18.03.11 Prader-Willi/Angelman syndrome

Question 1

What is the incidence of PWS?

Answer 1

1 in 15,000 to 1 in 30,000

Question 2

What is the phenotype of PWS in early infancy?

Answer 2

Severe hypotonia and feeding difficulties

Question 3

Give five phenotypic features of PWS in early childhood.

Answer 3

1. Hyperphagia which if left uncontrolled leads to obesity
2. Obesity can lead to development of non-insulin dependent diabetes mellitus
3. Characteristic facial features-narrow bifrontal diameter, almond shaped eyes, triangular mouth
4. Motor milestones and language development delayed, mild-moderate mental retardation
5. Distinctive behavioural phenotype -temper tantrums, obsessive-compulsive characteristics, skin-picking
6. Hypogonadism-manifests as genital hypoplasia, incomplete pubertal development, infertility
7. Short stature and small hands and feet for height age
8. Narcolespy/daytime sleepiness
9. Hypopigmentation (deletions)

Question 4

What is the incidence of Angelman Syndrome?

Answer 4

1 in 12,000 - 1 in 20,000

Question 5

Give five phenotypic features of Angelman syndrome.

Answer 5

1. Severe developmental delay
2. Poor/absent speech (minimal to no use of words)
3. Movement or balance disorder (gait ataxia or tremulous movement of limbs)
4. Learning difficulties
5. Epilepsy/seizures - onset usually when 1-3 years old
6. Disturbed sleep
7. Behavioural problems
8. Microcephaly (more severe in those with deletions)9. Facial dysmorphism – brachycephaly, prominent chin, wide mouth.
9. Hyperactivity
10. Tongue thrusting, excessive chewing/mouthing.
11. Abnormal EEG with characteristic pattern -striking high voltage slow-wave activity
12. Unique behaviour - happy demeanour, frequent laughing and smiling, hand-flapping, fascination with water
13. Hypopigmentation (deletions)

Question 6

Where is the PWAS locus?

Answer 6

~2Mb imprinted domain at 15q11-13

Question 7

Which genes in the PWAS locus are typically maternally expressed in the brain?

Answer 7

In the brain, UBE3A and ATP10C are only expressed from the maternal chromosome.

Question 8

Which genes in the PWAS locus are typically paternally expressed

Answer 8

There are four paternally expressed, protein-coding genes: SNURF-SNRPN, NDN, MAGEL2 and MKRN3;

Question 9

Describe the normal physiology of the paternal 15q11-13 contribution and how expression of UBE3A is affected.

Answer 9

Expression of UBE3A-AS prevents UBE3A expression in cis.

1. On the paternal chromosome, the promoters for the protein-coding genes are unmethylated allowing expression.
2. The protein coding genes have their own promoters. SNURF-SNRPN has several tissue-specific isoforms and various different transcript lengths.
3. Some of the larger SNURF-SNRPN transcripts include the snoRNAs as they do not have their own promoter and another non-coding RNA; UBE3A-AS (UBE3A-antisense).
4. Only the longest brain-specific transcript contains UBE3A-AS, which is an antisense RNA for the end of the UBE3A gene.
5. When UBE3A-AS is expressed, there is chromosome specific repression of UBE3A.
6. As UBE3A-AS is only expressed from the brain-specific transcript UBE3A will only be expressed from the maternal allele in the brain, but biallelically elsewhere.

Question 10

Describe the normal physiology of the maternal 15q11-13 contribution.

Answer 10

CpG islands at the promoters of the protein-coding genes, including SNURF-SNRPN, are methylated on the maternal chromosome.

Promoter methylation leads to a conformational change in the DNA which represses transcription by blocking access to the DNA from the transcription factors and the transcription machinery needed for expression.

Without SNURF-SNRPN expression, there is no snoRNA or UBE3A-AS and therefore UBE3A can be transcribed and expressed (from the maternal chromosome).

Question 11

Describe the role and expression of UBE3A.

Answer 11

UBE3A - biallelically expressed in most tissues, but preferentially expressed from maternal allele in brain

Produces the E6-associated protein (E6AP), an E3 ubiquitin ligase involved in protein degradation

Disruption could affect crucial neuronal processes of protein degradation and replacement and is crucial for development of normal synapses

in mice loss of E6AP expression results in increased levels of synaptic protein Arc and impaired synaptic function, this may explain some features of AS patients (Kühnle et al 2013).

Question 12

What is the genotype/phenotype correlation between deletions and PWAS?

Answer 12

PWS and AS caused by deletions have a more severe phenotype (and hypopigmentation – caused by deletion of the OCA2 gene) compared to those with UPD or imprinting defects

Question 13

Describe the structure of the PWAS ICR.

Answer 13

The ICR has a bipartite structure

AS ICR is more centromeric, the PWS ICR more telomeric (including the SNRPN promoter and exon 1)

Question 14

What is the effect and recurrence risk associated with ICR deletions?

Answer 14

Micro-deletions to the ICR have a recurrence risk of up to 50%, if they have been inherited from a parent

1. Deletions including the PWS ICR only cause PWS when inherited from the father (they are passed silently from mother to child)
2. Deletions of the AS ICR only cause AS when inherited from the mother (they are passed silently from father to child)

Question 15

What are the causes of PWS, their frequency and recurrence risks?

Answer 15

Caused by loss of expression from the paternally inherited allele.

