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Genetics of Learning Disorders > Prader-Willi and Angelman Syndromes > Flashcards

Prader-Willi and Angelman Syndromes Flashcards

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1
Q

PWS and AS are examples of disorder associated with…..?

A

PWS and AS are examples of disorder associated with imprinted regions of the genome.

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2
Q

What are ‘imprinted’ genes?

A

Imprinted genes are expressed from only one parental chromosome. The expression pattern can be tissue specific or in all cells. The term ‘imprinted’ is often used to mean ‘repressed’, but not exclusively.

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3
Q

How many genes in the human genome are known to be imprinted?

A

Currently only approximately 80 of the 30,000 to 35,000 genes in the human genome are known to be imprinted.

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4
Q

Name some diseases associated with imprinted loci other than Prader-Willi and Angelman.

A
  • Beckwith-Wiedermann syndrome (11p15)
  • Silver-Russell syndrome (11p15)
  • Transient neonatal diabetes mellitus (6p22)
  • UPD 14 (14q32)
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5
Q

What imprinted region is Beckwith-Wiedermann syndrome associated with?

A
  • Beckwith-Wiedermann syndrome (11p15)
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6
Q

What imprinted region is Silver-Russell syndrome associated with?

A
  • Silver-Russell syndrome (11p15)
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7
Q

What imprinted region is transient neonatal diabetes mellitus associated with?

A
  • Transient neonatal diabetes mellitus (6p22)
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8
Q

What imprinted region is - UPD 14 associated with?

A
  • UPD 14 (14q32)
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9
Q

Describe imprinting.

A
  • Imprinted genes are expressed by only one parental chromosome.
  • Imprinted genes are often found in clusters.
  • Imprinting control elements/imprinting centres (IC) control the expression of the cluster.
  • The IC is cis-acting and can act over a long distance.
  • Maternally and paternally imprinted genes can be within the same cluster or expression can be specific to one parental chromosome.
  • Expression is controlled by epigenetic modification. Usually methylation of cytosine in CpG dinucleotides.
  • The methylation pattern must then be maintained throughout development.
  • Imprints must be erased and reset during germ cell formation - e.g. for when a male passes on an allele inherited from his mother.
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10
Q

Imprinted genes are often found in clusters. What elemts control the expression of the cluster?

A
  • Imprinted genes are often found in clusters.
  • Imprinting control elements/imprinting centres (IC) control the expression of the cluster.
  • The IC is cis-acting and can act over a long distance.
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11
Q

How is expression controlled by imprinting?

A
  • Expression is controlled by epigenetic modification. Usually methylation of cytosine in CpG dinucleotides.
  • The methylation pattern must then be maintained throughout development.
  • Imprints must be erased and reset during germ cell formation - e.g. for when a male passes on an allele inherited from his mother. All gametes must have the correct pattern for the sex of that person.
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12
Q

Why must imprinting be reset at gametogenesis?

A
  • Imprints must be erased and reset during germ cell formation - e.g. for when a male passes on an allele inherited from his mother.
  • All gametes must have the correct pattern for the sex of that person.
  • For example, for each chromosome pair a male will have one chromosome with a maternal impinting pattern and one with a maternal chromosome pattern. However, all of his sperm will need to have the paternal pattern so that the resultant embryo will have the correct balance of maternal and paternal expression.
  • The males chromosme with the maternal pattern must therefore be reset to have the paternal pattern.
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13
Q

What region are both Prader-Willi and Angelman syndrome associated with?

A

The imprinted region associated with PWS and AS is at 15q11-q13.

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14
Q

Describe the 15q11-q13 region that is associated wiith both Prader-Willi and Angelman syndrome.

A
  • The imprinted region associated with PWS and AS is at 15q11-q13. This region contains a number of genes.
  • In the brain UBE3A and ATP10C are only maternally expressed.
  • SNURF-SNRPN, NDN, MAGEL2 and MKRN3 are protein coding genes expressed from the paternal chromosome.
  • All of the other paternally expressed genes are snoRNAs.
  • HB11-85 (SNORD116) is in a cluster has 29 copies and HB11-52 (SNORD115) has 48 copies.
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15
Q

What are the main genes only expressed from the paternal chromosome 15q11-13?

