RETT SYNDROMERETT SYNDROME

UNDERSTANDING THE ROLE OF UNDERSTANDING THE ROLE OF MECP2MECP2

BASIC NEUROSCIENCE NBL 120BASIC NEUROSCIENCE NBL 120

DECEMBER 2007DECEMBER 2007

OUTLINEOUTLINE

CLINICAL BACKGROUNDCLINICAL BACKGROUND

MOLECULAR IMPLICATIONSMOLECULAR IMPLICATIONS

PHENOTYPE-GENOTYPE RELATIONPHENOTYPE-GENOTYPE RELATION

FUNCTIONAL EFFECT OF MUTATIONSFUNCTIONAL EFFECT OF MUTATIONS

RETT SYNDROMERETT SYNDROMEA NEURODEVELOPMENTAL DISORDER OF A NEURODEVELOPMENTAL DISORDER OF

YOUNG FEMALES CHARACTERIZED BYYOUNG FEMALES CHARACTERIZED BY

PROFOUND COGNITIVE PROFOUND COGNITIVE IMPAIRMENTIMPAIRMENT

COMMUNICATION DYSFUNCTIONCOMMUNICATION DYSFUNCTION

STEREOTYPIC MOVEMENTSSTEREOTYPIC MOVEMENTS

PERVASIVE GROWTH FAILUREPERVASIVE GROWTH FAILURE

RETT SYNDROME RETT SYNDROME CONSENSUS CRITERIA - 2001CONSENSUS CRITERIA - 2001

Normal at birthNormal at birth Apparently normal early development (may Apparently normal early development (may

be delayed from birth)be delayed from birth) Postnatal deceleration of head growth in Postnatal deceleration of head growth in

mostmost Lack of achieved purposeful hand skillsLack of achieved purposeful hand skills Psychomotor regression: Emerging social Psychomotor regression: Emerging social

withdrawal, communication dysfunction, loss withdrawal, communication dysfunction, loss of learned words, and cognitive impairmentof learned words, and cognitive impairment

Stereotypic movements: Hand Stereotypic movements: Hand washing/wringing/squeezing; Hand washing/wringing/squeezing; Hand clapping/tapping/rubbing; Hand mouthingclapping/tapping/rubbing; Hand mouthing

Gait dysfunction: Impaired (dyspraxic) or Gait dysfunction: Impaired (dyspraxic) or failing locomotionfailing locomotion

RETT SYNDROMERETT SYNDROME TEMPORAL PROFILE TEMPORAL PROFILE

APPARENTLY NORMAL DEVELOPMENT APPARENTLY NORMAL DEVELOPMENT ARREST OF DEVELOPMENTAL ARREST OF DEVELOPMENTAL

PROGRESSPROGRESS FRANK REGRESSION WITH POOR FRANK REGRESSION WITH POOR

SOCIAL CONTACT AND FINGER SKILLSSOCIAL CONTACT AND FINGER SKILLS STABILIZATION: BETTER SOCIAL STABILIZATION: BETTER SOCIAL

CONTACT AND EYE GAZE, BUT CONTACT AND EYE GAZE, BUT GRADUAL SLOWING OF MOTOR GRADUAL SLOWING OF MOTOR FUNCTIONSFUNCTIONS

RETT SYNDROMERETT SYNDROMEWHAT DO WE KNOW?WHAT DO WE KNOW?

GENETIC DISORDER AFFECTING GENETIC DISORDER AFFECTING FEMALES PREDOMINANTLYFEMALES PREDOMINANTLY

OCCURRENCE >>99.9% SPORADICOCCURRENCE >>99.9% SPORADIC VARIABLE CLINICAL EXPRESSIONVARIABLE CLINICAL EXPRESSION PERVASIVE GROWTH FAILUREPERVASIVE GROWTH FAILURE SIGNIFICANT LONGEVITYSIGNIFICANT LONGEVITY CONSISTENT NEUROPATHOLOGYCONSISTENT NEUROPATHOLOGY MORE THAN 95% OF FEMALES MORE THAN 95% OF FEMALES

MEETING CONSENSUS CRITERIA HAVE MEETING CONSENSUS CRITERIA HAVE MUTATIONS IN MUTATIONS IN MECP2MECP2

Rett Syndrome in USARett Syndrome in USAIRSA Case Registry by State (May 2007)IRSA Case Registry by State (May 2007)

