1. Chris Campbell West Midlands Regional Genetics laboratory
Improving molecular diagnosis of Beckwith Wiedemann syndrome patients using methylation sensitive MLPA and pyrosequencing.
Chris Campbell
West Midlands Regional Genetics laboratory

2. 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.

3. Molecular mechanisms for BWS at 11p15.5
5-10%
Mutations in CDKN1C
(40% autosomal
dominant families)
50-60%
Hypomethylation
at KvDMR1
2-7%
Hypermethylation
at H19DMR Me
KCNQ1
H19
CDKN1C
IGF2
H19DMR
KVDMR1 Me
ICR2
ICR1
10-20%
Mosaic paternal
isodisomy at11p15.5
1-2%
Cytogenetic duplication,
translocation or inversion

4. Current testing strategy
Combined Bisulphite
Restriction Analysis
(COBRA) at KvDMR1
Loss of methylation
At KvDMR1
Molecular diagnosis of BWS
(low recurrence risk associated
with these mechanisms)
UPD analysis using
microsatelite markers
at 11p15.5
Mosaic paternal
isodisomy
Borderline
LOM ?

5. Aims
Validate the use of methylation sensitive MLPA (MS-MLPA) and pyrosequencing for BWS testing.
Comparison of the two methods with the existing COBRA method to develop a new testing strategy for the laboratory.
Retrospective analysis of patients with unusual results by previous testing.

6. Methylation sensitive MLPA
Commercial Kit from MRC Holland (ME030) which can detect most known genetic causes of BWS at 11p15.5.
Stuffer
primer
Hha1
primer
Denatured genomic DNA Me
Ligation and
digestion with
Hha1
Ligation
Hybridisation
PCR amplification
Methylation index
H19DMR +KvDMR1
Deletion/duplication
detection
Methylated DNA
All DNA

7. 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.

8. Dosage assay
Methylation assay

9. Results Positive Negative 42 31 8 17 H19DMR probes
Normal (42) 42
KvDMR1 +ve (31) 31
H19DMR (8) 8
UPD +ve (17) 17
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.

11. 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 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.
Mother
Father
TD and CC
Both with paternally derived duplications of the H19 region
Concomitant hypermethylation of the H19DMR

12. 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.

13. Example of a KvDMR1 run Example of a H19DMR run Normal KvDMR1+ve
CpG BC CpG CpG CpG CpG CpG CpG7
Normal
KvDMR1+ve
Example of a H19DMR run
CpG CpG CpG BC CpG4
Normal
H19+ve

14. Results Positive Negative Normal (84) 84 KvDMR1 +ve (31) 31 H19DMR (9)84
KvDMR1 +ve (31) 31
H19DMR (9) 9
UPD +ve (10) 10

15. MLPA Pyrosequencing Advantages Disadvantages
-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.
MLPA
Pyrosequencing
Disadvantages
Advantages

16. Future testing strategy
ME030 MS-MLPA Kit
MRC Holland
Dosage
Methylation
Deletion or
duplication
Hypermethylation
at H19DMR
Confirmation of
UPDs using
microsatelite markers
at 11p15.5 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)

17. 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)