Rett Syndrome

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. 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” Symptomsafter 7 to 18 months

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. MBDNan, 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. 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 Exons

11. Structure of Human MECP2Wan, M., et.al.

12. DetectionLewis, 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 cDNALewis, 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 ExperimentsLewis, 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 MECP2Reichwald, 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 MECP2Wan, M., et.al.

22. 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 TreatmentNo trxt has shown significant improvements

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.

25. Questions