Rna Splicing Analysis Of Exonic Variants In The Pathogenic Genes Of Bartter Syndrome

Objective:Bartter syndrome(BS)is a rare renal tubular disease caused by gene variants in SLC12A1,KCNJ1,CLCNKA,CLCNKB,BSND,or MAGED2 genes.There is growing evidence that many exonic variants can affect the pre-m RNA normal splicing and induce exon skipping by altering various splicing regulatory signals,which may be the underlying true pathogenic mechanism of many traditionally considered missense,nonsense,and synonymous variants.Therefore,the aim of this study was to comprehensively analyze the consequences of all exon variants associated with BS on pre-m RNA splicing,and to further explore the different forms of abnormal splicing caused by BS pathogenic variants.Methods:All missense,synonymous and nonsense variants associated with pathogenic genes in BS were selected from the Human Gene Mutation Database(HGMD)and literature.Bioinformatics programs BDGP,Mx Ent Scan,and HSF3.1 were used to analyze and screen variants that may affect classical splice sites(5’ donor splice sites and 3’ receptor splice sites)and splicing regulatory sequences [ exonic splicing enhancers(ESEs)and exonic splicing silencers(ESSs)].Finally,the candidate variants that may promote exon skipping were identified,and their effects on splicing were verified by minigene assay.Results:A total of 170 variants were analyzed using bioinformatics software.We finally selected 14 putative variants for minigene splicing assay.The results of RT-PCR experiments showed that 12 of the 14 candidate variants caused abnormal splicing changes,including 7 in SLC12A1 gene(c.728G>A,c.735C>G,c.904C>T,c.905G>A,c.1304C>T,c.1493C>T,c.2221A>T)and 5 in CLCNKB gene(c.226C>T,c.228A>C,c.229G>A,c.229G>C,c.1979C>A).These variants were distributed in 7 different exons and caused different degrees of exon skipping.1.Splicing outcome of sequences variations of SLC12A1:1.1 Variants of exon 5(c.728G>A,c.735C>G),exon 6(c.904C>T,c.905G>A),and exon 11(c.1493C>T)induced partial skipping of exons.BDGP indicated that c.728G>A may slightly reduce the score of the WT 3’ acceptor splice site from 0.99 to 0.98.The predictive results of HSF3.1 indicated that variants c.735C>G,c.904C>T,c.905G>A,and c.1493C>T might lead to inactivation of potential overlapping ESEs and generation of new ESSs.The result of the assay revealed that the splicing products detected in the mutant and WT minigenes were different.The control minigenes(p SPL3 Ex5,p SPL3 Ex6,p SPL3 Ex11)produced a unique band,whereas mutant minigenes(c.728G>A,c.735C>G,c.904C>T,c.905G>A and c.1493C>T)generated two different bands respectively.The larger amplicons were the exon-included transcripts,while the smaller amplicons included only the 3’ and 5’ p SPL3 exons.1.2 Missense variants c.1304C>T resulted in complete skipping of exon 10.Variant c.1304C>T is located at position 4 in exon 10.This variation reduces the score of the 3’ acceptor splice site from 0.87 to 0.84 with BDGP analysis.The results of RT-PCR showed a unique product of 263 bp in mutant minigene and a larger band of 415 bp in WT minigene.Sequencing analysis confirmed that this variant induced skipping of exon 10.The larger fragment corresponded to correctly spliced exons,while the smaller splice corresponded to a transcript without exon 10.1.3 Nonsense variant c.2221A>T resulted in complete skipping of exon 17.Nonsense variant c.2221A>T alters an AAG codon for Lys to a premature TAG stop codon.In silico analysis by HSF 3.1 software demonstrated that this variant not only disrupted four ESEs but also created two ESSs.RT-PCR analysis results of minigenes after transfection indicated that the mutant and WT minigenes generated different sized products.The control minigene produced a band of 404 bp,whereas the mutant minigene generated a unique product of 263 bp.The larger amplicon was the transcript containing exon 17,and the smaller was the transcript excluding exon 17.2.Splicing outcome of sequences variations of CLCNKB:2.1 Variations caused skipping of exon 2 compared with the WT constructs.Variants c.226C>T,c.228A>C,c.229G>A and c.229G>C were all predicted to decrease the score of the WT 5’ splice donor site with BDGP and Mx Ent Scan.RT-PCR analysis showed that all the mutant and WT minigenes produced two different lanes,located in 263 bp and 392 bp respectively.However,the amounts of the exon 2-skipping transcript of these mutants were significantly increased compared with those of the control plasmids.2.2 Nonsense variants c.1979C>A increased the amounts of the exon 18-excluded transcripts when compared with the WT constructs.The nonsense variant c.1979C>A was predicted that the variant not only eliminated five ESEs but also generated three novel ESSs by HSF3.1 analysis.As a result,two products of 263bp(corresponding to defected transcripts)and 350bp(corresponding to mature transcripts)were both found from the c DNA products of the WT minigenes in HEK293 T and Hela cells.However,the unique products of 263 bp were found in mutant minigenes,which suggested that this variant significantly enhanced pre-m RNA splicing,promoting the skipping of exon 18.Conclusions:This is a comprehensive study regarding alterations in pre-m RNA of exonic variants in BS pathogenic genes.Our results reinforce the necessity of assessing the consequences of exonic variants at the m RNA level.