Lynch Syndrome Case Study

1. Lynch Syndrome Case Study Louise Bourdon, KGC

2. Patient MF • Referred in 2003 aged 55 (by Family Cancer Clinic at St Mark’s Hospital) • Colon cancer aged 37 • Caecal cancer in her 40s • Oesophageal cancer in her 50s • Family history of CRC • Daughter died at 32yrs of age • Lung metastases • Primary tumour in bowel

3. Investigations Performed • Mutation screening of MLH1 and MSH2 genes • DGGEof all exons, and sequencing of any shifts • No mutation identified - Reported in October 2003 • IHC performed at University College London Hospitals • Loss of MSH2 and MSH6 • Followed by microsatellite studies at KGC - unstable • Suggestive diagnosis of Lynch - Reported May 2004 • MSH6 Mutation screen • DGGE of all exons, and sequencing of any shifts • No mutations identified - Reported September 2005 • MLPA for MLH1 and MSH2 also reported (MRC-Holland kit P003) • MSH6 MLPA (MRC-Holland kit P008) • Normal for MSH6 - Reported November 2006 • ?Heterozygous deletion of probe upstream of MSH2

4. MSH6 MLPA

5. Contact With MRC-Holland • July 2006 - Member of staff contacted MRC-Holland following apparent heterozygous deletion of one probe in the P008 kit (EPCAM probe – 15kb upstream of MSH2) • MRC-Holland emailed laboratory in December 2008 • Emailed reference of Nature Genetics paper1 • Discussed mechanism behind deletion and loss of MSH2 • Announce new probes in kit P072 being released in early 2009

6. EPCAM deletion analysis • Review of patients with loss of MSH2/MSH6 not originally screened with kit P072 • 5 patients screened • One patient clearly positive (MF) • Second patient failed quality control measures but appeared heterozygous for deletion • Second laboratory investigating same patient using long-range PCR confirmed presence of mutation • Recently confirmed MSH2 promoter methylation for both patients using MS-MLPA kit (ME011)

7. MSH6-EPCAM MLPA

8. MS-MLPA

9. EPCAM deletions • Families identified with deletions of last exon of EPCAM gene (originally called TACSTD1) • Two deletions identified to date • c.859-1462_*1999del in Dutch population1 • C.555+894_*14164del in Chinese population1 • Gene is directly upstream of MSH22 • Deletion includes polyadenylation signal and results in the transcription read-through into MSH2 • Promoter of MSH2 on same allele is methylated (can be detected using MS-MLPA kit (MRC-Holland kit ME011) • Loss of expression of MSH2 (resulting in negative IHC for MSH2 and MSH6)

10. EPCAM Deletion Families • One study noted that the phenotype for their cohort of EPCAM deletion families differed slightly from “classical” Lynch families3 • Diagnosed almost exclusively with CRC • MSH2 mutation carriers more frequently develop extracolonic tumours • Important to note that this is not reproducible in all studies

11. Promoter Hypermethylation • MLH1 promoter hypermethylation is a known phenomenon • Some cases show germline methylation (~5% MLH1 negative IHC patients) • MSH2 promoter hypermethylation known to segregate with EPCAM deletion • Only somatic cases identified (~6% MSH2 negative IHC) • No promoter methylation identified in lymphocyte DNA (possibly result of low levels of EPCAM transcription in this tissue2)

12. References • Ligtenberg, M.J. et al (2009) Heritable somatic methylation and inactivation of MSH2 in families with Lynch syndrome due to deletion of the 3’ exons of TACSTD1 Nat Genet41(1):112-117 • Niessen, R.C. et al (2009) Germline Hypermethylation of MLH1 and EPCAM deletions are a frequent cause of Lynch syndrome Genes, Chrom & Cancer48:737-744 • Kovacs, M.E. et al (2009) Deletions removing the last exon of TACSTD1 constitute a distinct class of mutations predisposing to Lynch syndrome Hum Mutat30:197-203