Dataset Information

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.

SUBMITTER: Wang S

PROVIDER: S-EPMC7674938 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Six Exonic Variants in the <i>SLC5A2</i> Gene Cause Exon Skipping in a Minigene Assay.

Wang Sai S Wang Yixiu Y Wang Jinchao J Liu Zhiying Z Zhang Ruixiao R Shi Xiaomeng X Han Yue Y Guo Wencong W Bottillo Irene I Shao Leping L

Frontiers in genetics 20201105

<h4>Background</h4>Familial renal glucosuria is a rare renal tubular disorder caused by <i>SLC5A2</i> 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 <i>SLC5A2</i> exonic variants can result in disease <i>via</i> interfering with the normal splicing process of the pre-mRN ...[more]

PMID: 33250922

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Project description:Genetic testing of BRCA1 and BRCA2 identifies a large number of variants of uncertain clinical significance whose functional and clinical interpretations pose a challenge for genetic counseling. Interestingly, a relevant fraction of DNA variants can disrupt the splicing process in cancer susceptibility genes. We have tested more than 200 variants throughout 19 BRCA2 exons mostly by minigene assays, 54% of which displayed aberrant splicing, thus confirming the utility of this assay to check genetic variants in the absence of patient RNA. Our goal was to investigate BRCA2 exon 16 with a view to characterizing spliceogenic variants recorded at the mutational databases. Seventy-two different BIC and UMD variants were analyzed with NNSplice and Human Splicing Finder, 12 of which were selected because they were predicted to disrupt essential splice motifs: canonical splice sites (ss; eight variants) and exonic/intronic splicing enhancers (four variants). These 12 candidate variants were introduced into the BRCA2 minigene with seven exons (14-20) by site-directed mutagenesis and then transfected into MCF-7 cells. Seven variants (six intronic and one missense) induced complete abnormal splicing patterns: c.7618-2A>T, c.7618-2A>G, c.7618-1G>C, c.7618-1G>A, c.7805G>C, c.7805+1G>A, and c.7805+3A>C, as well as a partial anomalous outcome by c.7802A>G. They generated at least 10 different transcripts: Δ16p44 (alternative 3'ss 44-nt downstream; acceptor variants), Δ16 (exon 16-skipping; donor variants), Δ16p55 (alternative 3'ss 55-nt downstream), Δ16q4 (alternative 5'ss 4-nt upstream), Δ16q100 (alternative 5'ss 4-nt upstream), ▾16q20 (alternative 5'ss 20-nt downstream), as well as minor (Δ16p93 and Δ16,17p69) and uncharacterized transcripts of 893 and 954 nucleotides. Isoforms Δ16p44, Δ16, Δ16p55, Δ16q4, Δ16q100, and ▾16q20 introduced premature termination codons which presumably inactivate BRCA2. According to the guidelines the American College of Medical Genetics and Genomics these eight variants could be classified as pathogenic or likely pathogenic whereas the Evidence-based Network for the Interpretation of Germline Mutant Alleles rules suggested seven class 4 and one class 3 variants. In conclusion, our study highlights the relevance of splicing functional assays by hybrid minigenes for the clinical classification of genetic variations. Hence, we provide new data about spliceogenic variants of BRCA2 exon 16 that are directly correlated with breast cancer susceptibility.

Dataset Information

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

Publications

Six Exonic Variants in the <i>SLC5A2</i> Gene Cause Exon Skipping in a Minigene Assay.

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