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Epigenetics and

Human Disease

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Angelman Syndrome & Prader-Willi Syndrome “Sister Syndromes”

•  ~1/20,000 births •  happy disposition •  smile often •  bouts of laughter •  minimal verbal skills •  balance problems and hand

flapping (Marionette Syndrome or Puppet Children)

•  seizures •  severe developmental delay •  microcephaly •  dysmorphic features

Angelman Syndrome

Angelman Syndrome & Prader-Willi Syndrome “Sister Syndromes”

•  ~1/10,000 births •  mild-moderate mental retardation •  sometimes unhappy disposition •  obsessive-compulsive behaviors •  infantile hypotonia (low muscle tone) •  obesity/hyperphasia •  developmental delay •  short stature •  hypogonadism/genital hypoplasia •  small hands/feet •  thin upper lip •  almond shaped eyes

Prader-Willi Syndrome

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•  “Sister syndromes” because they are caused by seemingly identical genetic abnormalities –  Deletion of chromosome 15q11-q13 or

–  Uniparental disomy

How can these abnormalities result in two very different phenotypes?

Angelman Syndrome & Prader-Willi Syndrome

Genomic Imprinting Disorders

Uniparental disomy (UPD) can lead to human diseases.

Maternal UPD Paternal UPD

normal

1st evidence for role of epigenetics in disease.

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Angelman Syndrome & Prader-Willi Syndrome Genomic Imprinting Disorders

Angelman Syndrome

Prader-Willi Syndrome

Paternal deletion

Maternal UPD

Maternal deletion

Paternal UPD

Imprint

•  The individual silenced gene that contribute to each disease are known –  Classical forward genetics - identified the genes lost to cause the phenotype –  Reverse genetics – how does disruption of these genes result in the phenotype

Genomic Imprinting Disorders

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Obesity and hyperphagia (excessive hunger) in Prader-Willi

•  Ghrelin (hunger hormone) –  Neuropeptide produced in

the GI tract –  Fluctuates with stomach

contents –  Increases hunger and

prepares GI for food intake

•  Patients display elevated ghrelin levels that drives the insatiable appetite and obesity.

•  Links missing genes and elevated ghrelin levels remains unclear

UPD reported for all human chromosomes except 3 & 19

Genomic Imprinting Disorders

Beckwith-Wiedemann Syndrome (BWS) - defects map to ~1-Mb H19-IGF2 region on chromosome 11 (with >12 imprinted genes)

Silver-Russell Syndrome (SRS) ~10% result from mat UPD for chromosome 7

Loss of imprinting can also lead to cancer.

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Trans Effects: Disorders affecting chromatin

Complex multisystem phenotypes result from mutations in proteins involved in chromatin structure and remodelling.

misregulation of multiple genes?

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Trans Effects: Rett Syndrome

neurodevelopmental disorder causing mental retardation, hand-wringing, language regression..... Like “autistic spectrum disorders”, Rett Syndrome manifests after a period of apparent normal development, disrupts social and language development.

Rett Syndrome occurs almost exclusively in females and shows a spectrum of phenotypic variability .... Why?

The gene responsible for this syndrome is found on the X chromosome (i.e. X-linked), thus typically lethal in males. The phenotypic variability arises because of the random inactivation of one of the X chromosomes in each cell in females.

Trans Effects: Rett Syndrome

Random inactivation of the X chromosomes in females can lead to a spectrum of phenotypic variability.

X x X x X x x X

X x X x x X x X

X x x X x X x X

less severe

more severe

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Trans Effects: Rett Syndrome

Mutation of the gene encoding a methyl-CpG binding domain (MBD) protein, MECP2, is the major cause of Rett Syndrome.

MECP2

MBD binds to symmetrically methylated CpG TRD interacts with histone deacetylases (HDACs)

Trans Effects: ICF Syndrome

ICF is an autosomal recessive disorder. Which parent contributed the mutation to the afflicted offspring?

Both the mother and father were carriers of the mutation and contributed it to the afflicted offspring.

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Trans Effects: ICF Syndrome

In 1999, three labs independently reported that ICF was due to mutations in the DNA methyltransferase, DNMT3B. DNMT3B is a de novo DNA methyltransferase and is known to localize to centromeric satellite sequences. DNMT3B also known to partner with a number of chromatin regulatory proteins.

Trans Effects:

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Phenotype: Mental retardation, affected males have connective tissue defects (eg. hyperextensible joints), large ears, long face, shyness, etc. Females typically less severe phenotype (severity correlated with degree of X-inactivation on abnormal chromosome).

Cis Effects: Fragile X Syndrome

- Most common inherited cause of intellectual disability

- X-linked

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Cis Effects: Fragile X Syndrome

Named for constriction seen cytologically on the X chromosome (Xq27.3). This cytologically visible constriction occurs in the 5‘UTR of the FMR1 gene.

The disease is manifested due to a lack of expression of the FMR1 gene, which encodes an RNA-binding protein (FMRP) thought to play a role as a translational suppressor through an RNAi-like pathway. What causes the repression of FMR1 expression in affected individuals?

Cis Effects: Fragile X Syndrome

5-54: normal phenotype -  Methylation: no -  Histone acetylation: yes -  Transcription: yes 55-200: mostly normal (develop distinct neurodegenerative syndrome: tremors & ataxia) 200+: Pathologic -  Methylation: yes -  Histone acetylation: no -  Transcription: no

Conclusion: The primary cause of Fragile X Syndrome is genetic but it depends on epigenetic processes.

