PWS/AS Flashcards
Where do PWS/AS map to?
15q11.2-q13
Why do PWS/AS arise?
Loss of expression of one or more imprinted genes
Why is prenatal diagnosis for PWS/AS rare>
Most cases are de novo- commoner to offer PND to parents with chromosome 15 translocations
How can uniparental disomy be ruled out in the foetus?
Genotyping at microsatellite markers
Features of PWS a) infancy b) beginning in childhood
a) hypotonia, feeding difficulties, poor growth and development
b) hyperphagia and obesity, learning disability, behavioural problems
Frequency of PWS
1 in 10,000 to 30,000 individuals
Expression of which genes are lost in PWS, and which chromosome are these genes usually expressed from?
MKRN3, MAGEL2, NECDIN, C15ORF2, SNURF-SNRPN and a family of five snoRNA genes
Paternal
Proportion of PWS which arises from maternal uniparental disomy?
20-25%
Which genes are lost in AS, and which chromosome are these usually expressed from?
UBE3A and ATP10
Maternal
Proportion of AS which arises from paternal uniparental disomy?
1-2%
What are the mechanisms of loss of expression in PWS/AS?
Deletion Uniparental disomy Defect in imprinting Point mutation Chromosomal translocation
What is the recurrence risk if the father has a 15;15 Robertsonian translocation?
Almost 100%
What technique is utilised to test for PWS/AS?
Methylation-specific MLPA
What needs to be included in any MLPA experiment?
Control (disease specific positive control and no-DNA negative control) and reference samples
How does MS-MLPA differ from normal MLPA?
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
MS-MLPA steps (5)
DNA denaturation and hybridization Ligation and digestion PCR Capillary electrophoresis to separate fragments Data analysis
Steps in analysing imprinting disorders using MS-MLPA
Analyse undigested samples in GeneMarker (as would normally for MLPA)- dosage analysis
Analyse undigested and digested samples together in GeneMarker (methylation analysis)
How does a normal person show up on MS-MLPA for PWS or AS testing?
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)
In PWS the methylation status is alwaus 1.0 whereas in AS the methylation status is always 0, explain?
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
In uniparental disomy the dosage is always…
1.0, because there are two alleles
Considerations when analysing MS-MLPA data? (3)
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
