Angelman Syndrome (AS) and UBE3A (E6-AP)

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Angelman Syndrome (AS) and UBE3A (E6-AP) Powered By Docstoc
					Epigenetics and Imprinting

Dr Una Fairbrother

   Describes the differential expression
    of genetic material at
    chromosomal/allelic level, depending
    on that material being maternal or
    paternal in origin.
What is the imprint at a
molecular level?
 An imprint is an heritable tag
 Usually associated with
  differential DNA methylation
 The met CpG usually on the silent
 In addition, histone aceylation
  and methylation has been
Imprinting involves 3 distinct
biological stages
  (a)Establishment of the imprint
  in gametes, according to the
  sex of the individual
 (b) Its maintenance during
  embryogenesis/adult somatic
 (c) Erasure in the early
Schematic of Imprinting
Why imprinting?

 The advantages of biparental
  inheritance seem clear from
  the recessive nature of many
  disease mutations.
 Imprinting could help to
  maintain sexual reproduction.
Imprinting in Development
In mammals, imprinted genes have a
 vital role in development of the
 embryo and neonate.
The division is not clear cut but;
placental development appears to
 rely more heavily on paternal
embryonic growth appears to rely
 more heavily on maternal
Imprinted Genes Present a
complex Picture Of
 In embryonic development,
  monoallelic expression is not
  always coincident with the
  onset of gene expression
 Can vary with development and
Imprinting, primary
 Some evidence suggests that
  the imprint itself does not have
  to be a primary inactivation
 The imprinting mechanism may
  involve additional trans-acting
Imprinting and Disease
   Approx 80 imprinted genes in the human
    genome poss up to 600!
   Non-mendelian inheritance pattern with
    parent of origin effects
   Best characterised syndromes:
   Angelman/Prader-Willi syndromes on 15q
    (UBE3A- AS)
   Beckwith-Wiedemann syndrome (BWS) 11p
Species   Chr   Gene
Human     01    ARHI, NOEY2
Human     01    TP73
Human     06    PLAGL1; ZAC; LOT1
Human     06    HYMAI
Human     06    IGF2R, M6PR (disputed)
Human     07    GRB10, MEG1
Human     07    PEG10
Human     07    DLX5
Human     07    MEST, PEG1, MESTIT1, PEG1-
Human     07    AS COPG2 (disputed
Human     07    PEG1-AS, MESTIT1
Human     07    CPA4
Human     11    WT1 (disputed)
Human     11    H19
Human     11    IGF2
Human     11    IGF2AS, PEG8
Human     11    INS, insulin
Human     11    TRPM5, LTRPC5, MTR1
Human     11    KCNQ1, KvLQT1
Human     11    KCNQ1OT1, LIT1, KvLQT1-AS,
Human     11
                KCNQ1DN, BWRT
Human     11
                CDKN1C, p57KIP2
Human     11
                SLC22A1L, IMPT1, BWR1A,
Human     11    ORCTL2, ITM, TSSC3, IPL,
Human     11    BWR1C, TSSC5, HET ,ZNF215
Human     11    2G3-8
Human     11    SDHD
Human   14   MEG3, GTL2
Human   14   DLK1, PEG9
        15   Paternally expressed non-
             coding transcripts on 15p
             (Prader-Willi syndrome region)
Human   15   MKRN3, ZNF127
Human   15   NDN
Human   15   MAGEL2, NDNL1
        15   SNRPN, SNURF
        15   PAR-SN
        15   HBII-13
        15   PAR5, D15S226E
        15   HBII-85, PWCR1
        15   IPW
        15   PAR1, D15S227E
        15   HBII-52
        15   UBE3A, E6-AP, UBE3A-AS
        15   ATP10A, ATP10C
        15   Elongin A3
Human   18   PEG3
Human   19   NNAT, Neuronatin
Human   20   GNAS1, Gs alpha, NESP55,
             XLalpha s, GNAS-AS
Human   20
Features Of Imprinted Genes
   No direct sequence homology has been
    found among imprinted genes.
   Sequences carrying mat and pat gametic
    imprints resemble CpG islands containing
    GC rich regions of between 200 and
    1500bp with a balanced CG:GC ratio.
   Imprinted sequences contain or are closely
    associated with a region of direct repeats
    ranging in size between 25 and 120bp.
    They are found in all imprinted genes
    analysed so far and are evolutionarily
Imprinting Clusters

