Prader-Willi and Angelman syndromes

Question 1

What is specific about the expression of imprinted genes?

Answer 1

They are expressed from only one parental chromosome

Question 2

What is the common type of epigenetic modification seen in control of expression of imprinted genes?

Answer 2

Methylation of cytosine in CpG dinucleotides

Question 3

When a male passes on an allele inherited from his mother, what must happen?

Answer 3

Imprints must be erased and reset during the germ cell formation

Question 4

What is the genomic region associated with PWAS?

Answer 4

15q11-q13

Question 5

Only one gene is associated with Angelman syndrome due to expression on maternal chromosome only. Which gene?

Answer 5

UBE3A - only expressed in the brain

Question 6

There are 4 protein coding genes expressed from the paternal chromosome - which is the main one?

Answer 6

SNURF-SNRPN

Question 7

What is the methylation status of the paternal and maternal chromosomes?

Answer 7

• Paternal: Generally unmethylated

- Maternal: CpG islands associated with paternally expressed, protein coding genes are methylated

Question 8

What is UBE3A-AS and what does it do?

Answer 8

• anti-sense RNA at end of UBE3A gene that will prevent expression of UBE3A in cis (same chromosome)

Question 9

How does the methylation on the maternal chromosome impact UBE3A expression?

Answer 9

• methylation of promoter regions blocks transcription factor binding and assembly of transcription machinery
• without expression from SNURF-SNRPN there is also no snoRNA or UBE3A-AS expression
• UBE3A is therefore expressed from the maternal chromosome

Question 10

Where are the PWS and AS imprinting centres located?

Answer 10

• PWS paternal imprinting centre is located at 5’ end of SNURF-SNRPN
• AS maternal imprinting centre is approx. 35kb upstream of this

Question 11

What is thought to happen with the imprinting centres at oogenesis/spermatogenesis?

Answer 11

• During oogenesis, factors bind to maternal IC and promote methylation of paternal IC. methylation spreads to other CpG islands in region
• During spermatogenesis maternal factors not present therefore paternal IC stays unmethylated

Question 12

What is the incidence rate of PWS/AS?

Answer 12

1 in 15-20,000

Question 13

What are Prader-Willi and Angelman syndromes due to?

Answer 13

Loss of paternal and maternal contributions from 15q11-q13, respectively

Question 14

What are the clinical features of PWS?

Answer 14

• mild to moderate mental retardation
• hypotonia
• failure to thrive/feeding problems in neonate
• hyperphagia/obesity in later development
• male hypogonadism
• short stature
• small hands and feet

Question 15

What are the clinical features of AS?

Answer 15

• severe mental retardation
• lack of speech
• hyperactivity
• happy demeanour and inappropriate laughter
• gait ataxia
• seizures
• microcephaly

Question 16

What are the potential disease mechanisms for PWS and AS?

Answer 16

• 15q11-q13 deletion (most common: ~70-80%)
• UPD: maternal for PWS (20-25%) or paternal for AS (3-7%)
• imprinting defect (~1-3%)
• UBE3A mutation (AS only: 14%)
• Unknown cause (AS only: ~10%)

Question 17

What is uniparental disomy and how does it impact on PWS/AS?

Answer 17

• both chromosomes are from same parent
• either two copies of same chromosome (isodisomy) or one copy of each chromosome from same parent (heterodisomy)
• Maternal UPD15 leads to PWS, paternal UPD15 leads to AS

Question 18

Is UPD more commonly seen in PWS or AS?

Answer 18

PWS

Question 19

What is the most common type of UPD in AS?

Answer 19

Paternal isodisomy

Question 20

What is the mechanism that results in paternal uniparental Isodisomy?

Answer 20

monosomy rescue (ND at meiosis II)

Question 21

What are the two mechanisms that can result in:

• maternal uniparental Isodisomy?
• maternal uniparental heterodisomy?

Answer 21

Both occur by trisomy rescue or gamete complementation with ND at either meiosis II (mUPID) or meiosis I (mUPHD)

Question 22

A carrier of what type of translocation involving chromosome 15 is at increased risk of having a child with PWS/AS? Why?

Answer 22

• Robertsonian translocation carrier

- Increased risk of monosomy 15 or trisomy 15 in zygote, which can lead to UPD15 via monosomy or trisomy rescue

Question 23

What is an imprinting defect?

Answer 23

Failure to set the correct parental expression pattern - often de novo with no obvious mutation

Question 24

10-15% of imprinting defects are Microdeletions of either imprinting centre - how are these affected differently?

Answer 24

• if maternal IC is deleted and chromosome is inherited maternally the child will have AS
• if paternal IC is deleted and chromosome is inherited paternally the child will have PWS
• However, a maternal IC deletion can be inherited silently from a male as the correct parental imprint is still established and vice versa

Question 25

What are the recurrence risks for an imprinting defect?

Answer 25

- Low recurrence risk for an imprinting defect without an IC deletion - If IC deletion is present, recurrence risk can be up to 50%

Question 26

What is PWS pathogenicity attributed to?