1. Paternally derived deletion (75-80%) <1% RR
2. matUPD (20-25%) <1% RR
3. imprinting defect on paternal allele (~1%) <1% RR
4. imprinting centre deletion (~10-15% of patients with an imprinting defect) Up to 50% RR if present in the father.

Question 16

What are the causes of Angelman Syndrome, their frequency and recurrence risks?

Answer 16

Caused by loss of expression from the maternally inherited allele.

1. Maternally derived deletion (70-75%) <1% RR
2. patUPD (3-7%) <1% RR
3. Imprinting defect on maternal allele (2-3%) <1% RR
4. Imprinting centre deletion (~10-15% of patients with an imprinting defect) RR up to 50% if present in the mother.
5. point mutation in UBE3A (~10%) 50% if present in the mother

Question 17

What proportion of Angelman Syndrome cases have no identifiable molecular abnormality. What is the recurrence risk in these cases?

Answer 17

~10% have no identifiable cause

RR is unknown, but may be up to 50%

Question 18

What is the mechanism underlying the recurrent 1.5Mb deletion in PWAS?

Answer 18

The deletions are thought to occur due to unequal cross over between low copy repeats of HERC2.

The 15q11-13 region contains three common break points:
BP1 and BP2 are both proximal of 15q11-q13
BP3 is distal of 15q11-q13.

The approximate frequency of deletions with these breakpoints is:
37% for BP1
60% for BP2
95% for BP3

Question 19

What genetic testing methods are in place for PWAS testing?

Answer 19

1. Cytogenetics - karyotype, FISH, array
2. Methylation studies -MS-MLPA, MS-PCR
3. Southern blotting
4. Microsatellite analysis
5. Sequence analysis of UBE3A

Question 20

What follow-up testing is required for patients with abnormal methylation but no UPD or deletion detected?

Answer 20

Referred to a specialist laboratory for analysis of the imprinting centre for the presence of a microdeletion.

If no IC del detected, an imprinting defect is assumed - low recurrence risk.

Question 21

What proportion of PWS cases have abnormal methylation?

Answer 21

> 99%

Consider an alternative diagnosis for patients with an absence of a methylation defect.

Question 22

Give two differential diagnoses for PWS.

Answer 22

1. Myotonic dystrophy (DM1) or spinal muscular atrophy (SMA1) for hypotonia
2. Craniopharyngioma and the results of its treatment
3. Hyperphagic short stature- an acquired condition which includes growth hormone insufficiency, hyperphagia and mild learning disabilities
4. Mat UPD 14-presents with neonatal hypotonia and later onset obesity
5. Imprinting defects or deletions in imprinted DLK1-MEG3 locus at 14q32.
6. Cohen syndrome, Bardet-Biedl syndrome- also present with obesity and developmental disability

Question 23

What proportion of Angelman syndrome cases have abnormal methylation?

Answer 23

80%

A diagnosis of AS cannot be excluded if abnormal methylation not detected.

Question 24

What proportion of Angelman syndrome cases due to an imprinting defect are mosaic? What is a consideration for these cases?

Answer 24

40%

May be missed by MS-PCR and Southern blotting as the maternal and paternal bands may appear of slightly different intensity in normal individuals

Question 25

When should sequence analysis of UBE3A be considered?

Answer 25

In patients without a methylation defect detected

UBE3A CNVs very rare

Question 26

What are the possible explanations for Angelman syndrome patients who do not have any detectable molecular mechanism of disease detected?

Answer 26

1. Undetected mutations in a regulatory region of UBE3A
2. Other unidentified mechanism(s)/gene(s) involved in UBE3A function
3. Incorrect clinical diagnosis.

Question 27

Give two differential diagnoses for Angelman syndrome.

Answer 27

1. Rett syndrome (in girls)
2. Mowat-Wilson syndrome
3. Pitt-Hopkins syndrome
4. Christianson syndrome (X-linked)
5. ATR-X
6. Mitochondrial encephalopathy
7. Lennox-Gastaut syndrome
8. MEF2C mutations
9. MTHFR deficiency
10. CDKL5 mutation

Question 28

What current treatments are available for PWAS patients?

Answer 28

These rely presently on the treatment of clinical features and symptoms and focus on improving quality of life Including:

1. Behavioral, physical, social therapies
2. Medical intervention for seizures (AS)
3. Use of specialised equipment for feeding difficulties and strict supervision on daily food intake (PWS)
4. Growth hormone therapy (can also improve mobility and behavioural problems)
5. Hyperphagia treatments – mostly in the research setting
6. Devices (also in research setting) to help improve appetite and behaviour in PWS include transcranial direct magnetic stimulation and vagal nerve stimulators.

Question 29

What future treatments may be possible for patients with Angelman syndrome?

Answer 29

UBE3A encodes E6-AP a protein ligase which is associated with protein degradation in proteasomes and is key to neuronal survival.

Genomic therapy could be used to restore UBE3A expression in neurons. This could be achieved by activating the expression of the paternally derived UBE3A gene. Two approaches are being developed, both involve inhibiting the expression of the UBE3A antisense strand on the paternal chromosome.

Artificial transcription factors (ATF) such as the CRISPR/Cas9 system can be used to target the SNRPN gene to inhibit the expression of UBE3A-ATS and thus enable the UBE3A gene to be expressed.

In an AS mouse model symptoms have be reduced by truncating UBE3A-ATS by inserting a poly(A) cassette. This enabled the paternal UBEA3

Topoisomerase inhibitors have also been shown to inhibit UBE3A-ATS expression in mice, however there are problems with specificity, toxicity and drug delivery