11. PWS and AS

Question 1

What is the phenotype of PWS?

Answer 1

Severe hypotonia, dev delay, characteristic facial features, hypogonadism, infertility, obesity

Question 2

What causes PWS? What causes AS?

Answer 2

PWS - loss of expression from PATERNAL allele

AS - loss of expression from MATERNAL allele

Question 3

Which genes are involved in PWS and AS and how are they expressed?

Answer 3

PWS - SNURF/SNRPN, MAGEL2 expressed from PATERNAL allele only. Loss of SNRPN enough to cause PWS

AS - UBE3A expressed from MATERNAL allele only in the brain. Encodes E6AP, essential for synapse development

Question 4

What is the normal methylation pattern of SNRPN?

Answer 4

Unmethylated and expressed on paternal allele

Methylated and not expressed on maternal allele

Question 5

What do changes in methylation pattern seen on MS-MLPA signify?

Answer 5

Hypermethylation of SNRPN = PWS
Hypomethylation of SNRPN = AS

Question 6

What are the molecular mechanisms behind PWS?

Answer 6

1. De novo 15q11-q13 deletion of paternal allele (70-75%)
2. Maternal UPD15 (25-30%)
3. Imprinting defect with or without paternal ICR deletion (1%)

Question 7

What are the molecular mechanisms behind AS?

Answer 7

1. De novo 15q11-q13 deletion of maternal allele (75%)
2. Paternal UPD15 (1-2%)
3. Imprinting defect with or without maternal ICR deletion (3%)
4. UBE3A mutation

Question 8

How are PWS and AS tested for?

Answer 8

MS-MLPA

Detects 15q11-q13 deletion or ICR deletion and determines methylation change at SNRPN

UPD by microsatellite analysis (requires parental DNA)

Question 9

On MS-MLPA, what does a normal copy number but a change in methylation signify

Answer 9

UPD or imprinting defect without an ICR deletion

Question 10

What does a normal copy number and normal methylation pattern signify on MS-MLPA

Answer 10

PWS unlikely

UBE3A sequencing for AS

Question 11

How does MS-MLPA work?

Answer 11

2-tube protocol:

1. Undigested reaction used for copy number determination.
2. Digested with HhaI - digests unmethylated double-stranded probe–DNA complexes - only get amplification from probes hybridised to methylated DNA

Question 12

Where is the PWS critical region?

What is the pattern of gene expression in normal individuals?

Answer 12

15q11-13

Genes are expressed from one parental allele only - functionally haploid

Question 13

What is the phenotype of AS?

Answer 13

Dev delay, speech impairment, gait ataxia, LD, epilepsy, microcephaly, ‘happy demeanour’

Question 14

What is the recurrence risk associated with different causes of PWS & AS?

Answer 14

Deletions, UPD, imprinting defect (without IC deletion) = <1%

IC deletion = 50% if inherited from father (PWS) or mother (AS)

Question 15

How does SNURF/SNRPN regulate UBE3A?

Answer 15

Paternally expressed SNURF/SNRPN transcript blocks UBE3A expression from paternal allele

Question 16

How does SNRPN hypomethlation cause AS?

Answer 16

SNRPN becomes unmethylated on maternal allele

Blocks maternal UBE3A expression

Question 17

What are the different recurrent deletions seen in PWS/AS?

Answer 17

Class 1 - from BP1-BP3, include NIPA1 & 2

Class 2 - from BP2-BP3

Question 18

How do recurrent deletions seen in PWS/AS arise?

Answer 18

NAHR mediated by LCRs flanking the PWS/AS critical region