Answer 26

- no individual protein coding genes linked to PWS and no point mutations in single genes have been found - loss of expression from SNORD116 snoRNA cluster now thought to underlie PWS phenotype

Question 27

What is AS pathogenicity attributed to?

Answer 27

- Loss of UBE3A expression in the brain - UBE3A protein is involved in ubiquitination pathway, targeting selected proteins for degradation - aberrant protein degradation interferes with correct neuronal development

Question 28

What are the three main cytogenetic techniques involved in the detection of PWS/AS?

Answer 28

1. Karyotype - detects most deletions and any rare trans. Slow compared to other techniques and won't detect UPD, imprinting defects or UBE3A mutations 2. FISH - uses SNRPN probe. Rapid technique but will still miss UPD, imprinting defects and UBE3A mutations. Probes can be designed specifically to detect IC deletions. 3. ArrayCGH - similar advantages and disadvantages to FISH. Possible to detect IC deletions with good probe depth.

Question 29

What four molecular methods are used for detection of large deletions, UPD or imprinting defects in PWS/AS?

Answer 29

- Southern blotting - methylation specific PCR - methylation specific MLPA - microsatellite analysis (UPD only)

Question 30

Describe the process of southern blotting for PWS/AS

Answer 30

- can distinguish between pat (unmethylated) and mat (methylated) copies of PWAS critical region using a XbaI/NotI digest - NotI only cuts at unmethylated CpGs, XbaI cuts either methylated or unmethylated DNA - normal patient has both mat and pat bands - PWS patient has heavy mat band (methylated: cut by Xbal) with absent pat band (unmethylated: cut by both). AS patient has reverse scenario

Question 31

What step is undertaken prior to methylation specific PCR that allows differentiation between methylated and unmethylated DNA?

Answer 31

- Treatment with sodium bisulphite - Unmethylated cytosines are converted to uracil - Methylated cytosines are unmodified and remain as cytosine - Final DNA sequence will now be different if the DNA was methylated or not

Question 32

How does methylation-specific PCR work?

Answer 32

- Two sets of primers in one reaction - one set specific to methylated DNA sequence (maternal primers; amplify 174bp band) - other set complementary to unmethylated DNA sequence (paternal primers; amplify 100bp band) - different product sizes allow differentiation of products from methylated/unmethylated DNA using gel electrophoresis

Question 33

What are the disadvantages of methylation specific PCR?

Answer 33

- possibility of false positive due to incomplete DNA modification (no product from paternal allele = false PWS) - false positive result due to SNP under primer site (can give either false PWS or AS) - lack of info about underlying mechanism: PCR can't identify if the methylation pattern is due to UPD, deletion or imprinting defect - will not detect mosaicism

Question 34

What is methylation specific multiplex ligation-dependent probe amplification (MS-MLPA)?

Answer 34

- standard detection technique for PWS/AS - modified MLPA that allows detection of copy number changes at 15q11-q13 and methylation pattern - allows detection of common large deletion and smaller IC deletions - cases with UPD, or imprinting defects without an IC deletion, will have normal copy number at 15q11-q13 but abnormal methylation - DNA doesn't require modification

Question 35

The PWAS methylation specific MLPA kit contains 48 probes. What do these entail?

Answer 35

- Use control probes from genomic regions that should have normal copy number - Use probes from commonly deleted 15q11-13 region and some from just outside this region - Probe sites with normal methylation pattern are used to quantify methylation of the region - Some probes act as methylation controls: e.g. methylated on both mat and pat chromosome so shouldn't be digested, or unmethylated so act as digestion controls

Question 36

What is the dosage quotient with regards to PWAS MS-MLPA copy number results?

Answer 36

- analysis software produces dosage quotient for each probe by comparing peak height in internal and external controls - dosage quotients give indication of copy number compared to normal control samples: - 1.0 (0.8-1.2) = 2 copies - 1.5 (1.35-1.65) = 3 copies - 0.5 (0.35-0.65) = 1 copy - 0 = 0 copies

Question 37

What are the disadvantages of methylation specific MLPA?

Answer 37

- sensitive to PCR contaminants and DNA quality - can't be used to detect UBE3A point mutations - can get false positives due to SNP under probe - cost (due to multitude of controls on each run) - still can't differentiate between UPD and an imprinting defect without an IC deletion

Question 38

What is microsatellite analysis for UPD detection in PWAS?

Answer 38

- performed if MS-MLPA shows abnormal methylation with normal copy number - need parental DNA samples - chr15 microsatellite markers (predominantly located in 15q11-q13 region) are genotyped for affected patient and both parents

Question 39

Give details on prenatal diagnosis of PWAS

Answer 39

- referral = parents who carry chr15 translocations or those for whom an IC deletion was identified in previous child - involves microsatellite analysis - MS-MLPA is possible but depends on DNA quality (methylation status at SNRPN exon 1 is established early in embryonic development)