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).