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Cath Willoughby SW Thames Molecular Genetics Diagnostic Laboratory - St George’s

Development of Methylation Specific High Resolution Melt analysis for detection of 11p15 methylation abnormalities and comparison to MS-MLPA. Cath Willoughby SW Thames Molecular Genetics Diagnostic Laboratory - St George’s. 11p15 region. CH 3. CH 3. IGF2. H19. KCNQ1OT1. KCNQ1 Ex11-16.

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Cath Willoughby SW Thames Molecular Genetics Diagnostic Laboratory - St George’s

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  1. Development of Methylation Specific High Resolution Melt analysis for detection of 11p15 methylation abnormalities and comparison to MS-MLPA. Cath Willoughby SW Thames Molecular Genetics Diagnostic Laboratory - St George’s

  2. 11p15 region CH3 CH3 IGF2 H19 KCNQ1OT1 KCNQ1 Ex11-16 KCNQ1 Ex1-10 CDKN1C Pat H19 KvDMR CH3 CH3 H19 IGF2 KCNQ1OT1 KCNQ1 Ex11-16 KCNQ1 Ex1-10 CDKN1C Mat H19 KvDMR Imprinted domain 1 Imprinted domain 2

  3. 11p15 abnormalities – Opposite syndromes • Beckwith-Wiedemann syndrome (BWS) • Macrosomia (Overgrowth) • Macroglossia (Large tongue) • Exomphalos (Abdominal contents develop outside body wall) • Hemihypertrophy (Body asymmetry) • Increased risk of Wilms’ Tumour

  4. 11p15 abnormalities – Opposite syndromes • Silver-Russell syndrome (SRS) • Undergrowth (Intrauterine growth retardation and poor postnatal growth) • Classical facial features including a triangular shaped face, prominent forehead and pointy chin • Hemihypertrophy (Body asymmetry) • Clinodactyly (Finger deflections)

  5. 11p15 abnormalities BWS • Sporadic Loss of methylation of KvDMR – 50-60% • Sporadic Gain of methylation of H19 – 2-7% • Paternal UPD - ~20% • CDNK1C mutations • + other rare causes CH3 CH3 IGF2 H19 KCNQ1OT1 KCNQ1 Ex11-16 Pat KCNQ1 Ex1-10 CDKN1C H19 KvDMR CH3 CH3 H19 IGF2 KCNQ1OT1 KCNQ1 Ex11-16 KCNQ1 Ex1-10 CDKN1C Mat H19 KvDMR

  6. CH3 CH3 11p15 abnormalities SRS • Sporadic Loss of methylation of H19 – Majority of cases • Maternal duplications -~4% • Maternal UPD – 1 reported case IGF2 H19 KCNQ1OT1 KCNQ1 Ex11-16 KCNQ1 Ex1-10 CDKN1C Pat H19 KvDMR CH3 CH3 H19 IGF2 KCNQ1OT1 KCNQ1 Ex11-16 KCNQ1 Ex1-10 CDKN1C Mat H19 KvDMR

  7. Techniques for diagnosis of BWS and SRS - Methylation-specific High Resolution Melt Analysis (MS-HRM) • Alders et al.,2008 Eur J Hum Genet. Advance online publication Oct 15 2008 CH3 CH3 CH3 C C C Pat G G G C C C Mat G G G Bisulphite modification of genomic DNA C C C Pat G G G T T T Mat A A A PCR across each imprinting centre (H19 and KvDMR) C C C ************************************ G G G A A A ************************************ T T T

  8. Techniques for diagnosis of BWS and SRS - Methylation-specific High Resolution Melt Analysis LightScanner

  9. Aims of the project • Develop and validate Methylation-specific High Resolution Melt Analysis (MS-HRM) of H19 and KvDMR for diagnostic testing of BWS and SRS referrals • Complete validation of Methylation-specific MLPA (MS-MLPA) (Scott et al.,2008 J Med Genet 45;106-13) • Compare MS-HRM and MS-MLPA by testing a cohort of patients

  10. 100% methylation control Normal methylation index = 0.5 Normal controls 0.37 0.03 Methylation Specific - High Resolution Melt Analysis (MS-HRM) Validation at KvDMR 100% methylated control DNA 14 normal samples 6 Loss of methylation samples (BWS) • Level of plateau in abnormal samples corresponds to previously determined methylation indices • Therefore this technique not only identifies loss of methylation but also indicates its degree

  11. Methylation Specific - High Resolution Melt Analysis (MS-HRM) Validation at H19 100% methylated control DNA 13 normal samples 4 hypermethylated samples (BWS) 5 Loss of methylation samples (SRS) 100% methylation control Normal controls

  12. Cohort study - samples

  13. Cohort study – Summary of results

  14. Significantly below 0.5 => loss of methylation Significantly above 0.5 => hypermethylation Cohort study – non concordant result Original MS-HRM analysis JS Pat UPD H19 LOM KvDMR LOM Repeat MS-HRM analysis

  15. Cohort study - results

  16. Cohort study - Further testing • Microsatellite analysis confirmed 3 cases of Paternal UPD Imprinted region p15.4 D11S1997 (D11S4957) D11S2071 D11S922 D11S2362 D11S1997 D11S1984

  17. Cohort study - Further testing • CDKN1C sequencing in 12 BWS 11p15 normal patients identified from the cohort - 1 previously identified probable mutation (c.956+1G>A)

  18. Summary • Developed MS-HRM for confirmation of MS-MLPA results • Sensitive technique for detection of methylation abnormalities at 11p15 • 99% concordance between MS-MLPA and MS-HRM • Offering testing of 11p15 for BWS, SRS and isolated features of disease using MS-MLPA as a 1st screen, supported by MS-HRM, microsatellite analysis and sequencing of CDKN1C.

  19. Acknowledgments • Marielle Alders – Department of Clinical Genetics, Academic Medical Centre, Amsterdam • Rohan Taylor, Liz Ormshaw, Alice Johnson-Marshall, Sally Cottrell, Nadiya Mahmud and the rest of the DNA laboratory at St George’s • Kate Tatton-Brown – St George’s • Naz Rahman,Richard Scott and Linda Baskcomb – The Institute of Cancer Research: Royal Cancer Hospital, Sutton

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