64 9 31 222

43232

7

14TOTAL 3712

87

27

14 13

10

2734 44

24 179

89

37720

50

40

33

66

178 87

66

193170

90

61

19

53

180

24

56

10742 101

5325

103

11

18

451425

90

26

1

Puerto Rico 10

Puerto Rico10

RETT SYNDROMERETT SYNDROMEVARIABLE CLINICAL VARIABLE CLINICAL

EXPRESSIONEXPRESSION PHENOTYPIC EXPRESSIONSPHENOTYPIC EXPRESSIONS

SEIZURES OR BEHAVIORAL PATTERNSSEIZURES OR BEHAVIORAL PATTERNS

BREATHING IRREGULARITIESBREATHING IRREGULARITIES

SLEEP CHARACTERISTICSSLEEP CHARACTERISTICS

SCOLIOSISSCOLIOSIS

AMBULATIONAMBULATION

LONGEVITY IN RETT LONGEVITY IN RETT SYNDROMESYNDROME

SURVIVAL FOLLOWS THAT OF ALL SURVIVAL FOLLOWS THAT OF ALL

FEMALES UNTIL AGE 10FEMALES UNTIL AGE 10

70% SURVIVAL TO AGE 35 70% SURVIVAL TO AGE 35

COMPARED TO 98% IN ALL FEMALES COMPARED TO 98% IN ALL FEMALES

AND 27% IN PERSONS WITH AND 27% IN PERSONS WITH

PROFOUND MOTOR AND COGNITIVE PROFOUND MOTOR AND COGNITIVE

IMPAIRMENT IMPAIRMENT

RETT SYNDROMERETT SYNDROMEBRAIN MORPHOLOGYBRAIN MORPHOLOGY

REDUCED BRAIN WEIGHTREDUCED BRAIN WEIGHT

REDUCED VOLUME OF SPECIFIC REGIONS REDUCED VOLUME OF SPECIFIC REGIONS

REDUCED MELANIN PIGMENTATIONREDUCED MELANIN PIGMENTATION

SMALL NEURONS; SIMPLIFIED SMALL NEURONS; SIMPLIFIED

DENDRITES WITH REDUCED SPINESDENDRITES WITH REDUCED SPINES

ABSENCE OF RECOGNIZABLE DISEASEABSENCE OF RECOGNIZABLE DISEASE

OTHER OTHER NEURODEVELOPMENTAL NEURODEVELOPMENTAL

DISORDERSDISORDERS DOWN SYNDROMEDOWN SYNDROME

REDUCED DENDRITIC BRANCHES AND SPINES REDUCED DENDRITIC BRANCHES AND SPINES AFTER EARLY INFANCYAFTER EARLY INFANCY

AUTISMAUTISM INCREASED PACKING DENSITYINCREASED PACKING DENSITY DECREASED CELL SIZEDECREASED CELL SIZE

ANGELMAN AND FRAGILE X SYNDROMES:ANGELMAN AND FRAGILE X SYNDROMES: REDUCED DENDRITIC ARBORIZATIONS REDUCED DENDRITIC ARBORIZATIONS AND AND

SPINESSPINES

• 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)

Normal DS MR FraX

wt

FMR1 KO mice

Rett Syndrome

Spine Dysgenesis in Mental Spine Dysgenesis in Mental RetardationRetardation

Rett syndrome is caused by Rett syndrome is caused by mutations in X-linked mutations in X-linked MECP2MECP2, , encoding methyl-CpG-binding encoding methyl-CpG-binding

protein 2protein 2Ruthie E. Amir, Ignatia B. van den Ruthie E. Amir, Ignatia B. van den Veyver, Mimi Wan, Charles Q. Tran, Uta Veyver, Mimi Wan, Charles Q. Tran, Uta Francke & Huda Y. Zoghbi Francke & Huda Y. Zoghbi Nature Nature GenetGenet 1999;23:185 1999;23:185

METHYL-CpG-BINDING PROTEIN 2METHYL-CpG-BINDING PROTEIN 2

ONE OF FAMILY OF METHYL-BINDING ONE OF FAMILY OF METHYL-BINDING PROTEINSPROTEINS

CAPABLE OF TRANSCRIPTIONAL CAPABLE OF TRANSCRIPTIONAL SILENCING OR REGULATIONSILENCING OR REGULATION

UBIQUITOUS IN MAMMALIAN TISSUESUBIQUITOUS IN MAMMALIAN TISSUES HIGHLY EXPRESSED IN MAMMALIAN HIGHLY EXPRESSED IN MAMMALIAN

BRAINBRAIN SPECIFIC TARGET GENES UNDEFINEDSPECIFIC TARGET GENES UNDEFINED MAY FUNCTION IN MAINTENANCE OF MAY FUNCTION IN MAINTENANCE OF

