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Improving molecular diagnosis of Beckwith Wiedemann syndrome
patients using methylation sensitive MLPA and
pyrosequencing.
Chris CampbellWest Midlands Regional Genetics laboratory
Beckwith Wiedemann syndrome (BWS)
• Incidence of 1 in 13,700
• Clinical features:
- Exomphalos, macroglossia and gigantism in the neonate.- Hemihyperplasia resulting in visceromegaly.- Increased risk of neoplasia specifically Wilm’s tumour.- Hypoglycemia at birth.
• Prognosis for long-term survival is favourable if neonatal problems are addressed.
• 85% of cases are sporadic and 15% are familial.
Molecular mechanisms for BWS at 11p15.5
Me
Me
KCNQ1 H19CDKN1C IGF2 H19DMRKVDMR1
ICR2 ICR1
10-20%Mosaic paternal
isodisomy at11p15.5
1-2%Cytogenetic duplication,translocation or inversion
50-60%Hypomethylation
at KvDMR1
2-7%Hypermethylation
at H19DMR
5-10%Mutations in CDKN1C
(40% autosomaldominant families)
Current testing strategy
BorderlineLOM ?
Combined Bisulphite Restriction Analysis
(COBRA) at KvDMR1
Loss of methylationAt KvDMR1
Molecular diagnosis of BWS(low recurrence risk associated
with these mechanisms)
UPD analysis usingmicrosatelite markers
at 11p15.5
Mosaic paternalisodisomy
Aims
1. Validate the use of methylation sensitive MLPA (MS-MLPA)
and pyrosequencing for BWS testing.
2. Comparison of the two methods with the existing COBRA method
to develop a new testing strategy for the laboratory.
3. Retrospective analysis of patients with unusual results by
previous testing.
Methylation sensitive MLPA
Commercial Kit from MRC Holland (ME030) which can detect most known genetic causes of BWS at 11p15.5.
Denatured genomic DNAMe
Stufferprim
er
primerHha1
Ligation and digestion with
Hha1
Ligation
Hybridisation
PCR amplification
Methylation indexH19DMR +KvDMR1
Deletion/duplication detection
Methylated DNAAll DNA
Validation
• MS-MLPA kit contains:
– 4 methylation sensitive probes specific for KvDMR1– 5 methylation sensitive probes specific for H19DMR
• 42 normal control were tested as well as BWS patients with known molecular mechanisms:
– 31 patients with hypomethylation at KvDMR1.– 8 patients with hypermethylation at H19DMR.– 17 patients with paternal isodisomy at 11p15.5.
Dosage assay Methylation assay
Results
Positive Negative
Normal (42) 0 42
KvDMR1 +ve (31) 31 0
H19DMR (8) 8 0
UPD +ve (17) 17 0
H19DMR probes
• 4 out of the 5 H19DMR ms-probes were unreliable showing wide standard deviations in the normal control cohort.
• These probes have been replaced in the latest version of the kit.
MLPA MI vs COBRA MI at KvDMR1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5
MLPA MI
CO
BR
A M
I
Deletions and duplications causing BWS
Dosage assay
PT
• Family history of exomphalos
• Deletion spanning KCNQ1 and CDKN1C.
• Further testing by MS-MLPA showed that her mother also carried the deletion.
• 50% recurrence risk
TD and CC
• Both with paternally derived duplications of the H19 region
• Concomitant hypermethylation of the H19DMR
FC
• Microsatellite analysis showed inheritance of two paternal alleles and one maternal allele at 3 markers.
• Large duplication on the paternal chromosome resulting in hypermethylation at H19DMR.
FC
Mother
Father
Pyrosequencing
Methylation analysis
• Bisulphite treatment of DNA creates a C / T at differentially methylated CpG sites.
• The ratio of C : (C + T ) is directly proportional to the degree of methylation at this site or Methylation index.
• Pyrosequencing is fully quantitative.
• 2 BWS pyrosequencing assays:– KvDMR1, analysing 7 CpG sites.– H19DMR, analysing 4 CpG sites.
150
200
E S C A C T C G C T C G T C T G T A G T A C T C
C:50.2%T:49.8%
C:47.1%T:52.9%
T:47.9%C:52.1%
C:49.9%T:50.1%
5 10 15 20
Example of a KvDMR1 run
150
E S A T C T G C T C G T C T G T A G T T A G T A G A T C T G T C T G T C T G T C T G T C T G
T:53.5%C:46.5%
C:0.0%T:100.0%
T:48.9%C:51.1%
T:49.0%C:51.0%
T:53.1%C:46.9%
T:50.6%C:49.4%
T:51.5%C:48.5%
T:49.3%C:50.7%
5 10 15 20 25 30 35 40
150
E S A T C T G C T C G T C T G T A G T T A G T A G A T C T G T C T G T C T G T C T G T C T G
T:84.5%C:15.5%
C:0.0%T:100.0%
T:80.0%C:20.0%
T:84.6%C:15.4%
T:82.9%C:17.1%
T:83.9%C:16.1%
T:86.2%C:13.8%
T:84.6%C:15.4%
5 10 15 20 25 30 35 40
CpG1 BC CpG2 CpG3 CpG4 CpG5 CpG6 CpG7
Normal
KvDMR1+ve
Example of a H19DMR run
120
140
160
E S C A C T C G C T C G T C T G T A G T C G A C T C
C:18.2%T:81.8%
C:15.0%T:85.0%
T:83.7%C:16.3%
T:94.1%C:5.9%
C:14.1%T:85.9%
5 10 15 20
120
140
160
E S C A C T C G C T C G T C T G T A G T C G A C T C
C:51.2%T:48.8%
C:48.3%T:51.7%
T:48.9%C:51.1%
T:92.6%C:7.4%
C:44.5%T:55.5%
5 10 15 20
CpG1 CpG2 CpG3 BC CpG4
Normal
H19+ve
Results
Pyrosequencing MI vs COBRA MI at KvDMR1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 10 20 30 40 50Pyrosequencing MI
CO
BR
A M
IPositive Negative
Normal (84) 0 84
KvDMR1 +ve (31) 31 0
H19DMR (9) 9 0
UPD +ve (10) 10 0
-Expensive (half volume reactions may be possible).
-Kit still under development by MRC Holland.
-Requires bisulphite treatment of the DNA.
-Some problems encountered with run failure.
-Deletion/duplication information.
-H19DMR/KvDMR1 defects detected in the same kit.
-Fast.
-Stand alone assays.
-Cheap.
-Could add H19 assay to existing protocol.
MLPAPyrosequencing
Disadvantages
Advantages
Future testing strategy
Molecular diagnosis of BWS(low recurrence risk associated
with these mechanisms)
ME030 MS-MLPA Kit MRC Holland
Molecular diagnosis of BWS(high recurrence risk associated
with these mechanisms)
MethylationDosage
or
Deletion or duplicationConfirmation of
UPDs usingmicrosatelite markers
at 11p15.5
Hypermethylation at H19DMR
Hypomethylationat KvDMR1
Acknowledgements
• Ana Bras-Goldberg and Richard Barber (Birmingham molecular genetics laboratory)
• Carol Hardy (Birmingham molecular genetics laboratory)
• Fiona Macdonald (Birmingham molecular genetics laboratory)
• Eammon Maher (Department of Medical and Molecular Genetics, University of Birmingham)
• Helen White (Salisbury molecular genetics laboratory) and Adam Smith (Hospital for Sick Children, Toronto)
