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Learning Disabilities > Prader-willi And Angelman > Flashcards

Prader-willi And Angelman Flashcards

1
Q

What are imprinted genes and how many known are there?

A

Expressed from only one parental chromosome - tissue specific or in all cells
~80 known

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2
Q

Give another example of imprinted disorders

A

Beckwith-Widemann syndrome (11p15) and silver Russel syndrome (11p15)

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3
Q

How do imprinting control elements/imprinting centres (IC) work?

A

They are cis acting and can act over a long distance

Control expression of a cluster of imprinted genes

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4
Q

Are maternally and paternally imprinted genes specific to on parental chromosome?

A

Yes, OR expression can be specific to one parental chromosome

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5
Q

What epigenetic modification usually creates imprinting?

A

Methylation of cytosine in CpG dinucleotides

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6
Q

What happens methylation status during germ cell formation?

A

Methylation is maintained throughout development and re-set during germ cell formation to develop new sex-specific imprint

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7
Q

PWAS region?

A

15q11-q13

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8
Q

What genes are only expressed from the maternal chromosome in the brain?

A

UBE3A and ATP10C

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9
Q

What genes are only expressed from the paternal chromosome?

A

SNURF-SNRPN, NDN,MAGEL2& MKRN3 all others are snoRNAs

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10
Q

Describe SNORD116&115

A

116 is Cluster of 29 copies and 115 is a cluster of 48 copies

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11
Q

Methylation status of paternal 15q11-13?

A

Generally unmethylated

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12
Q

True or false : there is a single promoter that controls all the protein coding genes of paternal 15q11-13

A

False: the protein coding genes have their own promotors

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13
Q

Describe transcription of SNURF-SRPN

A

Has several tissue specific promotors and alternatively spliced transcripts including one v.long brain specific transcript with UBE3A-AS

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14
Q

Why is paternal ube3a not expressed in the brain?

A

V. Long brain specific spur-SNrpn with ube3a at end

UBE3A is an antisense rna at the end of the UBE3A gene that prevents UBE3A expression in cis

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15
Q

Maternal allele methylation?

A

CpG islands associated with paternally expressed protein coding genes are methylated
UBE3A is expressed

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16
Q

How does methylation effect transcription?

A

Methylation of promoter regions prevents transcription factor binding and assembly of the transcription machinery

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17
Q

Where is the maternal and paternal IC?

A

Paternal IC is at the 5’ end of SNURf-SNRPN

Maternal IC is about 35kb upstream of the male/pws IC

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18
Q

What is the proposed method of methylation during oogenesis and spermatogenesis?

A

During oogenesis factors bind to the maternal IC and promote methylation of the paternal IC which spreads to the CpG islands in the region
During spermatogenesis maternal factors not there so paternal IC remains unmethylated

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19
Q

Cause and frequency of PWS?

A

Loss f the paternal contribution from 15q11-q13

1 in 15-20,000

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20
Q

Features of PWS?

A 
Mild to moderate mental retardation 
Hypotonia 
Feeding problems in early life
Hyerphagia and obesity in later development 
Male hypogonadism 
Short stature
Small hands and feet
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21
Q

Cause and frequency of AS?

A

Loss of maternal contribution of 15q11-13

1 in 15-20,000

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22
Q

Clinical features of angelman?

A 
Severe mental retardation 
Lack of speech 
Hyperactivity
Happy demenanour and inappropriate laughter 
Gait ataxia 
Seizures
Microcephaly
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23
Q

What causes the deletion and how large is it?

A

~ 4 mb
Usually de novo but small risk of gremlins mosaicism in parent
Common breakpoints- most likely due to non-allelic homologous recombination (NAHR)

24
Q

What is the difference between homodisomy and heterodisomy?

A

Homodisomy- two copies of the same chromosome

Heterodisomy- one copy of each chromosome from same parent

25
Q

Most common causes of UpD?

A

pWS more commonly due to maternal non-dysfunction at meiosis
AS- most common paternal isodisomy - sperm fertilises egg with no15 - monosomy rescue to maternal 15

Or heterodisomy can occurs for trisomy rescue of nondisjuction occurred in meiosis I

26
Q

Non disjunction at meiosis I vs non disjunction at meiosis II?

A

Non disjunction at meiosis I= heterodisomy

Non disjunction at meiosis II= isodisomy

27
Q

Besides non disjunction what else can cause UPD?

A

Mitotic error with rescue leading to isodisomy or heterodisomy - can lead to mosaicism in some cell lines

28
Q

What is the risk of robertsonian translocation involving chromosome 15

A

Increased risk of monopsony 15 or trisomy 15 in zygote - lead to UPD15 via monosomy or trisomy rescue

29
Q

What percentage of imprinting defects are microdeletion of either IC?

A

10-15%

30
Q

If there is a maternal IC and the chromosome is inherited maternally wha is the phenotype ?

