RESULTS
A total of 366 missense variants,
distributed in 26 exons of SLC12A3, were collected for bioinformatics
predictions. They were
analysed in silico with
BDGP for splice site prediction, and with ESE/ESS estimation algorithms
integrated in HSF. It is well-known that regulatory elements are
commonly exist in exons with weak splice sites (Wu, Zhang, & Zhang,
2005). Therefore, we sought to select variants located in exons that
have a weak 5′ or 3′ splice site. Except for variants located in the
first and last exon that could not be analyzed with the minigene
approach, four potential splicing variants were selected following the
screening criteria (BDGP and HSF score below 0.7 and -8, respectively).
We also selected six variants within two bases of 5’ or 3’ ends of the
exons, which were predicted to have a big influence on classic splicing
sites according to BDGP. As a result, a total of 10 candidate variants
(c.602G>A,
c.602G>T, c.1452C>G, c.1567G>A,
c.1667C>T, c.1925G>A, c.2548G>C,
c.2549G>C, c.2755A>T and
c.2863A>T) located in seven exons of the SLC12A3gene were included in this study and collected from literatures
(Balavoine et al., 2011; Colussi et al., 2007; Fujimura et al., 2019;
Jiang et al., 2015; Miao et al., 2009; Riveira-Munoz et al., 2007;
Vargas-Poussou et al., 2011). These candidate variants involved the
generation of ESSs and/or the destruction of ESEs, the activation of a
cryptic splice site, or the alteration of
canonical splice sites (Table 1).
Based on this bioinformatics data, we followed up with
minigene splicing assays in
vitro . Taking the corresponding
WT minigenes of each exon (pSPL3
Ex5, pSPL3 Ex12, pSPL3 Ex13, pSPL3
Ex15, pSPL3 Ex21, pSPL3 Ex22, pSPL3 Ex24) as the template, we
successfully constructed all candidate variants minigenes through
site-directed mutagenesis (Figure 1). Results of minigene analysis
indicated that 60% (6/10) of them effectively disturbed the normal
pre-mRNA splicing as expected
(Figure 2 A and C), and these were further confirmed by sequencing
analysis. Among ten candidates selected by BDGP and HSF, eight variants
(c.602G>A,
c.602G>T, c.1452C>G, c.1567G>A,
c.1667C>T, c.1925G>A, c.2548G>C
and c.2549G>C) led to exon skipping and two variants
(c.2755A>T and c.2863A>T)
caused no exon skipping. Although
two variants (c.1452C>G and c.1567G>A)
resulted in exon 12 skipping, it was regrettable that
the WT minigene of exon 12 (pSPL3
Ex12) completely caused exon
skipping, possibly due to
experimental technique.
1. Presumed missense variantsc.602G>A
(p.Gly201Asp) andc.602G>T
(p.Gly201Val) induced
skipping of exon 5compared with the WT
plasmids.
Missense variants c.602G>A (p.Gly201Asp) and
c.602G>T (p.Gly201Val) both affected the G at the first
nucleotide in exon 5 of SLC12A3.
Bioinformatics predictions from
BDGP showed that the variants reduced the score of the WT 3′acceptor
splice site from 0.83 to 0.57 and 0.35 respectively (Table
1). To observe the effects of the
two variants on pre-mRNA
splicing,
the
WT (pSPL3 Ex5) and mutants
(c.602G>A and c.602G>T) minigenes were
transfected individually into HEK 293T and Hela cells. The result of the
minigene assays revealed that
two different electrophoresis
bands were detected in the WT and the mutant (c.602G>A)
lane: a larger band of 403 bp and a smaller band of 263 bp. Meanwhile,
the mutant lane of c.602G>T revealed one unique band of 263
bp in line with skipping of exon 5 (Figure 2 A and
C).
Quantitative analysis of cDNA
products obtained from HEK 293T
and Hela cells indicated that there was an increase of
the exon 5-skipping transcript of
c.602G>A corresponded to the WT plasmids
(93.04% versus 9.90% in HEK
293T, P<0.0001; and 90.02% versus 7.56% in Hela cells,
P<0.0001; Figure 3).
2. Variantc.1667C>T
(p.Pro556Leu) gave rise toskipping of exon 13.
Variant c.1667C>T (p.Pro556Leu),
located at the third-to-last
nucleotide position of exon 13,
was
predicted by HSF to make a significant alteration of ESE / ESS motifs
ratio (-9), broking five ESEs (CGCC TG, CGCC TGG
(ESE_ASF), CGCC TGG (ESE_ASFB) , GCC TGG,
CC TGGT) and creating four ESSs (GCT TGG,
GCT TGGT, CT TGGT, T TGGTA) (Table 1, Table S3).
In addition, the splicing effect
of exon 13 is affected by a weak acceptor site, and the BDGP score of
this site is 0.13 (Table 1).