A
  • SNURF-SNRPN, NDN, MAGEL2 and MKRN3 are protein coding genes expressed from the paternal chromosome.
  • All of the other paternally expressed genes are snoRNAs.
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16
Q

What are the main genes only expressed from the maternal chromosome 15q11-13?

A
  • In the brain UBE3A and ATP10C are only maternally expressed.
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17
Q

Describe the paternal chromosome 15q11-13 region.

A
  • The paternal chromosome region is generally unmethylated.
  • The protein coding genes have their own promoters.
  • SNURF-SNRPN has several tissue specific promoters and alternatively spliced transcripts.
  • Some of the longer transcripts include the snoRNAs and a very long brain specific transcript also includes UBE3A-AS.
  • UBE3A-AS is an antisense RNA of the end of the UBE3A gene.
  • Expression of UBE3A-AS prevents UBE3A expression in cis. As UBE3A is only expressed by the brain-specific transcript UBE3A will only be expressed from the maternal allele in the brain but biallelically elsewhere.
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18
Q

Describe the maternal chromosome 15q11-13 region.

A
  • On the maternal chromosome the CpG islands associated with the paternally expressed, proteing coding genes are methylated.
  • Methylation of the promoter regions prevents transcription factor binding and assembly of the transcription machinery.
  • Without expression from SNURF-SNRPN there is also no snoRNA or UBE3A-AS expression.
  • UBE3A is therefore expressed from the maternal chromosome.
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19
Q

Why isn’t UBE3A expressed from the paternal 15q11-13?

A
  • Some of the longer transcripts include the snoRNAs and a very long brain specific transcript also includes UBE3A-AS.
  • UBE3A-AS is an antisense RNA of the end of the UBE3A gene.
  • Expression of UBE3A-AS prevents UBE3A expression in cis. As UBE3A is only expressed by the brain-specific transcript UBE3A will only be expressed from the maternal allele in the brain but biallelically elsewhere. - On the maternal chromosome the CpG islands associated with the paternally expressed, proteing coding genes are methylated.
  • Methylation of the promoter regions prevents transcription factor binding and assembly of the transcription machinery.
  • Without expression from SNURF-SNRPN there is also no snoRNA or UBE3A-AS expression.
  • UBE3A is therefore expressed from the maternal chromosome.
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20
Q

Describe imprinting at 15q11-q13 with regards to the imprinting centre and how imprinting takes place.

A
  • The paternal imprinting centre is located at the 5’ end of the SNURF-SNRPN.
  • This region is required to maintain the paternal expression pattern.
  • The maternal (Angelman syndrome) imprinting centre is approximately 35kb upstream of the paternal (Prader Willi syndrome) imprinting centre and is required to set up the maternal expression pattern.
  • It has been proposed that during oogenesis factors bind to the maternal imprinting centre and promte methylation of the paternal imprinting centre.
  • The methylation then spreads to the other CpG islands in the region.
  • During spermatogenesis the maternal factors are not present and therefore the paternal imprinting centre remains unmethylated.
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21
Q

Describe Prader-Willi syndrome.

A
  • Prader-Willi syndrome is due to a loss of the paternal contribution from 15q11-q13
  • Incidence is about 1 in 15-20,000
  • Mild to moderate mental retardation
  • Hypotonia
  • Failure to thrive and feeding problems in neonatal period
  • Hyperphagia and obesity in later development
  • Male hypogonadism
  • Short stature
  • Small hands and feet
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22
Q

Describe Angelman syndrome.

A
  • Angelman syndrome can be due to a loss of maternal contribution from 15q11-q13
  • Incidence is 1 in 15-20,000
  • Severe mental retardation
  • Lack of speech
  • Hyperactivity
  • Happy demeanour and inappropriate laughing
  • Gait ataxia
  • Seizures
  • Microcephaly
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23
Q

What is the most common cause of both Prader-Willi and Angelman syndrome?

