DISCUSSION
Alternative splicing, which is
present in over 90% of mammalian genes, plays an important role in
biodiversity and biocomplexity, and the
misregulation of splicing is
implicated in an increasing number of human diseases. About 10% of HGMD
reported human pathogenic variants have been found to influence pre-mRNA
splicing (Soemedi et al., 2017), which is likely an underestimate.
Therefore, it is necessary to study the effects of exonic variants in
the SLC12A3 gene of GS on potential splicing, with the discovery
of novel variants. In the absence of RNA samples to study alternative
splicing, minigene analysis, the effectiveness of which has been
confirmed by different studies (Fraile-Bethencourt et al., 2019;
Suarez-Artiles, Perdomo-Ramirez, Ramos-Trujillo, & Claverie-Martin,
2018; Takeuchi et al., 2015; Tournier et al., 2008), is still the most
direct and credible experimental approach to assess whether a variant
influences recognition of an exon and potentially causes phenotypic
changes (Bao, Moakley, & Zhang, 2019). In previous studies, we have
used this approach to assess the consequences on pre-mRNA splicing of
presumed mutations associated with different diseases (Han et al., 2019;
Wang et al., 2020; Zhang et al., 2021).
Gene splicing diversity is dramatically affected not only by canonical
splice signals such as splice sites recognized by the core spliceosomal
components, but also by a large number of regulatory elements in the
exon or flanking intronic sequences. Based on the above, we empirically
chose BDGP and HSF as
bioinformatics tools to predict
the effect of missense variants of SLC12A3 gene, and obtained
some satisfactory results through minigene assays. Variants located at
or near to classical splice sites (DS or AS) could result in skipping of
the corresponding exon either by generating new splice sites or by
inducing a significant reduction of splice site strength, as confirmed
by the minigene analysis of variants c.602G>A,
c.602G>T, c.1925G>A, c.2548G>C
and c.2549G>C in the study. Additionally, variants in the
exon regulated by a weak donor or acceptor splicing site could modify
splicing regulatory sequences (disrupt ESEs or create ESSs), leading to
the corresponding exon skipping, as revealed by the minigene analysis of
variants c.1667C>T in our study. Among the 10 candidate
variants selected by BDGP and HSF following the screening criteria, 6
variants causing exon skipping were predicted successfully, which
indicated a degree of concordance betweenin silico predictions and
the experimental results. Despite a considerable degree of inaccuracy,
it is still valuable to use these two bioinformatics tools to predict
the impact of SLC12A3 exonic variation on splicing based on this
or more stringent criteria.
According to the resulting data, among six variants that predicted
successfully, c.602G>T, c.1925G>A and
c.2548G>C were complete splicing mutations just generating
the corresponding exon-excluded transcripts, while the other variants
(c.602G>A, c.1667C>T and
c.2549G>C) still produced a little part of transcripts that
contained the corresponding variant and were the same size as WT
products. Therefore, these three
variants probably have a dual damaging effect: on the one hand, a
portion of SLC12A3 transcripts are deleterious due to the exon
skipping, which would result in loss of the corresponding proteins, and
on the other hand, the remaining mRNA is destructive due to the
replacement of a single amino acid, which could lead to the
malfunctioning of NCCT. The true
splicing effect of these variants needs to be confirmed by analyzing the
mRNA from the patient, which we did not have yet.
However, it is worth noting that bioinformatics software and minigene
assays, as effective tools for identifying splicing malfunctions, have
the limitation in detecting and simulating all splicing patterns
compared to the in vivo situation. Variants of
c.2755A>T and c.2863A>T, predicted by BDGP and
HSF to have a significant impact on ESE / ESS motifs, did not cause exon
24 skipping, and the WT minigene of exon 12 only produced the exon
12-excluded transcript. Coincidentally, the same results that candidate
variants did not alter pre-mRNA splicing were revealed in other studies
(Suarez-Artiles et al., 2018; Wang et al., 2020; Zhang et al., 2021) and
there was a previous study that suggested only 18 of the 20 wild-type
minigene constructs produced the desired wild-type transcripts in the
pSPL3 context, improving the awareness of the limitations of minigene
assays and emphasizing the importance of
sequence context in regulating
splicing (Lin et al., 2021). The reasons for these results may be
related to the defects of software, the limited transferability of
minigenes, the differential expression of splicing factors in cells, the
interference of the mRNA secondary structure, etc. The complete skipping
of wild-type exon 12 might also be associated with the inactivation of
splicing sites and the difficulty in recognizing splicing sites.
Moreover, it has been reported
that the role of splicing regulatory elements could be highly context
dependent (Goren et al., 2006), but the underlying mechanism has been
unclear, and our minigene splicing assays might disrupt this sequence
context effect leading to exon 12 skipping.
In addition, we have not yet prepared the expected cDNA constructs based
on the prediction of the single amino acid substitution and the
corresponding exon deletion, nor have we examined the functional
activities and the cell surface expression of these mutant NCCTs, which
need further investigation.
In order to verify the pathogenicity of exonic variants of theSLC12A3 gene in GS, variation analyses at both DNA and RNA levels
should be performed to predict the impact on mRNA and protein.
Minigene
splicing assays have revealed new insights and changed the
misinterpretation about the functional consequences of certain
variants.
It is worth mentioning that exon-skipping methods to correct variants
that disturb normal pre-mRNA
splicing have been effectively assessed in rare diseases (Veltrop &
Aartsma-Rus, 2014). Knowledge of the results of exonic splicing variants
may have potential therapeutic significance for GS, further emphasizing
the importance of minigene splicing analysis.