A DELETERIOUS PMS2-PMS2CL HYBRID ALLELE 827 fragment containing this intron 10 speciﬁcally from the deleterious hybrid allele. For this pur- pose, we used a gene-speciﬁc forward primer located in exon 10 and a pseudogene-speciﬁc reverse primer in exon 11. As shown in Figure 3B, sequencing of the amplicon revealed PMS2- speciﬁc single-nucleotide variants at all paralog- discriminating sites 50 of nucleotide c.1145-942 (inclusive) and PMS2CL-speciﬁc single-nucleotide variants 30 of nucleotide c.1145-790 (inclusive). These two nucleotides delimit the 153-bp break- point region, which includes three additional paralog-discriminating, single-nucleotide variants: one derived from PMS2CL (the adenine at posi- tion c.1145-921) and two from PMS2 (the guanine and inserted cytosine at positions c.1145-886 and c.1145-880_1145-879, respectively; Fig. 3B). This pattern of gene- and pseudogene-speciﬁc variants within the breakpoint region reﬂects the type of double Holliday junction resolution that leads to strand crossover in the recombination-initiated, double-strand break repair process (as well-illus- trated in Fig. 1 of Chen et al., 2007), thereby conﬁrming that recombination with crossover is the mechanism that generated this deleterious hybrid PMS2 allele. Because PMS2 and PMS2CL are oppositely ori- ented, interparalog crossover is more likely to occur intrachromosomally. This process would generate two reciprocal hybrids, each containing paralog-derived sequences (Fig. 3A). However, in the presence of a reciprocal hybrid PMS2CL al- lele, the deleterious hybrid PMS2 allele found in patient TR13 would have escaped detection by MLPA because of the equal numbers of gene- speciﬁc and pseudogene-speciﬁc sequences (2:2) in exons 11 and 12. PMS2-speciﬁc cDNA sequencing (Etzler et al., 2008; van der Klift et al., 2010) as well as approaches based on PMS2-speciﬁc long-range PCR (Vaughn et al., 2010) would detect the deleterious hybrid allele also in this conformation. However, high quality RNA and gDNA is needed for these assays. As such material is frequently not available of retro- spective patient’s cohorts, we developed a simple PCR assay that identiﬁes this deleterious hybrid allele even when it is accompanied by its recipro- cal hybrid PMS2CL allele. This assay can be used as screening tool in large retrospective cohorts. It is based on the use of primers that speciﬁcally amplify a 239-bp breakpoint-including fragment from the deleterious PMS2 hybrid allele (Fig. 3B). This reaction is duplexed with a control PCR that generates a 696-bp fragment using non- speciﬁc primers located in exon 15 (Ganster et al., 2010). After verifying its speciﬁcity in the analysis of DNA samples from 150 control indi- viduals (Ganster et al., 2010), all of which were negative for the 239-bp long PCR product (Fig. 3C), we used the new assay to retest the 10 patients in our cohort whose suspected PMS2 mutation had not been veriﬁed. Negative ﬁndings in all 10 cases exclude the possibility that a recip- rocal PMS2CL hybrid allele is masking the pres- ence in these individuals of the deleterious hybrid PMS2 allele found in patient TR13. DISCUSSION The high prevalence of clinically irrelevant PMS2 and PMS2CL hybrid alleles (found in 70% in the Caucasian population; van der Klift et al., 2010) has seriously hindered MLPA-based detection of copy number changes involving PMS2 exons 13–15. Our ﬁndings conﬁrm that PMS2 MLPA analysis can be substantially improved by the use of MRC Holland’s recently redesigned PMS2 MLPA kit (P008-B1), together with selected reference DNA samples known to carry two PMS2-derived and two PMS2CL- derived sequences in this region (Vaughn et al., 2011). The eight reference samples we veriﬁed for use in this study can be adopted as controls for this assay in any diagnostic laboratory. They are publicly available (from Coriell Cell Reposito- ries), and because they are derived from EBV- immortalized lymphocyte cell lines, their avail- ability is long-term. Routine use of these refer- ence DNAs can facilitate standardization of MLPA analysis. With this improved MLPA approach, we ana- lyzed germline DNA from 13 patients with colo- rectal tumors characterized by isolated PMS2 expression loss. The assay identiﬁed deleterious germline alterations involving the PMS2 locus in 3 (27%) of the 11 cases in which MLPA was the ﬁrst-line method of mutation analysis (23% of the entire cohort). These ﬁgures are consistent with previous MLPA ﬁndings in colon cancer series similar to our own. Deleterious PMS2 copy num- ber alterations were found in 11 (17%) of 65 patients tested in a US laboratory (Vaughn et al., 2010, 2011) whereas a Dutch group reports that up to 33% (15/45) of the analyzed patients carry germline PMS2 mutations that should be detecta- ble with the P008-B1 PMS2 MLPA kit (van der Klift et al., 2010; van der Klift and Tops, personal communications). Genes, Chromosomes & Cancer DOI 10.1002/gcc