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Microsatellite Instability (MSI) and Mutation Detection in HNPCC

Microsatellite Instability (MSI) and Mutation Detection in HNPCC. Jennie Bell West Midlands Regional Genetics Service. Overview. Introduction to HNPCC and MSI Current strategies Impact on mutation detection of MSI and IHC. Contribution of familial cancer syndromes to colorectal cancer.

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Microsatellite Instability (MSI) and Mutation Detection in HNPCC

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  1. Microsatellite Instability (MSI) and Mutation Detection in HNPCC Jennie Bell West Midlands Regional Genetics Service SCOBEC and Birmingham Cancer Study Day 6th June 2007

  2. Overview • Introduction to HNPCC and MSI • Current strategies • Impact on mutation detection of MSI and IHC SCOBEC and Birmingham Cancer Study Day 6th June 2007

  3. Contribution of familial cancer syndromes to colorectal cancer Lynch: Cancer:100, No1,2004 SCOBEC and Birmingham Cancer Study Day 6th June 2007

  4. Familial adenomatous polyposis (FAP) Thousands of polyps in colon Certain to become malignant by fourth decade Prevention of cancer depends on regular sigmoidoscopy Molecular testing can be used to indicate those requiring clinical screening SCOBEC and Birmingham Cancer Study Day 6th June 2007

  5. Extracolonic manifestations Congenital hypertrophy of the retinal pigment epithelium (CHRPE) Upper GI adenomas Desmoid tumour Epidermoid cysts Osteomas SCOBEC and Birmingham Cancer Study Day 6th June 2007

  6. The APC gene Cabrera CM, López-Nevot MA. APC and chromosome instability in colorectal cancer. Rev Esp Enferm Dig 2005; 97: 738-743. SCOBEC and Birmingham Cancer Study Day 6th June 2007

  7. Adenoma-Carcinoma Sequence Renata dos Santos CouraArq. Gastroenterol. vol.42 no.2  São Paulo Apr./June2005 SCOBEC and Birmingham Cancer Study Day 6th June 2007

  8. FAP vs HNPCC SCOBEC and Birmingham Cancer Study Day 6th June 2007

  9. HNPCC • Characterised on the basis of family pedigree - not on clinical grounds alone • Termed the Amsterdam criteria • One person affected<50 • At least two generations involved • One person must be first degree relative of another • FAP must be excluded SCOBEC and Birmingham Cancer Study Day 6th June 2007

  10. HNPCC • Early onset of colorectal cancer- fifth and sixth decade • Associated with a range of other cancers including endometrial cancer, transitional cell carcinomas SCOBEC and Birmingham Cancer Study Day 6th June 2007

  11. Phenotype is caused by at least 5 different genes MSH2 (16 exons) MLH1 (19 exons) PMS 2 (and ?1) MSH6 MSH3 Mismatch Repair Genes SCOBEC and Birmingham Cancer Study Day 6th June 2007

  12. Mismatch repair The EMBO Journal (1998) 17, 6427–6436 SCOBEC and Birmingham Cancer Study Day 6th June 2007

  13. Microsatellite instability (MSI) • Comparison of normal with tumour DNA • 7 microsatellite markers • Additional ‘peaks’ recorded as ‘unstable’ • >30% markers unstable = MSI-High • <30% markers unstable = MSI-Low SCOBEC and Birmingham Cancer Study Day 6th June 2007

  14. Example of MSI Tumour DNA Normal DNA SCOBEC and Birmingham Cancer Study Day 6th June 2007

  15. White Paper (2004) • Funding for 0.5 WTE for post in Department of Cellular Pathology at University of Birmingham Medical School for 2 years • Impact has been outstanding • MSI and IHC service working smoothly for last 12 months SCOBEC and Birmingham Cancer Study Day 6th June 2007

  16. Integration of MSI/IHC with Mutation Detection Blocks returned Dept. Cell. Pathol Mutation screen other path lab Blocks sent Blocks cut & IHC Request for blocks Genetics Lab Clinical Genetics IHC/Amsterdam pos IHC/Amsterdam neg Sections for MSI MSI positive MSI negative SCOBEC and Birmingham Cancer Study Day 6th June 2007

  17. Mutation detection • Two genes analysed • hMLH1 – 19 exons • hMSH2 – 16 exons • hMSH6 in development • Point mutations (missense, nonsense, splice site) • Small insertions and deletions • Large deletions and duplications SCOBEC and Birmingham Cancer Study Day 6th June 2007

  18. Automation Developments (2005) • Sequence based analysis using robotics • 2 x Beckman NX, 1 x Beckman FX • 2 x ABI 3730 genetic analysers • Mutation detection software (Mutation Surveyor) • MLPA SCOBEC and Birmingham Cancer Study Day 6th June 2007

  19. Mutation analysis Sample received Del/dup identified report MLPA Del/dup not identified Mutation identified Mutation not identified report report Sequence all exons presymptomatic testing available presymptomatic testing not available SCOBEC and Birmingham Cancer Study Day 6th June 2007

  20. Mutation screening 2002 - 2005 • 270 patients screened by a combination of dHPLC, sequencing and MLPA • 90 mutations reported • Mutation detection rate approx 35% SCOBEC and Birmingham Cancer Study Day 6th June 2007

  21. Automated strategy – Results since Jan 06 • 98 patients screened • 39 variants identified • 40% detection rate SCOBEC and Birmingham Cancer Study Day 6th June 2007

  22. Contribution of Gene Mutations SCOBEC and Birmingham Cancer Study Day 6th June 2007

  23. 1 23456 78910111213141516 Duplications (MLPA) Deletions (MLPA) Missense Splice site Frameshift/nonsense Positions of mutations identified hMSH2 SCOBEC and Birmingham Cancer Study Day 6th June 2007

  24. 1 23456 78910111213141516171819 Deletions (MLPA) Missense Splice site Frameshift/nonsense hMLH1 SCOBEC and Birmingham Cancer Study Day 6th June 2007

  25. Summary of identified variants SCOBEC and Birmingham Cancer Study Day 6th June 2007

  26. Reporting times • MSI • 4 weeks (once sections cut) • Mutation screen • within 8 weeks SCOBEC and Birmingham Cancer Study Day 6th June 2007

  27. Relationship between MSI/IHC and Mutation Screening 624 screened 135 MSI +ve 489 MSI neg 40 Mutation identified 18 Mutation not identified 77 Status unknown SCOBEC and Birmingham Cancer Study Day 6th June 2007

  28. How does MSI/IHC help? • Patient 1 • Missense p.Lys618Ala identified (exon 16 MLH1) by SSCP • Unclear pathogenic significance • Not able to offer presymptomatic testing to the family • MSI and IHC undertaken to gain further information SCOBEC and Birmingham Cancer Study Day 6th June 2007

  29. Tumour tissue exhibited MSI and loss of MSH2 antibody staining • Repeat mutation screen by dHPLC • Identified frameshift mutation in exon 1 of MSH2 • Clearly pathogenic • Presymptomatic testing available SCOBEC and Birmingham Cancer Study Day 6th June 2007

  30. How does MSI/IHC help? • Increasing information on MSI & IHC status • Can give supporting evidence to interpretation of missense variants • Identification of more mutations through screening more appropriate families SCOBEC and Birmingham Cancer Study Day 6th June 2007

  31. Presymptomatic testing • Aim of mutation screening is provide presymptomatic testing to at risk individuals • Use of MSI and IHC along with pedigree information targets appropriate families • Improved efficiency of mutation detection SCOBEC and Birmingham Cancer Study Day 6th June 2007

  32. Presymptomatic testing SCOBEC and Birmingham Cancer Study Day 6th June 2007

  33. The future of mutation detection • Functional assays to define effect of missense variants • RNA analysis to identify sequence variants within introns and exons that affect splicing machinery • MSI and IHC will focus this analysis to appropriate families SCOBEC and Birmingham Cancer Study Day 6th June 2007

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