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RETT SYNDROME

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

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  1. RETT SYNDROME UNDERSTANDING THE ROLE OF MECP2 BASIC NEUROSCIENCE NBL 120 DECEMBER 2007

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

  3. RETT SYNDROMEA NEURODEVELOPMENTAL DISORDER OF YOUNG FEMALES CHARACTERIZED BY • PROFOUND COGNITIVE IMPAIRMENT • COMMUNICATION DYSFUNCTION • STEREOTYPIC MOVEMENTS • PERVASIVE GROWTH FAILURE

  4. 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

  5. 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

  6. 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

  7. 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

  8. RETT SYNDROMEVARIABLE CLINICAL EXPRESSION • PHENOTYPIC EXPRESSIONS • SEIZURES OR BEHAVIORAL PATTERNS • BREATHING IRREGULARITIES • SLEEP CHARACTERISTICS • SCOLIOSIS • AMBULATION

  9. 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

  10. 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

  11. 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

  12. 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)

  13. 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

  14. 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

  15. 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

  16. MeCP2 HDAC Sin3A Function of MeCP2 TRD MBD Methylated CpG Chromatin

  17. HDAC Sin3A Mutated MeCP2 MeCP2 Methylated CpG Chromatin

  18. 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

  19. Mutations • Mutations in MeCP2 found in 70-95% “classical” Rett syndrome • Missense, nonsense, frameshift, large scale rearrangements

  20. 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

  21. 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

  22. 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

  23. 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

  24. 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

  25. 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

  26. Assessing function of mutant protein • Drosophila S cells • Not highly methylated • Transfect MeCP2 construct and methylated reporter plasmid • Assess transcription MeCP2 mmm CAT

  27. Mutations decrease MeCP2 repression in Drosophila cells • Transient transfection • MeCP2 construct • CAT reporter Relative CAT activity (%) Kudo et al., Brain and Dev 2001

  28. 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

  29. Mecp2 is a mobile protein in live cells

  30. Rett mutations increase mobility

  31. MOUSE MODELS • Knock-out mouse: Mecp2 deleted • Knock-in mouse: Insertion of human mutation in Mecp2

  32. KNOCK-OUT MUTANT • Note hind-limb clasping Guy et al. Nature Genetics 2001;27:322-326

  33. 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

  34. KNOCK-IN MUTANT • Note humped back and forelimb clasping Young and Zoghbi, Am J Hum Genet 2004;74:511-520

  35. 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

  36. 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

  37. 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

  38. 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

  39. North American Database

  40. North American Database

  41. North American Database

  42. 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

  43. 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

  44. That’s all folks!!!

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