1. Suggestions for PMS2 best practice Jenny Simmonds 30.11.2009

2. Background PMS2 located at 7p22 has 15 exons across 36 kb genomic DNA. Exons 1-5 have multiple transcribed pseudogenes and exons 9, 11-15 have a single highly homologous pseudogene (PMS2CL) UKGTN dossier submitted and accepted

3. PMS2 involvement in Lynch Syndrome (LS) Figures for PMS2 involvement vary but the following are published: –11.5% of tumours with abnormal MMR have isolated loss of PMS2 expression (Truninger et al, 2005) –4.3% of MSI-high tumours have loss of PMS2 (Gill et al, 2005) Limited information on penetrance for PMS2 mutation carriers Family histories more often meet Bethesda guidelines than Amsterdam II criteria (Truninger et al, 2005)

4. Mutation pick up rates Currently we are testing samples from three referral streams as set out in the gene dossier. 1. PMS2 only 53% (n = 30) 2. Combined PMS2 & MLH1 loss; no mutations in MLH1 0% (n = 19) 3. LS family history; MSI-H and no mutations in MLH1, MSH2 or MSH6 0% (n = 1)

5. Sequencing issues Exons 1 – 5: multiple transcribed pseudogenes Exons 6 – 8: no issues Exon 9: affected by PMS2CL Exon 10: no issues Exons 11-12: affected by PMS2CL Exons 13-15: affected by PMS2CL and risk of gene conversion

6. MLPA issues P008 kit contains probes for exons 1-2 and 5-15 but even MRC-Holland admit that the probes for exons 13-15 might not give reliable results Gene conversion thought to be relatively frequent Dosage changes for exons 13 and 14 observed in normal controls as well as HNPCC patients How do you know the dosage change is genuine??

7. Recommendations Primer design: aim for as many gene specific bases as possible, especially 3’ MLPA: exclude exons 13-15 from routine analysis MLPA: confirm single exon deletions by long range PCR where possible Sequencing: exclude exons 13 – 15 from routine analysis or exercise caution when interpreting variants