3 Results
A total of 113 different variants, including 20 of SLC4A1 , 39 ofATP6V1B1 , 48 of ATP6V0A4 , 2 of FOXI1, 3 of WDR72, 1 of
ATP6V1C2 gene, were collected for analysis by the bioinformatics
software. Potential splicing variants within three bases of the 5 ’or 3’
end of the exon, or predicted that affect splicing regulatory elements
(elimination of enhancers or creation of silencers) were selected for
further minigene assay to determine whether such variants affect
splicing. Some selected variants had combined effects that both generate
ESS and broke ESE (the total number of ESEs disruption and ESSs
generation is more than 6 for SLC4A1, 7 for ATP6V1B1 and ATP6V0A4). From
the results of these in silico analyses, the distribution of these
candidate variants enrolled in this study was as follows: 3 inSLC4A1(c.1437C>G,
c.1564G>A and c.1765C>T), 6 in ATP6V1B1(c.368G>T, C.370C>T, c.481G>A,
c.484G>T, c.1102G>A and
c.1251C>G) and 5 in ATP6V0A4 (c.52C>T,
c.322C>T, c.1571C>T, C.1572G>A,
c.2190C>G and c.2257C>T) as shown in Table 1,
and Figure 1 B-D. Different control minigenes were generated comprisingSLC4A1 WT sequences of exons 13 (pSPL3-SLC4A1 Ex13) and 14
(pSPL3-SLC4A1 Ex14), ATP6V1B1 of exons 5
(pSPL3-ATP6V1B1 Ex5), 6 (pSPL3-ATP6V1B1 Ex6), 11
(pSPL3-ATP6V1B1 Ex11), and 13 (pSPL3-ATP6V1B1 Ex13), andATP6V0A4 of exons 3 (pSPL3-ATP6V0A4 Ex3), 6
(pSPL3-ATP6V0A4 Ex6), 15 (pSPL3-ATP6V0A4 Ex15), and 20
(pSPL3-ATP6V0A4 Ex20) respectively. With the corresponding WT
minigenes as the template, all candidate variants minigenes successfully
generated through site-directed mutagenesis, except
c.1251C>G in ATP6V1B1 and c.2257C>T inATP6V0A4 was not introduced into the pSPL3-WT minigene due to
limitation of experimental technique.