Lecture 7 Flashcards
Symptoms of Angelman’s syndrome
- Developmental delay
- Speech impairments
- Jerky movements/hand flapping
- Happy disposition, laughing, smiling
- Microcephaly
- Seizures
- Abnormal EEG
Prader-Willi syndrome
- Hypotonia - low muscle tone
- Poor suckling reflex and feeding in infancy
- Insatiable appetite in later life
- Obesity
- Short stature
- Compulsive behaviour
- Strabismus (eye crossing)
- Hypogonadism
What causes PWS and AS?
- 75% of both PWS and AS cases are caused by a deletion of up to 6 Mbp on chromosome 15q11.2-q13.1
- Deletions can occurs as a result of aberrant meiotic recombination between direct repeats that flank the region
- Deletion can be detected by FISH or array CGH for diagnosis
How does genomic imprinting explain PWS/AS difference?
Usually, people inherit one intact paternal and one intact maternal chromosome 15
Genomic imprinting illustrates that some genes in this region are only expressed from the paternal or maternal chromosome.
Imprinting centre is methylated on maternal chromosome (cytosine residues methylated) - controls expression of genes.
Prader-Willi syndrome deletions
Deletion on paternal chromosome and maternal chromosome in tact
Maternal chromosome cannot express any genes from MKRN3 to SNORD109B
Angelman’s syndrome deletions
Deletion of UBE3A and ATP10A on maternal chromosome
What kind of trait is genomic imprinting?
Epigenetic trait - stably, heritable phenotype resulting from changes in chromosome without DNA sequence alterations
Means genes on one chromosome are silenced, no change to DNA sequence.
How do epigenetics modify gene expression
Methylation of cytosine residues in DNA
Modification of Histones
Regulation by lncRNA
Net effect of mechanisms to sequester DNA into heterochromatin
Epigenetic mechanisms ensure correct genes are turned on/off in tissue development
Methylation of cytosine bases at CpG site in Differentially methylated regions is responsible for imprinting
Embryos and somatic cells:
either maternal or paternal allele is methylated, rendering it inactive (no transcription or translation)
Methylation occurs at cytosine residues in CpG, where both strands are methylated.
Methyl transferases (de novo and maintenance), where de novo adds methyl group to unmethylated DNA, and maintenance methyl transferase adds methyl group to hemi methylated DNA
Gametes:
All oocytes have methylation pattern consistent with pattern on maternal chromosomes
All sperm have methylation pattern consistent with pattern on paternal chromosomes
Is genomic imprinting reversible
- Paternal methylation imprints are established in prospermatogonia in testes of fetus
Maternal methylation imprints established during oocyte maturation in meiosis
Genomic imprints persist in zygote
Genes in somatic cells have imprint that marks their origin as either the paternal or maternal chromosome
Methylation removed in primordial germ cells
What controls gene expression
Methylation status of the Imprinting centre controls gene expression
Methylation blocks transcription of MKRN3, MAGEL2, NDN, PWRN1, NPAP1, SNURF-SNRPN, SNHG14 transcript, SNORD116 and SNORD115 (Prader-Willi genes)
ATP10A and UBE3A still active
What causes PWS?
Loss of snoRNA gene expression
PWS arises when there is a deletion of 15q11.2-q13.1 in paternal chromosome (maternal chromosome imprinted)
PWS cases of rare small deletions implicate SNORD116 cluster disruption of small nucleolar RNAs
snoRNAs are located in introns of SNHG14 (small nucleolar host gene 14) ubiquitously expressed - highest expressions and longest transcripts in brain
snoRNAs mature rRNA for ribosome assembly and regulate mRNA levels and splicing
Paternal chromosome when IC is unmethylated - gene expression in non-neurons
- IC unmethylated
- Gene expression activated
- SNHG14 transcript terminates after SNORD116 locus in non-neurons
- UBE3A expressed
Paternal chromosome when IC is unmethylated - gene expression in neurons
- IC unmethylated
- Gene expression activated
- SNHG14 transcript extends through SNORD115 to generate antisense transcript to UBE3A
- UBE3A silenced
What causes Angelman’s syndrome
- 11% cases caused by point mutation in UBE3A allele
- UBE3A gene only imprinted in neurons
- AS caused by loss of UBE3A in brain due to deletion between 15q11.2-q13.1 or UBE3Q point mutation on maternal chromosome or paternal uniparental disomy
- UBE3A - E3 ubiquitin ligase that targets proteins for proteasomal degradation
- Ephexin 5 is a target - degradation required for synapse development
What causes 25% of PWS cases
Uniparental maternal disomy on chromosome 15
Equivalent to paternal chromosome deletion
No expression of PWS genes causes prader-willi syndrome
Array CGH and FISH will be normal
Diagnosis confirmed by methylation specific PCR
What causes 7% of AS cases
Uniparental paternal disomy for chromosome 15
Paternal imprints on both chromosomes
IC unmethylated
Equilvelent to maternal chromosome deletion
Diagnosing PWS and AS
Methylation specific PCR
DNA treated with sodium metabisulfite
PCR amplification of DNA
methylated or unmethylated DNA specific primer
How is X chromosome inactivation mediated by epigenetic mechanisms
- At blastocyst stage, each cell randomly silences one of the X chromosomes (always paternal X in kangaroos)
- Each cell randomly generates a clone with the same X active
- Biological females have a mosaic with either the maternal or paternal X active
How is the X chromosome silenced
lncRNA, Xist or Tsix determine X inactivation
Pathway:
Both chromosomes express Xist: RNA unstable
Antisense Tsix RNA is expressed from future active X chromosome. Xist transcript is negative,y regulated by antisense Tsix
Active X ceases Xist RNA synthesis
What are the consequences of X-inactivation for carriers
- Half cells lack normal gene expression
- Effect depends on gene product nature and number/location of X-inactivated cells
- Gene for secreted protein unlikely to have much of an effect
‘ - Cell autonomous requirement - tissues may be partially affected
- Requirement for cell survival - non-random X-inactivation
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