DEVELOPING AND MATURE NEURONSDEVELOPING AND MATURE NEURONS

MeCP2 DISTRIBUTION IN MeCP2 DISTRIBUTION IN HUMAN BRAIN DURING HUMAN BRAIN DURING

DEVELOPMENTDEVELOPMENT CAUDAL-ROSTRAL GRADIENT OF MeCP2 IN HUMAN CAUDAL-ROSTRAL GRADIENT OF MeCP2 IN HUMAN

BRAINBRAIN CORTICAL NEURONS LAST TO EXPRESSCORTICAL NEURONS LAST TO EXPRESS

Shahbazian et al. Shahbazian et al. Hum Mol GenetHum Mol Genet 2002;11:115 2002;11:115

MeCP2

MBD

TRD

Chromatin

Methylated CpG

Function of Function of MeCP2MeCP2

Sin3A HDAC

MeCP2

Methylated CpG

Chromatin

Mutated MeCP2

Sin3AHDAC

WHAT DO WE KNOW ABOUT WHAT DO WE KNOW ABOUT MECP2MECP2 AND RETT SYNDROME AND RETT SYNDROME ?? >95% OF CLASSIC RETT SYNDROME >95% OF CLASSIC RETT SYNDROME

CAUSED BY MUTATIONS IN CAUSED BY MUTATIONS IN MECP2 MECP2 8 MUTATIONS ACCOUNT FOR ~ 65% 8 MUTATIONS ACCOUNT FOR ~ 65%

OF THOSE IN RETT SYNDROMEOF THOSE IN RETT SYNDROME SPORADIC RS: MAJORITY APPEAR SPORADIC RS: MAJORITY APPEAR

TO BE OF PATERNAL ORIGINTO BE OF PATERNAL ORIGIN FAMILIAL RS (<<1% of total) FAMILIAL RS (<<1% of total)

MAJORITY DUE TO LARGE DELETIONMAJORITY DUE TO LARGE DELETION

MutationsMutations

Mutations in MeCP2 found in 70-95% Mutations in MeCP2 found in 70-95% “classical” Rett syndrome“classical” Rett syndrome

Missense, nonsense, frameshift, large scale Missense, nonsense, frameshift, large scale rearrangementsrearrangements

DOES MUTATION PREDICT DOES MUTATION PREDICT OUTCOME?OUTCOME?

Certain mutations (R133C, R294X, Certain mutations (R133C, R294X, and R306C and C-terminal truncations and R306C and C-terminal truncations are associated with “better outcome”are associated with “better outcome”– Lower severity scoresLower severity scores– Slower progressionSlower progression– Preserved speech variantsPreserved speech variants

Missense mutations in C-terminal Missense mutations in C-terminal region in males associated with XLMRregion in males associated with XLMR

RETT SYNDROME AND RETT SYNDROME AND MECP2MECP2 RETT SYNDROME IS A CLINICAL DIAGNOSISRETT SYNDROME IS A CLINICAL DIAGNOSIS RETT SYNDROME IS RETT SYNDROME IS NOTNOT SYNONYMOUS SYNONYMOUS

WITH WITH MECP2 MECP2 MUTATIONSMUTATIONS RETT SYNDROME MAY BE SEEN RETT SYNDROME MAY BE SEEN WITHWITH MECP2MECP2

MUTATIONSMUTATIONS RETT SYNDROME MAY BE SEEN RETT SYNDROME MAY BE SEEN WITHOUTWITHOUT

MECP2MECP2 MUTATIONS MUTATIONS MECP2MECP2 MUTATIONS MAY BE SEEN MUTATIONS MAY BE SEEN WITHOUTWITHOUT

RETT SYNDROMERETT SYNDROME

RETT SYNDROME AND RETT SYNDROME AND NORMAL NORMAL MECP2MECP2

LARGE SCALE DELETIONS MISSED LARGE SCALE DELETIONS MISSED BY CURRENT PCR METHODSBY CURRENT PCR METHODS

ALTERNATE SPLICE VARIANT, ALTERNATE SPLICE VARIANT, TERMED MeCP2BTERMED MeCP2B

OTHER GENES INCLUDING MeCP2 OTHER GENES INCLUDING MeCP2 DOWNSTREAM TARGETSDOWNSTREAM TARGETS

Mnatzakanian et al. Mnatzakanian et al. Nature Nature GenetGenet 2004;36:339-3412004;36:339-341