A
  • The most common cause of both Prader-Willi and Angelman syndrome is a large (about 4mb) deletion of 15q11-q13. Deletion on the paternal chromosome leads to Prader-Willi syndrome and deletion on the maternal chromosome leads to Angelman syndrome.
  • Approximately 4mb deletion of entire region.
  • Usually de novo, but small risk of germline mosaicism in parent.
  • Common breakpoints between patients.
  • Breakpoints have sequence homology.
  • Most likely due to non-allelic homologous recombination (NAHR).
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24
Q

Apart from a deletion of the 15q11-q13 region what other mechanisms can lead to Prader-Willi and Angelman syndrome?

A
  • Uniparental disomy (UPD) can also lead to a loss of one parent’s genetic contribution.
  • This is where both chromosomes originate from the same parent.
  • Can be 2 copies of the same chromosome (same chromosome duplicated = homodisomy) or one copy of each chromosome from the same parent (heterodisomy).
  • Maternal (m)UPD15 leads to Prader-Willi syndrome.
  • Paternal (p)UPD15 leads to Angelman-syndrome.
  • UPD is much more commonly seen in PW due to a higher non-disjunction rate in maternal non-disjunction at meiosis making an offspring with 2 maternal chromosome copies more likely.
  • Non-disjunction in maternal meiosis increases with maternal age and so UPD is far more prevalent in Prader-Willi cases in older mothers.
  • The UPD seen in Angelman cases is most commonly due to paternal isodisomy which arises when a normal sperm fertilises an egg nullisomic for chromosome 15. The single chromosome 15 is duplicated in a process called monosomy rescue. The nullisomic egg is a result of maternal nondisjunction.
  • All forms of UPD have a low recurrence risk if the parents are karyotypically normal.
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25
Q

What does maternal (m)UPD15 lead to?

A
  • Maternal (m)UPD15 leads to Prader-Willi syndrome.

- No paternal material to express has the same effect as deletion of the paternal segment!

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26
Q

What does paternal (p)UPD15 lead to?

A
  • Paternal (p)UPD15 leads to Angelman-syndrome.

- No maternal material to express has the same effect as deletion of the maternal segment!

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27
Q

Is UPD as and underlying mechanism more commonly found in Prader-Willi syndrome or Angelman syndrome?

A
  • UPD is much more commonly seen in PW due to a higher non-disjunction rate in maternal non-disjunction at meiosis making an offspring with 2 maternal chromosome copies more likely.
  • Non-disjunction in maternal meiosis increases with maternal age and so UPD is far more prevalent in Prader-Willi cases in older mothers.
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28
Q

What is the most common reason for the UPD seen in Angelman syndrome?

A
  • The UPD seen in Angelman cases is most commonly due to paternal isodisomy which arises when a normal sperm fertilises an egg nullisomic for chromosome 15. The single chromosome 15 is duplicated in a process called monosomy rescue. The nullisomic egg is a result of maternal nondisjunction.
  • Nondisjunction at meiosis 2 creating a nullisomic egg followed by isodisomy and monosomic rescue can lead to UPD.
29
Q

In addition to monosomy rescue what other rescue mechanism is a common event leading to UPD?

A
  • Trisomy rescue may also lead to UPD.
  • In this case 1 of the 3 copies of chromosome 15 is lost to give a viable chromosomally balanced embryo.
  • If 1 chromosome is deleted at random there is a 1 in 3 chance that the paternal copy will be deleted leaving only the 2 maternal copies and that the resulting embryo will have UPD.
30
Q

Can UPD arise as a result of paternal nondisjunction?

A

Yes, but maternal nondisjunction is a lot more common.

31
Q

How can heterodisomy (a form of UPD) arise due to trisomy rescue?

A

Heterodisomy can arise after trisomy rescue if the nondisjunction event occurs during meiosis 1. A cell that has duplicated it’s DNA and so contains 4 Chr15 copies may undergo a 4:0 split of chromosome between daughter cells. In normal meiosis 2 division will then result in eggs with 2 different maternal chromosome 15s. This egg is then fertilised so the zygote will initially contain 3 copies of chr 15 (2 different mat and 1 pat). If the zygote undergoes trisomic rescue then depending on which chromosome 15 gets deleted it may end up with maternal heterodisomy for chromosome 15.

32
Q

How can gamete complementation lead to UPD?

