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

11_05.jpg

11_05_2.jpg

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

12

DNA Methylation

• Beckwith-Wiedemann syndrome

13

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

14

Beckwith-Wiedemann syndrome

• Genetic causes of BWS:– Maternal DMR hypermethylation– UPD– Remainder unknown

15

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

18

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.

24

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

25

Prader-Willi and Angelman Syndrome

• Angelman syndrome

• Genes/proteins involved

• Prader-Willi syndrome

• Genes/proteins involved

15q11-13

26

Prader-Willi and Angelman Syndrome

• UBE3A is paternally silenced

• This primarily occurs in brain, other tissues show biallelic expression

27

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…

28

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

29

Prader-Willi and Angelman Syndrome

• Thus, two different diseases based on the cells “memory” of methylation – alter the memory, alter the phenotype

30

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

31

Prader-Willi and Angelman Syndrome

32

Uniparental Disomy

• Receive two chromosomes from one parent

• Eg. Paternal disomy – both of chromosome 15 are from father, thus both have silenced UBE3A

33

Uniparental Disomy

• How does it happen?

• Trisomic Rescue - majority• Monosomic Duplication

34

Meiosis I

35

Meiosis II

36

Meiosis I Non-disjunction

37

Meiosis I Non-disjunction

Fertilization

Trisomy Meiosis I non-disjunction always creates a problem

38

Meiosis II Non-disjunction

Fertilization Fertilization

Trisomy Normal

2/3 gametes following Meiosis II non-disjunction are normal

39

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

40

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

41

Trisomic Rescue following Meiosis II Non-disjunction

+

M,M1/3

M,P2/3

Two copies of identical chromosomes

Anaphase lag

42

Uniparental Disomy

• How does it happen?

• Trisomic Rescue - majority

• Monosomic Duplication

43

Monosomic Duplication

+

P,P

Two identical copies of paternal chromosome - isodisomy

44

Parental Origin Determines Phenotype

Prader-Willi Syndrome

M,M

{15

M,M

Prader-Willi Syndrome

P,P

Angelman Syndrome

45

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

46

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.

47

Trisomic rescue of Meiosis II non-disjunction can have other problems

Anaphase lag{

Pair of identicalchromosomes

(isochromosomes)

48

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