3.1 Splicing outcome of sequences variations of SLC4A1
3.1.1 Variant c.1765C>T (p.Arg589Cys) induced skipping of exon 14 compared with the WT plasmids.
Variant c.1765C>T (p.Arg589Cys), located at nucleotide position -36 from the 3’ end of exon 14, was predicted that the variant not only eliminates four ESEs (C GCAAGT, GC GCAA, TGC GCA, TGCTGC ) but also generate two overlapping ESSs (CTGT GC, TGCTGT ) by bioinformatics analysis with HSF (affected nucleotide is bold, shown at Table 1). To investigate the effect of c.1765C>T on pre-mRNA splicing, the WT (c.1765C) and mutant (c.1765C>T) minigene were created and were transfected separately into 293T cells. The result of the minigene assay revealed that two different electrophoresis bands were detected in both of WT and mutant minigene: a larger band of 400 bp and a smaller band of 263 bp (Figure 2A). Sequencing analysis showed that the larger splicing product contained exon 14 of SLC4A1 , whereas the smaller did not contain exon 14. The absence of exon 14 resulting in the loss of 174 bp does not alter the open reading frame. Analysis of cDNA extracted from the transfected HEK293T revealed that the amounts of the exon 14-skipping transcript of c.1765C>T were significantly increased with those of the WT plasmid (83.03% versus 43.93%, P<0.05), suggesting that the variant c.1765C>T disturbed the normal splicing in vitro (Figure 2D).