RETT SYNDROMERETT SYNDROME

UNDERSTANDING RETT UNDERSTANDING RETT SYNDROME AND THE ROLE OF SYNDROME AND THE ROLE OF

MECP2MECP2

NEUROSCIENCE NEUROSCIENCE

JANUARY 2009JANUARY 2009

OUTLINEOUTLINE CLINICAL BACKGROUNDCLINICAL BACKGROUND

MOLECULAR IMPLICATIONSMOLECULAR IMPLICATIONS

PHENOTYPE-GENOTYPE RELATIONPHENOTYPE-GENOTYPE RELATION

ANIMAL MODELSANIMAL MODELS

THERAPYTHERAPY

Rett syndrome is caused by mutations in X-linked Rett syndrome is caused by mutations in X-linked MECP2MECP2, encoding methyl-CpG-binding protein 2, encoding methyl-CpG-binding protein 2

Ruthie 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

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 - mainly in females Genetic disorder - mainly in females

Diagnosis based on meeting clinical Diagnosis based on meeting clinical criteriacriteria

Variable clinical expression Variable clinical expression

Significant longevitySignificant longevity

Consistent neuropathologyConsistent neuropathology

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 DENDRITES SMALL NEURONS; SIMPLIFIED DENDRITES

WITH REDUCED SPINESWITH REDUCED SPINES

ABSENCE OF RECOGNIZABLE DISEASEABSENCE OF RECOGNIZABLE DISEASE

• 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 RetardationSpine Dysgenesis in Mental Retardation

OTHER NEURODEVELOPMENTAL OTHER 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

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

MECP2MECP2 AND RETT SYNDROME AND RETT SYNDROME 90-95% of classic RTT due to mutations in 90-95% of classic RTT due to mutations in

MECP2 MECP2 4 missense and 4 nonsense mutations 4 missense and 4 nonsense mutations

account for ~ 65%; c-terminal truncations account for ~ 65%; c-terminal truncations and large deletions account for another 15-and large deletions account for another 15-18%18%

Sporadic RTT: majority of paternal originSporadic RTT: majority of paternal origin Familial RTT (<<1% of total): majority due Familial RTT (<<1% of total): majority due

to large deletions to large deletions Phenotypes extend beyond classic RTTPhenotypes extend beyond classic RTT

Normal*

Learning Disabilities*

Behavioral Phenotype* Autistic; OCD Hyperactive/aggressive 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

Females Males

MECP2 Duplication syndrome* +/- recurrent infections

X-linked cognitive impairment* +/- progressive spasticity; other NDD features/OCD; aggression

Rett syndrome XXY; somatic mosaicism

Severe encephalopathy*

*

Continuum of MECP2 Associated Phenotypes

*Likely under-represented

Medical IssuesMedical IssuesLongevityLongevityGrowthGrowthEpilepsyEpilepsyGI dysfunctionGI dysfunctionScoliosisScoliosisOsteopeniaOsteopeniaBreathing irregularitiesBreathing irregularitiesSleepSleepCardiac conductionCardiac conductionSexual MaturationSexual Maturation

LONGEVITY IN RETT LONGEVITY IN RETT SYNDROMESYNDROME

Unpublished study from Baylor College of Unpublished study from Baylor College of

Medicine: survival follows that of all Medicine: survival follows that of all

females until age 10; 70% survival to age females until age 10; 70% survival to age

35 versus 98% in all females35 versus 98% in all females

Recent study from Australia: 78% survival Recent study from Australia: 78% survival

to age 25 versus 99.9% in all females - to age 25 versus 99.9% in all females - Laurvick et al. J Pediatr 2006;148:347Laurvick et al. J Pediatr 2006;148:347

Figure 1

0. 00

0. 25

0. 50

0. 75

1. 00

YEARS

0 10 20 30 40 50 60 70

STRATA: BYEAR=1935- 1959 BYEAR=1960- 1969 BYEAR=1970- 1979BYEAR=1980- 1989 BYEAR=1990- 1999 BYEAR=2000- 2009

RETT SYNDROME – SURVIVAL BY DATE OF BIRTH

MOLECULAR IMPLICATIONSMOLECULAR IMPLICATIONS

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 HUMAN MeCP2 DISTRIBUTION IN HUMAN BRAIN DURING DEVELOPMENTBRAIN DURING DEVELOPMENT

