Health Technology Assessments undertaken by NGRL (Wessex) in ...

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Health Technology Assessments undertaken by NGRL (Wessex) in ...

New techniques for DNAmethylation analysisHelen WhiteNGRL (Wessex)Salisbury NHS Foundation TrustSCOBEC Training Day: Imprinting disorders


Prader Willi and Angelman SyndromesPWSTwo clinically distinct phenotypes that map to 15q11-q13• Caused by loss of the paternal (unmethylated) contribution• Paternal deletion (~70%)• Maternal UPD (~30% cases)• Mutation in the imprinting region causing abnormal methylation (


MS-MLPA


AS (UPD)2 alleles unmethylatedPWS (UPD)2 alleles methylatedNormal controlUndigestedDigested


Bisulphite Treatment• Bisulphite treatment causes ummethylated Cytosines to convert to Uracilwhile methylated cytosines remain unchanged.* **CAGCGATCACGACCTBisulphite Treatment* *UAGCGATUACGAUUTPCR* *TAGCGATTACGATTT( * =methylated )


SNRPN 1(Genomic)SNRPN 1(After bisulphite)


MS PCRMATPATPaternal 221-bp productMaternal 313-bp productCOMMONN AS PWS


Pyrosequencing


Pyrosequencing assay designYG YG YG YGR S T UBBStreptavidin


Principle of PyrosequencingPPidGTPC G T C C G G AG


PyrogramsT:65.2%C:34.8%T:100.0%C:0.0%T:64.2%C:35.8%T:70.6%C:29.4%C:34.8% C:0.0% C:35.8% C:29.4%T:67.3%C:32.7%C:32.7%120110100NormalE S T G T C C G T C T A G T T G A T C C G T C T G A T G C A G T C T G T5 10 15 20 25 30130120PWST:0.0%C:100.0%T:100.0%C:0.0%T:0.0%C:100.0%T:14.1%C:85.9%C:100% C:0.0% C:100% C:85.9%T:15.6%C:84.4%C:84.4%110100E S T G T C C G T C T A G T T G A T C C G T C T G A T G C A G T C T G T5 10 15 20 25 30120110AST:100.0%C:0.0%T:100.0%C:0.0%T:100.0%C:0.0%T:100.0%C:0.0%C:0.0% C:0.0% C:0.0% C:0.0%T:100.0%C:0.0%C:0.0%100E S T G T C C G T C T A G T T G A T C C G T C T G A T G C A G T C T G T5 10 15 20 25 30


Pyrosequencing ResultsA1034 Position 1 (R)A1034 Position Rn = 40120Result (%methylation)100806040200n = 33n = 76-20Sample No.120A1034 Position SA1034 Position 2 (S)n = 40Result (%methylation)100806040200n = 33n = 76-20Sample No.


Sample 58Position RNormalSequenceSample 58PWSBisulphitedsequenceT G T T T G A G G A G CG G T T A G T G A C G C G A TSample 58BisulphitedsequenceT G T T T G A G G A G TG G T T A G T G A C G C G A T


Advantages• AQ software allows accurate quantification of methylation at multiple CpG sites withinamplicon• PCR primers independent of methylation state• Confidence scores (passed, checked or failed) alert user to the quality of assay data• Reference peaks incorporated into the analysis add confidence to data collection –also bisulphite treatment controls are included• Results are presented in sequence context so sequence variants will be identified• Assays relatively inexpensive & rapid• Has potential to detect mosaicismDisadvantages• Further work is still necessary to establish the cause behind the abnormal methylatione.g. UPD, deletion or an imprinting mutation


High resolution melt curve analysis


High resolution melt curve analysis


High resolution melt curve analysisDouble stranded DNAHeterozygous mutation 62C>GWild type curveHeteroduplexes melt at lowertemperatures than homoduplexesHomozygous mutation (62GG)Single stranded DNA


21 sites can vary


Normal ControlsPWSAS


Advantages• Closed tube method• No post PCR processing and no separation step- improves analysis time- reduces contamination risk• Rapid• Inexpensive - requires the use of only PCR reagents and dsDNA binding dye• PCR primers independent of methylation state• Global information about methylation/sequence composition of ampliconDisadvantages• Further work is still necessary to establish the cause behind the abnormalmethylation e.g. UPD, deletion or an imprinting mutation


Mass Spectrometry


Advantages• PCR primers independent of methylation state• Quanitfication of methylation (to 5%) at all CpG sites within amplicons up to 600bp• Useful for large scale projects to identify ‘useful’ CpG sites• High throughput• High precisionDisadvantages• Expensive• Extensive post PCR handling – relies on success of multiple reactions• Need access to equipment and specialised data analysis software


Quality control issues


Quality and quantity of DNA Bisulphite treatment- quality- batches Preferential amplification Use of standard curves


Quality and concentration of DNA50ng 20ng 10ng 5ng 2.5ng


Bisulphite treatmentTotal conversionPyrosequencing has in built control sites to monitor whether bisulphiteconversion is completeMost techniques do not have controls to monitor thisT:65.2%C:34.8%T:100.0%C:0.0%T:64.2%C:35.8%T:70.6%C:29.4%C:34.8% C:0.0% C:35.8% C:29.4%T:67.3%C:32.7%C:32.7%120110Normal100E S T G T C C G T C T A G T T G A T C C G T C T G A T G C A G T C T G T5 10 15 20 25 30Batches


Preferential amplification and standard curves

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