Transcript Lecture 2 4285 2015 - Scheid Signalling Lab @ York University

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
Unmethylated
Usually not
affected
Usually not
affected
Full mutation
>200
Completely
methylated
~50%
affected
All affected
11_05.jpg
11_05_2.jpg
Skewed X-Chromosome inactivation in
a family with Fragile X
Southern Blot Analysis
Blood sample
Digest genomic DNA
with EcoRI and EagI
Electrophoresis and
transfer to membrane
Hybridize with FMR1
specific probe
“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.”
DNA Methylation
• Beckwith-Wiedemann syndrome
12
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
13
Beckwith-Wiedemann syndrome
• Genetic causes of BWS:
– Maternal DMR hypermethylation
– UPD
– Remainder unknown
14
15
The Journal of Pathology
Volume 211, Issue 3, pages 261-268, 18 DEC 2006 DOI: 10.1002/path.2116
http://onlinelibrary.wiley.com/doi/10.1002/path.2116/full#fig1
The Journal of Pathology
Volume 211, Issue 3, pages 261-268, 18 DEC 2006 DOI: 10.1002/path.2116
http://onlinelibrary.wiley.com/doi/10.1002/path.2116/full#fig3
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-GolabiBehmel syndromes
Jonathan Eggenschwiler,1 Thomas Ludwig,2 Peter Fisher,3 Philip A.
Leighton,4,5 Shirley M. Tilghman,4 and Argiris Efstratiadis1,6
18
Figure 2 The epigenetic progenitor model of cancer.
Feinberg AP et al. (2005) The epigenetic progenitor origin of human cancer
Nat Rev gene. 7: 21–33 doi:10.1038/nri1748
Prader-Willi and Angelman
Syndrome
Prader-Willi
Angelman
Mild mental retardation
endocrine abnormalities
Severe impairment and
loss of speech
seizures and ataxia
temper tantrums
unprovoked laughter
Obesity
1 in 15,000
hyperactivity
1 in 15,000
24
Prader-Willi and Angelman
Syndrome
• Angelman syndrome
• Genes/proteins involved
15q11-13
• Prader-Willi syndrome
• Genes/proteins involved
25
Prader-Willi and Angelman
Syndrome
• UBE3A is paternally silenced
• This primarily occurs in brain, other tissues
show biallelic expression
26
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…
27
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
28
Prader-Willi and Angelman
Syndrome
• Thus, two different diseases based on the
cells “memory” of methylation – alter the
memory, alter the phenotype
29
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
30
Prader-Willi and Angelman
Syndrome
31
Uniparental Disomy
• Receive two chromosomes from one
parent
• Eg. Paternal disomy – both of
chromosome 15 are from father, thus both
have silenced UBE3A
32
Uniparental Disomy
• How does it happen?
• Trisomic Rescue - majority
• Monosomic Duplication
33
Meiosis I
34
Meiosis II
35
Meiosis I Non-disjunction
36
Meiosis I Non-disjunction
Fertilization
Trisomy
Meiosis I non-disjunction
always creates a problem
37
Meiosis II Non-disjunction
Fertilization
Fertilization
Trisomy
Normal
2/3 gametes
following Meiosis
II non-disjunction
are normal
38
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
39
Trisomic Rescue following Meiosis I
Non-disjunction
+
Two copies of homologous, but
not identical, chromosomes
Maternal
Anaphase lag
Maternal
Paternal
M,M
1/3
M,P
1/3
M,P
1/3
40
Trisomic Rescue following Meiosis II
Non-disjunction
+
Two copies of
identical
chromosomes
Anaphase lag
M,M
1/3
M,P
2/3
41
Uniparental Disomy
• How does it happen?
• Trisomic Rescue - majority
• Monosomic Duplication
42
Monosomic Duplication
+
Two identical copies of
paternal chromosome isodisomy
P,P
43
Parental Origin Determines
Phenotype
15
{
Prader-Willi Syndrome
M,M
Prader-Willi Syndrome
M,M
Angelman Syndrome
P,P
44
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% AS
Maternal non-disjunction and trisomic rescue
leading to the pair of maternal chromosomes
45
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.
46
Trisomic rescue of Meiosis II nondisjunction can have other problems
{
Anaphase lag
Pair of identical
chromosomes
(isochromosomes)
47
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+/+
Uniparental
isodisomy and
reduction to
homozygosity
Pair of identical
Chromosome 7
Harboring CFTR mutation
48