Frequency of deletions of EPCAM (TACSTD1) in MSH2-associated Lynch syndrome cases

Abstract

Lynch syndrome is an autosomal dominant cancer predisposition syndrome characterized by loss of function of DNA mismatch repair enzyme MLH1, MSH2, MSH6, or PMS2. Mutations in MLH1 and MSH2 account for ∼80% of the inherited cases. However, in up to 20% of cases suspected of having a germline mutation in MSH2 due to loss of MSH2 expression, a germline mutation is not identified. Recent studies have shown that some Lynch syndrome cases are due to 3' EPCAM/TACSTD1 deletions that subsequently lead to MSH2 promoter hypermethylation. In this study, we examined the frequency of this novel mechanism for MSH2 inactivation in cases recruited through the Colon Cancer Family Registry and from the Mayo Clinic Molecular Diagnostics Laboratory. From the combined cohort, 58 cases were selected in which immunohistochemical staining suggested a mutation in MSH2 or MSH6, but no mutations were identified on follow-up testing. Of these 58 cases, 11 demonstrated a deletion of EPCAM/TACSTD1. Of cases with a deletion, the methylation status of the MSH2 promoter was confirmed in tumor tissue using methylation-sensitive PCR primers. One case showed MSH2 promoter hypermethylation in the absence of a detectable EPCAM/TACSTD1 deletion. These results indicate that approximately 20% to 25% of cases suspected of having a mutation in MSH2 but in which a germline mutation is not detected, can be accounted for by germline deletions in EPCAM/TACSTD1. These data also suggest the presence of other alterations leading to MSH2 promoter hypermethylation.

Figure 1

Analysis of EPCAM/TACSTD1 for large deletions and duplications by MLPA, two normal copies of each probe (green squares) and control probes (blue squares) are represented by a peak ratio of one. Loss of one allele at the probe site results in a decrease in the peak ratio to 0.5 and is represented by a red square. A: Shows a normal result, with all EPCAM/TACSTD1 probes showing normal dosage. B: Shows a deletion that includes exons 8 and 9 of EPCAM/TACSTD1 and two probes located downstream of the gene including a probe 3 kb away. C: Shows a deletion that includes exons 8 and 9 only.

Figure 2

Results of MSH2 hypermethylation test by methylation-specific PCR and fragment length analysis. A: Shows the results of the ABI-based fragment analysis for methylation-specific primers in region 1 of the MSH2 promoter. MSH2 hypermethylation can be seen in tumor and normal tissue in a case with a EPCAM/TACSTD1 deletion. In contrast, the negative control case (loss of MLH1 expression) does not show MSH2 methylation. B: Shows similar data for region 3 of the MSH2 promoter.