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. 2016 Nov 29;107(11):1677–1686. doi: 10.1111/cas.13073

Figure 5.

Figure 5

Gene testing of subject No. 6. (a) Immunochemistry (IHC) using four antibodies to mismatch repair (MMR) proteins. Note the loss of PMS2 protein expression in cancer cell nuclei. (b) RT‐PCR/direct sequencing analysis of the PMS2 gene in subject No. 6. The upper panel indicates the sequencing profile showing the c.2446delG (p.Val816Stop) frameshift mutation from a puromycin‐treated total RNA sample and the middle panel shows the sequencing profile without the puromycin treatment. Note that the signal ratios of the frameshift mutation to the wild‐type sequence were almost the same with or without the puromycin treatment, indicating the absence of nonsense‐mediated mRNA decay (NMD). (c) Long/nested PCR/direct sequencing analysis of the PMS2 gene. An analysis of genomic DNA by a long PCR/nested PCR/direct sequencing analysis showed the c.2446‐1G>A mutation in the splicing acceptor site of exon 15 (black arrow). Because exon 15 was the last exon of the PMS2 gene, a mutation in the exon–intron boundary activated cryptic splicing site and caused a 1‐bp‐deleted frameshift mutation in mRNA without NMD.