826 WERNSTEDT ET AL. Figure 3. Breakpoint analysis and allele-speciﬁc PCR detection of the deleterious hybrid PMS2 allele in patient TR13. (A) Schematic showing sequence exchange between PMS2 and PMS2CL due to intra- chromosomal recombination with crossover. Upper ﬁgure: Normal locations on 7p22 of gene (green); pseudogene (red); and 100-kb duplicons (gray hatching). Arrowheads indicate direction of transcrip- tion. The 0.7-Mb interduplicon sequence is shown as an arrow. Mid- dle ﬁgure: intrachromosomal recombination with crossover produces a hybrid PMS2CL allele (in the telomeric duplicon) containing a PMS2- derived sequence and a hybrid PMS2 allele (partially embedded in the centromeric duplicon) that contains a PMS2CL-derived sequence 30 of the recombination breakpoint (intron 10; IVS10). The interduplicon sequence has also been inverted. Lower ﬁgure: zoom showing the hybrid PMS2 allele with PMS2-derived (green) and PMS2CL-derived (red) sequences. (B) Zoom (not to scale) showing the breakpoint heterozygosity for all known PMS2CL-speciﬁc sequence variants in exons 11–14 and homozygos- ity for the PMS2CL-speciﬁc variant c.*92dupA in exon 15. When individual RT-PCR products were sequenced after cloning, the results con- ﬁrmed the presence of the deduced deleterious PMS2 hybrid allele (with sequences in exons 11– 15 that were all PMS2CL-derived). They also showed that the other PMS2 allele was a nonde- leterious hybrid containing the pseudogene- derived variant c.*92dupA. Moreover, cDNA sequencing of PMS2CL transcripts conﬁrmed the presence of a nonhybrid allele, together with the expected hybrid PMS2CL allele containing PMS2-speciﬁc sequences in exons 13–15 (data Genes, Chromosomes & Cancer DOI 10.1002/gcc region of the deleterious hybrid PMS2 allele. The heavy black line between exons 10 and 11 (rectangles) represents intron 10. Vertical bars represent PMS2-derived (green) and PMS2CL-derived (red) sequence variants. Horizontal arrows represent allele-speciﬁc primers spanning the breakpoint (gene-speciﬁc in green, pseudogene-speciﬁc in red). The resulting allele-speciﬁc PCR products (2,314 and 239 bp long) are indicated (light gray brackets), and the breakpoint region is enclosed in a dotted rectangle (light gray). (C) Results of the duplex PCR assay for the deleterious hybrid PMS2 allele in patient TR13 (P) and two of the 150 normal controls (C) tested. The 696-bp control PCR product was present in the patient and all 150 controls; the 239-bp PCR product speciﬁc for the deleterious hybrid PMS2 was present only in the patient sample. M: 100 bp DNA ladder; W: nega- tive (water) control. [Color ﬁgure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] not shown). This complex genotype (Fig. 2B) explains the gene:pseudogene copy number ratios observed in exons 11, 12, and 15 (1:3) and in exons 13 and 14 (2:2). Characterization of the Breakpoint of Sequence Exchange Between PMS2 and PMS2CL Leading to the Deleterious PMS2 Hybrid Allele with Recombination crossover between the paralogous sequences is the most likely mecha- nism underlying the formation of the deleterious hybrid PMS2 allele (Fig. 3A). Because the recom- bination breakpoint was expected to lie 50 of exon 11, in intron 10, we ampliﬁed a 2314-bp