Rett SyndromeRett SyndromeFemale Phenotypes With Female Phenotypes With

MECP2MECP2 Mutations Mutations

Rett SyndromeRett Syndrome Preserved Speech VariantPreserved Speech Variant Delayed Onset VariantDelayed Onset Variant Congenital Onset VariantCongenital Onset Variant Early Onset Seizure VariantEarly Onset Seizure Variant Autistic-like VariantAutistic-like Variant Angelman SyndromeAngelman Syndrome Mild Learning Disability Mild Learning Disability Normal CarriersNormal Carriers

Rett SyndromeRett SyndromeMale Phenotypes With Male Phenotypes With MECP2MECP2

MutationsMutations Fatal EncephalopathyFatal Encephalopathy

Rett/Klinefelter SyndromeRett/Klinefelter Syndrome

X-Linked MR/Progressive SpasticityX-Linked MR/Progressive Spasticity

Somatic Mosaicism/NDDSomatic Mosaicism/NDD

MECP2MECP2 Duplications and X-linked MR Duplications and X-linked MR

WHO SHOULD HAVE WHO SHOULD HAVE MECP2 MECP2 TESTING?TESTING?

FEMALES WITH TYPICAL AND VARIANT FEMALES WITH TYPICAL AND VARIANT RETT SYNDROME FEATURESRETT SYNDROME FEATURES

INFANTS, ESPECIALLY MALES, WITH INFANTS, ESPECIALLY MALES, WITH UNEXPLAINED PROGRESSIVE UNEXPLAINED PROGRESSIVE ENCEPHALOPATHY ENCEPHALOPATHY

MALES WITH RETT SYNDROME MALES WITH RETT SYNDROME FEATURESFEATURES

CHILDREN WITH ANGELMAN CHILDREN WITH ANGELMAN FEATURES AND NORMAL FEATURES AND NORMAL METHYLATIONMETHYLATION

CHILDREN WITH FAMILIAL XLMR AND CHILDREN WITH FAMILIAL XLMR AND NORMAL FRAGILE X TESTINGNORMAL FRAGILE X TESTING

Assessing function of Assessing function of mutant proteinmutant protein

DrosophilaDrosophila S cells S cells – Not highly methylatedNot highly methylated

Transfect MeCP2 Transfect MeCP2 construct and construct and methylated reporter methylated reporter plasmidplasmid

Assess transcriptionAssess transcription

MeCP2

CATCATmmm

Mutations decrease Mutations decrease MeCP2 repression in MeCP2 repression in

DrosophilaDrosophila cells cells

MeCP2R106WR133CF155ST158MR255X0

20

40

60

80

100

120

Rel

ativ

e C

AT

act

ivity

(%

)

• Transient transfection Transient transfection • MeCP2 constructMeCP2 construct• CAT reporterCAT reporter

Kudo et al., Brain and Dev 2001

FRAP = fluorescence recovery after photobleaching

Phair RD, and Misteli T. (2001) Nat Rev Mol Cell Biol. 2(12):898-907

A photobleaching technique to study protein movement in living cells

Immobile fraction

Mobile fractionT1/2

Mecp2 is a mobile protein in live cells

0 50 100 150 200 250 3000.0

0.2

0.4

0.6

0.8

1.0R

ela

tive In

tensi

ty

Time (seconds)

Mecp2A Mecp2B

Rett mutations increase Rett mutations increase mobilitymobility

0 20 40 60 80

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0R

ela

tive

Inte

nsi

ty

Time (seconds)

WT R106W R133C F155S T158M R255X GFP

MOUSE MODELSMOUSE MODELS

Knock-out mouse: Knock-out mouse: Mecp2Mecp2 deleted deleted

Knock-in mouse: Insertion of human Knock-in mouse: Insertion of human mutation in mutation in Mecp2Mecp2

KNOCK-OUT MUTANTKNOCK-OUT MUTANT

Note Note

hind-hind-limb limb claspingclaspingGuy et al. Guy et al.

Nature Nature Genetics Genetics 2001;27:322-2001;27:322-326326

KNOCK-OUT MUTANTKNOCK-OUT MUTANT Is Mecp2 knock-out reversible?Is Mecp2 knock-out reversible? Using estrogen receptor Using estrogen receptor

controlledcontrolled Mecp2 Mecp2 promoter: promoter:– Mecp2Mecp2 knock-out phenotype knock-out phenotype

reversed in both immature male reversed in both immature male and mature male and female mice and mature male and female mice with estrogen analog, tamoxifenwith estrogen analog, tamoxifen

– Rapid re-expression in immature Rapid re-expression in immature males resulted in death in 50%males resulted in death in 50%

