Microsatellite instability in colorectal-cancer patients with suspected genetic predisposition.

Hereditary non-polyposis colorectal cancer (HNPCC) is a dominantly inherited syndrome linked to DNA-mismatch-repair (MMR) gene defects, which also account for microsatellite instability (MSI) in tumour tissues. Diagnosis is based mainly on family history, according to widely accepted criteria (Amsterdam Criteria: AC). Aim of this work was to assess MSI in colorectal-cancer patients with suspected genetic predisposition, and to verify whether MSI represents a tool to manage MMR gene (hMSH2 and hMLH1) mutation analysis. We investigated 13 microsatellites (including the 5 NCI/ICG-HNPCC markers) in 45 patients with suspected hereditary predisposition (including 16 subjects from HNPCC families fulfilling the AC). We found MSI-H (high frequency of instability, i.e., in > or =30% of the markers) in 85% of the HNPCC patients and in 16% of the non-HNPCC subjects. The 5 NCI/ICG-HNPCC microsatellites proved to be the most effective in detecting MSI, being mononucleotide repeats the most unstable markers. We investigated the association between hMSH2- and hMLH1 gene mutations and MSI. Our results indicate that AC are highly predictive both of tumour instability and of MMR-gene mutations. Therefore, as the most likely mutation carriers, HNPCC subjects might be directly analyzed for gene mutations, while to test for MSI in selected non-HNPCC patients and to further investigate MMR genes in MSI-H cases, appears to be a cost-effective way to identify subjects, other than those from kindred fulfilling AC, who might benefit from genetic testing.

Mean age of tumor onset in hereditary nonpolyposis colorectal cancer (HNPCC) families correlates with the presence of mutations in DNA mismatch repair genes.

Fourteen Italian families affected with hereditary nonpolyposis colorectal cancer (HNPCC) were screened for germline mutations at three DNA mismatch repair (MMR) genes, MSH2, MLHI, and GTBP, by using a combination of different methods that included an in vitro synthesized protein assay, single-strand conformation polymorphism analysis, and direct sequencing. DNA alterations were observed in six instances, including a single base deletion in MSH2 exon 14, an A-to-G transition in the splice donor site of MLHI exon 6, and two missense mutations in MLHI exons 5 and 9. A previously reported common mutation affecting the splice donor site of MSH2 exon 5 was identified in two families. No mutations were detected in the GTBP gene. In total, eight of 16 Italian HNPCC families (50%), including two previously reported kindreds, were found to carry a mutation in MMR genes. We compared the mean age of colorectal cancer onset in the index cases (three patients for each family) between the two groups of kindreds, those with identified mutation vs. those without, and found that the first had a significantly lower value (43.0 vs. 53.7 years, P = 0.014). This finding suggests that HNPCC families with a more advanced age of tumor onset are less likely to be associated with known MMR genes.

Characterization of four microsatellites in an Italian population and their application to paternity testing.

Microsatellites have recently been used for linkage analysis of genetic diseases and for DNA fingerprinting in forensic medicine. In the present study the heterozygosity, PIC values and allele distributions of four microsatellites, D8S85, D8S88, D5S346 and D7S460, in an Italian population have been investigated. After amplification with primers specific for each locus, alleles were separated and detected by denaturing gel electrophoresis and ethidium bromide staining. High heterozygosity and PIC values were observed for all microsatellites in accordance with data in other Caucasian populations. However, different allele distributions for D8S85, D8S88 and D5S346, due to the presence of additional bands or to different frequencies, were found. D7S460, which has never been fully characterized before, appeared to have five alleles in the range 172 to 188 bp. When used for paternity testing, all microsatellites gave results which were consistent with those obtained with established markers, including apo B 3'HVR, D1S80 and COL2A1. This indicates that D8S85, D8S88, D5S346 and D7S460 may be useful as additional informative markers or for solving discrepancies in selected cases.

Molecular screening of families affected by familial adenomatous polyposis (FAP).

OBJECTIVES: To assess the risk of developing familial adenomatous polyposis (FAP) in presymptomatic individuals using APC gene flanking and intragenic polymorphic markers. SETTING: Twenty families enrolled in the Italian Registry of Polyposis comprising a total of 217 individuals, including 53 (24%) presymptomatic subjects with a 50% a priori risk of FAP, were analysed. Direct analysis techniques had previously failed to identify the FAP mutation in these families. METHODS: DNA isolated from peripheral mononuclear blood cells and tissue sections was analysed by the polymerase chain reaction and a panel of seven highly polymorphic markers--YN5.64, CB83, CB26, LNS, APC1458.5, MBC, 37AB. Amplification products were separated by a modified denaturing gel electrophoresis method. RESULTS: The haplotype associated with the disease was identified in 18 families (90%). The segregation of the FAP haplotype in these kindreds showed that 10 presymptomatic individuals had inherited the FAP mutation and carried a high risk of developing the disease. The remaining two families were not informative because of the lack of a sufficient number of probands or biological specimens. CONCLUSIONS: These data indicate that indirect analysis with linked DNA markers has a high rate of success in defining the risk of FAP of presymptomatic subjects, provided that a sufficient number of probands or samples is available. Uninformative families accounted for 10% of the total, indicating that linkage analysis may still have higher sensitivity than direct mutation analysis techniques. The combined use of both approaches should be implemented, however, to enhance further the application of molecular genetics to the screening of families with FAP.