144 nature genetics• volume 23• october 1999 correspondence teria (7/154, 4.5%). These kindreds display a very high frequency of atypical hyperplastic lesions and carcinomas of the endometrium: 73% in female MSH6 mutation carriers compared with 29% in MSH2 and 31% in MLH1. Moreover, delayed age of cancer onset and incomplete penetrance were characteristic clinical features of the MSH6 mutation carriers (see Table 3 and Fig. 1, http://genetics. nature. com/supplementary_info/). We investigated MSI in 16 endometrial, ovarian, rectal and urothelial tumours from 4 families using 40 microsatellite markers (Table 2). According to the recently established MSI criteria for colorectal tumours11, 9 of 16 tumours displayed a MSI-high phenotype, 3 were MSI-low and 4 were microsatellite stable (MSS). MSS tumours, however, did show instability of at least one mononucleotide marker. In fact, all tumours analysed showed instability of the MSH6 (C8) repeat, which is likely to represent a preferential target for somatic inactivation. Length variations of the MSH6 (C8) tract thus provide a strong indication to search for MSH6 germline mutations in kindreds without mutations in MSH2 or MLH1. In general, MSI was mainly observed at mononucleotide repeats, as expected from the biological function of the MSH6 product in human DNA mismatch repair12, 13. Only the rectal and urothelial carcinomas displayed a broader MSI phenotype, affecting mono-, di-, tri-and tetra-nucleotide repeat markers at a level comparable with that observed in colorectal tumours with MSH2 or MLH1 mutations (Table 2). Our data provide indirect evidence for a difference between the tumorigenic pathways in endometrial and colorectal cancer. The TGFBR2 (A) 10 intragenic repeat showed instability in carcinomas of the rectum and urothelium, although it was not altered among the endometrial and ovarian tumours (Table 2). In the endometrium, MSH6 inactivation may select for a different tumour-suppressor gene, thus triggering uterine tumorigenesis equivalent to the loss of MSH2 or MLH1, leading to instability at the TGFBR2 intragenic (A) 10 repeat in colorectal tumorigenesis14. In addition to the truncating mutations, we detected 6 missense MSH6 mutations of unknown significance in 11 families, of which 7 were AMS–families (see Table 4, http://genetics. nature. com/supplementary _info/). The clinical features of these families differ from those found in families with MSH6 truncating mutations. Colorectal cancer was the most frequent tumour type among families with missense substitutions (29/33 cancers), whereas of 64 tumours found in families with MSH6 truncating mutations, 31 were colorectal and 22 endometrial. Our results indicate that tumours of the endometrium represent the most common clinical manifestation of HNPCC among female MSH6 mutation carriers and that colorectal cancer cannot be considered an obligatory requisite to define HNPCC. In our total HNPCC cohort, the selection of which was exclusively based on clinical features and not on the MSI status of the corresponding tumours, MMR gene mutations were found in 49% of the AMS+ families and in 12% of the kindreds not complying with the criteria. Recently, the International Collaborative Group on HNPCC implemented the Amsterdam criteria by including extracolonic tumour types commonly observed in HNPCC (ref. 15). Of 83 mutation-positive families in our total cohort, 73 fulfil the modified Amsterdam criteria, thus confirming their validity for the selection of HNPCC families to be analysed for mutations in MMR genes. In view of our findings, MSH6 mutation analysis is recommended, particularly in those …