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

Mechanisms causing PWS/AS Disease Mechanism PWS AS Large Deletion 70%  70% UPD  30% (mat)  5% (Pat) IC mutation 1% 5% Chromosome rearrangement <1% Other - ~10% UBE3A mutation ~ 10% Unknown Previously PWAS testing carried out by S blotting – this only detects aberrent methylation, so will distingiush between AS/PWS, but it will not confirm the mechanism of disease. Aberrant CpG island methylation-S.Blot, MS-MLPA

MS-MLPA (ME0028) MS-MLPA, detects changes in: COPY NO. (Standard MLPA) Differences in methylation status at Chr15q11-13. (HhaI) MS-MLPA can detect copy number variation and differences in methylation status, which means that the disease mechanism can be determined.

Mechanisms causing PWS/AS & effect seen using MS-MLPA kit Disease Mechanism PWS AS MS-MLPA Change Large Deletion 70%  70% Copy no. + Ch3n UPD  30% (mat)  5% (Pat) Ch3n IC mutation 1% 5% Chromosome rearrangement <1% - Other ~10% UBE3A mutation ~ 10% Unknown Large deletions show a copy number change along with aberrant methylation UPD/IC mutations show a normal copy number, but aberrant methylation. MS-MLPA cannot distinguish between UPD and IC mutations. NB: other methods cannot cannot discern between these

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

Ligation & PCR as with standard MLPA MS-MLPA Procedure Overnight hybridisation Reaction split into 2 tubes Ligation & PCR as with standard MLPA Add HhaI, Ligate & PCR Overnight hybridisation occurs as normal, then each reaction is split in two, one for ligation as normal for the copy no test, the other for ligation and HhaI digestion for the methylation status. COPY NO. METHYLATION

HhaI HhaI cuts at unmethylated sites Only methylated (undigested) & ligated probes are exponentially amplified Methylated  signal Unmethylated  no signal

Understanding results-PWS Normal mUPD Doubling of Methylation dose m Ch3 Same (2) P IC m Ch3 Doubling of Methylation dose Same (2) Deletion (pat) m Ch3 P One methylation dose; like normal Reduced (1) Ch3 m P Expression Normal - Maternal allele is methylated, paternal unmethylated = copy no = 2, one methylation dose mUPD – Two copys of mat chr 15 present, both copies methylated, but two copies are present, IC – Mat/Pat alleles present, but IC mut causes aberrant methylation of pat allele = 2 copies but double methylation dose. Del – Reduced copy no, methylation dose is normal. One methylation dose Copy No: 2 NB: UPD and IC indistinguishable. Linked markers

Understanding results-AS Normal pUPD P No methylation signal Same (2) IC P m No methylation signal Same (2) Deletion (mat) m P No methylation signal Reduced (1) Ch3 m P Expression Normal - As previous slide mUPD – Two copys of pat chr 15 present, both copies unmathylated – no signal on MS-MLPA, but two copies are present, IC – Mat/Pat alleles present, but IC mut means that mat allele is unmethylated = 2 copies but no methylation signal Del – Reduced copy no, methylation signal is lost, as the methylated region of mat chr 15 is not present. One methylation dose Copy No: 2 NB: UPD and IC indistinguishable. Linked markers

Equal heights of probes in PWS/AS critical region = NO  Normal Results Copy no. Results 100% Control probes Now to show you some examples of results. Firstly typical results from a normal individual. And let’s consider the copy no. results first. The Y axis plots the copy number as a % figure and the X axis lists probes which map to different chromosomal regions. The blue + pink bars indicate copy number of control probes. The green bars indicate copy number of probes in order down chromosome 15q11-q13. The red dotted line indicates the normal level that should be present. Control probes (blue and pink) are the same height as the green test probes, indicating that there has not been any loss in copy number, i.e. no deletion. Chr15q11-13 probes: in order down csome 15q Control probes Equal heights of probes in PWS/AS critical region = NO 

Equal heights of probes in PWS/AS critical region = NO  Normal Results Copy no. Results Methylation Results 100% 0.5 Undigested Controls 1 Differentially Ch3td alleles 0.5 Digested Controls 0 Control probes Normal reults, copy number on left, methylation results right Dosage – Control probes in blue and red, critical region probes in green. All probes show at the same relative height – shown by the red 100% line therefore normal copy no. Methylation results. The green bars are our digest control probes. These should not be digested and always be present at the 100% level. The red arrows indicate digest control probes: these should ideally be completely digested. The white bars are the important ones to focus on. These are the differentially methylated probes and should show a half height pattern: representing maternal methylated alleles and unmethylated paternal alleles. Chr15q11-13 probes: in order down csome 15q Control probes Equal heights of probes in PWS/AS critical region = NO 

Understanding Results Deletions: 50% drop in relative peak height. Stats-cfn betwn patient & normal controls 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 Deletions show as an approximate 50% drop in relative peak height, when compared to control probes and normal control samples. Methylation abnormalities are calculated by a pairwise comparison between the cut and uncut results from the same patient. This means that although a PWS deletion is normally methylated, it always gives a methylation ratio of 1 because there is no second allele to compare itself to. AS always gives a methylation ratio of 0. Both results  conclusive diagnosis

Deletion Results PWS  AS  Copy no. Results Methylation Results 50% Half heights of probes in PWS/ AS Critical region =  Methylation Results What results may we get from deletion patients then? The left of the slide summarises results from a PWS patient and the right results from an AS  patient. Looking at the top row, you can see that the green probes, i.e. probes mapping into the 15q11q-13 region show half heights, indicating that there is a . The origin of this  can be deduced looking at the methylation data below. In cases of a paternal  causing PWS, only maternal alleles are present and these are fully methylated (represneted by the white bars in the bottom left chart) In cases of a maternal  causing AS, only paternal alleles are present and these are unmethylated (no white bars) 0.5 Doubling of methylation signal Loss of methylation signal

UPD/ IC Results PWS UPD/ IC AS UPD/ IC Copy no. Results 100% Equal heights of probes in PWS/ AS Critical region = NO  Methylation Results This slide summarises typical from IC/ UPD cases. Again, The left of the slide summarises results from a PWS patient and the right, results from an AS patient. The copy no. results indicate that there has been no , with all probe bars showing equal heights. However, the methylation data is abnormal. In PWS cases, the effective doubling in methylation dose indicates that there are 2 copies of maternal (methylated) alleles (caused by UPD or an IC mutation). In AS cases, the methylation signal disappears indicative of there being 2 copies of paternal (unmethylated) alleles (caused by UPD or an IC mutation). 0.5 Doubling of methylation signal Loss of methylation signal