1. Rett Syndrome Presented by: Ashley Owen University of Wisconsin-Eau Claire November 30, 2004

2. What is RTT?  Neurodevelopmental disorder cased by mutations in the methyl-CpG-binding protein 2 (MECP2) and is characterized by the loss of acquired skills after a period of normal development in infant girls.

3. Symptoms after 7 to 18 months  Mild learning difficulties  Disturbances with breathing and cardiac rate  Bowel immobility  Screaming fits  Autistic features  Microcephaly  Seizures  Hand stereotypes Washing, clapping, mouthing  Decrease in head growth  Small statue  Teeth grinding  “eye pointing”

4. Inheritance  Prevalence 1/10,000-15,000 females  X-linked dominant mutation De novo Inherited from parent with the disease causing mutation and germline mosaicism Mother would have XCI and be unaffected

5. Males  47, XXY Identified as RTT Somatic mosaicism  XCI  46, XY Severe neonatal encephalopathy Leads to death “Disease of the brain”

6. Gene of Interest: MECP2  Methyl-CpG-Binding Protein  Two domains Methyl-CpG-binding domain (MBD) Transcriptional repression domain (TRD)  Location: Xq28 Pericentromeric heterochromatin  Transcriptional silencing/repression, epigenetic regulation, nuclear structure (chromatin) 5-methylcytosine rich heterochromatin

7. MBD Nan, X., Meehan, R.R., & Bird, A.  Located between amino acid 89 and 162  Symmetrical methylated CpG dinucleotides  Binds to minor groove of beta DNA

8. MBD (cont.) Nan, X., Meehan, R.R., & Bird, A.  Dimerization is not required for binding Monomer  MBD and TRD are important for XCI Methyl dependent repression

9. TRD  Interacts with co-repressor Sin3A Recruits histone deactylases

10. Exons  Exon 1 Non-coding 5‘ untranslated region (UTR)  Exon 2 Coding sequence  Exon 3 Coding sequence  Exon 4 Non-coding 3‘ UTR Coding sequence Polyadenylation creates different protein lengths

11. Structure of Human MECP2 Wan, M., et.al.

12. Detection Lewis, J.D., et.al.  Looking for MECPs expression clone Methylated and unmethylated probe  Differ from MECP1 MECP1 is a 120kb Tissue distribution Testis Anion/cation ion exchange column Binding specificities 12 methyl-CpGs vs. a pair

13. Obtaining cDNA Lewis, J.D., et.al.  Partial amino acid sequence as a primer  340 bp fragment from original PCR to complete library λZAPll  ORF 492 amino acids and all 6 major peptides

14. Translational Experiments Lewis, J.D., et.al.  SDS-polyacrylamide  Relationship between ORF and MECP2 Showed an 81kd sequence cDNA fused into E.Coli with ß-galactosidase gene Bound to methylated probe  Conclusion: ORF codes for MECP2

15. Localization Lewis, J.D., et.al.  Immunofluorescence Ab76 serum  Stained in heterochromatin areas  Parallel satellite DNA in mice Contains 8 CpGs sights  Associated with pericentromeric heterochromatin

16. Comparison of Mouse and Human MECP2 Reichwald, K., et.al. Identity between the mouse and human gene is 68% (average)

17. Mechanism  Expressed during organogenesis during embryonic life and in the hippocampus during adult life Other methylated binding proteins take over in other cells during adulthood  Believed to be involved in XCI and genetic imprinting

18. Tissue Specific  Two transcripts 1.9kb ~10kb  Difference in tissue expression Difference in translatability Half life is similar

19. Mutations  99.5% are sporadic  Nonsense, missense/frameshifts, deletions Majority are nonsense  Detected using PCR and restriction enzyme analysis  Occur in CpG sites Hypermutable Methylated in germline and prone to deamination (C to T)

20. Mutations (Cont.) Wan, M., et.al.  See word document

21. Structure of Human MECP2 Wan, M., et.al.

22. Treatment No trxt has shown significant improvements  Previous trxts L-carnitine Fatty acid metabolism Respiratory features Ketogenic diet Control of seizures Lacked vitamins Naltrexone* Oral opiate antagonist Respiratory features, EEG patterns  Ongoing trial Folate-Betaine Methyl-donor group Alter gene expression Recruit other methyl binding groups  Current trxt Supportive/symptomatic therapy Occupational/physical therapy

23. Social and Ethical Dilemmas  Money…testing in general is expensive  Have to show clinical signs/family member  Diagnostic testing Test for mutation prenatally Possible involvement with other disorders  Mental retardation in males

24. References  Hagberg, B.A. and Skjeldal, O.H. (1994). Rett variants: A suggested model for inclusion criteria. Pediatric Neurology, 11, 5-11.  Lewis, J.D., et.al. (1992). Purification, sequence, and cellular localization of a novel chromosomal protein that binds to methylated DNA. Cell, 69, 905-914.  Nan, X., Meehan, R.R., & Bird, A. (1993). Dissection of the methyl-CpG binding domain from the chromosomal protein MeCP2. Nucleic Acids Research, 21, 4886-4892.  Percy, A. K. (2002). Clinical trials and treatment prospects. Mental Retardation and Developmental Disabilities Research Reviews, 8, 106-111.  Reichwald, K., et.al. (2000). Comparative sequence analysis of the MECP2-locus in human and mouse reveals new transcribed regions. Mammalian Genome, 11, 182-190.  Rett Syndrome, RTT. (2004). Online mendelian inheritance in man, 9 Oct. 2004.  Shahbazian, M.D., Antalffy, B., Armstrong, D.L. & Zoghbi, H.Y. (2002). Insight into rett syndrome: MECP2 levels display tissue- and cell-specific difference and correlate with neuronal maturation. Human Molecular Genetics, 11, 115-124.  Wan, M. et.al. (1999). Rett syndrome and beyond: Recurrent spontaneous and familial MECP2 mutations at CpG hotspots. American Journal of Human Genetics, 65, 1520-1529.  Zoghbi, H.Y. (2004). Rett Syndrome. GeneReviews, www.genetests.org, 29 Sept. 2004.www.genetests.org

25. Questions