Six Exonic Variants in the SLC5A2 Gene Cause Exon Skipping in a Minigene Assay

Abstract

Background: Familial renal glucosuria is a rare renal tubular disorder caused by SLC5A2 gene variants. Most of them are exonic variants and have been classified as missense variants. However, there is growing evidence that some of these variants can be detrimental by affecting the pre-mRNA splicing process. Therefore, we hypothesize that a certain proportion of SLC5A2 exonic variants can result in disease via interfering with the normal splicing process of the pre-mRNA. Methods: We used bioinformatics programs to analyze 77 previously described presumed SLC5A2 missense variants and identified candidate variants that may alter the splicing of pre-mRNA through minigene assays. Results: Our study indicated six of 7 candidate variants induced splicing alterations. Variants c.216C > A, c.294C > A, c.886G > C, c.932A > G and c.962A > G may disrupt splicing enhancer motifs and generate splicing silencer sequences resulting in the skipping of exon 3. Variants c.305C > T and c.1129G > A probably disturb splice sites leading to exon skipping. Conclusion: To our knowledge, we report, for the first time, SLC5A2 exonic variants that produce alterations in pre-mRNA. Our research reinforces the importance of assessing the consequences for putative point variants at the mRNA level. Additionally, we propose that minigenes function analysis may be valuable to evaluate the impact of SLC5A2 exonic variants on pre-mRNA splicing without patients' RNA samples.

Identification of seven exonic variants in the SLC4A1 , ATP6V1B1 , and ATP6V0A4 genes that alter RNA splicing by minigene assay

“…In many cases, however, RNA samples from the affected individual are unavailable, and mRNAs are difficult to detect due to the activation of the nonsense‐mediated decay of the mRNA pathway (Théry et al, 2011). Fortunately, a functional splicing assay based on the minigene assay was confirmed as an effective, reliable, and relatively simple tool to functionally assay potential splicing (Fraile‐Bethencourt et al, 2018), which has been extensively validated in our previous studies (Wang et al, 2020; Zhang, Wang, Wang, et al, 2018; Zhao et al, 2016).…”

“…To investigate the effect on the splicing process of the candidate variants, in vitro analysis was performed using a minigene splicing assay based on the pSPL3 exon trapping vector, as shown in Figure 1a. Minigene constructions were described as previously reported (Wang et al, 2020; Zhang, Wang, Wang, et al, 2018). Briefly, for each variation, the fragments with the wild‐type (WT) alleles involving interested exon, flanked by approximately 50–200 nucleotides of upstream intronic sequence and downstream intronic sequence, were cloned into the splicing vector pSPL3 using specific primers linking the XhoI and NheI restriction enzyme sites (XhoI: TGGAGC^TCGAG; NheI: AATTTG^CTAGC).…”

Identification of eight exonic variants in the SLC4A1, ATP6V1B1 and ATP6V0A4 gene that alter RNA splicing by minigene assay

“…In many cases, however, RNA samples from the affected individual are unavailable, and mRNAs are difficult to detect due to the activation of the nonsense-mediated mRNA decay pathway [24] . Fortunately, a functional splicing assay based on the minigene assay was confirmed as an effective, reliable and relatively simple tool to functionally assay potential splicing [25] , which has been extensively validated in our previous studies [20,21,26] .…”

“…To investigate the effect on the splicing process of the candidate variants, in vitro analysis was performed using a minigene splicing assay based on the pSPL3 exon trapping vector shown at Figure 1 A. Minigene constructions were described as previously reported [20,21] . Briefly, for each variation, the fragments with the wild-type (WT) alleles involving interested exon, flanked by approximately 50-200 nucleotides of upstream intronic sequence and downstream intronic sequence, were cloned into the splicing vector pSPL3 using specific primers linking the XhoI and NheI restriction enzyme sites (XhoI: TGGAGCˆTCGAG; NheI: AATTTGˆCTAGC).…”

Unraveling synonymous and deep intronic variants causing aberrant splicing in two genetically undiagnosed epilepsy families

“…Disease-causing variations are identified mainly at the genomic level. The effects of variation on mRNA and encoded protein can only be predicted by DNA sequence, very few cases have been experimentally confirmed the effects of variants at both DNA and RNA levels [ 15 ]. Current bioinformatics filtering strategies and clinical interpretation guidelines tend to focus on the impact of variants at the amino acid level.…”