   Imprinted genes are clustered
   Best understood on chromosome 15 and 11
   Complex coordinated regulation based on
    an imprinting centre (IC) or an imprinting
    control element (ICE)
   Coodinates in cis expression of
    neighbouring genes in one domain
Action of Imprinting Centres
   Allele discriminating action of imprinting
    centres based on epigenetic modifications
    of chromatin
   Typically via methylation of cytosines
    (DNA)and histone acetlylation and
   Acetylation of histones associated with
    transcriptionally active chromatin
   Methylation of histones and DNA
    associated with inactivation of
Imprinting and Transcription

   Methylation and acetylation lead to allele
    specific accessibility to transcriptional
   And to DNA binding proteins acting as
    enhancers or insulators
   Setting and stability of imprinted gene
    expression controlled by ICs with multiple
    levels of DNA and chromatin modifications
More complexity: IGF2

  IGF2 (chromosome 11) has been shown to
   be imprinted very early on, in 8 cell
   human embryos
  recruits different promoters to confer
   mono or biallelic expression at different
   developmental stages.
….And KVLQT1
  KVLQT1 (chromosome 11) has been
   reported as having a number of
   alternatively spliced transcripts
  shows both mono and biallelic expression
   patterns in different tissues
  Important in identification of imprinted
   genes associated with imprinted
  Obviously evolutionary conservation is a
   good way to identify fundamentally
   important genes and sequence motifs
  A horse mare-donkey cross gives rise to
   a mule but a donkey-stallion gives rise to
   a hinny
  The callipyge gene phenotype “beautiful
   buttocks” is only expressed when
   paternally inherited Hum Mol Genet
   1998 7:No10 review
Imprinting in animal models
    Once thought to be restricted to
      mammals, genomic imprinting has been
      documented in angiosperm plants
      1970, zebrafish 1995, insects, and C.
      elegans 2004
  There may be genomic imprinting in
   drosophila, but transgenes have shown
   that imprinting switch regions act as
   silencers in flies
  In marsupials methylation on the X chr
   preferentially inactivates paternal X
  Mouse studies are one of the commonest
   in the literature
Of Mice and Men
 Mouse models have been helpful in
  identifying the genes involved in
  Prader-Willi and Angelman
 The PWS mouse model has a partial
  maternal duplication of the region
  of mouse chromosome 7 homologous
  to human 15q11-13
 The AS mouse model has a paternal
  duplication for the same region
Imprinting in Mice Not
 There are differences in
  imprinting between mice and
 Imprinting of H19, Igf2,
  p57kip2 and Snrpn is the same
 IGF2R appears to be
  monoallelic in mice and biallelic
  in humans, though this may be
    Imprinting on Human
    Chromosome 15
   IC regulates chromatin structure, DNA
    methylation and gene expression in a 2Mb
    region in 15q11-13
   Mutations in IC cause chromosome to be
    stuck as a single gender and not assume
    the sex imprint of its ‘host’.
   >7 imprinted transcripts in AS region,
    paternally expressed
   UBE3A is only coding transcript maternally
    Angelman Syndrome (AS)
   incidence of 1/20,000 live births
   severe mental retardation
   Lack of speech (commonly only a three to
    six word vocabulary)
   Ataxic gait
   Hand flapping
   Happy disposition including inappropriate
   Diagnostic EEG
   Others
Inheritance pattern of AS

