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

3. APC Gene 2843 500100015002000 3159/9a101112131415 (aa) Ex Classic FAP MUTATION CLUSTER REGION 106113095bp mutation Hot Spots AFAP 436 AFAP 1596 Grady and Markowitz B-CATENIN BINDING DOMAIN B-CATENIN DEGRADATION DOMAIN MICROTUBULE BINDING DOMAIN FUNCTONAL DOMAINS ARMADILLO REPEATS EB1 BINDING DOMAIN

4. APC Mutations Mutation Type No. Mutations (%) Small deletions 356 (41%) Missense/nonsense 235 (27%) Small insertions 131 (15%) Gross deletions 54 (6%) Splicing 49 (5%) Small indels 17 (2%) Regulatory 3 (<1%) Molecular genetic testing using range of techniques including MLPA, PTT and DNA sequencing Mutation detection rate – up to 90%

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

6. APC Gene AFAPFAP with CHRPEAFAP 4361596 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 2843 500100015002000 13159/9a101112131415 (aa) Ex ARMADILLO REPEATS c.1409-5 A>G CCCTTTTTAA TTAG GGGGACTAC G A -Splice acceptor site Exon 11

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

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

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

10. AFAP APC Gene 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 500100015002000 3159/9a101112131415 (aa) 4 c.423G>T p.R141S

11. 109bp Ex3Ex4Ex5 DNA AAGAGAG GTCAATTGCTTCTTGCT T Intron 3Exon 4 c.423G>T 301 192 Ex3Ex5 Ex3Ex4 RNA II.2 N II.4III.3 Molecular analysis Exon skipping confirmed by sequence analysis of the cDNA product across junction.

12. Summary Case 1 – Atypical FAP Case 1 – Atypical FAP c.1409-5 A>G mutation – c.1409-5 A>G mutation – Splice-site prediction programmes Splice-site prediction programmes RNA studies RNA studies Selection of an alternative splice site. Selection of an alternative splice site. Case 2 – AFAP Case 2 – AFAP c.423 G>T (p.R141S) missense mutation – c.423 G>T (p.R141S) missense mutation – Amino acid conservation Amino acid conservation Segregation with disease Segregation with disease Splice site prediction programmes Splice site prediction programmes RNA studies RNA studies Exon skipping possibly due to possible effect on exonic splicing enhancer motifs (ESEs). (Aretz et al., 2004). 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 Splicing mutations can cause FAP and warrant further investigation CMGS Best Practice Guidelines on the interpretation and reporting of unclassified variants CMGS Best Practice Guidelines on the interpretation and reporting of unclassified variants