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 intrachromosomal recombination with crossover. Upper figure: Normal locations on 7p22 of gene (green); pseudogene (red); and 100-kb duplicons (gray hatching). Arrowheads indicate direction of transcription. The 0.7-Mb interduplicon sequence is shown as an arrow. Middle 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 3′ 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 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: negative (water) control. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]