   AS gene expressed MATERNALLY
Chromosome 15 deletion in As
and PWS
   Initially identified by the same
    cytogenetic lesion i.e. the deletion of
Prader-Willi Syndrome (PWS)
   Clinically distinct syndrome
   frequency of about 1 in 25,000 and
   probably the most common syndromal cause
    of human obesity Clinical features
   Mild mental retardation
   Obesity
   Short stature
AS Oppositely Imprinted to PWS
How Does AS Arise?
   AS caused by loss of function of a
    gene/genes from the maternal chromosome
   (ICH study) 60% AS patients have large
    cytogenetic deletion
   4% have uniparental disomy
   4% have mutations in the imprinting centre
   5% remaining, screened for mutations in
    the AS gene, UBE3A (E6AP) (50% familial,
    10 sporadic)
AS Oppositely Imprinted to PWS
Recurrence risk
   typical large deletions are de novo and are
    expected to have less than 1% risk
   paternal UPD, (no parental translocation),
    less than 1%
   transmission of a structurally or
    functionally unbalanced chromosome
    complement can lead to 15q11-q13 deletions
    or to UPD and will result in case-specific
    recurrence risks.
   no large deletion or UPD, probably 50% due
    to maternal IC or UBE3A mutation see
   Risks confounded by mosaicism
UBE3A (E6AP) and AS
   UBE3A (ubiquitin protein ligase) complex
    expression patterns.
    5’ end of gene alternatively spliced in a
    variety of human tissue cell lines and in
    human foetal tissues including brain
   > 7 isoforms have been identified;
    monoallelic expression is tissue specific
    and isoform specific
   Human fibroblast and lymphoblast tissues
    and also adult mouse tissues show biallelic
    Monoallelic expression of
   Monoallelic expression of an isoform found
    in mouse brain tissue
   depression of expression is also evident in
    the hippocampus of a paternal UPD mouse
   Evidence for decrease of expression in
    human brain
   Clinical description of AS indicates a
    developmental brain disorder and so
    differential expression of the gene would
    be expected in brain tissue.
UBE3A Has Even More
Complex Splicing
   Normal UBE3A decreased in AS brain
   A new larger transcript decreased in
    PW brain (PATERNAL)
   Larger transcript is antisense and
    spans half UBE3A with additional
    down stream sequence
   Also another sense strand was detected
    which lies in between the 2nd and 3rd
    polyadenylation signal of UBE3A
   Imprinted and transcribed in the same way
    as UBE3A
   All other tissues antisense transcript not
   The expression of the antisense transcript
    in brain may force the UBE3A transcript
    to be monoallelic in brain
   Some as yet uncharacterised AS patients
    could have a mutation in either the down
    stream sense transcript or the antisense
    transcript, see overhead*
Mouse models

 Mouse models have been used to
  identify imprinted regions by
  engineering UPDs for mouse
  chromosomes, in part or in full
 Extensive mapping of imprinted
  mouse genes and their human
  homologues has been undertaken
Similarities to Human
 AS mouse: Neuro behavoural
  differences, mild ataxia, abnormal
  limb clasping, hyperactivity,
  diminished brain weight (10%
 The PWS mouse: increase in
  postnatal loss, small skeleton,
  grossly obese by 6 months
Differences to Human
 AS mouse no detection of language
  difficulties (obviously), mouse also
  showed gross obesity - could be
  present in a subset of AS patients
  as late onset
 PWS mouse no detection of mild
  mental retardation.
Usefulness of AS Mouse
 When gene was identified, AS
  mouse brains could be looked at
  and a monoallelically expressed
  transcript of UBE3A was found
  to be decreased as compared to
  the normal and the PWS mouse
    Outstanding questions
   How and when during germline development are old
    imprints removed and new ones introduced?
   Which demethylating activities and chromatin
    factors are involved?
   How does the spreading of epigenetic information
    in clusters work, - germline-specific, postzygotic
    phenomenon or both?
   How are imprints maintained when there is
    genome-wide active and passive demethylation in
    the early embryo?
   How many fundamentally different arrangements
    of imprinted genes and imprinting control elements
    are there in the genome?
   How conserved is imprinting between mammalian
   How precisely do imprinted genes affect
    extraembryonic and embryonic development, and
    the nutritional exchange with the mother?
   Are there interactions of imprinted genes
    (particularly antagonistic ones) in known, or in
    novel, physiological pathways?
   In addition to growth and behaviour, are there
    other developmental processes and mechanisms in
    which imprinted genes have a decisive role, and
    how will these fit with evolutionary theories?

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