Correlation Between Familial Renal Glucosuria Pathogenic SLC5A2 Gene And The Renal Threshold For Glucose Excretion And RNA Splicing Analysis

Objective:Familial renal glucosuria(FRG)is a rare renal tubular disease characterized by isolated persistent glucosuria without abnormal glucose metabolism and any other symptoms of proximal tubular malfunctioning.The vast majority of FRG cases are caused by SLC5A2(OMIM182381)pathogenic variants.The purpose of this study was to further expand the mutation analysis of SLC5A2 gene in FRG and investigate the relationship between genotype and the renal threshold for glucose excretion(RTG).Based on the lack of mRNA levels of the SLC5A2 exonic variants described,this study also explored the effect of point mutations on pre-mRNA splicing.Methods:1 Quantitative test for 24-hour urine glucose and RTG were determined in 9 families(totaling 25 subjects).All coding regions,including intron-exon boundaries,were analyzed with PCR followed by direct sequence analysis.The differences in renal glucose thresholds between patients with different genotypes(heterozygotes and compound heterozygotes;c.886(-10_-31)del heterozygotes and other heterozygotes)were compared.2 Bioinformatics programs(BDGP and HSF 3.1)was used to analyze 77 previously described presumed SLC5A2 missense variants and candidate variants were selected based on their potential effects on splice sites or splicing regulatory elements,such as exonic splicing enhancers/silencers(ESEs/ESSs).These variants were analyzed in a minigene assay.Results:1 By direct sequencing analysis,twelve mutations in SLC5A2 were identified in nine FRG families,including ten novel missense variants(c.331T>C p.(Trp111 Arg);c.374T>C p.(Met125Thr);c.394C>T p.(Argl32Cys);c.612G>C p.(Gln204His);c.829C>T p.(Pro277Ser);c.880G>A p.(Asp294Asn);c.1129G>A p.(Gly377Ser);c.1194C>A p.(Phe398Leu);c.1540C>T p.(Pro514Ser)and c.1573C>T p.(His525Tyr)).Variant c.886(-10_-31)del that is specific to Chinese population was found in 5 out of 9 families,with a mutation rate of 28%(5/18).The compound heterozygotes presented with much lower RTG values(1.28 ± 0.10 mmol/L),compared with the carriers of heterozygous variants(5.14 ±0.77 mmol/L)(P<0.001);c.886(-10_-31)del heterozygotes had significant lower RTG values than others(4.43 ± 0.37 vs 5.7 ± 0.51 mmol/L;P<0.001).2.1 A total of 77 missense variants compiled in the SLC5A2 database were analyzed using the bioinformatics tools BDGP and HSF.We finally selected nine putative missense variants for minigene analysis.Four control minigenes containing exons 3,4,6 and 9 and their corresponding intronic regions were constructed in this study.Seven mutant minigenes were generated by site-directed mutagenesis using their corresponding wild-type minigenes as a template.The results of the RT-PCR experiments showed that three of them,c.216C>A,c.294C>A and c.1129G>A,affected normal pre-mRNA splicing.2.2 Variant c.216C>A is located on the 5' end of exon 3 at position+18 within a sequence containing three potential overlapping ESE sites(according to HSF).Bioinformatics analysis of this variant indicated the disruption of these ESEs and the generation of three new ESSs.Presumed missense variant c.294C>A affects nucleotide-10 from the 3'end of exon 3.Analysis with HSF that this variant inactivates two potential ESEs and generates four potential overlapping ESSs.RT-PCR analysis of RNA from transfected minigenes showed the control minigene produced two different bands of 368 bp and 263 bp,respectively,whereas both mutant minigenes generated a unique product of 263 bp.Sequencing analysis of this band lack the entire exon 3.We postulate both variants result in the skipping of exon 3 due to the disruption of ESEs and the generation of ESSs.2.3 Putative SLC5A2 missense variant c.1129G>A results from substitution at the last nucleotide of exon 9 upstream of 5'splice site.Bioinformatic analysis with BDGP demonstrated that this variant marginally decreased the score of the 5' splice site.We used a minigene containing exon 9 and flanking intronic sequences to examine the experimental effect of it.The result of RT-PCR analysis showed differences between the splicing products generated by the mutant minigene and the wild-type minigene.The wild-type lane showed two different products of 263 bp and 371 bp,respectively.In contract,the mutant lane showed a unique product of 263 bp corresponding to skipping of exon 9 in the mRNA.Therefore,variant c.1129G>A abrogates the donor splice site and results in skipping of exon 9.Conclusions:1 This study found ten novel SLC5A2 mutations may be lead to FRG,and confirmed that c.886(-10-31)del is a high-frequency mutation unique to Chinese population again.Compound heterozygotes had much lower RTG values than simple heterozygotes.Mixed-meal tolerance test is a simple method for determining RTG in FRG patients.2 This is the first reported SLC5A2 exonic variants producing an impact on pre-mRNA splicing and we suggest minigene analysis could be a valuable tool for assessing the impact of SLC5A2 exonic variants on pre-mRNA splicing in the absence of patient RNA samples.