A
  • Gamete complementation is a theoretical possible mechanism for UPD and it is likely to be very rare.
  • This is where a nullisomic gamete from one parent and a complementary disomic gamete from the other parent form an embryo. The embryo therefore has UPD.
  • Depending on the timing of the nondisjunction event the results are either iso- or heterodisomy.
  • Nondisjunction at meiosis 2 produces isodisomy.
  • Nondisjunction at meiosis 1 produces heterodisomy.
33
Q

How can mitotic errors lead to UPD?

A
  • Mitotic errors can also lead to situations of nullisomy or trisomy which can be rescued resulting in either isodisomy or heterodisomy.
  • Depending on the timing of the mitotic error within development this may result in somatic mosaicism for UPD.
34
Q

What mechanisms that can result in UPD?

A
  • Nondisjunction
  • Monosomic rescue
  • Trisomic rescue
  • ?Gamete complementation

Combinations of nondisjunction at meiosis 1 or 2 which can be followed by rescue mechansims can result in UPD.

35
Q

What type of translocations is chromosome 15 most likely to be involved in? Why is this of note with regards to Prader-Willi and Angelman syndrome?

A
  • Chromosome 15 is commonly involved in Robertsonian translocations.
  • This type of translocation has no phenotypic impact on the carrier but there is an increased risk of UPD (and therefore PW or AS) in their children.
  • The translocation leads to an unusual trivalent chromosome at meiosis.
  • The trivalent is most likely to segregate to give balanced gametes, however, adjacent segregation will produce disomic or monosomic gametes leading to trisomy or monosomy in the embryo. Trisomy or monosmy rescue can then produce UPD.
36
Q

What is an imprinting defect? Describe imprinting defects.

A
  • An imprinting defect is a fault with the set up or maintenance of the expression pattern.
  • 10-15% of imprinting defects have an underlying microdeletion of one of the imprinting centres. The remainder have no clear mutation but the result is still an incorrect imprint being formed. Without a deletion the event is likely to have been sporadic and has a low recurrence risk. However, microdeletions can be inherited with a recurrence risk of up to 50%.
  • A microdeletion of the maternal IC will cause Angelman syndrome if inherited maternally. Without the IC the individual will express the paternal genes (with pat copy essentially now have 2 x paternal pattern chromosome 15s). However, if the same microdeletion is inherited paternally (i.e. a paternal chromosome with no maternal IC) then the individual will have normal expression of mat and pat chromosomes. The mat copy will have the mat IC to suppress paternal expression and we don’t want pat expression suppressed by the mat IC in the pat copy anyway! The opposite applies to paternal IC inheritance.
  • The unusual expression of 15q11 may allow the deletion to be transmitted silently depending on sex (e.g. transmission of a mat IC deletion can be inherited silently from a male as the correct paternal imprint is still established).
37
Q

What does the maternal imprinting centre do?

A

Regulates silencing of the paternal expression profile - essentially silences genes that are not supposed to be maternally expressed.

38
Q

What does the paternal imprinting centre do?

A

Regulates silencing of the maternal expression profile - essentially silences genes that are not supposed to be paternally expressed.

39
Q

How is it possible for an imprinting centre deletion to be transmitted silently?

A
  • The unusual expression of 15q11 may allow the deletion to be transmitted silently depending on sex (e.g. transmission of a mat IC deletion can be inherited silently from a male as the correct paternal imprint is still established).
40
Q

What will a maternally inherited microdeletion of the maternal imprinting centre result in?

A
  • A microdeletion of the maternal IC will cause Angelman syndrome if inherited maternally. Without the IC the individual will express the paternal genes (with pat copy essentially now have 2 x paternal pattern chromosome 15s). However, if the same microdeletion is inherited paternally (i.e. a paternal chromosome with no maternal IC) then the individual will have normal expression of mat and pat chromosomes. The mat copy will have the mat IC to suppress paternal expression and we don’t want pat expression suppressed by the mat IC in the pat copy anyway! The opposite applies to paternal IC inheritance.
41
Q

What single gene point mutation has been found to give rise to Prader-Willi syndrome?

A

No single gene point mutation has been found to give rise to Prader-Willi syndrome.

