L7, Epigenetics

Question 1

Characteristics of Angelmann syndrome: (Symptoms)

Answer 1

• Developmental delay
• Severe speech impairment
• Jerky movements and hand flapping
• Happy disposition
• Microcephaly (small head)
• Seizures, abnormal EEG (electrical activity in brain measured using electrodes)

Question 2

Prader Willi characteristics:

Answer 2

• Hypotonia
• Poor suckling reflex in infancy vs insatiable appetite in later life
• Obesity and short stature
• Compulsive behaviour like skin picking
• Strabismus (eye crossing)
• Hypogonadism, poor sexual development
• Lower than normal level of growth hormone

Question 3

Deletion in PWS and AS: Origin, cause and detection

Answer 3

• Around 75% of PWS and AS cases originate in a deletion of up to 6 Mbp on chromosome 15q11.2q13.1
• -> Deletions are either break point 1 to 3 (type I) or 2 to 3 (type II)
• Deletion can occur as a result of aberrant meiotic recombination between direct repeats that flank the region
• Deletion can be detected by FISH or array CGH for diagnoses

Question 4

How is genomic imprinting carried out under normal circumstances?

Answer 4

• Imprinting centre (IC) coordinates the expression of certain genes on exclusively paternal or maternal chromosome
• IC is methylated on maternal chromosome
• Normally, an individual will thus inherit one intact maternal and one intact paternal chromosome 15

Question 5

Effect of genomic imprinting under Chr 15 deletions:

Answer 5

• Paternal deletion -> PWS (One or more of genes normally expressed on parental chr. are turned off in maternal)
• Maternal deletion -> AS (Converse situation)

Question 6

Why is genomic imprinting an epigenetic trait?

Answer 6

• Stably heritable phenotype, no change to DNA sequence

Question 7

Molecular basis for genomic imprinting:

Answer 7

• Methylation of cytosine bases at CpG sites in differentially methlyated regions (DMRs) is responsible for imprinting (carried out by DNMTs) -> maintenance methyltransferase recognises hemi-meth. sites -> full methylation
• Either maternal or paternal allele is methylated in embryo and somatic cells
• As such, all oocytes in next generation have maternal pattern and vice versa for sperm
• Imprint is erased in subsequent germ cells of following generation (primordial germ cells of offspring)

Question 8

Maternal transcript in 15q11-13

(Effect on transcription of particular genes)

Answer 8

1. Imprinting centre is methylated
2. Methlyation of IC blocks transcription of PWS genes flanking IC site (heterochromatic)
3. ATP10A and UBE3A genes remain active

Question 9

Loss of snoRNA expression in PWS:

Answer 9

• Studies of PWS patients implicate disruption of SNORD 116 cluster of snoRNAs (small nucleolar)
• snoRNAs are located in introns of SNHG14, ubiquitously expressed, normally highest expression levels and longest transcripts in brain
• Function in maturation of rRNA for ribosome assembly and in regulation of mRNA levels and splicing

Question 10

Chromosome 15 (paternal) methylation status effect on gene expression in non-neurons:

Answer 10

1. IC unmethylated
2. Gene expression activated
3. SNHG14 transcript terminates after SNORD116 locus in no-neurons
4. UBE3A gene expressed

Question 11

Chromosome 15 (paternal) methylation status effect on gene expression in neurons:

Answer 11

1. IC unmethylated
2. Gene expression activated
3. SNHG14 transcript extends through SNORD115 to generate antisense transcript to UBE3A
4. UBE3A expression silenced

Question 12

Loss of UBE3A in neurons:

Answer 12

• 11% of AS cases caused by point mutations in maternal UBE3A allele
• Rest are either uniparental disomy or normal 14q11.2-13.1 deletion
• E3 ubiquitin ligase; targets proteins for proteasomal degradation -> may target ephexin 5

Question 13

Uniparental disomy:

Answer 13

• Both members of a chromosome pair are inherited from one parent, and the other parent’s chromosome for that pair is missing

Question 14

Uniparental disomy in PWS and AS:

Prevalence and diagnosis

Answer 14

• 25% of PWS cases, ~7%
• Equivalent to deletion of the paternal or maternal chromosome
• Array CGH and FISH will be normal -> diagnosis must be confirmed by methylation specific PCR

Question 15

Methylation specific PCR:

Answer 15

• Bisulfite treatment
• Defining methylation status of IC
• Sodium bisulfite treatment to purified genomic DNA -> methylated sequence protects cytosine residues from deamination whereas unmethylated residues are deaminated to uracil
• PCR amplification carried out from these templates; complementary to different primers (different sizes)
• Only definitive method for diagnosis in the case of uniparental disomy

Question 16

Disorders involving imprinted regions:

Answer 16

• Prader willi
• Angelman
• Beckwith-Wiedemann (Foetal overgrowth) 11

Question 17

How do epigenetic mechanisms mediate dosage compensation?

Answer 17

• Dosage compensation: Random silencing of one of X chromosomes (except in kangaroos -> paternal always silenced)
• One of the chromosomes is silenced by lncRNA (Xist)
• Initially, both chromosomes express unstable Xist RNA -> stochastic choice of one to express Tsix first (antisense) -> degradation of Xist
• On other chromosome, Xist binds to Xic and coats whole chromosome -> recruitment of factors to sequester as inactive heterochromatin -> Barr body

Question 18

How may X-inactivation vary in effect for carriers of X-linked disease:

Answer 18

• Half of cells will lack expression of normal gene; effects depend on nature of gene product and extent/localisation of X-inactivation
• Genes for secreted protein (e.g. blood clotting factor) unlikely to have much effect
• Cell autonomous requirement (e.g. Dystrophin) tissues may be partially effected
• Required for cell survival -> non-random X-inactivation (e.g. X-linked SCID)

Question 19

+ Why might a PWS patient display especially fair skin

Answer 19

• OCA2 gene is next to region associated with the gene
• If the deletion encompasses this, it will lose OCA2 expression
• OCA2 is involved in pigmentation