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MS-MLPA Methylation Specific Multiplex Ligation Probe Amplification for detection of PWS/AS MRC-Holland Somai Man/Kate Gibson Oxford.

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Presentation on theme: "MS-MLPA Methylation Specific Multiplex Ligation Probe Amplification for detection of PWS/AS MRC-Holland Somai Man/Kate Gibson Oxford."— Presentation transcript:

1 MS-MLPA Methylation Specific Multiplex Ligation Probe Amplification for detection of PWS/AS MRC-Holland Somai Man/Kate Gibson Oxford

2 MRC-Holland b.v. Mechanisms causing PWS/AS Aberrant CpG island methylation-S.Blot, MS-MLPA Disease Mechanism PWSAS Large Deletion 70% UPD  30% (mat)  5% (Pat) IC mutation1%5% Chromosome rearrangement <1% Other-~10% UBE3A mutation ~ 10% Unknown

3 MRC-Holland b.v. MS-MLPA (ME0028) MS-MLPA, detects changes in: COPY NO. (Standard MLPA) Differences in methylation status at Chr15q (HhaI)

4 MRC-Holland b.v. Mechanisms causing PWS/AS & effect seen using MS-MLPA kit Disease Mechanism PWSASMS-MLPA Change Large Deletion 70% Copy no. + Ch3n UPD  30% (mat)  5% (Pat) Ch3n IC mutation1%5%Ch3n Chromosome rearrangement <1% - Other-~10% UBE3A mutation ~ 10% Unknown - NB: other methods cannot cannot discern between these

5 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

6 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

7 MRC-Holland b.v. HhaI cuts at unmethylated sites Only methylated (undigested) & ligated probes are exponentially amplified HhaI Methylated  signal Unmethylated  no signal

8 MRC-Holland b.v. Understanding results-PWS Normal P m Ch3 Expression One methylation dose Copy No: 2 mUPD Doubling of Methylation dose m 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

9 MRC-Holland b.v. Understanding results-AS Normal Pm Ch3 Expression One methylation dose Copy No: 2 pUPD P P 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

10 MRC-Holland b.v. Control probes Chr15q probes: in order down csome 15q Control probes Copy no. Results 100% Equal heights of probes in PWS/AS critical region = NO  Normal Results

11 MRC-Holland b.v. Normal Results Control probes Chr15q probes: in order down csome 15q 0.5 Copy no. ResultsMethylation Results Undigested Controls 1 Differentially Ch3td alleles 0.5 Digested Controls 0 Control probes Equal heights of probes in PWS/AS critical region = NO  100%

12 MRC-Holland b.v. Understanding Results Deletions: 50% drop in relative peak height. Stats-cfn betwn patient & normal controls Both results  conclusive diagnosis Methyl n abnormalities: difference in dose of methyl d allele Simple pairwise cfn betwn cut & uncut results from same patient Irrespective of mutation mechanism, methyln ratio for PWS= 1.0, AS= 0

13 MRC-Holland b.v. Deletion Results PWS  AS  50% Half heights of probes in PWS/ AS Critical region =  Doubling of methylation signalLoss of methylation signal Copy no. Results Methylation Results 0.5

14 MRC-Holland b.v. UPD/ IC Results PWS UPD/ IC AS UPD/ IC Doubling of methylation signalLoss of methylation signal Copy no. Results Methylation Results 100% 0.5 Equal heights of probes in PWS/ AS Critical region = NO 


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