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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.
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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 SYNDROMEA NEURODEVELOPMENTAL DISORDER OF YOUNG FEMALES CHARACTERIZED BY • PROFOUND COGNITIVE IMPAIRMENT • COMMUNICATION DYSFUNCTION • STEREOTYPIC MOVEMENTS • PERVASIVE GROWTH FAILURE
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/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 SYNDROMEWHAT DO WE KNOW? • 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 USAIRSA Case Registry by State (May 2007) 18 87 14 13 89 19 10 90 11 64 27 9 31 179 222 24 103 193 27 178 87 170 26 34 44 40 45 66 24 42 107 142 377 5 50 66 33 101 20 90 56 53 25 43 53 61 1 232 7 180 TOTAL 3712 14 Puerto Rico 10 Puerto Rico 10
RETT SYNDROMEVARIABLE 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 SYNDROMEBRAIN 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 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 1999;23:185
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
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
MeCP2 HDAC Sin3A Function of MeCP2 TRD MBD Methylated CpG Chromatin
HDAC Sin3A Mutated MeCP2 MeCP2 Methylated CpG Chromatin
WHAT DO WE KNOW ABOUT MECP2 AND RETT SYNDROME? • >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 • RETT SYNDROME IS A CLINICAL DIAGNOSIS • RETT SYNDROME IS NOT SYNONYMOUS WITH MECP2 MUTATIONS • RETT SYNDROME MAY BE SEEN WITHMECP2 MUTATIONS • RETT SYNDROME MAY BE SEEN WITHOUTMECP2 MUTATIONS • MECP2 MUTATIONS MAY BE SEEN WITHOUT RETT SYNDROME
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 2004;36:339-341
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
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
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
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 • Transient transfection • MeCP2 construct • CAT reporter Relative CAT activity (%) 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
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;27:322-326
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;74:511-520
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 • 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
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
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 Acknowledgements