Mutation analysis of the MSH6 gene in 52 MLH1/MSH2-negative, HNPCC suspect Italian patients

The HNPCC syndrome bears high cancer risk at colorectum, endometrium and other organs, due to MMR defects. Testing in clinical contexts is frequently limited to MLH1/MSH2, since only a minority of cases are thought to depend on MSH6 and other genes. Within a program aimed to prevent inherited cancer in the Piedmont area, we have implemented the mutation analysis of MSH6 on a set of 88 suspect HNPCC probands, in which 52 were previously found to be MLH1 and MSH2 mut- negative, with the aim to define proper criteria for MSH6 testing. Cases were included on the basis of recognized clinical criteria and on preliminary analyses on the tumor for IHC expression of MMR proteins and microsatellite instability. All 10 MSH6 exons were amplified in 19 amplimers from leukocyte DNA, and analysed by DHPLC. Screening for major deletions was performed by MLPA. Two point mutations (K537fs and Y1286delinsIN), one deletion of exons 2-4 and two variants of unknown pathogenic significance were found. In all three MSH6-mut patients the tumor was MSI-H; in one of them we demonstrated a double-negative IHC pattern (MSH2- and MSH6-). Within the limits of our small sample, MSH6-mut patients showed less typical family history. The inclusion of MSH6 in HNPCC genetic testing can significantly improve sensitivity. A MSH6 and/or MSH2 negative IHC, better than any clinical feature, appears to efficiently pinpoint cases for MSH6 screening among MSH2-neg cases. Supported by Regione Piemonte and Compagnia di S.Paolo, Torino