RETT SYNDROME

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RETT SYNDROME

• UNDERSTANDING THE ROLE OF MECP2
• BASIC NEUROSCIENCE NBL 120
• DECEMBER 2007

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OUTLINE

• CLINICAL BACKGROUND
• MOLECULAR IMPLICATIONS
• PHENOTYPE-GENOTYPE RELATION
• FUNCTIONAL EFFECT OF MUTATIONS

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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 AFFECTING FEMALES PREDOMINANTLY
• OCCURRENCE gtgt99.9 SPORADIC
• VARIABLE CLINICAL EXPRESSION
• PERVASIVE GROWTH FAILURE
• SIGNIFICANT LONGEVITY
• CONSISTENT NEUROPATHOLOGY
• MORE THAN 95 OF FEMALES MEETING CONSENSUS
  CRITERIA HAVE MUTATIONS IN MECP2

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Rett Syndrome in USAIRSA Case Registry by State
(May 2007)
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87
14
13
89
19
10
90
11
64
27
9
31
222
179
24
103
193
27
178
87
170
26
34
44
40
45
66
24
107
42
142
377
5
50
66
33
101
20
90
56
53
25
43
61
53
1
232
7
180
TOTAL 3712
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Puerto Rico 10
Puerto Rico 10
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RETT SYNDROMEVARIABLE CLINICAL EXPRESSION

• PHENOTYPIC EXPRESSIONS
• SEIZURES OR BEHAVIORAL PATTERNS
• BREATHING IRREGULARITIES
• SLEEP CHARACTERISTICS
• SCOLIOSIS
• AMBULATION

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LONGEVITY IN RETT SYNDROME

• SURVIVAL FOLLOWS THAT OF ALL FEMALES UNTIL AGE 10
• 70 SURVIVAL TO AGE 35 COMPARED TO 98 IN ALL
  FEMALES AND 27 IN PERSONS WITH PROFOUND MOTOR
  AND COGNITIVE IMPAIRMENT

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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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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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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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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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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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Function of MeCP2
TRD
MBD
Methylated CpG
Chromatin
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Mutated MeCP2
MeCP2
Methylated CpG
Chromatin
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WHAT DO WE KNOW ABOUT MECP2 AND RETT SYNDROME ?

• gt95 OF CLASSIC RETT SYNDROME CAUSED BY MUTATIONS
  IN MECP2
• 8 MUTATIONS ACCOUNT FOR 65 OF THOSE IN RETT
  SYNDROME
• SPORADIC RS MAJORITY APPEAR TO BE OF PATERNAL
  ORIGIN
• FAMILIAL RS (ltlt1 of total) MAJORITY DUE TO LARGE
  DELETION

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Mutations

• Mutations in MeCP2 found in 70-95 classical
  Rett syndrome
• Missense, nonsense, frameshift, large scale
  rearrangements

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DOES MUTATION PREDICT OUTCOME?

• Certain mutations (R133C, R294X, and R306C and
  C-terminal truncations are associated with
  better outcome
• Lower severity scores
• Slower progression
• Preserved speech variants
• Missense mutations in C-terminal region in males
  associated with XLMR

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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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RETT SYNDROME AND NORMAL MECP2

• LARGE SCALE DELETIONS MISSED BY CURRENT PCR
  METHODS
• ALTERNATE SPLICE VARIANT, TERMED MeCP2B
• OTHER GENES INCLUDING MeCP2 DOWNSTREAM TARGETS
• Mnatzakanian et al. Nature Genet
  200436339-341

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Rett SyndromeFemale Phenotypes With MECP2
Mutations

• Rett Syndrome
• Preserved Speech Variant
• Delayed Onset Variant
• Congenital Onset Variant
• Early Onset Seizure Variant
• Autistic-like Variant
• Angelman Syndrome
• Mild Learning Disability
• Normal Carriers

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Rett SyndromeMale Phenotypes With MECP2 Mutations

• Fatal Encephalopathy
• Rett/Klinefelter Syndrome
• X-Linked MR/Progressive Spasticity
• Somatic Mosaicism/NDD
• MECP2 Duplications and X-linked MR

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WHO SHOULD HAVE MECP2 TESTING?

• FEMALES WITH TYPICAL AND VARIANT RETT SYNDROME
  FEATURES
• INFANTS, ESPECIALLY MALES, WITH UNEXPLAINED
  PROGRESSIVE ENCEPHALOPATHY
• MALES WITH RETT SYNDROME FEATURES
• CHILDREN WITH ANGELMAN FEATURES AND NORMAL
  METHYLATION
• CHILDREN WITH FAMILIAL XLMR AND NORMAL FRAGILE X
  TESTING

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Assessing function of mutant protein

• Drosophila S cells
• Not highly methylated
• Transfect MeCP2 construct and methylated reporter
  plasmid
• Assess transcription

MeCP2
mmm
CAT
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Mutations decrease MeCP2 repression in Drosophila
cells

• Transient transfection
• MeCP2 construct
• CAT reporter

Relative CAT activity ()
Kudo et al., Brain and Dev 2001
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A photobleaching technique to study protein
movement in living cells
Immobile fraction
T1/2
Mobile fraction
FRAP fluorescence recovery after photobleaching
Phair RD, and Misteli T. (2001) Nat Rev Mol Cell
Biol. 2(12)898-907
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Mecp2 is a mobile protein in live cells
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Rett mutations increase mobility
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MOUSE MODELS

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

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KNOCK-OUT MUTANT

• Note
• hind-limb clasping
• Guy et al.
• Nature Genetics 200127322-326

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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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Longevity Study

• Data gathered on 1928 girls and women
• Completion of data gathering and filling in
  missing data consumed most of winter
• Analysis of longevity underway
• Databank very informative
• Appears representative

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North American Database
Total enrolled 1928
Typical 1648 (85.5 )
Atypical 259 (13.4 )
Not RS (MECP2 positive) 21 (1.1 )
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North American Database
Group Total Mutation No mutation Unknown
Typical 1648 791 (91) 79 (9) 778
Atypical 259 94 (58) 68 (42) 97
Not RS 21 21 (100) 0 0
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North American Database
Mutation Type N France (Philippe et al.)
Missense 356 38.8 35.6
Nonsense 323 35.2 37.3
Frameshift 161 17.5 12.0
Complex deletion 59 6.4 5.8
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DATABASE RESOURCES

• RettBase Dr. John Christodoulou
• MECP2 Mutation Repository
• mecp2.chw.edu.au
• InterRett Dr. Helen Leonard
• Clinical information repository from parents and
  physicians
• www.ichr.uwa.edu.au

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Acknowledgements

• IRSA
• UAB
• Jane Lane
• Suzie Geerts
• Jerry Childers
• duPont Hospital for Children
• Carolyn Schanen
• NIH
• NICHD/ORD/NCRR

• Greenwood Genetic Center
• Steve Skinner
• Fran Annese
• Baylor College of Medicine
• Daniel Glaze
• Jeff Neul
• Huda Zoghbi
• Judy Barrish
• Girls and women with RS and their families

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Thats all folks!!!