Unbalanced germ-line expression of hMLH1 and hMSH2 alleles in hereditary nonpolyposis colorectal cancer

We analyzed the hMLH1 and hMSH2 genes in 30 unrelated hereditary nonpolypos is colorectal cancer (HNPCC) patients using mutational and immunohistochemi cal analyses combined whenever possible with primer extension assays, desig ned to estimate hMLH1 and hMSH2 transcript expression in peripheral blood l ymphocytes, Single-strand conformational polymorphism screening and PCR-dir ect sequencing revealed seven hMLH1 and five hMSH2 sequence variants in 14 unrelated HNPCC patients, including three definite pathogenic mutations, fo ur amino acid substitutions of uncertain pathogenic significance, and five polymorphisms. Immunohistochemistry indicated the lack of either hMLH1 or h MSH2 protein expression in tumors from 13 patients, and the absence of both hMLH1 and hMSH2 immunostaining was observed in the tumor from one addition al case. The lack of hMLH1 or hMSH2 immunostaining was associated with the presence of microsatellite instability in the corresponding tumor and was a lso observed in tumors from patients negative for pathogenic mutations by m utational screening. There was a marked unbalance in the allelic expression of either hMLH1 or hMSH2 transcripts in three of eight unrelated HNPCC pat ients that could be analyzed, although a less marked unbalance was detected in two additional patients. Tumors from patients with germ-line unbalance in hMLH1 or hMSH2 transcript expression did not express the corresponding m ismatch repair protein and displayed microsatellite instability. Our result s indicate that constitutional alterations in hMLH1 and hMSH2 transcript ex pression may represent genetic markers for HNPCC carrier status also in cas es in which mutational analysis did not detect a definite pathogenic varian t. This suggests that transcript deregulation may represent a relevant mode of germ-line inactivation for mismatch repair genes.