Genomic DNA (gDNA) of patient was extracted from the peripheral blood using MagPure Buffy Coat DNA Midi KF Kit (Magen, China). Then, gDNA was broken into 100–500 bp fragments using enzyme kit (BGI, China), 280–320 bp fragments were collected by magnetic beads. Agilent 2100 bioanalyzer and BMG were used to estimate the enrichment degree, and qualified products were collected to make DNA nanoballs and quantified according to different library quantities. DNA nanoballs were sequenced with PE100 + 100 on MGISEQ-2000. The average sequencing depth of the target region is ≥ 200×, more than 96% of the locus have a coverage depth of >20×.

A 49-year-old male was presented to our hospital with the major complaints of fatigue, polydipsia, polyuria, and repeated muscle weakness, but without a history of salt craving, constipation, physical, and intellectual disability over a 30-year period. Due to the diagnosis of renal tubular acidosis 25 years earlier, the patient had token potassium sodium hydrogen citrate granules orally for a long time to maintain the blood potassium at 2.0–2.5 mmol/L. Two months earlier, the patient was admitted to the endocrinology department due to fatigue and developed hypokalemia. The patient was supplied with potassium chloride sustained release tablets. During the follow-up, serum potassium levels were maintained between 2.3 and 2.55 mmol/L. He was hospitalized in our department due to recurrent fatigue.

The patient had a height of 169 cm, weight of 69 kg, and a heart rate of 78 beats per minute with no growth retardation. His respiratory rate was 18 breaths per minute, temperature was 37.0°C, and 24-h urine volume was 1.9–2.4 L. We monitored the blood pressure of this patient twice a day, with the systolic blood pressure in the range of 95–123 mmHg, and the diastolic blood pressure in the range of 60–76 mmHg. The laboratory investigation revealed hypokalemia, hypocalciuria and normal blood pressure, without obvious metabolic alkalosis, hypomagnesemia, hypochloremia or RAAS activation (Table 1). The fractional excretion rate of potassium (FEK%) was significantly increased to 30.25% (normal range, 8%–12%) and spot potassium-creatinine ratio elevated to 7.33, suggesting that hypokalemia resulted from renal potassium loss (Table 1). Urinalysis showed microalbuminuria, normal urinary immunoglobulin IgG, elevated β2-microglobulinuria, and elevated urinary retinol binding protein, which indicated proximal tubular injury. Other possible causes of hypokalemia, such as thyrotoxic periodic paralysis, renal tubular acidosis, and hypercortisolism, were excluded. 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). Nevertheless, the renin-angiotensin-aldosterone system was normal, hence primary aldosteronism was excluded.

The electrocardiogram (ECG) showed normal sinus rhythm, abnormality of T-waves but no prolongation of the QT interval. The urinary ultrasound showed kidneys with normal size and without obvious abnormality in the ureters (Supplementary Figure S2). The pure tone audiometry of the patient was normal. The ophthalmology examination indicated that sclerochoroidal calcifications could be excluded. A renal biopsy was performed, and the renal pathology revealed a mild injury of the renal tubular epithelium (Figure 1). The light microscope showed focal granular degeneration, cell swelling, brush edge falling off, and without tubular atrophy and tubulitis. Vacuolar degeneration of the renal tubular epithelial cells was observed, and no special lesions were found in the renal interstitium.

The patient was diagnosed with GS based upon clinical features and biochemical parameters according to the criteria (Blanchard et al., 2017), whereas the confirmation of clinically suspected GS rested on genetic testing. The blood potassium was maintained at 3.3–3.5 mmol/L by clinical follow-up, with the addition of 3 g potassium chloride tablets in three divided doses and 40 mg spironolactone in two divided doses. Subsequently, the patient’s symptoms dramatically improved.

For a precise diagnosis, we performed whole-exome sequencing and identified a novel compound heterozygous variant in the SLC12A3 gene. One of the variants, SLC12A3 (NM_001126108.1, c.965-1_976delGCGGACATTTTTGinsACCGAAAATTTT) is caused by the deletion of 13 nucleotides GCGGACATTTTTG, and the insertion of 12 nucleotides ACCGAAAATTTT at nucleotide positions 965-976 of the coding sequence. Another variant SLC12A3 (NM_001126108.1, c.1112T>C, p.Ile371Thr) is caused by the substitution of nucleotide T with C. Sanger sequence were used to verify the mutations (Figures 2A, B).

Meanwhile, the sister of the proband had normal manifestations, no variants were found. Notably, a heterozygous variant (NM_001126108.1, c.965-1_976delGCGGACATTTTTGinsACCGAAAATTTT) was identified in the SLC12A3 gene in the proband’s daughter. The daughter is 24-year-old, appeared healthy, was found to have normal plasma potassium (4.2 mmol/L) and normal serum creatinine (67 μmol/L) levels. The serum potassium, sodium, chloride, calcium, and magnesium levels of the family members were unremarkable. The pedigree structure of the family was drawn according to the clinical manifestations and the sequencing results (Figure 2C).

Variants were located in the intracellular and extracellular carboxyterminal domain of the NCC protein (Figure 3A). The variant (NM_001126108.1, c.965-1_976delGCGGACATTTTTGinsACCGAAAATTTT) in the SLC12A3 gene is a known GS associated variant (Shao et al., 2008; Wang et al., 2021). However, the variant SLC12A3 (NM_001126108.1, c.1112T>C, p.Ile371Thr) is not included in the databases (HGMD Professional and ClinVar). According to the American College of Medical Genetics and Genomics (ACMG) guidelines, the variation was judged to be of undetermined significance. The web-based software Variant 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. Since the daughter of the proband carries only one of the two SLC12A3 variants, we can assume that these two variants are in-trans in the proband. In addition, we used CADD to predict the variant SLC12A3 (NM_001126108.1, c.1112T>C, p.Ile371Thr). The CADD-phred score is 26.8. We used mutation taster online website to predict and find that the variant is disease causing, and the prob is 0.999999999971332 (Figure 3B). Thus, the two variants may support the GS phenotype.

Sequence alignment of SLC12A3 protein revealed that the isoleucine of p.371 was conserved among different species (Figure 3C). We used bioinformatics techniques to perform protein function prediction and secondary structure simulation (Supplementary Table S1). We used the SWISS-MODEL workspace (http://swissmodel.expasy.org) to characterize the effects of the novel variants, SLC12A3 (NM_001126108.1, c.965-1_976delGCGGACATTTTTGinsACCGAAAATTTT) and (NM_001126108.1, c.1112T>C, p.Ile371Thr) on the protein structure of NCC, which demonstrated that the alterations caused by the variants modified the protein structure, and might even affected the function in terms of NCC physiology. The variant 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).