MS-MLPA Methylation Specific Multiplex Ligation Probe Amplification for detection of PWS/AS MRC-Holland Somai Man/Kate Gibson Oxford
MS-MLPA
Methylation Specific Multiplex Ligation Probe
Amplification for
detection of PWS/AS
MRC-HollandSomai Man/Kate Gibson
Oxford
MRC-Holland b.v.
MRC-Holland b.v.
Mechanisms causing PWS/AS
Aberrant CpG island methylation-S.Blot, MS-MLPA
DiseaseMechanism
PWS AS
Large Deletion 70% 70%
UPD 30% (mat)
5% (Pat)
IC mutation 1% 5%
Chromosome rearrangement
<1% <1%
Other - ~10% UBE3A mutation~ 10% Unknown
MRC-Holland b.v.
MRC-Holland b.v.
MS-MLPA (ME0028)
MS-MLPA, detects changes in:
• COPY NO. (Standard MLPA)
• Differences in methylation status at Chr15q11-13. (HhaI)
MRC-Holland b.v.
MRC-Holland b.v.
Mechanisms causing PWS/AS & effect seen using MS-MLPA kit
DiseaseMechanism
PWS AS MS-MLPA Change
Large Deletion 70% 70% Copy no. + Ch3n
UPD 30% (mat)
5% (Pat) Ch3n
IC mutation 1% 5% Ch3n
Chromosome rearrangement
<1% <1% -
Other - ~10% UBE3A mutation~ 10% Unknown
-
NB: other methods cannot cannot discern between these
MRC-Holland b.v.
MRC-Holland b.v.
PWS/AS MS-MLPA Probe Mix
• Test probes: specific for sequences within & near 15q11-13; 5 contain a HhaI recognition site (SNRPN & NECDIN)
• Control probes: for copy number & HhaI digestion
MRC-Holland b.v.
MRC-Holland b.v.
MS-MLPA Procedure
Overnight hybridisation
Reaction split into 2 tubes
Ligation & PCR as with standard MLPA
COPY NO.
Add HhaI, Ligate & PCR
METHYLATION
MRC-Holland b.v.
MRC-Holland b.v.
• HhaI cuts at unmethylated sites• Only methylated (undigested) &
ligated probes are exponentially amplified
HhaI
•Methylated signal•Unmethylated no signal
MRC-Holland b.v.
MRC-Holland b.v.
Understanding results-PWS
Normal
Pm
Ch3
Expression
One methylation dose
Copy No: 2
mUPD
Doubling of Methylation dose
m
Ch3 Ch3
m
Same (2)
Deletion (pat)
m
Ch3
P
One methylation dose; like normal
Reduced (1)
P
IC
m
Ch3
Doubling of Methylation dose
Same (2)
Ch3
NB: UPD and IC indistinguishable. Linked markers
MRC-Holland b.v.
MRC-Holland b.v.
Understanding results-AS
Normal
Pm
Ch3
Expression
One methylation dose
Copy No: 2
pUPD
PP
No methylation signal
Same (2)
IC
Pm
No methylation signal
Same (2)
Deletion (mat)
m P
No methylation signal
Reduced (1)
NB: UPD and IC indistinguishable. Linked markers
MRC-Holland b.v.
MRC-Holland b.v.
Control probes
Chr15q11-13 probes: in order down csome 15q
Control probes
Copy no. Results
100%
Equal heights of probes in PWS/AS critical region = NO
Normal Results
MRC-Holland b.v.
MRC-Holland b.v.
Normal Results
Control probes
Chr15q11-13 probes: in order down csome 15q
0.5
Copy no. Results Methylation Results
UndigestedControls
1
Differentially Ch3td alleles
0.5
Digested Controls
0Control probes
Equal heights of probes in PWS/AS critical region = NO
100%
MRC-Holland b.v.
MRC-Holland b.v.
Understanding Results
• Deletions: 50% drop in relative peak height. Stats-cfn betwn patient & normal controls
• Both results conclusive diagnosis
• Methyln abnormalities: difference in dose of
methyld allele•Simple pairwise cfn betwn cut & uncut results from same patient•Irrespective of mutation mechanism, methyln ratio for PWS= 1.0, AS= 0
MRC-Holland b.v.
MRC-Holland b.v.
Deletion Results
PWS AS
50%
Half heights of probes in PWS/ AS Critical region =
Doubling of methylation signal Loss of methylation signal
Copy no. Results
Methylation Results
0.5
MRC-Holland b.v.
MRC-Holland b.v.
UPD/ IC Results
PWS UPD/ IC AS UPD/ IC
Doubling of methylation signal Loss of methylation signal
Copy no. Results
Methylation Results
100%
0.5
Equal heights of probes in PWS/ AS Critical region = NO