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Genetics Of Learning Disorders > Prader-Willi and Angelman syndromes > Flashcards

Prader-Willi and Angelman syndromes Flashcards

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1
Q

What is specific about the expression of imprinted genes?

A

They are expressed from only one parental chromosome

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2
Q

What is the common type of epigenetic modification seen in control of expression of imprinted genes?

A

Methylation of cytosine in CpG dinucleotides

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3
Q

When a male passes on an allele inherited from his mother, what must happen?

A

Imprints must be erased and reset during the germ cell formation

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4
Q

What is the genomic region associated with PWAS?

A

15q11-q13

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5
Q

Only one gene is associated with Angelman syndrome due to expression on maternal chromosome only. Which gene?

A

UBE3A - only expressed in the brain

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6
Q

There are 4 protein coding genes expressed from the paternal chromosome - which is the main one?

A

SNURF-SNRPN

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7
Q

What is the methylation status of the paternal and maternal chromosomes?

A
  • Paternal: Generally unmethylated

- Maternal: CpG islands associated with paternally expressed, protein coding genes are methylated

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8
Q

What is UBE3A-AS and what does it do?

A
  • anti-sense RNA at end of UBE3A gene that will prevent expression of UBE3A in cis (same chromosome)
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9
Q

How does the methylation on the maternal chromosome impact UBE3A expression?

A
  • methylation of promoter regions blocks transcription factor binding and assembly of transcription machinery
  • without expression from SNURF-SNRPN there is also no snoRNA or UBE3A-AS expression
  • UBE3A is therefore expressed from the maternal chromosome
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10
Q

Where are the PWS and AS imprinting centres located?

A
  • PWS paternal imprinting centre is located at 5’ end of SNURF-SNRPN
  • AS maternal imprinting centre is approx. 35kb upstream of this
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11
Q

What is thought to happen with the imprinting centres at oogenesis/spermatogenesis?

A
  • During oogenesis, factors bind to maternal IC and promote methylation of paternal IC. methylation spreads to other CpG islands in region
  • During spermatogenesis maternal factors not present therefore paternal IC stays unmethylated
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12
Q

What is the incidence rate of PWS/AS?

A

1 in 15-20,000

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13
Q

What are Prader-Willi and Angelman syndromes due to?

A

Loss of paternal and maternal contributions from 15q11-q13, respectively

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14
Q

What are the clinical features of PWS?

A
  • mild to moderate mental retardation
  • hypotonia
  • failure to thrive/feeding problems in neonate
  • hyperphagia/obesity in later development
  • male hypogonadism
  • short stature
  • small hands and feet
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15
Q

What are the clinical features of AS?

A
  • severe mental retardation
  • lack of speech
  • hyperactivity
  • happy demeanour and inappropriate laughter
  • gait ataxia
  • seizures
  • microcephaly
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16
Q

What are the potential disease mechanisms for PWS and AS?

A
  • 15q11-q13 deletion (most common: ~70-80%)
  • UPD: maternal for PWS (20-25%) or paternal for AS (3-7%)
  • imprinting defect (~1-3%)
  • UBE3A mutation (AS only: 14%)
  • Unknown cause (AS only: ~10%)
17
Q

What is uniparental disomy and how does it impact on PWS/AS?

A
  • both chromosomes are from same parent
  • either two copies of same chromosome (isodisomy) or one copy of each chromosome from same parent (heterodisomy)
  • Maternal UPD15 leads to PWS, paternal UPD15 leads to AS
18
Q

Is UPD more commonly seen in PWS or AS?

A

PWS

19
Q

What is the most common type of UPD in AS?

A

Paternal isodisomy

20
Q

What is the mechanism that results in paternal uniparental Isodisomy?

A

monosomy rescue (ND at meiosis II)

21
Q

What are the two mechanisms that can result in:

  • maternal uniparental Isodisomy?
  • maternal uniparental heterodisomy?
A

Both occur by trisomy rescue or gamete complementation with ND at either meiosis II (mUPID) or meiosis I (mUPHD)

22
Q

A carrier of what type of translocation involving chromosome 15 is at increased risk of having a child with PWS/AS? Why?

A
  • Robertsonian translocation carrier

- Increased risk of monosomy 15 or trisomy 15 in zygote, which can lead to UPD15 via monosomy or trisomy rescue

23
Q

What is an imprinting defect?

A

Failure to set the correct parental expression pattern - often de novo with no obvious mutation

24
Q

10-15% of imprinting defects are Microdeletions of either imprinting centre - how are these affected differently?

A
  • if maternal IC is deleted and chromosome is inherited maternally the child will have AS
  • if paternal IC is deleted and chromosome is inherited paternally the child will have PWS
  • However, a maternal IC deletion can be inherited silently from a male as the correct parental imprint is still established and vice versa
25
Q

What are the recurrence risks for an imprinting defect?

A
  • Low recurrence risk for an imprinting defect without an IC deletion
  • If IC deletion is present, recurrence risk can be up to 50%
26
Q

What is PWS pathogenicity attributed to?