Guy et al. Guy et al. ScienceScience 2007;315:1143-1147 2007;315:1143-1147

KNOCK-IN MUTANTKNOCK-IN MUTANT

Note Note humped humped back and back and forelimb forelimb claspingclaspingYoung and Young and Zoghbi, Am J Zoghbi, Am J Hum Genet Hum Genet 2004;74:511-2004;74:511-520520

KNOCK-IN MUTANTKNOCK-IN MUTANT

Impaired hippocampus-Impaired hippocampus-dependent social, spatial, and dependent social, spatial, and contextual fear memory contextual fear memory

Impaired long-term potentiation Impaired long-term potentiation and depressionand depression

Reduced post-synaptic densitiesReduced post-synaptic densities No change in No change in BDNFBDNF expression expression

Moretti et al. J Neurosci 2006;26:319-Moretti et al. J Neurosci 2006;26:319-327327

KNOCK-IN MUTANTKNOCK-IN MUTANT

Enhanced anxiety and fear based Enhanced anxiety and fear based on:on:– Elevated blood corticosterone Elevated blood corticosterone

levels levels – Elevated corticotropin-releasing Elevated corticotropin-releasing

hormone in hypothalamus, central hormone in hypothalamus, central nucleus of amygdala, and bed nucleus of amygdala, and bed nucleus of stria terminalisnucleus of stria terminalis

– MeCP2 binds to MeCP2 binds to CrhCrh promoter promoter methylated regionmethylated region

McGill et al. McGill et al. PNASPNAS 2006;103:18267-18272 2006;103:18267-18272

KNOCK-IN MUTANTKNOCK-IN MUTANT

Implications of Implications of CrhCrh over- over-expression:expression:– Anxiety plays central role in clinical RSAnxiety plays central role in clinical RS– Amygdala has direct input into Amygdala has direct input into

hypothalamus and brainstem hypothalamus and brainstem autonomic nuclei correlating with autonomic nuclei correlating with clinical problems of respiration, GI clinical problems of respiration, GI function, and peripheral sympathetic function, and peripheral sympathetic NS NS

– Suggests strategies for therapeutic Suggests strategies for therapeutic interventionintervention

Longevity StudyLongevity Study

● Data gathered on 1928 girls and Data gathered on 1928 girls and womenwomen

● Completion of data gathering and Completion of data gathering and filling in missing data consumed most filling in missing data consumed most of winterof winter

● Analysis of longevity underwayAnalysis of longevity underway● Databank very informativeDatabank very informative● Appears representative Appears representative

North American North American DatabaseDatabase

Total enrolledTotal enrolled 19281928

TypicalTypical 1648 (85.5 %)1648 (85.5 %)

AtypicalAtypical 259 (13.4 %)259 (13.4 %)

Not RS (Not RS (MECP2 MECP2 positive)positive)

21 (1.1 %)21 (1.1 %)

North American North American DatabaseDatabase

GroupGroup TotaTotall

MutatioMutation n

No No mutatiomutatio

nn

UnknowUnknownn

TypicalTypical 16416488

791 791 (91%)(91%)

79 (9%)79 (9%) 778778

AtypicAtypical al

259259 94 94 (58%)(58%)

68 68 (42%)(42%)

9797

Not RSNot RS 2121 21 21 (100%)(100%)

00 00

North American North American DatabaseDatabase

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

DATABASE RESOURCESDATABASE RESOURCES

– RettBase: Dr. John ChristodoulouRettBase: Dr. John ChristodoulouMECP2MECP2 Mutation Repository Mutation Repositorymecp2.chw.edu.aumecp2.chw.edu.au

– InterRett: Dr. Helen LeonardInterRett: Dr. Helen LeonardClinical information repository Clinical information repository from parents and physiciansfrom parents and physicians

www.ichr.uwa.edu.auwww.ichr.uwa.edu.au

AcknowledgementsAcknowledgements● IRSAIRSA

● UABUAB – Jane LaneJane Lane– Suzie GeertsSuzie Geerts– Jerry ChildersJerry Childers

● duPont Hospital for duPont Hospital for ChildrenChildren– Carolyn SchanenCarolyn Schanen

● NIHNIH – NICHD/ORD/NCRRNICHD/ORD/NCRR

● Greenwood Genetic Greenwood Genetic CenterCenter– Steve Skinner Steve Skinner – Fran AnneseFran Annese

● Baylor College of Baylor College of MedicineMedicine– Daniel GlazeDaniel Glaze– Jeff NeulJeff Neul– Huda ZoghbiHuda Zoghbi– Judy BarrishJudy Barrish

● Girls and women Girls and women with RS and their with RS and their familiesfamilies

That’s all folks!!!That’s all folks!!!