Some point variants lead to aberrant pre-mRNA splicing. (A) A scheme of modified exon 1 construct for minigene splicing assay. The exon 1 of F9 was fused with the exon 3 of F9 as shown (sequence in supplemental File 1), and the modified sequence was subcloned into the multicloning site (MCS) of pSPL3 vector, which was named as E231. The c.88 indicated the position of the last nucleotide of 3′ site of exon 1. (B-C) Comparison of sequencing chromatograms between canonical and 2 major aberrant splicing products in c.87A>G (B) and c.82T>G (C). Sequence changes were indicated by red arrow lines. WT indicated the canonical splicing product in E231. (D) Statistics of splicing products. Approximately 100 sequencing results of each construct were analyzed. The splicing products were classified into 2 groups: canonical splicing product (N) and aberrant splicing product (Ab). The percentage of each group was calculated. The χ2 analysis was performed using GraphPad Prism 7. ns indicates no significant difference (P > .05); ∗P < .05; ∗∗P < .01. AmpR, Ampicillin resistance gene; MCS, multicloning site; SA, exon with splice acceptor site; SD, exon with splice donor site.
