Improving molecular diagnosis of
Beckwith Wiedemann syndrome
patients using
methylation sensitive MLPA and
pyrosequencing.
Chris Campbell
West 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
5-10%
Mutations in CDKN1C
(40% autosomal
dominant families)
50-60%
2-7%
Hypomethylation
at KvDMR1
Hypermethylation
at H19DMR
Me
CDKN1C
KVDMR1
KCNQ1
IGF2
H19DMR H19
Me
ICR2
ICR1
10-20%
1-2%
Mosaic paternal
isodisomy at11p15.5
Cytogenetic duplication,
translocation or inversion
Current testing strategy
UPD analysis using
microsatelite markers
at 11p15.5
Mosaic paternal
isodisomy
Combined Bisulphite
Restriction Analysis
(COBRA) at KvDMR1
Loss of methylation
At KvDMR1
Molecular diagnosis of BWS
(low recurrence risk associated
with these mechanisms)
Borderline
LOM ?
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.
Hha1
Denatured genomic DNA
Me
Hybridisation
Ligation and
digestion with
Hha1
Ligation
PCR amplification
All DNA
Deletion/duplication
detection
Methylated DNA
Methylation index
H19DMR +KvDMR1
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
0
42
31
0
H19DMR (8)
8
0
UPD +ve (17)
17
0
Normal (42)
KvDMR1 +ve (31)
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
0.7
COBRA MI
0.6
0.5
0.4
0.3
0.2
0.1
0
0
0.05
0.1
0.15
0.2
0.25
MLPA MI
0.3
0.35
0.4
0.45
0.5
Deletions and duplications causing BWS
Dosage assay
FC
PT
• Family history of
• Microsatellite analysis showed
exomphalos
inheritance
of two paternal alleles and
one maternal allele at 3 markers.
• Deletion spanning KCNQ1
FC and CDKN1C.
• Further testing by MSMLPA showed that her
mother also carried the
deletion.
Father
• 50% recurrence risk
TD and CC
Mother
• Both with paternally derived
duplications of the H19
region
• Concomitant
• Large
duplication on the paternal
hypermethylation
chromosome resulting inof the
hypermethylation
H19DMR at H19DMR.
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.
Example of a KvDMR1 run
Normal
CpG1
BC
CpG2
CpG3
CpG4
CpG5
BC
CpG4
KvDMR1+ve
Example of a H19DMR run
Normal
CpG1
H19+ve
CpG2
CpG3
CpG6
CpG7
Results
Positive
Negative
Normal (84)
0
84
KvDMR1 +ve (31)
31
0
H19DMR (9)
9
0
UPD +ve (10)
10
0
Pyrosequencing MI vs COBRA MI at KvDMR1
0.7
COBRA MI
0.6
0.5
0.4
0.3
0.2
0.1
0
0
10
20
30
Pyrosequencing MI
40
50
Pyrosequencing
Advantages
-Fast.
-Stand alone assays.
-Cheap.
-Could add H19 assay to
existing protocol.
Disadvantages -Requires bisulphite
treatment of the DNA.
-Some problems
encountered with run
failure.
MLPA
-Deletion/duplication
information.
-H19DMR/KvDMR1
defects detected in the
same kit.
-Expensive (half volume
reactions may be
possible).
-Kit still under
development by MRC
Holland.
Future testing strategy
ME030 MS-MLPA Kit
MRC Holland
Dosage
Methylation
Confirmation of
UPDs using
microsatelite markers
at 11p15.5
Hypermethylation
at H19DMR
Deletion or
duplication
or
Hypomethylation
at KvDMR1
Molecular diagnosis of BWS
(low recurrence risk associated
with these mechanisms)
Molecular diagnosis of BWS
(high recurrence risk associated
with these mechanisms)
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)