1 / 19

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.

velvet
Download Presentation

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

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related