RETT SYNDROME
UNDERSTANDING THE ROLE OF
MECP2
BASIC NEUROSCIENCE NBL 120
DECEMBER 2007
OUTLINE

CLINICAL BACKGROUND

MOLECULAR IMPLICATIONS

PHENOTYPE-GENOTYPE RELATION

FUNCTIONAL EFFECT OF MUTATIONS
RETT SYNDROME
A NEURODEVELOPMENTAL DISORDER OF
YOUNG FEMALES CHARACTERIZED BY

PROFOUND COGNITIVE IMPAIRMENT

COMMUNICATION DYSFUNCTION

STEREOTYPIC MOVEMENTS

PERVASIVE GROWTH FAILURE
RETT SYNDROME
CONSENSUS CRITERIA - 2001
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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/squeezing; Hand
clapping/tapping/rubbing; Hand mouthing
Gait dysfunction: Impaired (dyspraxic) or
failing locomotion
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

RETT SYNDROME
WHAT DO WE KNOW?


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GENETIC DISORDER AFFECTING FEMALES
PREDOMINANTLY
OCCURRENCE >>99.9% SPORADIC
VARIABLE CLINICAL EXPRESSION
PERVASIVE GROWTH FAILURE
SIGNIFICANT LONGEVITY
CONSISTENT NEUROPATHOLOGY
MORE THAN 95% OF FEMALES MEETING
CONSENSUS CRITERIA HAVE MUTATIONS
IN MECP2
Rett Syndrome in USA
IRSA Case Registry by State (May 2007)
87
14
13
18
89
10
64
27
27
9
34
179
31
24
178 87
44
40
377
56
90
50
20
232
7
66
33
53
170
24 107
4
2
66
25 43
1
222 1
193
101
53
61
1
9
103
2
6
45
5 142
9
0
1
180
Puerto Rico
Puerto
Rico 10
10
14
TOTAL 3712
RETT SYNDROME
VARIABLE CLINICAL EXPRESSION

PHENOTYPIC EXPRESSIONS

SEIZURES OR BEHAVIORAL PATTERNS

BREATHING IRREGULARITIES

SLEEP CHARACTERISTICS

SCOLIOSIS

AMBULATION
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
RETT SYNDROME
BRAIN MORPHOLOGY

REDUCED BRAIN WEIGHT

REDUCED VOLUME OF SPECIFIC REGIONS

REDUCED MELANIN PIGMENTATION

SMALL NEURONS; SIMPLIFIED
DENDRITES WITH REDUCED SPINES

ABSENCE OF RECOGNIZABLE DISEASE
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
Spine Dysgenesis in Mental
Retardation
Normal DS
MR
FraX
FMR1 KO mice
wt
Rett Syndrome
• Down’s 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)
Rett syndrome is caused by mutations
in X-linked MECP2, encoding methylCpG-binding protein 2
Ruthie E. Amir, Ignatia B. van den Veyver,
Mimi Wan, Charles Q. Tran, Uta Francke &
Huda Y. Zoghbi Nature Genet 1999;23:185
METHYL-CpG-BINDING PROTEIN 2
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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
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 2002;11:115
Function of MeCP2
MeCP2 Sin3A HDAC
TRD
MBD
Methylated
CpG
Chromatin
Mutated MeCP2
HDAC
Sin3A
MeCP2
Methylated
CpG
Chromatin
WHAT DO WE KNOW ABOUT
MECP2 AND RETT SYNDROME ?

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>95% 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 (<<1% of total)
MAJORITY DUE TO LARGE DELETION
Mutations


Mutations in MeCP2 found in 70-95% “classical”
Rett syndrome
Missense, nonsense, frameshift, large scale
rearrangements
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
RETT SYNDROME AND MECP2
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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
RETT SYNDROME AND
NORMAL MECP2
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LARGE SCALE DELETIONS MISSED BY
CURRENT PCR METHODS
ALTERNATE SPLICE VARIANT,
TERMED MeCP2B
OTHER GENES INCLUDING MeCP2
DOWNSTREAM TARGETS
Mnatzakanian et al. Nature Genet
2004;36:339-341
Rett Syndrome
Female Phenotypes With MECP2
Mutations
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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
Rett Syndrome
Male Phenotypes With MECP2
Mutations

Fatal Encephalopathy

Rett/Klinefelter Syndrome

X-Linked MR/Progressive Spasticity

Somatic Mosaicism/NDD

MECP2 Duplications and X-linked MR
WHO SHOULD HAVE MECP2
TESTING?
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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
Assessing function of
mutant protein

Drosophila S cells
– Not highly methylated


Transfect MeCP2
construct and
methylated reporter
plasmid
Assess transcription
MeCP2
mmm
CAT
Mutations decrease MeCP2
repression in Drosophila
cells
Relative CAT activity (%)
120
Relative CAT activities (%)
100
80
60
• Transient transfection
• MeCP2 construct
• CAT reporter
40
20
0
MeCP2 R106W R133C F155S T158M R255X
Kudo et al., Brain and Dev 2001
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
Mecp2 is a mobile protein in live cells
1.0
Relative Intensity
0.8
0.6
0.4
Mecp2A
0.2
Mecp2B
0.0
0
50
100
150
200
Time (seconds)
250
300
Rett mutations increase mobility
1.0
0.9
Relative Intensity
0.8
0.7
0.6
WT
R106W
0.5
R133C
F155S
0.4
T158M
R255X
0.3
GFP
0
20
40
60
Time (seconds)
80
MOUSE MODELS

Knock-out mouse: Mecp2 deleted

Knock-in mouse: Insertion of human
mutation in Mecp2
KNOCK-OUT MUTANT
 Note
hind-limb
clasping
Guy et al.
Nature
Genetics
2001;27:322326
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 2007;315:1143-1147
KNOCK-IN MUTANT

Note
humped
back and
forelimb
clasping
Young and
Zoghbi, Am J
Hum Genet
2004;74:511520
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 2006;26:319-327
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 2006;103:18267-18272
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
Longevity Study
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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
North American Database
Total enrolled
1928
Typical
1648 (85.5 %)
Atypical
259 (13.4 %)
Not RS (MECP2 positive)
21 (1.1 %)
North American Database
Group
Total Mutation
No
Unknown
mutation
Typical 1648
791
(91%)
79 (9%)
778
Atypical
259
94
(58%)
68
(42%)
97
Not RS
21
21
(100%)
0
0
North American Database
Mutation
Type
N
%
Missense
356
38.8
% France
(Philippe
et al.)
35.6
Nonsense
323
35.2
37.3
Frameshift
161
17.5
12.0
Complex
deletion
59
6.4
5.8
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
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
That’s all folks!!!