Patient code Age Sex CH1 CH2 CH3 CH4 CH5 CH6 CH7 CH8 CH9 CH10 CH11 CH12 TR13 35 29 40 48 53 77 73 38 80 29 51 44 56 M M F M M M M F M F F M M A DELETERIOUS PMS2-PMS2CL HYBRID ALLELE TABLE 1. Patients with Colorectal Tumors Site C A A A SF A A C A S SF R C/R Stagea T3N1M0 T3N0M0 T3N0M0 T2N0M0 T3N0M0 T1N0M0 T3N0M0 T3N0M0 T3N1M0 T2N0M0 T3N0M0 Advanced adenoma T3N0M0/T1N0M0 Gradea 3 2 2 3 2 2 2 2 3 2 2 Severe dysplasia G3/G2 MSI P Na Na P P P P P Na P P AB P 821 Revised AC and BG BG1 and BG4 BG1 BG1 BG1 and BG5 AC Na Na BG1 Na BG1 AC BG1 and BG5 BG2 and BG5 Note: CH6 ¼ 52557 and CH7 ¼ 61263 in Truninger et al. (2005). MSI, microsatellite instability at BAT26 and dinucleotide repeats on 7p22; P, present; AB, absent; m, male; f, female; C, cecum; A, ascending; SF, splenic ﬂexure; S, sigmoid colon; R, rectum; AC, revised Amsterdam Criteria; BG, revised Bethesda Guidelines; Na, not available. aTNM and tumor grade classiﬁcation according to Sobin and Wittekind (2002). characterized with other methods. Two were deletions (one of which encompassed the entire PMS2 gene and at least two other genes with tumor-suppressing functions); the third was an alteration that has not been previously described: a deleterious hybrid PMS2 allele produced by recombination with crossover between PMS2 and PMS2CL. MATERIAL AND METHODS Patient and Reference DNA Samples This study included gDNA samples from 13 patients with colorectal tumors displaying isolated PMS2 expression loss (Table 1). One (patient TR13) was recruited in the Department of Gas- troenterology of the Santa Chiara Hospital in Trento, Italy. The other 12 were enrolled at the Institute of Molecular Cancer Research, Uni- versity of Zurich, Switzerland. All patients gave written informed consent to molecular genetic testing. In the 10 cases in which family history was available, the pedigree fulﬁlled the revised Am- sterdam II criteria (Vasen et al., 1999) (n ¼ 2) or one or more of the criteria contained in the re- vised Bethesda Guidelines (Umar et al., 2004) (n ¼ 8). In 11 of the 13 patients, MLPA was the ﬁrst-line method of mutation analysis. The other two (CH6 and CH7) had undergone gDNA sequencing in a previous study (Truninger et al., 2005), which was negative for point mutations in PMS2. DNA remaining after the completion of this study was used now for MLPA analysis. To identify appropriate reference DNA sam- ples that could be used by all laboratories for PMS2 MLPA analysis, we obtained 24 DNA sam- ples from Coriell Cell Repositories (Camden, NJ) and analyzed them by MLPA. Results were com- pared with those for DNA samples from ﬁve con- trols whose genotypes had been determined in a previous study (Ganster et al., 2010). Candidate samples found to contain two PMS2-speciﬁc cop- ies and two PMS2CL-speciﬁc copies of each sequence bound by the probes for exons 11-15 were selected as reference samples for this study. We also used 150 DNA samples from individu- als with different ethnic backgrounds (Ganster et al., 2010) as negative controls in the allele-spe- ciﬁc PCR assay described below. MLPA The P008-B1 PMS2 MLPA kit (MRC-Holland) was used according to the manufacturer’s instruc- tions to detect single and multiple exon deletions (as well as possible duplications) in the PMS2 gene. The redesigned assay includes 24 PMS2- speciﬁc probes (exons 1-15), ﬁve PMS2CL-speciﬁc probes (exons 11-15), and ﬁve nonspeciﬁc probes (exons 12-15) that hybridize to both PMS2 and PMS2CL. Before MLPA analysis, all gDNA sam- ples were puriﬁed with the QIAamp DNA Micro kit (Qiagen, Hilden, Germany), and a total of 50- 100 ng of gDNA was used per MLPA reaction. Each MLPA run included ﬁve appropriate refer- ence DNA samples (selected as described above; Table 2). MLPA results were analyzed with the Genes, Chromosomes & Cancer DOI 10.1002/gcc