As a result of minigene splicing
assays, the WT lane showed two different electrophoresis bands,
representing a mature mRNA and a defected mRNA without exon 13 (36.26%
in HEK 293T and 32.96% in Hela cells). Mutant (c.1667C>T)
also generated two different transcripts, including the larger one with
the variant and the shorter one with exon 13 skipping compared with the
normal transcript (Figure 2 A and C). The analysis of the PCR products
suggested that the rate of transcript without exon 13 to the full
transcripts was 69.46% in HEK 293T and 61.21% in Hela cells,
respectively (P<0.0001, Figure 3).
3.Variantc.1925G>A
(p.Arg642His) led to exon
15 skipping.
Variant c.1925G>A (p.Arg642His) caused by the last
nucleotide substitution in exon 15, was demonstrated that it reduced the
score of the WT donor site from 0.99 to 0.68 with BDGP and also broken
the WT donor site with HSF (Table
1, Table S3), most probably affecting splicing. Taken together, we used
the WT (pSPL3 Ex15) and control
mutant minigene to examine the experimental effect of variant
c.1925G>A. The final PCR analysis result revealed that the
WT lane respectively showed 2 different fragments of 363 bp and 263 bp,
in contrast the mutant lane just showed one unique fragment of 263 bp
corresponding to the lack of exon 15 of mRNA (Figure 2 A and C).
Therefore, variant c.1925G>A (p.Arg642His) disturbed the
5’DS and led to exon 15 skipping.
4. Variant c.2548G>C (p.Gly850Arg) prevented
incorporation of exon 21 into the mature
mRNA.
Variant c.2548G>C (p.Gly850Arg) was identified at the last
nucleotide of exon 21 and
was
predicted to decrease the score of the
WT
5’DS from 0.85 to 0.22 with BDGP
(Table 1). Simultaneously, HSF predicted that Variant
c.2548G>C could lead to the deletion of exon 21 by
inactivating the splicing donor site (Table S3). Through the WT (pSPL3
Ex21) and mutant (c.2548G>C) minigene analysis, the
satisfactory result showed that a
short band of 263 bp was detected in the mutant lane and a long band of
365 bp was detected in the wild-type lane, indicating that the mutant
only produced the exon 21-excluded
transcript compared with the original exon 21-included transcript of the
WT (Figure 2 A and C).
5. Variantc.2549G>C
(p.Gly850Ala)resulted
in skipping of exon 22.
Variant c.2549G>C
(p.Gly850Ala) affected the G at
the first nucleotide in exon 22. Bioinformatic analysis of BDGP revealed
that the score of the acceptor site is 0.52, however, it could not be
analyzed after the nucleotide substitution (Table 1). The minigene
assays result was identified that cDNA products generated by the
mutant and WT
(pSPL3 Ex22) minigenes were
different. The lane of WT demonstrated one fragment of 375 bp that
contains exon 22, whereas mutant c.2549G>C generated two
different fragments of 263 bp and 375 bp, respectively (Figure 2 A and
C). Because the WT minigene only generated the transcript with exon 22,
it was obvious that the mutant (c.2549G>C) resulted in
skipping of exon 22.
6.Variants
of c.2755A>T (p.Arg919Trp) and c.2863A>T
(p.Ile955Phe) in exon 24did not alter pre-mRNA
splicing .
The pre-mRNA splicing of exon 24 is regulated by a weak donor site, and
the BDGP score of this site is 0.35 (Table 1).
Variants c.2755A>T,
identified at the 8th nucleotide of exon 24, was
predicted by HSF to make a
significant alteration of ESE / ESS motifs ratio (-8) (Table 1 and Table
S3). Variant
c.2863A>T was located
at the 21th-to-last nucleotide position of exon 24 and was predicted to
alter splicing with a HSF score of -10 (Table 1 and Table S3). However,
analysis of the minigenes demonstrated that both the WT (pSPL3 Ex24) and
mutants (c.2755A>T and c.2863A>T) just
generate the exon 24-included
transcript (Figure 2 B and D), indicating that the two variants did not
alter pre-mRNA splicing.
7. The WT
minigene of exon 12 (pSPL3 Ex12) andmutants(c.1452C>G
and c.1567G>A) only produced the exon 12-excluded
transcript.
Variant c.1452C>G (p.Cys484Trp), caused by the 9th
nucleotide substitution in exon 12, was predicted to
disturb pre-mRNA splicing with a
HSF score of -9 (Table 1 and Table S3). Variant c.1567G>A
(p.Ala523Thr) affected the G at the last nucleotide of exon 12, and was
predicted to decrease the score of the donor site from 1 to 0.45 with
BDGP (Table 1). Regrettably, the result of the
minigene assays showed that a band
of 263 bp (the exon 12-excluded transcript) was both detected in the WT
and the mutant lane (Figure 2 B and D). Therefore, although these two
mutants (c.1452C>G and c.1567G>A) resulted in
exon 12 skipping, there was no significant difference from the WT
minigene (pSPL3 Ex12).