A Novel Alu Element Insertion in ATM Induces Exon Skipping in Suspected HBOC Patients

Figure 3

Minigenes with AluYa5 element insertion predominantly expressed exon 54-lacking mutant transcripts. (a) Schematic representation of the mutant (+ Alu) and wild-type (- Alu) minigenes. The pET01 plasmid-descendent Rous sarcoma virus (RSV) promoter, exons (pEx1-2), and introns are depicted in grey. The AluYa5 element inserted in ATM exon 54 is indicated as red arrow. (b) Gel electrophoresis of RT-PCR products after transfection of HEK293T and MCF7 cells with mutant (+ Alu) and wild-type (- Alu) minigenes show the full-length (FL) transcript of 388 bp and the exon 54-deprived mutant transcript (Δex54) of 305 bp. (c) Sanger sequencing electropherograms across the junction of the plasmid-descendent exon 1 (pEx1) and ATM exon 55 after TA cloning of the mutant transcript. (d) Capillary electrophoresis of RT-PCR products from HEK293T cells transfected with mutant (+ Alu, top) or wild-type (- Alu, bottom) minigenes depicting the full-length (FL) and mutant (Δex54) transcripts. (e) Capillary electrophoresis results shown as bar diagrams depicting the percentage of full-length (FL) and mutant (Δex54) transcripts in HEK293T and MCF7 cells after transfection with mutant (+ Alu) or wild-type (- Alu) minigenes. Nonsense-mediated decay was inhibited by cycloheximide treatment. Data are represented as of three independent experiments. Bp: basepairs; EVC: empty vector control; ex: exon; nt: nucleotides.

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