42
Q

What single gene point mutation has been found to give rise to Prader-Willi syndrome?

A
  • Point mutations in UBE3A can also cause Angelman syndrome.
  • Loss of the maternally expressed ATP10C gene at 15q11-13 is not thought to have an impact on the phenotype of AS. Pathogenesis is mostly due to the loss of UBE3A expression in the brain. A deactivating point mutation in UBE3A therefore has the same effect as losing the maternal imprint.
  • Angelman syndrome patients with UBE3A mutations will show a normal methylation pattern at 15q11-q13. This is detectable by sequencing UBE3A.
  • UBE3A point mutations can be inherited silently from males, but will lead to AS if inherited maternally.
  • There is a recurrence risk of up to 50%.
43
Q

What percentage of PWS cases are caused by a large deletion of 15q11-q13?

A

75-80%

44
Q

What percentage of PWS cases are caused by mUPD?

A

20-25%

45
Q

What percentage of PWS cases are caused by imprinting defect?

A

1%

46
Q

What percentage of AS cases are caused by a large deletion of 15q11-q13?

A

70-75%

47
Q

What percentage of AS cases are caused by pUPD?

A

3-7%

48
Q

What percentage of AS cases are caused by imprinting defect?

A

2-3%

49
Q

What percentage of AS cases are caused by UBE3A mutation?

A

14%

50
Q

What percentage of AS cases are of unknown cause?

A

approximately 10%

51
Q

What percentage of AS cases are mosaic for an imprinting defect?

A

approximately 1%

52
Q

Describe the pathogenesis of Prader-Willi syndrome.

A
  • None of the individual protein coding genes at 15q11-q13 have been linked to PWS.
  • No point mutations in the single genes have been found.
  • Knockout mice for each of these genes may show some subtle features of the PWS phenotype, but do not mimic the entire human phenotype.
  • Loss of expression from the SNORD116 snoRNA cluster is now thought to underlie the PWS phenotype.
  • PWS patients have been found with translocations removing the SNORD116 from the SNURF-SNRPN promoter, while maintaining the correct imprinting pattern of the coding genes.
  • A microdeletion of the SNORD116 cluster has also been in a child with PWS.
  • SNORD116 has a possible role in the regulation of alternative splicing.
53
Q

Describe the pathogenesis of Angelman syndrome.

A
  • Pathogenesis of Angelman syndrome is linked to loss of UBE3A expression in the brain.
  • Ubiquitin-protein ligase E3A is involved in the ubiquitination pathway, which targets selected proteins for degradation.
  • Aberrant protein degradation interferes with correct neuronal development.
  • Exact mechanism is still unknown.
  • Possible general repression of transcription associated with loss of UBE3A in certain brain cells.
54
Q

What techniques may be used to confirm a diagnosis or Prader-Willi or Angelman syndrome?

A

1) . Karyotyping:
- Will detect most 15q11-q13 deletions and any rare translocations interrupting 15q11-q13
- Slow in comparison to other techniques
- Will not detect UPD, imprinting defects or UBE3A mutations

2) . FISH:
- Use SNRPN probe
- Rapid technique
- Will still miss UPD, imprinting defects and UBE3A mutations
- Probes can be designed specifically to detect IC deletions

3) . aCGH:
- Similar advantages and disadvantages to FISH
- Possible to detect IC deletions with good probe depth

55
Q

What are the advantages and disadvantages of using karyotyping to confirm a diagnosis or Prader-Willi or Angelman syndrome?

A

1) . Karyotyping:
- Will detect most 15q11-q13 deletions and any rare translocations interrupting 15q11-q13
- Slow in comparison to other techniques
- Will not detect UPD, imprinting defects or UBE3A mutations

56
Q

What are the advantages and disadvantages of using FISH to confirm a diagnosis or Prader-Willi or Angelman syndrome?

A

2) . FISH:
- Use SNRPN probe
- Rapid technique
- Will still miss UPD, imprinting defects and UBE3A mutations
- Probes can be designed specifically to detect IC deletions

3) . aCGH:
- Similar advantages and disadvantages to FISH
- Possible to detect IC deletions with good probe depth

57
Q

What methylation specific method that requires Southern blotting can be used to detect PW and AS?

