Abstract
Gitelman syndrome (GS) is a kind of salt-losing tubular disease, most of
which is caused by SLC12A3 gene
variants, and missense variants
account for the majority. Recently, the phenomenon of exon skipping, in
which exonic variants disrupt normal
pre-mRNA splicing, has been
related to a variety of diseases. The purpose of this study was to
identify the effect of previously presumed missense SLC12A3variants on pre-mRNA splicing using bioinformatics tools and minigenes.
The results revealed that, among ten candidate variants, six variants
(c.602G>A,
c.602G>T, c.1667C>T, c.1925G>A,
c.2548G>C and c.2549G>C) led to complete or
incomplete exon skipping by affecting exonic splicing regulatory
elements and/or disturbing canonical splice sites. It is worth
mentioning that this is the largest study on pre-mRNA splicing ofSLC12A3 exonic variants. In addition, our study emphasizes the
importance of detecting splicing function at the mRNA level in GS and
indicates that minigene analysis is a valuable tool for splicing
functional assays of variants in vitro .
Keywords: SLC12A3 ; Gitelman syndrome; exonic variant;
minigene assay; exon splicing.
INTRODUCTION
Gitelman syndrome (GS) is a kind
of autosomal recessive and salt-losing tubular disorder characterized by
hypokalemic metabolic alkalosis in conjunction with significant
hypomagnesemia and hypocalciuria (Parmar, Muppidi, & Bashir, 2021;
Simon et al., 1996; Vargas-Poussou et al., 2011). The vast majority of
GS phenotype is related to inactivating variants
in the SLC12A3 gene (OMIM
600968), located on chromosome 16q13 with a total of 26 exons and
encodes the thiazide-sensitive NaCl cotransporter (NCCT) in the distal
convoluted tubules of the nephron (Takeuchi et al., 2015; Vargas-Poussou
et al., 2011). Mutations in the SLC12A3 gene can lead to
the malfunctioning of NCCT, which
in turn, disrupt the absorption of sodium and chloride ions from the
tubular lumen (Parmar et al., 2021).
With the development and application of high-throughput sequencing
technology based on large-scale parallel sequencing, an expanding list
of rare variations about specific genetic diseases has been discovered
in the clinic. To date, a total of 492 GS-related gene variants have
been documented in the Human Gene Mutation Database (HGMD, Professional
2020.10), including missense/nonsense variants (309, 63%), splicing
(69, 14%), small deletions (56, 11%), small insertions (26, 5%),
small indels (5, 1%), gross deletions (22, 4%), gross
insertions/duplications (4, 1%) and complex rearrangements (1, 1%).
The conclusion that transcripts from at least 74% of all multiexon
genes were alternatively spliced has been drawn by using exon-exon
junction microarrays to analyze lots of RNA samples in a study (Johnson
et al., 2003).Evidently, the relatively large number of exons (26) that
encode NCCT suggests the existence of alternative splicing for SLC12A3.
Precursor messenger RNAs (Pre-mRNAs) splicing, removing non-coding
insertion segments called introns and joining exons composed of
protein-coding regions together with two terminal untranslated regions,
is an important process of generating mature translatable mRNAs in
eukaryotic gene expression (Baralle & Baralle, 2018). To stimulate
pre-mRNAs splicing, a large ribonucleoprotein (RNP) complex known as the
spliceosome can recognize numerous splicing signals, which include the
conserved splice sites (5′ splice
donor site and 3′ splice acceptor
site), the branch site, polypyrimidine track, exonic/intronic splicing
enhancers (ESEs/ISEs) and silencers (ESSs/ISSs), as well as other
regulatory components or the RNA
secondary structure (Dufner-Almeida, do Carmo, Masotti, & Haddad,
2019). As we know, exonic point
variants, refer to missense, synonymous, silent, or nonsense variants,
and certain point variants, can result in abnormal pre-mRNAs splicing by
disrupting splicing signals (Baeza-Centurion, Miñana, Valcárcel, &
Lehner, 2020; Cartegni, Chew, & Krainer, 2002).
We found that in the last large
sample study reported in 2015 (Takeuchi et al., 2015), 88 missense
variants were only predicted by ESEfinder software as candidate variants
that could induce exon skipping in the SLC12A3 gene. Moreover, most
novel
variants analyses were performed
to predict the impact on mRNA and protein at the genome level and only
in a few cases at both DNA and RNA levels (Ko & Kim, 2021; Takeuchi et
al., 2015). Herein, we aimed to explore the functional effects of more
than 300 previously described SLC12A3 missense variants that have
not been functionally validated on pre-mRNAs splicing by bioinformatics
tools and minigene assays.
MATERIALS AND
METHODS