Download
rt pcr studies on brca patients n.
Skip this Video
Loading SlideShow in 5 Seconds..
RT-PCR studies on BRCA patients PowerPoint Presentation
Download Presentation
RT-PCR studies on BRCA patients

RT-PCR studies on BRCA patients

214 Views Download Presentation
Download Presentation

RT-PCR studies on BRCA patients

- - - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript

  1. RT-PCR studies on BRCA patients Emma Sach04/04/2008

  2. Introduction Two cohorts; • Patients with unclassified variant (UV) identified during routine screening • BRCA1/BRCA2 sequencing of coding regions and MLPA • Patients with strong family histories of male breast cancer and no pathogenic mutations in routine screening

  3. Patients with UV – cohort selection • Identification of UV • Predicted effect on splicing • Fruitfly • ESE Finder • Literature • Request follow up blood sample for RNA studies • Sample received with consent

  4. 1. Patients with UV – protocol • Sample - 5-10ml EDTA • WCC - 10x106 cells extracted • RNA extraction - Stage 1 - Qiagen RNeasy mini kit - Stage 2 - QIAcube • cDNA preparation- Invitrogen Superscript III • PCR - flanking exonic primers - use at least three normal controls • Resolve products - agarose gel • Clean up products - QIAquick gel extraction kit • Sequence

  5. Acceptor Donor 1. Patients with UV – cohort

  6. Example 1 • BRCA2 c.681+2dupT • Fruitfly predicted the splice donor was abolished • Predicted effect on splicing Normal Prediction

  7. Example 1- Results C B • r.860_909del D A

  8. D A D A A D Normal 12 1 3 1 4 66 Prediction D Example 2 • BRCA1 c.4476+6T>C • Previously reported on BIC and in the literature as causative of a splice variant • Fruitfly predicted donor site score reduced by 0.07

  9. Example 2- Method

  10. D A D D A A Predict ion 12 1 3 1 4 66 Actual D Example 2- Results r.[4186_4357del,4186_4358delins4358-2784_4358-2719]

  11. Acceptor Donor 1. Patients with UV- Results

  12. 1. Patients with UV – tools for prediction • Useful - Fruitfly, literature and sequence predictions • Not useful - ESE Finder

  13. 2. Male patients with strong family history • 2 male patients • Strong FH of male breast cancer • No pathogenic mutations detected in BRCA1/BRCA2 by sequencing of coding regions or by MLPA • Designed primers to cover the whole gene • overlapping fragments • all products less than 1kb • 7 PCRs

  14. 2. Male patients with strong family history - Results • Number of products • Sequenced all of them • Used 5 NC for each PCR try and confirm all variants were normal • Chromosome 20 • Redesign primers

  15. 2. Male patients with strong family historyPCR of exons 18-24 50bp HA CC MGH NC1 2 3 4 5 6 -ve Ins 64bp (21A) Normal 18-24 Del ex 22 ins 21A Del 22 • Present in several NC • All previously reported in the literature • Illustrates problems with RNA work

  16. 2. Male patients with strong family history- Isoforms

  17. 2. Male patients with strong family historyDiscussion • What causes the variability of product presence and intensity? • Time between sample collection to extraction • Patient age • Do studies to monitor these effects • Try and classify all isoforms then can exclude them easily in future studies • More normal controls • How can we source these?

  18. Conclusion • Follow up of UV in individual cases is useful • In this project 3 out of 9 (33%) UV were confirmed to result in aberrant splicing • Whole gene screen • A lot of work • Need better knowledge of naturally occurring isoforms

  19. Yvonne Wallis Claire Morgan Richard Barber Rachel Doak Fiona Macdonald Jennie Bell CCs family for their donation BRCA team Sequencing team Molecular Oncology Acknowledgements