820 WERNSTEDT ET AL. of its heterodimeric partner MLH1 and of the other MMR proteins) was approximately as com- mon as loss of MSH2 (Truninger et al., 2005). In this study and others (Clendenning et al., 2008; Senter et al., 2008), heterozygous PMS2 muta- tions displayed lower penetrance than mutations involving other MMR genes, as reﬂected by later onset of colorectal cancer (average: 59 years vs. 45 years in MLH1/MSH2 mutation carriers) and weaker family histories of Lynch-syndrome asso- ciated cancers. Consequently, patients with these mutations were unlikely to meet the Amsterdam II criteria (Vasen et al., 1999). Mutational analysis of PMS2, which is located on chromosome 7 (band p22), is complicated by the existence of 15 PMS2 pseudogenes on the same chromosome. One of these, a transcribed pseudogene known as PMS2CL, lies on 7p22 itself, just 0.7 Mb centromeric to the functional gene. PMS2CL is the result of the inverted dupli- cation of a 100-kb repeat element that includes the 30 region of PMS2 containing exons 9–15. The pseudogene contains six of these PMS2 exons (9 and 11-15) but lacks exon 10 owing to an Alu-mediated 2.7-kb deletion (De Vos et al., 2004). Recombination involving these two duplicons has led to considerable sequence homogenization at their centers and to a lesser extent at their extremities (Hayward et al., 2007). Recombina- tion with crossover and/or gene-conversion between the paralogs situated at the inner ends of the duplicons produces hybrid PMS2 alleles that contain PMS2CL-derived sequences (as deﬁned by NCBI RefSeq NC_000007.13) as well as hybrid PMS2CL alleles with sequences derived from PMS2 (according to RefSeq NM_000535.5). The transfer involves mainly sequences lying between intron 12 and the 30 ends of the two paralogs – in terms of coding sequences, those of exons 13–15 (Hayward et al., 2007; Ganster et al., 2010; van der Klift et al., 2010). The PMS2CL sequences of these exons are almost always syn- onymous variants (with respect to those of the functional gene). The only exception is a mis- sense alteration (p.N775S) in PMS2CL exon 14. However, hybrid PMS2 alleles containing this variant represent 4–25% of all PMS2 alleles found in the general population, depending on the eth- nic group (Ganster et al., 2010). Therefore, the vast majority of hybrid PMS2 alleles identiﬁed thus far are classiﬁed as nondeleterious. Because of the very high prevalence of hybrid PMS2 and PMS2CL alleles (van der Klift et al., Genes, Chromosomes & Cancer DOI 10.1002/gcc 2010), the 30 regions of the gene and pseudogene cannot be reliably distinguished on the basis of sequence differences with respect to their respec- tive NCBI RefSeqs. This is a serious limitation for genomic DNA (gDNA)-based assays used for PMS2 mutation analyses [e.g., exon sequencing to detect point mutations and multiplex ligation- dependent probe ampliﬁcation (MLPA) for the identiﬁcation of deletions/ampliﬁcations]. The shortcomings of gDNA-based sequencing of PMS2 exons 11-15 can be effectively circum- vented with recently developed RNA-based strat- egies, such as complementary DNA (cDNA) sequencing (Etzler et al., 2008; van der Klift et al., 2010), or with long-range PCR (Vaughn et al., 2010). New solutions are also needed for the detection of copy number changes, above all deletions, which appear to account for up to 33% of all PMS2 mutations (van der Klift et al., 2010; Vaughn et al., 2010). The MLPA assay kit used until recently for this purpose (the P008-A1 PMS2 MLPA kit, MRC Holland, Amsterdam, The Netherlands) contained only PMS2-speciﬁc probes for exons 13-15, and this led to both false- negative and false-positive results for these exons due to the presence of hybrid alleles in many gDNA samples. To address these problems, the PMS2 MLPA kit has recently been redesigned. The new version (kit P008-B1) contains gene- and pseudogene-speciﬁc probes for exons 11-15, as well as nonspeciﬁc probes for exons 12-15, which hybridize to gene and pseudogene sequen- ces and thus detect a total of four DNA copies in the genome. These modiﬁcations have substan- tially improved the reliability of the assay, speciﬁ- cally from exons 12/13 downwards (Vaughn et al., 2011). Owing, however, to wide interindividual variability in the distribution of PMS2 and PMS2CL sequences downstream of exon 12, copy number variations (CNVs) can be accurately detected only when each run includes reference DNA samples known to contain two gene-speciﬁc copies and two pseudogene-speciﬁc copies of exons 11-15 sequences. This study illustrates the pivotal role played by these reference DNA samples and describes eight publicly available samples that can be used with the new PMS2 MLPA kit. We used this approach to identify the presence of PMS2 deletions in germline DNA from 13 patients whose colorectal tumors were suspected to be caused by PMS2- related Lynch syndrome. In three cases, MLPA yielded evidence of deleterious PMS2 alterations, which were subsequently veriﬁed and further