824 WERNSTEDT ET AL. Figure 1. PMS2 MLPA results for reference DNA candidates and DNA samples from patients with colorectal tumors displaying isolated PMS2 expression loss. (A) Schematic showing ligation sites for the probes in the redesigned (P008-B1) PMS2 MLPA kit. Paralog-discrimi- nating ligation sites are shown in boldface. Rectangles: exons 11–15 of PMS2 (and PMS2CL); lines: introns. (B) MLPA results (given as copy numbers of the DNA sequences bound by the MLPA probes) for 24 reference DNA sample candidates (obtained from Coriell Cell Reposi- tories) and 13 patient DNA samples. Results are grouped for exons 1– 7 (E1-E7) and 9 and 10 (E9 and E10) and reported individually for exons 8 (E8) and exons 11 (E11) to 15 (E15), the latter being shared Alterations Identiﬁed by PMS2 MLPA Analysis of gDNA from Patients with PMS2-Deﬁcient Colorectal Tumors As shown in Figure 1B, aberrant MLPA signals indicating a deleterious PMS2 alteration were de- tected in three (23%) of the 13 patients with PMS2- deﬁcient colorectal tumors (Table 1). In Patient CH8, the assay revealed loss of one PMS2-speciﬁc DNA copy of exons 1-12 and the presence of two PMS2CL- speciﬁc DNA copies in exons 11 and 12 (Fig. 1B; Supporting Information Fig. S2). We found reduced probe signals indicative for three (instead of four) cop- ies of the sequences bound by the nonspeciﬁc probes for exons 12, 13, 14, and 15. Paralog-speciﬁc probe signals for the latter three exons disclosed that the three copies were all PMS2-derived (Fig. 1B, Supporting Information Fig. S2). This MLPA pattern is most likely the result of a deletion encompassing the entire PMS2 gene and the concomitant presence of two hybrid PMS2CL alleles containing PMS2- speciﬁc sequences in exons 13-15. The latter alleles, together with the remaining nonhybrid, wild-type PMS2 allele, would account for the three gene-spe- ciﬁc signals in exons 13-15. Genes, Chromosomes & Cancer DOI 10.1002/gcc by the paralogs. The paralog-discriminating sequence variants at the liga- tion sites of the probes in exons 11–15 are color-coded: green, PMS2- speciﬁc probes; red, PMS2CL-speciﬁc probes; black, nonspeciﬁc probes. Numbers in cells (0–4): copies of exon sequences in sample. Paired gray cells: uneven distribution of PMS2- and PMS2CL-speciﬁc signals in samples with a total of four copies, which reﬂects the presence of non- deleterious hybrid alleles (PMS2 or PMS2CL). Orange cells: aberrant MLPA results in patient CH8 (reﬂecting deletion of all PMS2 exons); patient CH11 (exon 8 deletion); and patient TR13 (who had a deleteri- ous PMS2 hybrid allele; see text for details). [Color ﬁgure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] Genomic array analysis (Supporting Informa- tion Fig. S3) conﬁrmed the presence of a large ( 125 kb) deletion containing the entire PMS2 gene, three other genes (ANKRD61, AIMP2 [JTV1], and EIF2AK1), and a portion of the cod- ing region of RSPH10B. The distal breakpoint of the deletion was located downstream to PMS2, in the center of the telomeric duplicon, between probes CN_1240840 and CN_1240842 [at nucleo- tides 5,970,140 and 5,970,432 (NCBI build 36.1/ hg18), corresponding to between 6,003,614 and 6,003,906 in GRCh37/hg19]. The proximal breakpoint lay within the interval of nucleotides 6,095,502–6,097,302; probes CN_1240873 and CN_1240874 (corresponding to 6,121,772– 6,137,980 in hg19, respectively) which is located within the 0.7-Mb sequence separating the telo- meric and centromeric duplicon. Interestingly, this breakpoint falls within the range reported for a deletional CNV that has been observed in a number of studies (e.g., Kidd et al., 2008). In patient CH11, MLPA showed loss of one DNA copy at the site hybridized by the exon 8 probe (Fig. 1B). Sequencing of exon 8 from