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