45) 49 31 4
Lrg del (49) 57 55 12
c-ter del (53) 69 51 9
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Medical IssuesFrequency () by Mutation Type
Mutation (n) Constipation GERD Fractures
R106W (20) 75 50 20
R133C (28) 75 43 7
T158M (64) 78 56 16
R168X (55) 73 55 16
R255X (60) 75 52 17
R270X (34) 74 62 27
R294X (37) 81 49 19
R306C (45) 62 47 13
Lrg del (49) 69 55 20
c-ter del (53) 75 38 13
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MOUSE MODELS

• Knock-out mouse Mecp2 deleted
• Knock-in mouse Insertion of mutation in Mecp2

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KNOCK-OUT MUTANT

• Is Mecp2 knock-out reversible?
• Using estrogen receptor controlled Mecp2
  promoter
• Mecp2 knock-out phenotype reversed in both
  immature male and mature male and female mice
  with estrogen analog, tamoxifen
• Rapid re-expression in immature males resulted in
  death in 50
• Guy et al. Science 20073151143-1147

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KNOCK-IN MUTANT

• Note humped back and forelimb clasping
• Young and Zoghbi, Am J Hum Genet 200474511-520

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KNOCK-IN MUTANT

• Impaired hippocampus-dependent social, spatial,
  and contextual fear memory
• Impaired long-term potentiation and depression
• Reduced post-synaptic densities
• No change in BDNF expression
• Moretti et al. J Neurosci 200626319-327

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KNOCK-IN MUTANT

• Enhanced anxiety and fear based on
• Elevated blood corticosterone levels
• Elevated corticotropin-releasing hormone in
  hypothalamus, central nucleus of amygdala, and
  bed nucleus of stria terminalis
• MeCP2 binds to Crh promoter methylated region
• McGill et al. PNAS 200610318267-18272

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KNOCK-IN MUTANT

• Implications of Crh over-expression
• Anxiety plays central role in clinical RS
• Amygdala has direct input into hypothalamus and
  brainstem autonomic nuclei correlating with
  clinical problems of respiration, GI function,
  and peripheral sympathetic NS
• Suggests strategies for therapeutic intervention

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But overexpression is detrimental
Collins et al., HMG 2004
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THERAPY
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Therapeutic interventions in Rett syndrome

• Genetic
• Gene therapy
• Post-transcriptional repair
• Protein
• Reintroduce protein
• Modifiers of mutant MECP2 function

http//fajerpc.magnet.fsu.edu/Education/2010/
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New treatments on the horizon?

• Gene repair, replacement strategies
• Gene therapy
• Reintroduction of protein
• Can we modulate expression of the gene/protein?
• Up-regulation in normal cells in heterozygotes
• Reactivate normal gene
• Post transcriptional repair
• Treatment being tested in CF and Duchenne
  muscular dystrophy of a drug that allows the cell
  to read through a nonsense mutation
• Targeted treatments based on what Mecp2 does in
  neurons
• Can we improve protein function?
• Can we make another protein take over?
• Can we intercede on specific downstream targets?
• Can we modulate neurophysiology?

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Challenges across the board for therapies in Rett
syndrome

• No clear-cut reliable outcome measure for all the
  girls.
• Natural history study may be key.
• ?EEG or other objective physiologic measure.
• Girls will be heterogeneous in genetic background
  and XCI.
• Even in cultured cells, outcome measures are
  tricky.
• But the mice can help us here..

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What about gene therapy for Rett syndrome?

• Disorders due to mutations in one or more genes
• Single-gene disorders are more easily treated
• The responsible gene is known
• /- The role of the protein encoded by the gene
  is known
• Adding a normal gene will fix the problem
• The affected tissues are known and accessible

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Gene therapy
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Post-transcriptional repair aminoglycosides

• Aminoglycoside antibiotics allow read-through
  of premature stop signals
• Nonsense mutations
• Problems with toxicity
• Need to be given IV
• Introduce a missense mutation at the stop site

http//www.ptcbio.com/3.1.1_genetic_disorders.aspx
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Post-transcriptional repair?

• PTC124 New Agent developed by PTC Therapeutics
  with similar action
• Orally available
• Less toxic
• Being tested in cystic fibrosis and Duchenne
  Muscular Dystrophy with very promising results
• Phase 2 trials- increased walking duration in
  DMD, improved chloride conductance in CF

http//www.ptcbio.com/3.1.1_genetic_disorders.aspx
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Therapies for Rett Syndrome

• Nutrition
• Physical Therapy
• Occupational Therapy
• Communication Therapy
• Hippotherapy
• Music Therapy
• Aquatherapy

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TOE WALKING
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ANKLE-FOOT DYSTONIA
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A successful intervention
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Conclusions

• Current investigations into neurobiological
  underpinnings of CNS dysfunction opening doors to
  new therapies in immediate future.
• Repurposing existing drugs
• Development of new agents
• Gene based therapies are potential avenues of
  treatment in the future.
• Role of environmental enrichment (supports early
  intervention programs)
• Need to examine treatments in female mice
• Cogent planning needed for clinical trials for
  selected agents

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The Team

• Baylor College of Medicine
• Daniel Glaze
• Kay Motil
• Jeff Neul
• Judy Barrish
• Carmel Lusk
• Greenwood Genetic Center
• Steve Skinner
• Fran Annese
• Joy Graham
• Lauren McNair

• UAB
• Alan Percy
• Jane Lane
• Suzie Geerts
• Jerry Childers
• Russell Kirby
• NIH/NCRR/NICHD
• DTCC Hye-Seung Lee
• Girls and women with RS and their families

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Rollin with Curly