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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:

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Chris Campbell West Midlands Regional Genetics laboratory

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  1. 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, macroglossiaand 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. CDKN1C KCNQ1 KVDMR1 IGF2 H19DMR H19 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 Me ICR2 ICR1 10-20% Mosaic paternal isodisomy at11p15.5 1-2% Cytogenetic duplication, translocation or inversion

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

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

  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. Methylation assay Dosage assay

  9. Results • 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.

  10. 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. • 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 Father • TD and CC • Both with paternally derived duplications of the H19 region • Concomitant hypermethylation of the H19DMR Mother Deletions and duplications causing BWS Dosage assay

  11. 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.

  12. Example of a KvDMR1 run Normal KvDMR1+ve CpG1 BC CpG2 CpG3 CpG4 CpG5 CpG6 CpG7 Normal CpG1 CpG2 CpG3BC CpG4 H19+ve Example of a H19DMR run

  13. Results

  14. Pyrosequencing MLPA Advantages -Fast. -Stand alone assays. -Cheap. -Could add H19 assay to existing protocol. -Deletion/duplication information. -H19DMR/KvDMR1 defects detected in the same kit. Disadvantages -Requires bisulphite treatment of the DNA. -Some problems encountered with run failure. -Expensive (half volume reactions may be possible). -Kit still under development by MRC Holland.

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

  16. 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)

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