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Epigenetics and Cancer •  Malignant Neoplasia (harmful new growth) •  Should cancer be classified by tissue type

or causitive mutations? –  Leukemia

•  Acute or Chronic –  Lymphoid or Myeloid

»  B cell or T cell •  Pre-B or Pro-B o  Translocation (1:19, 9:22, etc)

–  C-Kit (receptor tyrosine kinase) mutations are causitive in AML, GIST and melanoma

•  Cancer is incredibly diverse. What characteristic are shared by all cancers? 23

Epigenetics and Cancer

•  Cancer results from the semi-heritable deregulation of genes that normally control the “hallmarks of cancer”

•  Gene disregulation is typically divided into activated “oncogenes” (dominant) and silenced “tumor suppressors” (recessive)

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The interplay between genetics and epigenetics is complex and multifaceted

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Gene expression changes are often mediated by mutation of chromatin modifiers

M: Missense F: Frameshift N: Nonsense S: Splice site T: Translocation D: Duplication PTD: Partial Tandem Duplication

Chromosomal translocations are a hallmark of childhood acute leukemias

•  MLL translocations (chromosome 11) are the defining mutation of a therapeutic subtype (poor prognosis)

•  MLL (mixed-lineage leukemia) methylates H3K4. K4 methylation is associated with active transcription.

•  >50 fusion partners have been identified in MLL translocations. All result in disruption of AT-hook and KMTase domain

•  Models of MLL-translocated ALL have been elusive and the role in mutant MLL in leukemogenesis remain largely unclear

Look, AT. Science. 1997.

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Gene expression changes are often mediated by mutation of chromatin modifiers

DNA methylation can lead to cancer in various ways

Global hypomethylation hallmark of human cancer and may lead to genomic instability (deletions/inversions/duplications/translocations) - Heterochromatin is typically refractory to Double-strand breaks and less sensitive to DNA

damage

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DNA methylation can lead to cancer in various ways

Presence of a methyl group on the C favors formation of carcinogenic adducts on adjacent G (eg. benzo(a) pyrene in smoke).

DNA methylation can lead to cancer in various ways

Methyl groups change the absorption spectrum of C (moving it into range of sunlight) leading to pyrimidine dimers in skin.

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DNA methylation can lead to cancer in various ways

Methylation is inherently mutagenic due to the spontaneous deamination of cytosines. Why mC and not Cs?

Spontaneous deamination of cytosines vs. methylated cytosines

cytosine uracil methylated cytosine thymine (not repaired as efficiently)

The most common single nucleotide mutation found in the genome and accounts for the paucity of the CpG dinucleotide in vertebrate genomes - so TpG overrepresented. Remember that DNA methylation is found predominantly (if not exclusively) at CpGs in vertebrates.

The CpG dinucleotide occurs in roughly the expected frequency at regions called “CpG islands”, which are found in the promoter regions of many genes. CpG islands are thought to retain their expected frequency of CpGs because they are typically not methylated.

>50% of p53 mutations occur at methylated CpG sites

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DNA methylation can lead to cancer in various ways

Hypermethylation of the CpG islands associated with tumor suppressor genes is typical. Leading to the transcriptional silencing.

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Hypermethylation induced silencing of key tumor suppressors

p16, associated with failure of cells to reach “mortality checkpoint” leading to progressive abnormalities

E-cadherin (metastasis suppressor - promotes cell-cell adhesion)

Chfr (checkpoint regulating - chromosomal instability)

O6-MGMT (DNA repair - alkylation damage leads to G->A)

GST-Pi (DNA repair - oxidative damage at adenines)

SFRPs (secreted frizzled related protein - affects Wnt signalling

HIC-1 (hypermethylated in cancer) (Zn-finger repressor)

Rb retinoblastoma protein (cell cycle regulator) gene - interacts with many chromatin remodellers.

MLH1 (mismatch repair) gene (multiple genetic alterations)

Epigenetic therapy BEFORE SAHA

mass compresses lung (black)

AFTER 4mo. SAHA treatment

Shrinkage of tumor; lung expands

Mesothelioma

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Can the environment influence the epigenome? Evidence for epigenetic-environment interactions

Control Hypertension

Hypertensive Mother = Hypertensive F1

Hypertensive Parent = Hypertensive F2

No differences

Can the environment influence the epigenome? Evidence for epigenetic-environment interactions

Diet of pregnant mother mouse (e.g. folate, vitB12) affects DNA methylation of agouti gene in offspring affecting coat color, as well as obesity thus susceptibility to diabetes and cancer.

Similarly, men with uremia have reduced DNA methylation that can be reversed with dietary folate. Uremia: A toxic condition resulting from kidney disease in which there is retention in the bloodstream of waste products normally excreted in the urine.

*Pregnant women take supplementary folic acid for proper fetal neural tube development.

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A way to think about interactions among genetic, epigenetic and environmental factors

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The mechanism of silencing is unknown but once methylation established, it plays “dominant” role

?

FSHD (Facioscapulohumeral Dystrophy) An autosomal dominant muscular dystrophy characterized by wasting

of muscles of face, upper arm, shoulder, etc.

Major locus maps to heteorchromatic subtelomeric region of 4q35 near D4Z4, an array of 3.3 kb GC-rich units.

Normally 11-150 units; 1-10 units on FSHD chromosomes

Contraction of repeats is necessary for FSDH

WHY? Best guess: 27bp sequence in D4Z4 binds “D4Z4-repressing complex” (DRC) and this is required to increased expression of genes in the region, including FRG1&2 and adenine nucleotide tranporter-1 (ANT1).

Cis Effects: