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Novel splice-site mutations as the cause of FAP-related cancer in two families

Novel splice-site mutations as the cause of FAP-related cancer in two families. K Sweet , B McIlhatton, V McConnell, W Logan and C Graham Regional Molecular Genetics Laboratory, Belfast. APC-Associated polyposis conditions. Familial adenomatous polyposis (FAP) >100 adenomatous colonic polyps

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Novel splice-site mutations as the cause of FAP-related cancer in two families

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  1. Novel splice-site mutations as the cause of FAP-related cancer in two families K Sweet, B McIlhatton, V McConnell, W Logan and C Graham Regional Molecular Genetics Laboratory, Belfast

  2. APC-Associated polyposis conditions • Familial adenomatous polyposis (FAP) • >100 adenomatous colonic polyps • Often thousandsof colonic polyps • Polyps present throughout the colon • Early age of onset • Germline mutations in APC gene • Autosomal dominant • Attenuated FAP (AFAP) • Fewer colonic polyps (<100) • Later age of onset

  3. AFAP Classic FAP AFAP 436 1596 MUTATION CLUSTER REGION 1061 1309 5bp mutation Hot Spots ARMADILLO REPEATS B-CATENIN BINDING DOMAIN FUNCTONAL DOMAINS MICROTUBULE BINDING DOMAIN B-CATENIN DEGRADATION DOMAIN EB1 BINDING DOMAIN Grady and Markowitz APC Gene 2843 (aa) 500 1000 1500 2000 Ex 1 3 5 9/9a 10 11 12 13 14 15

  4. APC Mutations Molecular genetic testing using range of techniques including MLPA, PTT and DNA sequencing Mutation detection rate – up to 90%

  5. Case 1 ? II:1 II:2 II:3 II:4 II:5 II:6 II:7 9 sibs III:1 • Age 47- Adenocarcinoma • APC Ex 15 PTT - Negative • Colonoscopy - > 100 + polyps located proximal and distal to tumour • APC Ex 1-14 screen c.1409-5A>G in intron 10 • Suspected family history of colon cancer on maternal side

  6. 2843 (aa) 500 1000 1500 2000 Ex 1 1 3 5 9/9a 10 11 12 13 14 15 ARMADILLO REPEATS c.1409-5 A>G APC Gene AFAP FAP with CHRPE AFAP 436 1596 Exon 11 G CCCTTTTTAA TTAG GGGGACTAC A • Splice acceptor site • Fruitfly and SpliceSiteFinder prediction programmes predicted use of alternative splice site. Score – 80.6 • Previously reported in large European study – Pathogenicity undetermined Friedl & Aretz, 2005

  7. c.1409-5A>G DNA cDNA Splice acceptor site G CCCTTTTTAA A TTAG GGGGACTAC Ex 11 Intron 10 Alternative splice site 4bp insertion – (p.G470Vfsx15) RNA CCCTTTTTAAGTTAG GGGGACTAC N N M AATGAACTAGTTAGGGGGACTACAGGC,,,,TGA 254bp Ex11 Ex10 250bp Term Molecular analysis

  8. Case 2 Breast Ca II:1 II:2 II:3 II:4 II:5 II:6 ? Colon Ca Melanoma Jejunal Ca ? Colon Ca • MYH gene - Negative III.2 • APC Ex 15 PTT - Negative • Age 55 - Carcinoma of sigmoid colon • APC Ex 1-14 Screen • Age 62 - Multiple polyps throughout colon c.423 G>T (p.R141S) Exon 4 APC gene

  9. Case 2 Breast Ca II:1 II:2 II:3 II:4 II:5 II:6 AFAP AFAP III:2 III:3 III:4 III:5 AFAP AFAP c.423 G>T Splicing mutation PRESENT

  10. 1 3 5 9/9a 10 11 12 13 14 15 c.423G>T p.R141S APC Gene AFAP AFAP (aa) 500 1000 1500 2000 4 • Conserved amino acid sequence • Predicted to disrupt splice-site – (Fruitfly and SpliceSiteFinder and prediction programmes) • Reported by Aretz et al., 2004. Human Mutation 24(5): 370-80

  11. Ex4 Ex3 RNA II.2 II.2 II.4 III.3 III.3 N Ex3 Ex5 Ex3 Ex4 301 Ex5 192 Molecular analysis Intron 3 Exon 4 c.423G>T DNA AAGAGAG GTCAATTGCTTCTTGCT T Ex5 109bp Exon skipping confirmed by sequence analysis of the cDNA product across junction.

  12. Summary • Case 1 – Atypical FAP • c.1409-5 A>G mutation – • Splice-site prediction programmes • RNA studies • Selection of an alternative splice site. • Case 2 – AFAP • c.423 G>T (p.R141S) missense mutation – • Amino acid conservation • Segregation with disease • Splice site prediction programmes • RNA studies • Exon skipping possibly due to possible effect on exonic splicing enhancer motifs (ESEs). (Aretz et al., 2004).

  13. To conclude… • Splicing mutations can cause FAP and warrant further investigation • CMGS Best Practice Guidelines on the interpretation and reporting of unclassified variants

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