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

CORTICAL NEURONS LAST TO EXPRESSCORTICAL NEURONS LAST TO EXPRESSShahbazian et al. Shahbazian et al. Hum Mol GenetHum Mol Genet 2002;11:115 2002;11:115

Mecp2 plays complex role in the Mecp2 plays complex role in the nucleus with differential effects on nucleus with differential effects on

transcriptiontranscription

Chahrour et al. Science 2008

Yasui et al. PNAS 2007

MeCP2

Methylated CpG

Chromatin

Mutated MeCP2

Sin3AHDAC

PHENOTYPE-GENOTYPE PHENOTYPE-GENOTYPE RELATIONRELATION

Does mutation predict Does mutation predict outcomeoutcome

R133C, R294X, and R306C mutations R133C, R294X, and R306C mutations and c-term truncations are associated and c-term truncations are associated with lower severity scores, slower with lower severity scores, slower progression, preserved speech progression, preserved speech

Classic RTT: missense and nonsenseClassic RTT: missense and nonsense

Variant RTT: nonsenseVariant RTT: nonsense

Non-RTT: missense and frameshiftNon-RTT: missense and frameshift

Medical IssuesMedical IssuesFrequency (%) by Mutation TypeFrequency (%) by Mutation Type

Mutation (n)Mutation (n) SeizuresSeizures ScoliosisScoliosis Sc SurgerySc Surgery

R106W (20)R106W (20) 7878 4545 1515

R133C (28)R133C (28) 5050 2525 00

T158M (64)T158M (64) 7474 5050 1616

R168X (55)R168X (55) 5454 3535 1111

R255X (60)R255X (60) 4949 4242 77

R270X (34)R270X (34) 5353 5050 1212

R294X (37)R294X (37) 6969 4343 33

R306C (45)R306C (45) 4949 3131 44

Lrg del (49)Lrg del (49) 5757 5555 1212

c-ter del (53)c-ter del (53) 6969 5151 99

Medical IssuesMedical IssuesFrequency (%) by Mutation TypeFrequency (%) by Mutation Type

Mutation (n)Mutation (n) Constipation Constipation GERDGERD FracturesFractures

R106W (20)R106W (20) 7575 5050 2020

R133C (28)R133C (28) 7575 4343 77

T158M (64)T158M (64) 7878 5656 1616

R168X (55)R168X (55) 7373 5555 1616

R255X (60)R255X (60) 7575 5252 1717

R270X (34)R270X (34) 7474 6262 2727

R294X (37)R294X (37) 8181 4949 1919

R306C (45)R306C (45) 6262 4747 1313

Lrg del (49)Lrg del (49) 6969 5555 2020

c-ter del (53)c-ter del (53) 7575 3838 1313

MOUSE MODELSMOUSE MODELS

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

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

KNOCK-OUT MUTANTKNOCK-OUT MUTANTIs 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 MUTANTNote 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 MUTANTImpaired 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 depressionReduced post-synaptic densitiesReduced post-synaptic densitiesNo 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 MUTANTEnhanced 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 MUTANTImplications 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

But overexpression is But overexpression is detrimentaldetrimental

Collins et al., HMG 2004

THERAPYTHERAPY

Therapeutic Therapeutic interventions in Rett interventions in Rett

syndromesyndromeGeneticGeneticGene therapyGene therapyPost-transcriptional Post-transcriptional

repairrepair

ProteinProteinReintroduce proteinReintroduce proteinModifiers of mutant Modifiers of mutant

MECP2 functionMECP2 function

http://fajerpc.magnet.fsu.edu/Education/2010/

New treatments on the New treatments on the horizon?horizon?

Gene repair, replacement strategiesGene repair, replacement strategies Gene therapyGene therapy Reintroduction of protein Reintroduction of protein

Can we modulate expression of the gene/protein?Can we modulate expression of the gene/protein? Up-regulation in “normal” cells in heterozygotesUp-regulation in “normal” cells in heterozygotes Reactivate normal geneReactivate normal gene

Post transcriptional repairPost transcriptional repair Treatment being tested in CF and Duchenne muscular Treatment being tested in CF and Duchenne muscular

dystrophy of a drug that allows the cell to “read dystrophy of a drug that allows the cell to “read through” a nonsense mutationthrough” a nonsense mutation

Targeted treatments based on what Mecp2 does in Targeted treatments based on what Mecp2 does in neuronsneurons Can we improve protein function?Can we improve protein function? Can we make another protein take over?Can we make another protein take over?

