Genetic Diagnosis And Clinical Characterization Of Familial Renal Glucosuria

BACKGROUNDFamilial renal glucosuria?FRG?is a mendelian disorder caused by mutations in the gene SLC5A2 encoding the sodium glucose cotransporter 2?SGLT2?.Patients with FRG have impaired tubular reabosorption of glucose and can have glucosuria in the absence o,f hyperglycemia.SGLT2 inhibitors have gaining popularity as anti-diabetic medications,underlying the importance to further characterize patients with SGLT2 mutations.AIM1.Develop genetic diagnosis of familial renal glucosuria,followed by in vitro functional tests to confirm the pathogenicity of new variants.Screen for other genes associated with renal glucosuria through next generation sequencing?NGS?.2.Evaluate renal glucosuria quantitatively using renal threshold for glucose?RTG?.3.Characterize renal reabsorption of various substrates in genetically diagnosed FRG patients.METHODS1.For those who have renal glucosuria with no known cause,amplify and sequence the 14 exons of SLC5A2 and screen other genes associated with renal glucosuria using Tubule Panel,a custom-designed NGS panel for tubular transport defects.Confirm pathogenicity of identified variants by in vitro functional tests in HEK-293T cells,including a localization test of SGLT2-EGFP and a transport activity assay using 2-NBDG,a fluorescent glucose analog.2.Measure renal threshold for glucose?RTG?using an extended oral glucose tolerance test?OGTT?.Calculate RTG from the blood glucose-time curve and the urine glucose excretion.3.Characterize renal reabsorption of uric acid,phosphate,and amino acid in FRG patients.Measure the fractional excretion of uric acid,24h xcretion of uric acid,tubular reabsorption of phosphate?TRP?,TmP/GFR and test for the presense of aminoaciduria.RESULTS1.In 10 families of FRG,we identified 13 variants,of which 7 have not been reported in the literature,including 3 missense variants?A212S,G484D,and R564W?,a nonsense variant W649X,a frameshift variant p.Ser592CysfsTer6,a splice site variant c.574+1G>C,and an intronic variant c.469-124₄69-107del.Next generation sequencing with Tubule Panel identified a new frameshift variant p.Asn26ThrfsTer3 9 in PDZK1IP1,the gene encoding MAP 17.2.When mutant SGLT2 were fused to EGFP and examined under the confocal microscope,G484D and R564W lost their co-localization with the plasma membrane while A212S still had the pacthy expression patterns on the plasma membrane similar to the wild-type.Transport activity were impaired for all four variants examined?A212S,G484D,R564W and W649X?,with 15.4%-21.5%SGLT2-mediated glucose uptake of the wild-type.3.In FRG patients,we measured renal threshold for glucose?RTG?using an extended oral glucose tolerance test,and evaluated tubular reabsorption of several solutes.The four FRG patients didn't show significant difference in fractional excretion of uric acid or 24h uric acid excretion compared to 22 healthy controls.2 of the FRG patients had higher than normal TRP,and 1 had higher than normal TmP/GFR.Among the 10 FRG pateints,9 were tested for aminoaciduria,of which one patient had weakly positive aminoaciduria and another patient had suspicious positive aminoaciduria.CONCLUSION1.We developed genetic and clinical diagnostic tools for familial renal glucosuria,identified 7 new variants of SLC5A2 and confirmed the pathogenicity of the missense and nonsense variants by in vitro functional tests.2.We identified a new variant of PDZK1IP1 in an FRG patient,suggesting a possible role ofPDZK1IP1 in FRG.3.We measured renal threshold for glucose through an extended oral glucose tolerance test.We observed some changes in phosphate and amino acid reabsorption in FRG patients,warranting confirmation in a larger sample.