A

Angelman syndrome

31
Q

True or false:paternal IC deletion will pass silently in females until inherited from dad

A

True

32
Q

What percentage of AS caused by UBE3A mutation?

A

14%

33
Q

What is the phenotype of a loss or ATP10C gene?

A

No impact on AS phenotype

34
Q

What % of mUPD cause PwS and what % of pUPD causes AS?

A 
MUPD= 20-25%
PUPD= 3-5%
35
Q

What gene is believed to underline PWS phenotype ?

A

sNORD116 snoRNA now believed to cause phenotype

SNORD116 possible role in the regulation of alternate splicing

36
Q

Role of UBE3A?

A

Ubiquitin protein ligament E3A involved in the ubiqutination pathway which targets certain proteins for degradation
Aberrant protein degradation interferes with correct neuronal development

37
Q

Karyotype for PWAS?

A

Will detect most 15q11-q13 deletions and rare translocations but slow and ninUPD, imprinting or UBE3A mutations

38
Q

FISH for PWAS?

A

SNrPN probe, and probes designed for IC

Fast but no UPD, imprinting defects or UBE3 mutations

39
Q

ACGH for PWAS?

A

Similar pros and cons as FIsH- can detect IC deletions if prone depth is good

40
Q

Southern blot for PWAS?

A

Xbal/NotI - notI only cuts UNMETHYLATED CpG dinucleotides

Probe = KB17 which binds snrpn gene promoter and coding exon 1

41
Q

Southern blot results for normal, pws and as?

A

Normal = two bands one 4.2kb and other 0.9kb
Pws= 0.9
As=4.2

42
Q

Pros and cons to southern blot?

A

Will detect large deletions UpD or imprinting defects but will not inform which is cause
Good quality blot can detect mosaicism
Labour intensive, time consuming and large amount of DNA required

43
Q

How does methylation specific pcr work and what is use of bisulphate treatment?

A

1)Prior to pcr treated with bisulphate- 2)unmethylated cytosines are converted to uracil and methylated cytosines are unmodified
3) uracil matches with thymine
Primers designed for both = 2 bands mean normal

44
Q

Disadvantages to ms-pcr

A

False pos due to snps under primer site

False PWS pos due to incomplete DNA modification - showing no paternal allele

45
Q

How does ms-mlpa probes designed ?

A

-each probe is made as a pair of oligonucleotides that hybridises to region of interest and one or both are followed by a stuffer fragment to differentiate probes and also sequence specific to u oversaw primers at ends of each probe
48 probes in Mrc holland kit

46
Q

How does ms-mlpa work?

A

1)DNA denatured
2)probe added and held at 60c for 16h
3) thermostable ligament added 48c for 30min
4)95c for 5 min to inactivate ligase
5) reaction split
6) methylation sensitive enzyme eg hha1 added- unmethylated DNA is Digested and NOT amplified (paternal)
Methylated is amplified (maternal)
7) not treated half used for copy number change detection
8) fluorescent labelled pcr primers and reagents added to lighted products

47
Q

How to interpret ms-mlpa?

A

Peak heights from undigested reaction are compared to digested- should be about half

48
Q

How many probes in kit?

A

14 controlprobes for areas of genome that should have 2 copies
31 probes from 15q11-13 (5 are normally maternally methylat d and paternally unmethylated and contain hhaI site)
3 probes just outside deleted region

49
Q

What methylation controls are present?

A

4 probes - 1 is methylated on the maternal and paternal chromosome so should not be digested
3 are always unmethylated so act as digestion controls

50
Q

What is the dosage quotient (DQ)?

A

Compares peak height to internal and external controls

  1. 8-1.2 = 2 copies
  2. 35-1.65= 3 copies
  3. 35-0.65= 1 copy
51
Q

What would be the ms-mlpa results for normal, AS and PWS?

A

Normal methylation pattern- 0.5 ratio
As= no methylation of 15q sites so ratio of 0
pWS= all methylated so ratio =1

52
Q

Disadvantages of ms-mlpa?

A

1) Sensitive to pcr contaminants
2) sensitive to DNA quality
3) snps under probe site
4) costs £10 per reaction but as need to run so many controls only economical if batch
5) can’t differentiate between UPD and an imprinting defect

53
Q

When would you request microsatellite analysis for UPD?

A

Ms-mlpa shows abnormal methylation with normal copy number - request parent DNA

54
Q

How to analyse for UPD?

A

Fluorescently labelled primers for markers

Require at least two fully informative markers to report as UpD

55
Q

Common pws referral?

A

Babies with hypotonia

Children with phenotype - over eating etc

56
Q

When is prenatal testing offered?

A
  • Parents with chromosome 15 translocations

- IC deletion identified in another child

57
Q

Is ms-mlpa offered as prenatal?

A

Ms-mlpa is possible as methylation is established early in embryonic development but limited by DNA quality- microsatellite usually offered

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