A DELETERIOUS PMS2-PMS2CL HYBRID ALLELE 825 Figure 2. MLPA results showing the deleterious hybrid PMS2 allele in patient TR13. (A) Bars in upper and lower histograms represent MLPA probes. Probe labels appear under the lower histogram. Probes for exons (E) 11–15 are designated gene-speciﬁc (G), pseudogene-speciﬁc (P), or nonspeciﬁc (GþP). Upper histogram: Lavender bars indicate mean probe signals with standard deviations for ﬁve of the selected refer- ence DNAs reported in Table 2; green bars: probe signals for patient DNA. Numbers below the green bars indicate the amplicon size (in nts) of the corresponding MLPA probe. Lower histogram: the bar for each gDNA excluded sequence alterations at this site, whereas sequencing of an RT-PCR product com- prising PMS2 exons 6–9 revealed heterozygous loss of exon 8 in the patient’s transcripts (data not shown). Collectively, these results conﬁrmed the presence of a genomic deletion involving PMS2 exon 8. To characterize better this deletion, we ampli- ﬁed patient gDNA using a forward primer (PMS2_7f) located in intron 6 and a reverse primer (PMS2/4) in exon 9. The amplicon pro- duced a single band in agarose gel electrophoresis and was 3,400 shorter than the expected prod- uct of wild-type allele ampliﬁcation ( 2,200 vs. 5,597 bp), which indicates that only the deletion- carrying allele is ampliﬁed under the PCR condi- tions we used. Sequencing of this amplicon with an internal primer showed that the breakpoints of this 3,351-bp deletion (i.e., c.803þ384_904- 1533del) were located within two identically ori- ented AluSb elements in introns 7 and 8. MLPA results for the third patient (TR13) revealed one gene-speciﬁc and three pseudogene- speciﬁc DNA copies at all probe sites in exons 11, 12, and 15 (Fig. 1B). In exons 13 and 14, the patient harbored two PMS2- and PMS2CL-speciﬁc copies (Figs. 1B and 2A). These ﬁndings are in- dicative of a hybrid PMS2 allele containing a probe represents the probe signal for patient DNA as a percentage of the mean signal for the reference DNAs. Lavender bars represent per- centages ranging from 75 to 125% (red dotted lines); larger discrepancies between patient and reference samples are represented by violet bars. (B) Number of DNA copies at sites hybridized by probes for exons 11– 15 and deduced patient genotypes. Green ‘ g’ : PMS2-speciﬁc sequence; red ‘ p’ : PMS2CL-speciﬁc sequence; D: absent in PMS2CL (exon 10) (see text for explanation of these results). [Color ﬁgure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] PMS2CL-derived sequence, which starts at the beginning of exon 11 (position c.1360_1361, where the ﬁrst PMS2-speciﬁc MLPA probe in exon 11 is located; see Fig. 1A) and extends at least 1,099 bp downstream from exon 12 (speciﬁ- cally, to position c.2174þ1097_2174þ1099, where the gene-speciﬁc and pseudogene-speciﬁc intron 12 probes are located; see Fig. 1A). Compared with its PMS2 paralog, the PMS2CL exon 11 sequence contains two frame-shift variations (c.1730dupA and c.1863_1864delTA). As each of these variations produces a premature stop codon when introduced into PMS2, the hybrid allele can be classiﬁed as deleterious. We suspected that the PMS2CL-derived sequence in this hybrid allele extended even far- ther downstream, into or past exon 15, and that the equal distribution of gene- and pseudogene- speciﬁc sequences (2:2) in exons 13 and 14 might reﬂect the presence of a hybrid PMS2CL allele containing PMS2-speciﬁc sequences in these exons. To conﬁrm this hypothesis, we sequenced PMS2 transcripts from the patient that had been ampliﬁed as described by Etzler et al. (2008) using a forward primer located in exon 10 and a reverse primer downstream of the termination codon. Direct sequencing of the RT-PCR prod- ucts (Supporting Information Fig. S4) showed Genes, Chromosomes & Cancer DOI 10.1002/gcc