PWS/AS

Question 1

Where do PWS/AS map to?

Answer 1

15q11.2-q13

Question 2

Why do PWS/AS arise?

Answer 2

Loss of expression of one or more imprinted genes

Question 3

Why is prenatal diagnosis for PWS/AS rare>

Answer 3

Most cases are de novo- commoner to offer PND to parents with chromosome 15 translocations

Question 4

How can uniparental disomy be ruled out in the foetus?

Answer 4

Genotyping at microsatellite markers

Question 5

Features of PWS a) infancy b) beginning in childhood

Answer 5

a) hypotonia, feeding difficulties, poor growth and development
b) hyperphagia and obesity, learning disability, behavioural problems

Question 6

Frequency of PWS

Answer 6

1 in 10,000 to 30,000 individuals

Question 7

Expression of which genes are lost in PWS, and which chromosome are these genes usually expressed from?

Answer 7

MKRN3, MAGEL2, NECDIN, C15ORF2, SNURF-SNRPN and a family of five snoRNA genes

Paternal

Question 8

Proportion of PWS which arises from maternal uniparental disomy?

Answer 8

20-25%

Question 9

Which genes are lost in AS, and which chromosome are these usually expressed from?

Answer 9

UBE3A and ATP10

Maternal

Question 10

Proportion of AS which arises from paternal uniparental disomy?

Answer 10

1-2%

Question 11

What are the mechanisms of loss of expression in PWS/AS?

Answer 11

Deletion
Uniparental disomy
Defect in imprinting
Point mutation
Chromosomal translocation

Question 12

What is the recurrence risk if the father has a 15;15 Robertsonian translocation?

Answer 12

Almost 100%

Question 13

What technique is utilised to test for PWS/AS?

Answer 13

Methylation-specific MLPA

Question 14

What needs to be included in any MLPA experiment?

Answer 14

Control (disease specific positive control and no-DNA negative control) and reference samples

Question 15

How does MS-MLPA differ from normal MLPA?

Answer 15

In MS-MLPA, one part of the experiment is run as a normal MLPA to provide copy number information

Other part of experiment uses probes which target DNA sequences containing cleavage site for methylation-sensitive restriction enzyme HHal. When hybridized to an un-methylated target, the probes will be ligated and simultaneously digested by HHal. In contrast when the target sequence is methylated, the methyl group prevents HHal digestion. Amplification products are separated in a capillary and analysed by Gene Marker

Question 16

MS-MLPA steps (5)

Answer 16

DNA denaturation and hybridization
Ligation and digestion
PCR
Capillary electrophoresis to separate fragments
Data analysis

Question 17

Steps in analysing imprinting disorders using MS-MLPA

Answer 17

Analyse undigested samples in GeneMarker (as would normally for MLPA)- dosage analysis

Analyse undigested and digested samples together in GeneMarker (methylation analysis)

Question 18

How does a normal person show up on MS-MLPA for PWS or AS testing?

Answer 18

Dosage analysis 1.0 (because should be the same as reference sample)

Methylation analysis 0.5 (because only one of their copies should be unmethylated, hence only 50% of the probes get amplified by PCR)

Question 19

In PWS the methylation status is alwaus 1.0 whereas in AS the methylation status is always 0, explain?

Answer 19

PWS- the imprinting centre is methylated- get signal from maternal allele

AS- imprinting centre is unmethylated- restriction enzyme will cut- therefore no signal from the unmethylated paternal alelle

Question 20

In uniparental disomy the dosage is always…

Answer 20

1.0, because there are two alleles

Question 21

Considerations when analysing MS-MLPA data? (3)

Answer 21

Single nucleotide change close to the probe ligation site can prevent ligation and result in reduced probe signal- can look like a deletion

When probes for non-neighbouring exons show decreased or increased signal, results are unlikely to be reliable

If one or more reference probes show an abnormal copy number in the same sample then the test should be repeated