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FAP somatic mosaic cases

FAP somatic mosaic cases. Kennedy Galton Centre Zoe Allen. FAP. Somatic mosaicism estimated to account for ~ 20% sporadic FAP cases Aretz et al (2007), Human mutation, 28(10):985-992 Hes et al (2008), Gut, 57(1):71-76 Rohlin et al (2009), Human mutation, 30:1012-1020

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FAP somatic mosaic cases

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  1. FAP somatic mosaic cases Kennedy Galton Centre Zoe Allen

  2. FAP • Somatic mosaicism estimated to account for ~ 20% sporadic FAP cases • Aretz et al (2007), Human mutation, 28(10):985-992 • Hes et al (2008), Gut, 57(1):71-76 • Rohlin et al (2009), Human mutation, 30:1012-1020 • Limitations of previous technology - unable to detect low level mutations • Two cases of somatic mosaicism detected in KGC in 2009 • Poster presented at BSHG 2009

  3. FAP sequencing • Large gene • 15 exons • 32 PCR fragments • Sequenced on ABI 3730 using BigDye v1.1 • dHPLC/CSCE/HRM problematic – very polymorphic gene • Too large for pyrosequencing

  4. Case 1 • Attenuated FAP phenotype • Multiple colonic adenomas • Colon removed at age 20 • No family history • Low level mutation detected using sequencing • c.646C>T (p.Arg216X) • Exon 6

  5. Resolving case 1 • DNA extracted from tubular adenoma • Amplified and sequenced • Smaller fragment • Tumour DNA degraded/fragmented • Mutation present at 50% level as expected Lymphocyte DNA Tumour DNA

  6. Reporting case 1 • Interim report “DNA sequencing identified a suspected mutation, c.646C>T …. If this is a mutation, it appears to be present at a low level suggesting tissue mosaicism This finding needs to be investigated further before the patient is informed We have initiated testing on tumour DNA from X.”

  7. Reporting case 1 • Final report “The c.646C>T mutation has been confirmed in the tumour sample. The strength of this mutant signal in tumour DNA (approx 50%) compared with lymphocyte DNA (approx 10-15%) is consistent with tissue mosaicism This confirms a diagnosis of FAP in x which is likely to have arisen de novo. This could be confirmed by analysis of parental samples. If appropriate, predictive testing could be offered to any of x’s children (who would be at 50% risk) and siblings (who would be at a low risk assuming the mutation is de novo).”

  8. Estimating sequencing sensitivity • Man made mosaics made for different mutation types (splice, missense, frameshift)

  9. Case 2 • Multiple adenomas present – first seen age 55 • Attenuated phenotype • No family history • PTT and SSCP screen –ve (2002) • Low level mutation detected using sequencing • c.1660C>T (p.Arg554X) • Exon 13

  10. Resolving case 2 • Similar to case 1 • Requested tumour blocks – not yet received in lab • Trying to optimise COLD PCR to preferentially amplify the low level variant • COLD PCR principle • Small PCR frag (<200bp) • PCR as normal for 10 cycles • Cycle for a further x no of cycles • denature DNA, let it form heteroduplexes while cooling • Then denature only the heteroduplexes before primer annealing and extension • Has been applied to KRAS/BRAF testing successfully in literature

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