A
  • Restriction digest with methylation sensitive enzymes and then Southern blotting can be used can be used to detect the abnormal methylation pattern. This technique allows for the detection of the maternal or paternal methylation pattern and can therefore show the presence of both in an unaffected patient or the loss of either in an affected patient. Loss of one pattern could be due to a large deletion, UPD, or imprinting defect.
  • In the Notts lab the Xba1 and Not1 enzymes are used prior to blotting.
  • Xba1 will cut the DNA whether it is methylated or not to create a large 4.2kb fragment containing the kb17 probe sites.
  • Not1 will only digest its’ target sites if they are unmethylated. As the paternal chromosome is unmethylated Not1 will cut the DNA fragment containing the probe site (digested to 4.2 kb by Xba1) to 0.9kb.
  • The mathernal chromosome is unmethylated and so will not be digested by Not1 and will remain at 4.2kb.
  • When run on a blot a normal patient will have both 4.2kb and 0.9kb bands as both the maternal and paternal chromosomes are present in the sample. A patient with PW will only show the 4.2kb maternal band. A patient with AS will only show the 0.9kb band.
  • Digested DNA is hybridised with KB17 probe: binds SNRPN gene promoter and non-coding exon 1.
58
Q

What kind of DNA will Xba1 cut?

A
  • Xba1 will cut the DNA whether it is methylated or not to create a large 4.2kb fragment containing the kb17 probe sites.
59
Q

What kind of DNA will Not1 cut?

A
  • Not1 will only digest its’ target sites if they are unmethylated. As the paternal chromosome is unmethylate Not1 will cut the DNA fragment containing the probe site down to 0.9kb.
60
Q

Will Not1 digest the maternal chromosome?

A

No. Not1 only digests unmethylated DNA and the maternal chromosome is methylated.

61
Q

What is the advantage of focussing PW/AS detection methods on the methylation patterns?

A
  • By analysing the methylation pattern of 15q11-q13 cases of PWS and AS with large deletions, UPD or imprinting defects can all be detected.
  • A good quality blot will even detect the rare cases of mosaicism.
  • Southern blotting is labour intensive, time consuming, uses a large amount of DNA and can involve the use of radioactively labelled probes.
  • Methylation-specific (MS) PCR is an alternative technique that can be used to identify the majority of PWS and AS cases.
  • Prior to PCR the DNA is treated with sodium bisulphite to allow differentiation between methylated and unmethylated DNA.
62
Q

What method other than restriction digest with methylation-specific enzymes followed by blotting can be used to detect methylation methylation patterns when testing for PW and AS? Describe this method.

A
  • Methylation-specific (MS) PCR is an alternative technique that can be used to identify the majority of PWS and AS cases.
  • Prior to PCR the DNA is treated with sodium bisulphite to allow differentiation between methylated and unmethylated DNA.
  • The bisulphite treatment process is also known as DNA modification. The unmethylated cytosines are converted to uracil whereas the methylated cytosines are unmodified and remain as cytosine.
  • The final DNA sequence will now be different if the DNA was methylated or not.
  • Uracil acts as Thymine in base pairing and primers can be designed to take this into account.
  • MS-PCR utilises the now different sequence of methylated and unmethylated DNA to amplify different products from each. The PCR uses 2 sets of primers, one specific to the DNA sequence for the methylated DNA and one for the unmethylated DNA. The primer pairs are designed to produce products of different sizes to enable identification.
  • Primers from the methylated DNA will not amplify the unmethylated DNA due to the presence of uracil bases that interfere with the primer binding and vice versa.
  • PCR products are then run on a 2.5% agarose gel. The presence of 2 bands indicates normal imprinting at 15q11-q13 (mat and pat). Loss of a band represents the loss of that methylation pattern.
  • PCR is much quicker to perform than restriction methods.
63
Q

What does bisulphite treatment convert unmethylated cytosines to?

A

What does bisulphite treatment convert unmethylated cytosines to?

64
Q

What does bisulphite treatment convert methylated cytosines to?

A
  • These will not be converted and will remain as cytosines.
65
Q

What does MS-PCR utilise to differentiate methylated and unmethylated sequences after bisulphite modification?

