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