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

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% 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

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 ?

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.

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

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 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.

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

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 Example of a H19DMR run Normal KvDMR1+ve CpG1 BC CpG2 CpG3 CpG4 CpG5 CpG6 CpG7 Normal KvDMR1+ve Example of a H19DMR run CpG1 CpG2 CpG3 BC CpG4 Normal H19+ve

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

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

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)

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)