Rett Syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2

1. Rett Syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2 Presented By: Vanessa Bowers Amir, R., I.B. Van den Veyver, M. Wan, C.Q. Tran, U. Francke, and H.Y. Zoghbi. 1999. Rett Syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nature Genetics. 23:185-188.

2. What is Rett Syndrome • A progressive neurodevelopmental disorder that begins at early infancy • Caused by a mutation in the methyl-cysteine binding protein 2 gene (MeCP2) • Most common cause of mental retardation in females • Shows high lethality in hemizygous males (causing miscarriage, still birth, or early death)

3. Symptoms & Stages of Rett Syndrome(RTT) • Patients with RTT appear to be born healthy and develop normally until about 6-18 months of age • Several stages then follow: • After initial regression, the condition stabilizes and female patients usually survive into adulthood

4. What is the MeCP2 gene? • MeCP2 gene is located on the X-chromosome (mapped to the region Xq28) • It is a transcriptional repressor that prevents unscheduled transcription throughout the body • The MeCP2 gene encodes the MeCP2 protein which binds methylated cytosine residue of DNA dinucleotides and mediates transcriptional silencing through the interaction with histone deacetylase and the corepressor mSIN3A • These cytosine residues are subject to transcriptional silencing after DNA methylation

5. Why is transcriptional silencing important in the body? • It is a mechanism of transcriptional control where DNA in no longer accessible for future transcription • This is important because in different stages of development, certain genes need to be silenced or turned off and others need to be turned on in order for proper development • In RTT, the mutation of the MeCP2 gene doesn’t allow for normal transcriptional silencing, therefore causing the disease

6. What does the MeCP2 protein look like? • MeCP2 protein is a chain of 486 amino acids • Two functional domains: - an 85 amino acid methyl-CpG binding domain (MBD) - a 104 amino acid transcription repression domain (TRD) • The MBD is essential for the binding of the protein to methyl cytosine at the beginning of genes in the body • The TRD recruits other repressor proteins (mSIN3a, histone deascetylase) to inhibit transcription

7. RTT is caused by mutations in the MeCP2 gene, which causes changes in the corresponding MeCP2 protein Mutations occur in either of the parents reproductive cells – sperm or egg Mutations are usually sporadic and not familial Where does RTT originate?

8. There are two scenarios for the origin of mutations in Rett Syndrome:1) The father has a mutation on his X chromosome or 2) The mother has a mutation on one of her x chromosomes Scenario 1 Scenario 2

9. Rett Syndrome is Neurodevelopmental Disorder • MeCP2 is very abundant and highly expressed in the brain. In the developing cerebral cortex, the appearance of MeCP2 correlates with neuronal maturation. • With this high abundance of MeCP2 within the brain, these mutations of the MeCP2 protein affect the development of neurons and therefore cause many of the neurological symptoms that are related to Rett Syndrome

10. The Experiment • 21 sporadic and 8 familial RTT patients’ genomic DNA were screened by conformation-sensitive gel electrophoresis • Sporadic patients: no known family members expressed the disorder • Familial patients were: 5 pairs of full sisters 2 pairs of half sisters 1 pair of second half cousins

11. Results • Among sporadic patients: 3 missense, 1 nonsense, and 1 frameshift mutations were identified among patients 6, 22, 24, 29, and 39 • In two half sisters, the same missense mutation was found in each, but was not present in the mother

12. Quick Definitions • Missense mutation - A mutation that changes a codon so that it codes for a different amino acid • Nonsense mutation - A mutation in which one of the three terminator codons in the RNA (TAG, TAA, TGA), used to signal the end of a polypeptide, appears in the middle of a genetic message, causes premature termination of transcription, and releases incomplete, nonfunctional polypeptides from the ribosome. • Frameshift mutation - A mutation resulting from an addition or subtraction that is not an exact multiple of 3 base pairs in a coding sequence. From the point of mutation onwards, codons are read out of phase; the reading frame of the gene is changed, and a completely different set of amino acids are made into protein.

13. Patient 39: a missense mutation occurred at amino acid 133, arginine was replaced by cysteinePatient 24: a missense mutation occurred at amino acid 155, thymine was replaced by cytosinePatient 6: a missense mutation occurred at amino acid 158, cytosine was replaced by thymine

14. Patient 22: a nonsense mutation occurred with a substitution of cytosine to thymine, which converted a CGA codon to a TGA codon

15. Patient 29: A frameshift mutation occurred from an insertion at codon 208 which shifted the reading frame and introduced a stop codon after 27 amino acids

16. Familial Patients Results • In two half sisters with the same mother, the same missense mutation of cytosine replaced by thymine was identified • No mutation was found in the mother (she is an obligate carrier) Sister Mother Sister

17. Discussion • These missense mutations disrupt the structure of the methyl binding region (MBR) of the MeCP2 protein • The nonsense and frameshift mutations disrupt the transcription repression domain (TRD) • These disruptions of the MBD and the TRD of the MeCP2 protein interferes with their functions of transcriptional silencing and cause Rett syndrome

18. Conclusion Rett syndrome patients display some type of mutation (missense, nonsense, or frameshift). These mutations affect the MBD or TRD domains of the MeCP2 protein, which cause defects that don’t allow for the proper functioning of the MeCP2 gene as a transcriptional silencer. Without proper functioning of this gene, patients will develop Rett Syndrome.

19. QUESTIONS?