Functional characterization of novel compound heterozygous SLC12A3 mutations in a patient with Gitelman syndrome

Abstract Gitelman syndrome (GS) is an autosomal recessive renal tubulopathy caused by loss-of-function mutations in the SLC12A3 gene, which encodes the thiazide-sensitive sodium-chloride cotransporter (NCC). Diagnosis can be challenging due to clinical and genetic heterogeneity, and novel variants of uncertain significance (VUS) require functional validation for definitive classification. We report a 6-year-old boy presenting with growth retardation who was incidentally found to have severe hypokalemia (2.11 mmol/L), metabolic alkalosis, and hypocalciuria—classic hallmarks of GS. Whole-exome sequencing identified novel compound heterozygous SLC12A3 variants: a missense mutation c.908G > A (p.Gly303Glu) and a frameshift deletion c.2895delT (p.Tyr966Thrfs*14). To investigate pathogenicity, we conducted in vitro functional studies using Wild-type (WT) and mutant (MUT1: p.Gly303Glu; MUT2: p.Tyr966Thrfs*14) NCC constructs transfected into HEK293T cells. Bioinformatics analysis showed both mutations affect highly conserved residues and were predicted pathogenic. Immunofluorescence microscopy revealed that while WT-NCC localized to the plasma membrane, both mutant proteins were retained in the endoplasmic reticulum, indicating defective trafficking characteristic of Class II mutations. Western blot analysis demonstrated reduced protein levels for both mutants compared to WT, suggesting instability and rapid degradation. Critically, a functional assay showed intracellular ion homeostasis in cells expressing mutant proteins. Our findings confirm that c.908G > A (p.Gly303Glu) and c.2895delT (p.Tyr966Thrfs*14) are pathogenic variants leading to protein misfolding, impaired trafficking, and accelerated degradation, resulting in loss of NCC function. This study expands the mutational spectrum of Gitelman syndrome and highlights the value of functional analysis for classifying VUS.