A DELETERIOUS PMS2-PMS2CL HYBRID ALLELE 823 was used according to the manufacturer’s instruc- tions for genomic copy number proﬁling. Probe allocation was based on the March 2006 human reference sequence (NCBI Build 36.1). Raw probe data ﬁles were processed within the R sta- tistical software framework (http://www.R-project. org) with additional packages from the aroma. affymetrix project (http://aroma-project.org). Copy numbers were estimated with the CRMAv2 method (Bengtsson et al., 2009), which includes allelic cross-talk calibration, normalization for probe sequence effects, and normalization for PCR fragment-length effects. Copy numbers were segmented using the Circular Binary Seg- mentation method (Venkatraman and Olshen, 2007). Normalized probe and segmentation data were visualized with custom software developed for the Progenetix project (Baudis and Cleary, 2001). Breakpoint Analysis of the Deleterious Hybrid PMS2 Allele and Development of an Assay for its Detection The sequence exchange breakpoint responsible for the deleterious hybrid PMS2 allele in case TR13 was expected to lie in intron 10. There- fore, we speciﬁcally ampliﬁed a 2,314-bp PCR fragment from gDNA containing the deleterious hybrid allele using a PMS2-speciﬁc primer located in exon 10 (PMS2B_F) and a PMS2CL- speciﬁc primer in exon 11 (PMS2CLc.1238CC_ R). The fragment was then sequenced with seven internal sequencing primers (Supporting Informa- tion Table S1). We developed a simple PCR-based assay for detecting the deleterious hybrid PMS2 allele. Pri- mers ﬂanking the identiﬁed breakpoint were designed to speciﬁcally amplify sequences from the hybrid allele. These primers, that is, a PMS2- speciﬁc forward primer (PMS2inIVS10_gen_1f) and a PMS2CL-speciﬁc reverse primer (PMS2i- nIVS10_psgen_1r), generate a 239-bp hybrid allele-speciﬁc PCR product (Supporting Informa- tion Table S1). They were used in a duplex PCR reaction with primers that generate a 696-bp con- trol PCR product (PMS2in14_1F and PMS2B_ Rnew) (Supporting Information Table S1). NCBI Reference Sequences NCBI reference sequences (RefSeqs) NG_ 008466.1 and NM_000535.5 were used for the human PMS2 gene and human PMS2 mRNA, respectively. RefSeq NC_000007.13 was used for the human PMS2CL pseudogene. The sequence variants that discriminate between the PMS2 and PMS2CL paralogs are those published by Hay- ward et al. (2007); they are given as variants with respect to the PMS2 RefSeq. All variants are described in accordance with the Human Genome Variation Society (http://www.hgvs.org/ mutnomen) guidelines, with the A of the ATG start codon as position c.1. RESULTS Publicly Available Reference DNA Samples for PMS2 MLPA Analysis Because of the high prevalence of nondeleteri- ous hybrid PMS2 and PMS2CL alleles, the distri- bution of PMS2 and PMS2CL-speciﬁc sequences downstream of exon 12 displays high interindivid- ual variability (Hayward et al., 2007;Ganster et al., 2010; van der Klift et al., 2010). Randomly chosen reference samples of DNA are thus likely to differ widely in terms of the distribution of gene- and pseudogene-derived sequences in this region. This is reﬂected by high standard devia- tions for the reference DNA signals generated with all the paralog-discriminating probes located downstream of exon 12 (Supporting Information Fig. S1). It is important to note that an unequal distribution of gene-derived and pseudogene- derived sequences in the reference DNA set will reduce the accuracy of copy number assessments at these loci in patient DNA samples. For this reason, reference DNAs must harbor two PMS2- speciﬁc copies and two PMS2CL-speciﬁc copies of each sequence bound by paralog-discriminat- ing probes for exons 11-15. To obtain a set of reference samples that could be used by all laboratories performing PMS2 MLPA, we evaluated 24 DNA samples from EBV-immortalized lymphocytes, which can be obtained from Coriell Cell Repositories. MLPA ﬁndings for these candidates were compared with those for ﬁve previously characterized DNA sam- ples (Ganster et al., 2010) that met the criteria listed above. Sixteen (67%) of the candidates showed MLPA signals reﬂecting an unequal dis- tribution of gene-derived and pseudogene- derived sequences (Fig. 1). The other eight (33%) met the prerequisites for suitable reference samples (Table 2). Each subsequent MLPA experiment was performed with a control set con- sisting of ﬁve of these samples (randomly chosen for each experiment). Genes, Chromosomes & Cancer DOI 10.1002/gcc