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

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

5. RETT SYNDROME A NEURODEVELOPMENTAL DISORDER OF YOUNG FEMALES CHARACTERIZED BY  PROFOUND COGNITIVE IMPAIRMENT  COMMUNICATION DYSFUNCTION  STEREOTYPIC MOVEMENTS  PERVASIVE GROWTH FAILURE

6. 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

7. 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

9. RETT SYNDROME WHAT 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

10. Rett Syndrome in USA IRSA Case Registry by State (May 2007) 64 9 31222 43 232 7 14 TOTAL 3712 87 27 1413 10 27 3444 24 179 89 377 20 50 40 33 66 17887 66 193 170 90 61 1919 53 180 24 56 1074242 101 53 25 103 1 18 45 142 5 9090 2626 1 Puerto Rico 10 Puerto Rico 10

11. RETT SYNDROME VARIABLE CLINICAL EXPRESSION  PHENOTYPIC EXPRESSIONS  SEIZURES OR BEHAVIORAL PATTERNS  BREATHING IRREGULARITIES  SLEEP CHARACTERISTICS  SCOLIOSIS  AMBULATION

12. 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

13. 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

15. 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

16. 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 Retardation

17. 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

18. 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

19. 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

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

21. MeCP2 Methylated CpG Chromatin Mutated MeCP2 Sin3A HDAC

22. 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

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

24. DOES MUTATION PREDICT OUTCOME? Certain mutations (R133C, R294X, and R306C and C-terminal truncations are associated with “better 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 Missense mutations in C-terminal region in males associated with XLMR

25. RETT SYNDROME AND MECP2  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

26. 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

27. Rett Syndrome Female Phenotypes With MECP2 Mutations Rett Syndrome Rett Syndrome Preserved Speech Variant Preserved Speech Variant Delayed Onset Variant Delayed Onset Variant Congenital Onset Variant Congenital Onset Variant Early Onset Seizure Variant Early Onset Seizure Variant Autistic-like Variant Autistic-like Variant Angelman Syndrome Angelman Syndrome Mild Learning Disability Mild Learning Disability Normal Carriers Normal Carriers

28. Rett Syndrome Male Phenotypes With MECP2 Mutations Fatal Encephalopathy Fatal Encephalopathy Rett/Klinefelter Syndrome Rett/Klinefelter Syndrome X-Linked MR/Progressive Spasticity X-Linked MR/Progressive Spasticity Somatic Mosaicism/NDD Somatic Mosaicism/NDD MECP2 Duplications and X-linked MR MECP2 Duplications and X-linked MR

29. 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

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

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

32. 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 fraction T 1/2

33. Mecp2 is a mobile protein in live cells

34. Rett mutations increase mobility

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

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

37. KNOCK-OUT MUTANT Is Mecp2 knock-out reversible? Is Mecp2 knock-out reversible? Using estrogen receptor controlled Mecp2 promoter: 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

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

39. KNOCK-IN MUTANT Impaired hippocampus-dependent social, spatial, and contextual fear memory Impaired hippocampus-dependent social, spatial, and contextual fear memory Impaired long-term potentiation and depression Impaired long-term potentiation and depression Reduced post-synaptic densities Reduced post-synaptic densities No change in BDNF expression No change in BDNF expression Moretti et al. J Neurosci 2006;26:319-327

40. KNOCK-IN MUTANT Enhanced anxiety and fear based on: 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

41. KNOCK-IN MUTANT Implications of Crh over-expression: 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

42. 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

43. North American Database Total enrolled 1928 Typical 1648 (85.5 %) Atypical 259 (13.4 %) Not RS (MECP2 positive) 21 (1.1 %)

44. North American Database GroupTotalMutation No mutation Unknown Typical1648 791 (91%) 79 (9%) 778 Atypical259 94 (58%) 68 (42%) 97 Not RS 21 21 (100%) 00

45. North American Database Mutation Type N% France (Philippe et al.) Missense35638.835.6 Nonsense32335.237.3 Frameshift16117.512.0 Complex deletion 596.45.8

46. 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

47. 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

49. That’s all folks!!!