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

IGF2 H19 CDKN1C H19 Imprinted domain 1 Imprinted domain 2 KCNQ1 Ex1-10 KCNQ1OT1 KCNQ1 Ex11-16 IGF2 H19 CDKN1C H19 KCNQ1 Ex1-10 KCNQ1OT1 KCNQ1 Ex11-16 KvDMR Pat Mat CH 3 11p15 region KvDMR

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

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)

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 IGF2 H19 CDKN1C H19 KCNQ1 Ex1-10 KCNQ1OT1 KCNQ1 Ex11-16 IGF2 H19 CDKN1C H19 KCNQ1 Ex1-10 KCNQ1OT1 KCNQ1 Ex11-16 KvDMR Pat Mat CH 3 KvDMR

11p15 abnormalities Sporadic Loss of methylation of H19 – Majority of cases Maternal duplications -~4% Maternal UPD – 1 reported case SRS IGF2 H19 CDKN1C H19 KCNQ1 Ex1-10 KCNQ1OT1 KCNQ1 Ex11-16 IGF2 H19 CDKN1C H19 KCNQ1 Ex1-10 KCNQ1OT1 KCNQ1 Ex11-16 KvDMR Pat Mat CH 3 KvDMR

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 Bisulphite modification of genomic DNA C G T A Pat Mat C G C G CH 3 C C G C G G T A T A C G C G C G Pat Mat PCR across each imprinting centre (H19 and KvDMR) A T C G ************************************ A T A T C G C G

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

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

Methylation Specific - High Resolution Melt Analysis (MS-HRM) Validation at KvDMR 100% methylated control DNA 14 normal samples 6 Loss of methylation samples (BWS) Normal methylation index = 0.5 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 100% methylation control Normal controls

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

Cohort study - samples Referral reason Total per referral type BWS33 Exomphalos3 Wilms Tumour 5 Hemihypertrophy6 SRS35 Total samples tested 82

Cohort study – Summary of results Total samples tested 82 Concordant between MS-MLPA and MS- HRM 78 (99%) Concordant between MS-MLPA, MS-HRM and a previous report 31 Non-concordant between MS-MLPA and MS-HRM 1 Fail3

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

Cohort study - results Referral reason ResultTotalExpectedMechanism Total per mechanism Expected Positive12/33(36%)>85% Pat UPD 2(16%)20% BWSNormal21/33 KvDMR hypomethylation 10(84%)>50% Fail0/33 H19 hypermethylation 02-11% Positive0/3(0%)10-20% Pat UPD 0 ExomphalosNormal3/3 KvDMR hypomethylation 0 Fail0/3 H19 hypermethylation 0 Positive0/5(0%)3% Pat UPD 0 Wilms Tumour Normal3/5 KvDMR hypomethylation 0 Fail2/5 H19 hypermethylation 0 Positive4/6(66%)25% Pat UPD 3(75%)60% HemihypertrophyNormal2/6 KvDMR hypomethylation 1(25%)22% Fail0 H19 hypermethylation 011% Positive3/35(8.5%)20-65% Mat UPD 0Rare SRSNormal31/35 H19 hypomethylation 3(100%)~99% Fail1/35

Cohort study - Further testing Microsatellite analysis confirmed 3 cases of Paternal UPD D11S1984 D11S1997 (D11S4957) p15.4 D11S922D11S2362D11S1997 Imprinted region D11S2071

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)

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 1 st screen, supported by MS-HRM, microsatellite analysis and sequencing of CDKN1C.

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