PWS/AS

Question 1

Cause of PWS; general

Answer 1

Loss of function of paternally expressed 15q11q13 genes

Question 2

Cause of AS; general

Answer 2

Loss of function of maternally expressed 15q11q13 genes

Question 3

Cause of PWS; breakdown of %

Answer 3

de novo deletion of pat 15q11q13 = 70-80%
maternal UPD = 20-25%
imprinting defects = ~1%
(imprinted centre deletion = 10-15% of defects)

Question 4

Cause of AS; breakdown of %

Answer 4

de novo deleion of mat 15q11q13 = 70-75%
UBE3A mut = 10%
No identifiable abnormality = 10%
paternal UPD = 3-7%
imprinting defect 2-3%
(imprint centre deletion = 10-15% of defects)

Question 5

Genes and expression of 15q11q13 region

Answer 5

paternal allele; SNRF-SNRPN promoters generally unmethylated = ON = many transcripts - one of which includes UBE3A-AS, which suppresses UBE3A expression in cis.
matnernal allele; SNRF-SNRPN promoters generally methylated = OFF = no UBE3A-AS = UBE3A can be expressed

Question 6

How do deletions at 15q11q13 arise?

Answer 6

region flanked by sequences of high homology - NAHR

Question 7

How does UPD arise?

Answer 7

1) homodisomy =
a) monosomic and trisomic rescue following meiosis II nondisjunction, consider robertsonian translocations too
b) gamete complementation
c) mitotic error

2) heterodisomy =
a) trisomic rescue following meiosis I, consider robertsonian translocations too
b) gamete complementation
c) mitotic error

Question 8

Why is UPD seen more in PWS than AS

Answer 8

Maternal age effect

Question 9

What are imprinting defects?

Answer 9

Failure to set the correct pattern of expression

Question 10

Underlying pathogenesis of PWS

Answer 10

SNORD116 small nucleolar RNA cluster (cluster of 29 tandem copies) thought to have role in regulation of alternative splicing

Question 11

Underlying pathogenesis of AS

Answer 11

Loss of UBE3A expression in brain. UBE3A targets selected proteins for degradation > aberrant protein degradation interferes with correct neuronal development

Question 12

Principles of MS-PCR

Answer 12

DNA bisulphite modification
2 sets of primers; 1 set for unmethylated sequence, 1 for meth seq
seperate by gel electrophoresis > different product sizes

Question 13

Limitations of MS-PCR

Answer 13

1. SNP under primer binding site could also false positive result
2. incomplete DNA modification could cause false pos PWS
3. Doesnt provide info about underlying mechanism such as UPD, deletion, IC defect etc
4. Doesnt detect mosaicism

Question 14

Principles of MS-MLPA

Answer 14

Perform MLPA as normal then split ligated products into two reactions; treat with methylation sensitive restriction enzyme . Unmethylated DNA digested > no product
Methylated DNA remains > product
then follow with final PCR step
[SEE NOTES FOR GOOD DIAGRAM]

Question 15

Limitations of MS-MLPA

Answer 15

• sensitive to PCR contaminants
• sensitive to DNA quality
• SNP under probe bidning site could cause allele drop out - false positive deletion
• doesnt detect UBE3A muts
• cannot differentiate between UPD or IC defect (other than IC deletion)