Rett Syndrome
Presented by: Ashley Owen
University of Wisconsin-Eau Claire
November 30, 2004
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.
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”
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
Males
 47, XXY

Identified as RTT

Somatic mosaicism
 XCI
 46, XY

Severe neonatal encephalopathy
Leads to death
 “Disease of the brain”

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
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
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
TRD
 Interacts with co-repressor Sin3A

Recruits histone deactylases
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
Structure of Human MECP2
Wan, M., et.al.
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
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
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
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
Comparison of Mouse and
Human MECP2
Reichwald, K., et.al.
•Identity between the mouse and human gene is 68% (average)
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
Tissue Specific
 Two transcripts


1.9kb
~10kb
 Difference in tissue
expression


Difference in
translatability
Half life is similar
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)

Mutations(Cont.)
Wan, M., et.al.
 See word document
Structure of Human MECP2
Wan, M., et.al.
Treatment
No trxt has shown significant improvements
 Previous trxts

L-carnitine





Control of seizures
Lacked vitamins
Naltrexone*


Oral opiate antagonist
Respiratory features,
EEG patterns
Folate-Betaine

Fatty acid metabolism
Respiratory features


Ketogenic diet


 Ongoing trial
Methyl-donor group
Alter gene expression
Recruit other methyl
binding groups
 Current trxt


Supportive/symptomatic
therapy
Occupational/physical
therapy
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
References
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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.
Questions