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. 2019 Jul 11;105(3):625–630. doi: 10.1016/j.ajhg.2019.06.011

Figure 2.

Figure 2

Effect of the ANAPC1 Splicing Variant on Protein Levels and the Mechanism of Pseudoexon Activation in Fibroblasts Derived from Individuals 1 and 2

(A) Realtime PCR analysis demonstrating significant difference (p < 0.001) in messenger RNA from individuals 1 and 2 compared to unrelated controls.

(B) An immunoblot showing a decrease in ANAPC1 protein levels in fibroblasts from individuals 1 and 2 compared to unrelated controls.

(C) Realtime PCR with primers spanning exons 22 and 23 of ANAPC1 and subsequent agarose gel electrophoresis revealing the presence of an additional fragment in fibroblasts from affected individuals. Sanger sequencing of the additional fragment revealed the presence of a 95 bp nucleotide that is incorporated between exon 22 and 23 of ANAPC1 cDNA. Analysis of the pseudoexon revealed two stop codons leading to the nonsense-mediated decay pathway, accounting for the decrease in RTS type 1 individual transcript (A) and protein (B) levels.