NGRL (Wessex) Reference and Control Materials Update May 2007

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1 NGRL (Wessex) Reference and Control Materials Update May 2007 Helen White, PhD

2 Plasmid based control material Sept 2002 May 2007 Breast cancer and HNPCC Mutation Scanning Generic controls for Mutation Scanning Genomic DNA (lymphoblastoid cell lines) Normal controls Myotonic dystrophy Friedreich ataxia RNA standards (Freeze dried cell lines) Quantification of BCR/ABL transcript

3 Development of plasmid based reference material

4 Production and Evaluation of Reference Reagents for hereditary breast cancer and HNPCC Mutation Detection Sept Oct Nov Dec Jan Feb Mar Apr May June July Aug Sept Oct Nov Dec Jan Feb Mar BRCA2 Exons 2-27 N = 58 hmlh1 N =34 BRCA2 Exon 10 trunc N =1 MECP2 N =4 BRCA2 Exon 11 N = 13 BRCA1 Exon 11 N = 6 BRCA1 Exons 2-24 N = 45 MSH2 N = 32 Design of insert, PCR of wt fragment, cloning, mutagenesis and sequence verification Total N = 193 Digestion and dilution of plasmids, construction of plasmid mixes and QC testing Progressive return of questionnaires

5 Performance Indicator Field Trial of Plasmid Based Reagents for Mutation Scanning of BRCA1, BRCA2, hmlh1 and hmsh2 (May 2003 October 2005) Ontario (x2) Winnipeg Edmonton Omaha Glasgow Germany Czech Republic Belgium Aberdeen Dundee Edinburgh (x2) Newcastle Distributed to 34 individuals in 26 labs Six mutation scanning techniques Belfast Liverpool Leeds Dublin Manchester & Crewe Nottingham Oxford Leicester Cardiff St George s Salisbury Guy s Northwick Park LGC BRCA & HNPCC BRCA Only HNPCC Only dhplc Sequencing SSCP/HD CSCE PTT MALDI-TOF

6 Feedback from Field Trial Data returned from 20 field trial participants 80% used the controls in routine testing 65% used the reagents to develop new assays or validate existing screens 30% altered diagnostic protocols as a results of using the controls 95% found plasmid DNA to be an acceptable alternative to genomic DNA 100% thought that the reagents were a useful resource 85% agreed that the reagents should be produced as reference material BRCA Plasmids have undergone modification compatible with standardised primers (NGRL designed and tested) acceptable format for certification (puc18) additional polymorphism controls also produced

7 Development of Generic Mutation Scanning Control Material 52 plasmid reagents constructed Used to determine the sensitivity and specificity of mutation scanning techniques type of base substitution GC content of the amplicon location of the mutation in the fragment

8 Performance Indicator Field Trial of Generic Reference Reagents (May November 2005) Position 3:..nnnAGnnn....nnnAGnnn.. Position 2: Position 1:..nnnAGnnn.. Mutation created A > C A > T G > A G > C Sequence generated nnncgnnn nnntgnnn nnnaannn nnnacnnn Heteroduplex produced C:T & G:A T:T & A:A A:C & G:T C:C & G:G Distributed to 16 labs and tested with 5 mutation detection techniques: Sequencing dhplc TGCE CSCE MALDI-TOF

9 Results from Field Trial Reagents performed well in most laboratories Comments from participants have confirmed that the reagents are a useful resource Sensitivity and specificity of mutation detection showed marked variation ranging from 75% - 100% and % respectively EMQN trial of limited numbers this year

10 Development of Control DNA from lymphoblastoid cell lines

11 Generation of lymphoblastoid cell lines I Normal controls Blood samples transferred to NIBSC 2 male 2 female Consent obtained to be used as normal control material for ANY genetic test EBV transformed cell lines produced by NIBSC

12 Friedreich Ataxia 3 cell lines established Generation of lymphoblastoid cell lines II Normal allele / Expanded allele (2.3kb) Expanded allele (1.4kb) / Expanded allele (2.6kb) Expanded allele (1.5kb) / Expanded allele (2.5kb) Myotonic dystrophy 3 cell lines established Normal allele (11 rpts) / Expanded allele (57 rpts) Normal allele (12 rpts) / Expanded allele c. 0.46kb Normal allele (13 rpts) / Expanded allele c. 0.8kb After consultation with DM testing labs a larger expansion will also be included This cell line is now established Cell lines to be passed to NIBSC for storage / WHO certified at a later date?

13 Project for the Standardisation of BCR-ABL RQ-PCR Methods

14 Formulation for primary and / or secondary reagents CML cells (primary or K562) diluted in normal leucocytes Cell line mixtures Armored RNAs

15 Evaluation of cell lines K-562 is fine for BCR-ABL KG-1, HL-60 possible for non-bcr-abl (at least the isolates we have tested). Proceeding to test pilot batch of freeze dried samples: 4 dilutions spanning 10%-0.01%; 3x10 6 cells/vial; 60 vials/dilution K-562 diluted in two BCR-ABL negative lines (HL-60 and KG-1) Grow ups and dilutions at NGRL (Wessex) Freeze dried at NIBSC Initial testing performed by NGRL (Wessex) BCR/ABL levels same before and after freeze drying Preliminary testing and trial of resuspension protocols carried out by Mannheim and Marseille Larger international trial planned (14 labs worldwide)

16 Armored RNAs Pros Easily available in large quantities Stable Good track record for calibration of RNA virus detection assays Easy to adjust BCR:ABL:GUS ratio Flexible: can use directly for reverse transcription or put through RNA extraction Cons (? Unproven for BCR-ABL)

17 Armoured RNAs: current status Survey of primer sets performed July 2006 Plasmids made (BCR, ABL, GUS, b2a2, b3a2) that cover the regions targeted by all members of the international group Sequence verified; sent (essentially gifted) to Asuragen Nov 2006 Armored RNAs available for initial testing May 2007 Evaluation round To be prepared in Salisbury; protocol to be agreed with Asuragen BCR-ABL + BCR+ ABL+ GUS armored RNA mixtures alone (in Trizol or GTC?) Mixtures in non-human cell line lysate to provide complex background (Sf9 insect cells) Potential for selection for development as primary reagents? How? Marketed as secondary reagents by Asuragen?

18 Current position NGRL (Wessex) funding was renewed April 2007 end March 2012 Workpackage covers production of controls material Lower level of funding and reduced staffing No infrastructure, funding or staffing for certification of reagents Seeking commercial partners for production and certification of plasmid reagents Cell line production possible in collaboration with NIBSC? Future plans Completion of BCR/ABL programme Oversight committee Updated survey with NGRL (M) Plasmids for large gene screens Acquired abnormalities e.g. V617F JAK2