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