PMC10291089_01
Renal pathology (A) Light microscope observations of PAS staining showing the focal granular and vacuolar degeneration of tubular epithelial cells; the brush border of some renal tubules fell off and the lumen expanded (arrow), and no special lesions were found in renal interstitium (B) Light microscope observations of PAS staining showing that the juxtaglomerular apparatus proliferation was not obvious (arrow) (C) Light microscope observations of MASSON staining showing exfoliated tubular epithelial cells and protein tubular formation in some kidney tubules (arrow) (D) Electron microscopy showing no obvious abnormalities, electron dense deposits or significant GBM thickening (280-450 nm).
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F1
The enhanced computed tomography of adrenal gland showed possible right adrenal myelolipoma and left adrenal outer limb nodular protrusion, and hyperplasia was also considered (Supplementary Figure S1). A renal biopsy was performed, and the renal pathology revealed a mild injury of the renal tubular epithelium (Figure 1).
Sanger sequencing diagram of SLC12A3 gene and pedigree of the family structure (A-B) The proband's NGS revealed two heterozygous variants in the SLC12A3 gene (c.1112T>C and c.965-1_976delGCGGACATTTTTGinsACCGAAAATTTT), which were confirmed by sanger sequencing (C) The pedigree of the family structure. The marked symbols show patients who carried compound heterozygous variants of SLC12A3. The variants of c.1112T>C were presented in black, and c.965-1_976delGCGGACATTTTTGinsACCGAAAATTTT was shown in grey. Circles present females, and squares present males. The arrow shows proband. The deceased parents of proband and II-1 showed normal phenotypes, without any features of Gitelman syndrome.
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F2
The pedigree structure of the family was drawn according to the clinical manifestations and the sequencing results (Figure 2C).
PMC10291089_02
Sanger sequencing diagram of SLC12A3 gene and pedigree of the family structure (A-B) The proband's NGS revealed two heterozygous variants in the SLC12A3 gene (c.1112T>C and c.965-1_976delGCGGACATTTTTGinsACCGAAAATTTT), which were confirmed by sanger sequencing (C) The pedigree of the family structure. The marked symbols show patients who carried compound heterozygous variants of SLC12A3. The variants of c.1112T>C were presented in black, and c.965-1_976delGCGGACATTTTTGinsACCGAAAATTTT was shown in grey. Circles present females, and squares present males. The arrow shows proband. The deceased parents of proband and II-1 showed normal phenotypes, without any features of Gitelman syndrome.
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F2
The pedigree structure of the family was drawn according to the clinical manifestations and the sequencing results (Figure 2C).
Predicted topological localization of NCC variants and effect of SLC12A3 variants on the modeled structure of NCC protein, evaluation of the pathogenic potential of c.1112T>C variant in SLC12A3
(A) Schematic diagram of NCC protein with the intercellular N- and C-terminal domains and transmembrane segments. The sites of detected variants are denoted by arrows in our study (B) The web-based software Variant Taster showed that this variant was disease causing, and the variant at this position was reported (C) Analysis of amino acid conservation based on the NCBI database. Evolutionary conservation of Ile371Thr using the ClustalX program. The black frame marks the Ile371Thr site (D-E) Compared to the wild-type, c.1112T>C (p.Ile371Thr) is only a point variant, with no significant change in the protein sequence (F) c.965-1_976delGCGGACATTTTT causes nonsense mediated mRNA degradation (NMD), because the protein sequence becomes shorter due to the early appearance of the stop codon, hence the structure is smaller.
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F3
Variants were located in the intracellular and extracellular carboxyterminal domain of the NCC protein (Figure 3A). Taster predicted that this variant was disease causing and capable of triggering amino acid sequence changes, frameshift, and splice site changes (Figure 3B). Two pathogenic in-trans variants in a single gene can produce the phenotype of this disease. We used mutation taster online website to predict and find that the variant is disease causing, and the prob is 0.999999999971332 (Figure 3B). Sequence alignment of SLC12A3 protein revealed that the isoleucine of p.371 was conserved among different species (Figure 3C). SLC12A3 (NM_001126108.1, c.1112T>C, p.Ile371Thr) is only a point variant, with no significant change in the protein structure compared to wild type (Figures 3D, E). SLC12A3 (NM_001126108.1, c.965-1_976delGCGGACATTTTTGinsACCGAAAATTTT) cause protein structure changes into a smaller structure, may affect SLC12A3 protein function (Figure 3F).