A
  • MS-PCR utilises the now different sequence of methylated and unmethylated DNA to amplify different products from each. The PCR uses 2 sets of primers, one specific to the DNA sequence for the methylated DNA and one for the unmethylated DNA. The primer pairs are designed to produce products of different sizes to enable identification.
  • Primers from the methylated DNA will not amplify the unmethylated DNA due to the presence of uracil bases that interfere with the primer binding and vice versa.
  • PCR products are then run on a 2.5% agarose gel. The presence of 2 bands indicates normal imprinting at 15q11-q13 (mat and pat). Loss of a band represents the loss of that methylation pattern.
  • PCR is much quicker to perform than restriction methods.
66
Q

What are the disadvantages of MS-PCR for PW and AS testing?

A

1) . Possibility of a false-positive due to incomplete DNA modification:
- No product from the paternal allele (false PWS)
- Need to confirm positive results by repeating the modification and PCR

2) . False positive result due to SNP under the primer site:
- A polymorphism under any of the primer sites could prevent binding of the primer and a product being produced
- Could give either false PWS or AS

3) . Lack of information about underlying mechanism:
- The PCR can’t identify if the methylation pattern is due to UPD, deletion or imprinting defect
- Imprinting defect can give high recurrence risk

4) . Will not detect mosaicism:
- Low level of a normal chromosome will produce PCR product
- False negative result

67
Q

What is a more recent method of detecting Prader-Willi and Angelman syndrome than MS-PCR that is now used as the standard detection technique?

A
  • Methylation specific MLPA (MS-MLPA) is now used as the standard detection technique for PWS and AS.
  • Modified MLPA that allows detection of copy number changes at 15q11-q13 and the methylation pattern.
  • Allows detection of the 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 and abnormal methylation.
  • DNA does not require modification. Utilises a methylation sensitive enzyme.
  • Requires at least 3 normal controls.
  • AS positive, PWS positive and water controls are also used.
68
Q

How does MS-MLPA work? What is the differentiating step that makes MS-MLPA different from normal MLPA?

A
  • Each probe is designed as a pair of oligonucleotides.
  • The hybridisation sequences are specific to adjacent regions of the site of interest.
  • Each half of the oligonucleotide pair is usually at least 21 nucleotides in length to enable strong and specific binding.
  • To allow size differentiation of the probe kit a stuffer fragment is bound to one or both halves of the probe pair.
  • The stuffer fragment varies in length so that each probe in the kit will give a different sized PCR product.
  • At the ends of each probe are sequences specific to primers supplied in the kit.
  • All the probes in a kit contain the same primer sequence to allow easy and efficient multiplex amplification.
  • The PWAS kit contains 48 probes.

1) . Sample DNA (150ng) is denatured at 98C for 5 mins.
2) . Probe mix and buffer is added and the reaction is held at 60C for 16 hours to allow probe hybridisation.
3) . The hybridisation sequence of the probes bind to the target sequences of the sample DNA.
4) . A thermostable ligase is added to the reaction.
5) . 48C for 30 mins.
6) . If the 2 halves of the probe have bound to the target sequence then the adjacent hybridisation sequences are ligated together.
7) . 98C for 5 mins to inactivate the ligase.
8) . At this point the MS-MLPA differs from a normal MLPA. Rather than going straight to the PCR reaction the reaction containing the ligated products is split in half. One half of the ligation product is treated with a methylation sensitive restriction enzyme (e.g.Hha). The other half of the ligation products are not treated with this enzyme. Unmethylated DNA is digested and therefore not amplified. Methylated DNA is not digested and will be amplified in the PCR setup. For example, with a normal methylation pattern the probes bound to the maternal chromosome will remain undigested whereas those bound to the paternal will be digested. The half of the ligation product treated with a methylation sensitive restriction enzyme will be used for methylation detection. The untreated half will be used for copy number analysis.
9) . PCR buffers and primers are then added to both halves of the reaction and the PCR reaction is performed. One of the generic primers if fluorescently labelled to allow the products to be sized by capillary electrophoresis.

Genetics of Learning Disorders (10 decks)

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