Can we intercede on specific downstream targets?Can we intercede on specific downstream targets?Can we modulate neurophysiology?Can we modulate neurophysiology?

Challenges across the board Challenges across the board for therapies in Rett syndromefor therapies in Rett syndrome

No clear-cut reliable outcome measure No clear-cut reliable outcome measure for all the girls.for all the girls. Natural history study may be key.Natural history study may be key. ?EEG or other objective physiologic ?EEG or other objective physiologic

measure.measure. Girls will be heterogeneous in genetic Girls will be heterogeneous in genetic

background and XCI.background and XCI. Even in cultured cells, outcome Even in cultured cells, outcome

measures are tricky.measures are tricky. But the mice can help us here…..But the mice can help us here…..

What about gene therapy for What about gene therapy for Rett syndrome?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

Gene therapyGene therapy

Post-transcriptional repair: Post-transcriptional repair: aminoglycosidesaminoglycosides

http://www.ptcbio.com/3.1.1_genetic_disorders.aspx

Aminoglycoside Aminoglycoside antibiotics allow “read-antibiotics allow “read-through” of premature through” of premature stop signalsstop signalsNonsense mutationsNonsense mutationsProblems with Problems with

toxicitytoxicityNeed to be given IVNeed to be given IV

Introduce a missense Introduce a missense mutation at the “stop” mutation at the “stop” sitesite

Post-transcriptional Post-transcriptional repair?repair?

PTC124: New Agent PTC124: New Agent developed by PTC developed by PTC Therapeutics with Therapeutics with similar actionsimilar action Orally availableOrally available Less toxicLess toxic Being tested in cystic Being tested in cystic

fibrosis and Duchenne fibrosis and Duchenne Muscular Dystrophy with Muscular Dystrophy with very promising resultsvery promising results

Phase 2 trials- increased Phase 2 trials- increased walking duration in walking duration in DMD, improved chloride DMD, improved chloride conductance in CFconductance in CF

http://www.ptcbio.com/3.1.1_genetic_disorders.aspx

Therapies for Rett Therapies for Rett SyndromeSyndrome

NutritionNutrition

Physical TherapyPhysical Therapy

Occupational TherapyOccupational Therapy

Communication TherapyCommunication Therapy

HippotherapyHippotherapy

Music TherapyMusic Therapy

AquatherapyAquatherapy

TOE WALKING

ANKLE-FOOT DYSTONIA

A successful intervention

ConclusionsConclusionsCurrent investigations into neurobiological Current investigations into neurobiological underpinnings of CNS dysfunction opening underpinnings of CNS dysfunction opening doors to new therapies in immediate future.doors to new therapies in immediate future.– Repurposing existing drugsRepurposing existing drugs– Development of new agentsDevelopment of new agents

Gene based therapies are potential avenues Gene based therapies are potential avenues of treatment in the future.of treatment in the future.Role of environmental enrichment (supports Role of environmental enrichment (supports early intervention programs)early intervention programs)Need to examine treatments in female miceNeed to examine treatments in female miceCogent planning needed for clinical trials for Cogent planning needed for clinical trials for selected agentsselected agents

The TeamThe TeamBaylor College of Baylor College of MedicineMedicine– Daniel GlazeDaniel Glaze– Kay MotilKay Motil– Jeff NeulJeff Neul– Judy BarrishJudy Barrish– Carmel LuskCarmel Lusk

Greenwood Genetic Greenwood Genetic CenterCenter– Steve Skinner Steve Skinner – Fran AnneseFran Annese– Joy GrahamJoy Graham– Lauren McNairLauren McNair

UABUAB– Alan PercyAlan Percy– Jane LaneJane Lane– Suzie GeertsSuzie Geerts– Jerry ChildersJerry Childers– Russell KirbyRussell Kirby

NIH/NCRR/NICHD NIH/NCRR/NICHD

DTCC –DTCC – Hye-Seung LeeHye-Seung Lee

Girls and women with Girls and women with RS and their familiesRS and their families

…Rollin’ with Curly