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

IGF2H19 CDKN1CH19

Imprinted domain 1 Imprinted domain 2

KCNQ1 Ex1-10 KCNQ1OT1 KCNQ1 Ex11-16

IGF2H19 CDKN1CH19KCNQ1 Ex1-10 KCNQ1OT1 KCNQ1 Ex11-16

KvDMRPat

Mat

CH3CH3

CH3CH3

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

IGF2H19 CDKN1CH19KCNQ1 Ex1-10 KCNQ1OT1 KCNQ1 Ex11-16

IGF2H19 CDKN1CH19KCNQ1 Ex1-10 KCNQ1OT1 KCNQ1 Ex11-16

KvDMRPat

Mat

CH3CH3

CH3CH3

KvDMR

11p15 abnormalities

•Sporadic Loss of methylation of H19 – Majority of cases

•Maternal duplications -~4%

•Maternal UPD – 1 reported case

SRS

IGF2H19 CDKN1CH19KCNQ1 Ex1-10 KCNQ1OT1 KCNQ1 Ex11-16

IGF2H19 CDKN1CH19KCNQ1 Ex1-10 KCNQ1OT1 KCNQ1 Ex11-16

KvDMRPat

Mat

CH3CH3

CH3CH3

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 15 2008

Bisulphite modification of genomic DNA

CG

TA

Pat

Mat

CG

CG

CH3 CH3CH3

CCGC

GG

TA

TA

CG

CG

CGPat

Mat

PCR across each imprinting centre (H19 and KvDMR)

AT

CG

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AT

AT

CG

CG

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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)

0.03

0.37

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 reasonReferral reason Total per referral typeTotal per referral type

BWSBWS 3333

ExomphalosExomphalos 33

Wilms TumourWilms Tumour 55

HemihypertrophyHemihypertrophy 66

SRSSRS 3535

Total samples testedTotal samples tested 8282

Cohort study – Summary of results

Total samples Total samples testedtested 8282

Concordant between Concordant between MS-MLPA and MS-MS-MLPA and MS-HRMHRM

78 (99%)78 (99%)

Concordant between Concordant between MS-MLPA, MS-HRM MS-MLPA, MS-HRM and a previous reportand a previous report

3131

Non-concordant Non-concordant between MS-MLPA between MS-MLPA and MS-HRMand MS-HRM

11

Fail Fail 33

Cohort study – non concordant result

JS

Pat U

PD

H19 L

OM

KvD

MR

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 reasonReferral reason ResultResult TotalTotal ExpectedExpected MechanismMechanismTotal per Total per

mechanismmechanism ExpectedExpected

PositivePositive 12/33(36%)12/33(36%) >85%>85% Pat UPDPat UPD 2(16%)2(16%) 20%20%

BWSBWS NormalNormal 21/3321/33 KvDMR hypomethylationKvDMR hypomethylation 10(84%)10(84%) >50%>50%

FailFail 0/330/33 H19 hypermethylationH19 hypermethylation 00 2-11%2-11%

PositivePositive 0/3(0%)0/3(0%) 10-20%10-20% Pat UPDPat UPD 00

ExomphalosExomphalos NormalNormal 3/33/3 KvDMR hypomethylationKvDMR hypomethylation 00

FailFail 0/30/3 H19 hypermethylationH19 hypermethylation 00

PositivePositive 0/5(0%)0/5(0%) 3%3% Pat UPDPat UPD 00

Wilms TumourWilms Tumour NormalNormal 3/53/5 KvDMR hypomethylationKvDMR hypomethylation 00

FailFail 2/52/5 H19 hypermethylationH19 hypermethylation 00

PositivePositive 4/6(66%)4/6(66%) 25%25% Pat UPDPat UPD 3(75%)3(75%) 60%60%

HemihypertrophyHemihypertrophy NormalNormal 2/62/6 KvDMR hypomethylationKvDMR hypomethylation 1(25%)1(25%) 22%22%

FailFail 00 H19 hypermethylationH19 hypermethylation 00 11%11%

PositivePositive 3/35(8.5%)3/35(8.5%) 20-65%20-65% Mat UPDMat UPD 00 RareRare

SRSSRS NormalNormal 31/3531/35 H19 hypomethylationH19 hypomethylation 3(100%)3(100%) ~99%~99%

FailFail 1/351/35

Cohort study - Further testing

•Microsatellite analysis confirmed 3 cases of Paternal UPD

D11S1984

D11S1997 (D11S4957)p15.4

D11S922 D11S2362 D11S1997

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 1st 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