Figure 13.6 Microsatellite alterations in cases with LOH or with microsatellite instability The normal pattern is shown in the leftmost lane. If LOH occurs, one or the other allele is lost, leading to loss of the according band (lanes 2 and 3). If microsatellite instability occurs, larger or smaller bands are found, which do not occur in the normal tissue. Normal bands may be retained (lane 5) or disappear (lane 4).

also to follow chromosomal loss or recombination in tumor cells (Figure 13.6). Typically, allelic loss resulting from deletion or recombination is seen as loss or strongly diminished intensity of one band resulting from PCR amplification of a microsatellite. In HNPCC tumors, additional bands appear in addition to the two present in normal tissue. These result from the expansion or contraction of a microsatellite repeat, corresponding to an insertion or deletion mutation. This phenomenon has been termed 'microsatellite instability' (abbreviated MSI).

Microsatellite instability and increased incidence of point mutations in HNPCC result from inactivation of genes involved in mismatch repair. Mismatches between DNA strands can occur as a consequence of several mechanisms and are repaired by according mechanisms (^3.1). One repair system most active during DNA replication consisting of several different proteins recognizes base mismatches and differences in the number of bases between opposite DNA strands, such as occuring by base misincorporation or 'slipping' of the DNA polymerase. These mismatches are first recognized by protein heterodimers consisting of MSH2 and MSH6 or MSH3 and MSH6, respectively. Further components of the complex, including PMS1, PMS2, and MLH1, are then directed towards the mismatch and remove the mismatched nucleotides (cf. Figure 3.4). A repair DNA polymerase is recruited to synthesize the correct sequence and a DNA ligase seals the corrected strand.

Patients with HNPCC carry mutations in one gene encoding a component of this repair system. Mutation in the MSH2 and MLH1 genes are most frequent, more rarely the PMS1, PMS2 or MSH6 genes are mutated. In rarer cases, the MYH or MBD4 genes (^3.1) are affected, but in «30% of all patients showing the characteristics of HNPCC, the underlying mutations are not known. Obviously, they could affect additional, still unknown components of the repair system.

Importantly, the mutation inherited in one allele is not sufficient to confer the MSI phenotype. Rather, mutation or epigenetic inactivation of the remaining allele has to take place before the function of the repair system is seriously compromised. So, the first step in tumor formation is probably the accidental loss of this second allele. In the affected cell, this creates a state of greatly enhanced mutability, specifically a strongly enhanced rate of point mutations. Eventually, these point

Figure 13.7 A comparison of cancer development in FAP and HNPCC The width of the arrows reflects the degree of acceleration of the process.

mutations will affect genes crucial for the development of colon cancer. This sequence of events would explain convincingly why only single or a few carcinomas arise in HNPCC and the process does not stop in most cases at the stage of adenomas as in FAP (Figure 13.7).

Interestingly, MSI is also observed in sporadic cancers arising in patients without a family history. These cancers are also defective in mismatch repair. In many such cases, the inactivation of the mismatch repair system is caused by inactivation of the MLH1 gene through promoter hypermethylation.

Conceptually, one can consider the genes harboring inherited mutations in HNPCC as 'caretaker' tumor suppressors (^5.4), since mutations in these genes do not lead to tumor growth directly, but increase the risk of mutations in genes that control cellular proliferation, differentiation and survival. To some extent, therefore, HNPCC cancers contain mutations in the same genes as other colon carcinomas. For instance, the tumor suppressor genes APC and TP53 are inactivated and the proto-oncogenes KRAS and CTNNB1 are activated by point mutations.

However, some genes are preferential mutation targets in colon cancers arising in the context of HNPCC and of sporadic MSI compared to other colon cancers. A prominent example is the TGFBRII gene which contains eleven successive adenines in its coding regions. Typical slippage mutations in this minirepeat leading to frameshift mutations are highly prevalent in HNPCC cancers. Another gene frequently affected by frameshift mutations encodes the pro-apoptotic protein BAX (^7.2).

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