systemic injection of mecp2bnull/y mice with scaav9/mecp2 virus results in mecp2 expression in...
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Systemic injection of Mecp2Bnull/y mice with scAAV9/MeCP2 virus results in MeCP2 expression in different cell types in brain.
Garg S K et al. J. Neurosci. 2013;33:13612-13620
©2013 by Society for Neuroscience
MeCP2 expressed from virus binds to DNA, restores normal neuronal somal size, and improves survival.
Garg S K et al. J. Neurosci. 2013;33:13612-13620
©2013 by Society for Neuroscience
Inappropriate Silencing of Genes
• Fragile-X Syndrome
Fragile-X Syndrome
Length Methylation Females Males
Stable 6 to ~45 Unmethylated Not affected Not affected
Gray zone ~45 to ~55 Unmethylated Not affected Not affected
Premutation ~55 to ~200 UnmethylatedUsually not
affectedUsually not
affected
Full mutation >200Completely methylated
~50% affected
All affected
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Skewed X-Chromosome inactivation in a family with Fragile X
Southern Blot Analysis
“A normal female will show an unmethylated 2.8-kb band and a 5.2-kb methylated band that correspond to the normal FMR1 gene present in the active and inactive X chromosome, respectively.”
Blood sample
Digest genomic DNA with EcoRI and EagI
Electrophoresis and transfer to membrane
Hybridize with FMR1 specific probe
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DNA Methylation
• Beckwith-Wiedemann syndrome
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DNA Methylation
• Beckwith-Wiedemann syndrome – Above average birth weight– Increase growth after birth (>95% growth
curve)– Enlarged organs– Hypoglycemic following birth– Increase risk of cancers
• Imprinting defect located at 11p15.5
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Beckwith-Wiedemann syndrome
• Genetic causes of BWS:– Maternal DMR hypermethylation– UPD– Remainder unknown
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The Journal of PathologyVolume 211, Issue 3, pages 261-268, 18 DEC 2006 DOI: 10.1002/path.2116http://onlinelibrary.wiley.com/doi/10.1002/path.2116/full#fig1
The Journal of PathologyVolume 211, Issue 3, pages 261-268, 18 DEC 2006 DOI: 10.1002/path.2116http://onlinelibrary.wiley.com/doi/10.1002/path.2116/full#fig3
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Genes Dev. Vol. 11, No. 23, pp. 3128-3142, December 1, 1997
Mouse mutant embryos overexpressing IGF-II exhibit phenotypic features of the Beckwith-Wiedemann and Simpson-Golabi-Behmel syndromes
Jonathan Eggenschwiler,1 Thomas Ludwig,2 Peter Fisher,3 Philip A. Leighton,4,5 Shirley M. Tilghman,4 and Argiris Efstratiadis1,6
Feinberg AP et al. (2005) The epigenetic progenitor origin of human cancerNat Rev gene. 7: 21–33 doi:10.1038/nri1748
Figure 2 The epigenetic progenitor model of cancer.
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Prader-Willi and Angelman Syndrome
Prader-Willi Angelman
Mild mental retardation Severe impairment and loss of speech
endocrine abnormalities seizures and ataxia
temper tantrums unprovoked laughter
Obesity hyperactivity
1 in 15,000 1 in 15,000
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Prader-Willi and Angelman Syndrome
• Angelman syndrome
• Genes/proteins involved
• Prader-Willi syndrome
• Genes/proteins involved
15q11-13
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Prader-Willi and Angelman Syndrome
• UBE3A is paternally silenced
• This primarily occurs in brain, other tissues show biallelic expression
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Prader-Willi and Angelman Syndrome
• What happens in each pathologies?
• If the maternal copy of chromosome 15 is missing, then genes normally expressed from this parental origin are not expressed
• Consequences…
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Prader-Willi and Angelman Syndrome
• If paternal chromosome 15 is missing, then only the maternally expressed proteins are made
• Consequence: UBE3A is ok, but other genes in the region are not expressed…Prader-Willi syndrome
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Prader-Willi and Angelman Syndrome
• Thus, two different diseases based on the cells “memory” of methylation – alter the memory, alter the phenotype
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Prader-Willi and Angelman Syndrome
• How do you “lose” chromosome 15?
– Microdeletion of 15q11-13 on one chromsome – 70%
– Single gene mutation – 15% of AS
– Defect in imprinting centre (IC) – 5%
– Uniparental Disomy – 30% of PWS, 5% AS
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Prader-Willi and Angelman Syndrome
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Uniparental Disomy
• Receive two chromosomes from one parent
• Eg. Paternal disomy – both of chromosome 15 are from father, thus both have silenced UBE3A
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Uniparental Disomy
• How does it happen?
• Trisomic Rescue - majority• Monosomic Duplication
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Meiosis I
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Meiosis II
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Meiosis I Non-disjunction
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Meiosis I Non-disjunction
Fertilization
Trisomy Meiosis I non-disjunction always creates a problem
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Meiosis II Non-disjunction
Fertilization Fertilization
Trisomy Normal
2/3 gametes following Meiosis II non-disjunction are normal
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Trisomy
• Trisomy for most autosomal chromosomes is lethal
• BIG exception: Trisomy 21, smallest autosomal chromosome, fewest genes, not lethal
• Under rare conditions, some autosomal trisomies can escape – Trisomic Rescue
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Trisomic Rescue following Meiosis I Non-disjunction
Two copies of homologous, but not identical, chromosomes+
Maternal
Maternal
Paternal
M,M1/3
M,P1/3
M,P1/3
Anaphase lag
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Trisomic Rescue following Meiosis II Non-disjunction
+
M,M1/3
M,P2/3
Two copies of identical chromosomes
Anaphase lag
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Uniparental Disomy
• How does it happen?
• Trisomic Rescue - majority
• Monosomic Duplication
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Monosomic Duplication
+
P,P
Two identical copies of paternal chromosome - isodisomy
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Parental Origin Determines Phenotype
Prader-Willi Syndrome
M,M
{15
M,M
Prader-Willi Syndrome
P,P
Angelman Syndrome
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Prader-Willi and Angelman syndrome
• Non-disjunction is more common in Meiosis I in females
• In human females, Meiosis I starts before birth but is arrested at diplotene stage (late prophase I)
• Oocytes sit like this for decades• Complete meiosis II once each month• While arrested at the diplotene stage, the tetrad chromosomes
are held together by chiasmata (formed during recombination)• If a pair of chromosomes don’t undergo recombination, the lack
of chiasmata can contribute to non-disjunction
• Uniparental Disomy – 30% of PWS, 5% ASMaternal non-disjunction and trisomic rescue leading to the pair of maternal chromosomes
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Uniparental Disomy and Human Disease
Eric Engel. Some lessons from uniparental disomy (UPD) in the framework of contemporary cytogenetics and molecular biology.Atlas Genet Cytogenet Oncol Haematol. December 2003.
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Trisomic rescue of Meiosis II non-disjunction can have other problems
Anaphase lag{
Pair of identicalchromosomes
(isochromosomes)
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Uniparental disomy as a mechanism for human genetic disease.Spence JE, Perciaccante RG, Greig GM, Willard HF, Ledbetter DH, Hejtmancik JF, Pollack MS, O'Brien WE, Beaudet AL.
Am J Hum Genet. 1988 Feb;42(2):217-26.
CFTR-/+CFTR+/+
CFTR-/- {Pair of identicalChromosome 7
Harboring CFTR mutation
Uniparental isodisomy and reduction to homozygosity