Fig. 1

TP63 variant form and the splicing effect of the 14-bp deletion (A) Sanger sequencing validated the TP63 variant in the patient with POI. The red arrow indicates the variant c.1742_1746 + 9del. (B) The variant influenced the mRNA splicing of the exon 13 and intron 13, so the WT minigene plasmid containing exon 12, intron 12, exon 13, intron 13, and exon 14 of the TP63 gene was constructed, and then the c.1742_1749 + 9del 14-bp deletion variant was introduced into the WT plasmid to obtain the mutational plasmid (Mut minigene). (C) The pcDNA3.1 empty vector (NC), WT, and Mut minigene plasmids were transfected into 293FT cells, and then the RNA was collected for the RT-PCR. The forward primer in exon 12 (12F), the reverse primer in exon 14 (14R), the forward primer in exon 12 (12F), and the reverse primer in exon 13 (13R) were used. The RT-PCR results indicated that 442 -bp bands were compatible with exon 13 skipping under Mut minigene conditions, while in the WT minigene, 536-bp bands corresponding to the correctly spliced product obtained using 12F and 14R primer pairs. For the 12F and 13R primer pairs, a 239-bp band product was obtained, corresponding to the correctly spliced product under WT minigene condition. There was no band compatible with exon 13 skipping under Mut minigene conditions. We conclude that the RT-PCR obtained represented the Mut minigene performed as the exon-12–14 transcript. (D) The cells transfected with Mut minigene plasmids were tested by Sanger sequencing, and the results showed the 14-bp deletion variant induced exon 13 skipping