A
  • no individual protein coding genes linked to PWS and no point mutations in single genes have been found
  • loss of expression from SNORD116 snoRNA cluster now thought to underlie PWS phenotype
27
Q

What is AS pathogenicity attributed to?

A
  • Loss of UBE3A expression in the brain
  • UBE3A protein is involved in ubiquitination pathway, targeting selected proteins for degradation
  • aberrant protein degradation interferes with correct neuronal development
28
Q

What are the three main cytogenetic techniques involved in the detection of PWS/AS?

A
  1. Karyotype - detects most deletions and any rare trans. Slow compared to other techniques and won’t detect UPD, imprinting defects or UBE3A mutations
  2. FISH - uses SNRPN probe. Rapid technique but will still miss UPD, imprinting defects and UBE3A mutations. Probes can be designed specifically to detect IC deletions.
  3. ArrayCGH - similar advantages and disadvantages to FISH. Possible to detect IC deletions with good probe depth.
29
Q

What four molecular methods are used for detection of large deletions, UPD or imprinting defects in PWS/AS?

A
  • Southern blotting
  • methylation specific PCR
  • methylation specific MLPA
  • microsatellite analysis (UPD only)
30
Q

Describe the process of southern blotting for PWS/AS

A
  • can distinguish between pat (unmethylated) and mat (methylated) copies of PWAS critical region using a XbaI/NotI digest
  • NotI only cuts at unmethylated CpGs, XbaI cuts either methylated or unmethylated DNA
  • normal patient has both mat and pat bands
  • PWS patient has heavy mat band (methylated: cut by Xbal) with absent pat band (unmethylated: cut by both). AS patient has reverse scenario
31
Q

What step is undertaken prior to methylation specific PCR that allows differentiation between methylated and unmethylated DNA?

A
  • Treatment with sodium bisulphite
  • Unmethylated cytosines are converted to uracil
  • Methylated cytosines are unmodified and remain as cytosine
  • Final DNA sequence will now be different if the DNA was methylated or not
32
Q

How does methylation-specific PCR work?

A
  • Two sets of primers in one reaction
  • one set specific to methylated DNA sequence (maternal primers; amplify 174bp band)
  • other set complementary to unmethylated DNA sequence (paternal primers; amplify 100bp band)
  • different product sizes allow differentiation of products from methylated/unmethylated DNA using gel electrophoresis
33
Q

What are the disadvantages of methylation specific PCR?

A
  • possibility of false positive due to incomplete DNA modification (no product from paternal allele = false PWS)
  • false positive result due to SNP under primer site (can give either false PWS or AS)
  • lack of info about underlying mechanism: PCR can’t identify if the methylation pattern is due to UPD, deletion or imprinting defect
  • will not detect mosaicism
34
Q

What is methylation specific multiplex ligation-dependent probe amplification (MS-MLPA)?

A
  • standard detection technique for PWS/AS
  • modified MLPA that allows detection of copy number changes at 15q11-q13 and methylation pattern
  • allows detection of common large deletion and smaller IC deletions
  • cases with UPD, or imprinting defects without an IC deletion, will have normal copy number at 15q11-q13 but abnormal methylation
  • DNA doesn’t require modification
35
Q

The PWAS methylation specific MLPA kit contains 48 probes. What do these entail?

A
  • Use control probes from genomic regions that should have normal copy number
  • Use probes from commonly deleted 15q11-13 region and some from just outside this region
  • Probe sites with normal methylation pattern are used to quantify methylation of the region
  • Some probes act as methylation controls: e.g. methylated on both mat and pat chromosome so shouldn’t be digested, or unmethylated so act as digestion controls
36
Q

What is the dosage quotient with regards to PWAS MS-MLPA copy number results?

A
  • analysis software produces dosage quotient for each probe by comparing peak height in internal and external controls
  • dosage quotients give indication of copy number compared to normal control samples:
  • 1.0 (0.8-1.2) = 2 copies
  • 1.5 (1.35-1.65) = 3 copies
  • 0.5 (0.35-0.65) = 1 copy
  • 0 = 0 copies
37
Q

What are the disadvantages of methylation specific MLPA?

A
  • sensitive to PCR contaminants and DNA quality
  • can’t be used to detect UBE3A point mutations
  • can get false positives due to SNP under probe
  • cost (due to multitude of controls on each run)
  • still can’t differentiate between UPD and an imprinting defect without an IC deletion
38
Q

What is microsatellite analysis for UPD detection in PWAS?

A
  • performed if MS-MLPA shows abnormal methylation with normal copy number
  • need parental DNA samples
  • chr15 microsatellite markers (predominantly located in 15q11-q13 region) are genotyped for affected patient and both parents
39
Q

Give details on prenatal diagnosis of PWAS

A
  • referral = parents who carry chr15 translocations or those for whom an IC deletion was identified in previous child
  • involves microsatellite analysis
  • MS-MLPA is possible but depends on DNA quality (methylation status at SNRPN exon 1 is established early in embryonic development)

Genetics Of Learning Disorders (11 decks)

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