830 WERNSTEDT ET AL. and PMS2CL in opposite duplicons and separated by oppositely oriented 0.7-Mb sequences. Figure These considerations may have also diagnostic implications. If the mechanism illustrated in Fig- ure 4B is responsible for its formation, the delete- rious hybrid PMS2 allele found in patient TR13 might have arisen very recently. The same is true if it was generated as shown in Figure 4A with si- multaneous intrachromosomal and interchromoso- mal recombination events. In either case, the deleterious hybrid allele would be very rare in our proband’s population. In contrast, if the intra- chromosomal and interchromosomal events depicted in 4A occurred successively rather than simultaneously, they were probably separated by an interval of more than 130 genera- tions (assuming an average recombination rate of Mb 1 for the 0.7-Mb sequence between 1.1 cM the duplicons; Kong et al., 2002). And during this interval, the reciprocal hybrid alleles would have spread through a considerable portion of the pop- ulation, in tandem. This latter mutational conﬁg- uration, however, would escape detection by MLPA analysis (see ‘‘Results’’ Section), but, it could be identiﬁed by PMS2-speciﬁc cDNA sequencing or by the deleterious hybrid allele- speciﬁc multiplex assay we developed (Figs. 3B and 3C). Future studies using one or both of these assays to test patients with colon cancers charac- terized by isolated loss of PMS2 expression and with negative results at ﬁrst-line mutation analy- sis will shed light on the true frequency of this deleterious hybrid allele and the possibility that it represents a founder mutation in the popula- tion inhabiting the eastern Alpine valleys of Italy (where patient TR13 comes from). In turn, iden- tiﬁcation of this allele in other patients could pro- vide us with additional information on the recombination mechanism that might have gener- ated it. In conclusion, using the P008-B1 PMS2 MLPA kit and a set of selected reference DNAs that are universally available, we identiﬁed deleterious PMS2 alterations in 23% of our PMS2-deﬁcient colorectal cancer patients (27% of those under- going ﬁrst-line testing). The alterations found included two deletions (conﬁrming the high fre- quency of this type of alteration) and a deleteri- ous hybrid PMS2 allele, which is particularly interesting since almost all PMS2-PMS2CL hybrids described thus far have been nondeleteri- ous. These ﬁndings suggest that MLPA should be considered a possible ﬁrst-line method for Genes, Chromosomes & Cancer DOI 10.1002/gcc PMS2 mutation analysis, especially when high- quality RNA is not available for direct cDNA sequencing. 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