Novel Pathogenic Genotype in SLC12A3 Associated to Gitelman Syndrome: A Case Report

ABSTRACT

Gitelman syndrome (GS) is considered one of the most common hereditary renal tubular disorders, characterised by hypokalemia, hypomagnesemia, hypocalciuria, and metabolic alkalosis. The primary cause of this disorder resides in the SLC12A3 gene, which encodes the NaCl cotransporter in the distal convoluted tubule, and for which more than 500 mutations associated with GS have been described. We present the case of a 51‐year‐old female referred for evaluation of recurrent hypokalemia and hypomagnesemia, with no clinical symptoms. The blood test also revealed hypocalciuria and metabolic alkalosis. Oral supplementation with potassium and magnesium was prescribed. A next‐generation sequencing (NGS) test was performed on her and her child (no other relatives alive), who was also asymptomatic with no obvious electrolytic abnormalities. Two mutations confirmed as pathogenic were found in the SLC12A3 gene (NM_000339.3) of the mother, c.704C>G and c.704C>T, as well as a new heterozygous variant in trans not reported before, c.704C>A p.(Thr235Lys), and identified as a variant of uncertain significance (VUS). This new VUS (c.704C>A p) was also present in the child, increasing evidence of its potential pathogenicity. The new SLC12A3 gene variant could represent a pathogenic mutation associated with GS. The use of NGS‐based panel is recommended to cover the large genotypic variability associated with this disease, in an attempt to identify novel SLC12A3 gene variants of potential pathogenicity.

1. Introduction

Hypokalemia is a common issue frequently encountered in daily clinical practice. Although it is a mild and asymptomatic disturbance for most patients, when aggravated, it can lead to potentially life‐threatening arrhythmias. Potassium (K⁺) is an electrolyte involved in multiple physiopathological processes, and its renal management is essential for homeostasis. Among the potential causes of hypokalemia is Gitelman Syndrome (GS).

GS is a rare renal tubular disorder typically identified during adolescence or adulthood. Patients may be asymptomatic or exhibit a wide range of non‐specific symptoms, such as fatigue, salt craving, thirst, nocturia, muscle weakness, cramps, tetany, paralysis and rhabdomyolysis [1, 2]. It follows an autosomal recessive inheritance pattern, and the prevalence of heterozygotes in Caucasian populations is estimated at 1% [3]. Therefore, GS is considered one of the most common hereditary renal tubular disorders [4].

Mutations in the Solute Carrier Family 12 Member 3 gene (SLC12A3) (OMIM* 600968), located on chromosome 16q13 and encoding the thiazide‐sensitive NaCl cotransporter (NCC), have been reported as the primary aetiology of this disorder [5, 6]. This NCC is a 1021‐amino acid polypeptide with 12 transmembrane domains that contribute to 5%–10% of renal sodium (Na⁺) reabsorption and it is located in the apical membrane of the distal convoluted tubule (DCT) [7]. Altered function of the NCC increases the amount of Na⁺ in the collecting duct, resulting in increased urinary excretion of K⁺, hydrogen (H⁺), and magnesium (Mg²⁺), leading to hypokalemia, metabolic alkalosis, hypomagnesemia, hypocalciuria, and hyperreninemic hyperaldosteronism [8].

We are presenting the case of a female with hypokalaemia and hypomagnesaemia.

2. Case Presentation

A 51‐year‐old female was referred to nephrology outpatient clinics for evaluation of recurrent hypokalemia and hypomagnesemia for the past 4 years. Her medical history included chronic headaches and minor depressive syndrome. After diagnosis, management, and specialised follow‐up, the patient currently remains asymptomatic. The main treatment was oral potassium OD and celecoxib OD.

Belonging to a small family, the patient is the only child of deceased parents with no known personal or family history of kidney disease. They are from Timișoara, Romania. Her grandparents are from the Romanian towns of Gătaia and Bodo, respectively. She has an only child with no phenotypic symptoms associated with kidney disease. There is no known data on her belonging to an endogamous population group or family history of consanguinity.

The physical examination was unremarkable, with blood pressure values of 128/80 mmHg, a heart rate of 85 bpm, a weight of 69 kg, and a height of 169 cm. In addition, the attending physician requested a comprehensive analysis including a complete blood count, blood gas analysis, biochemistry, spot urine and 24‐h urine collection. The analytical findings are reflected in Table 1.

TABLE 1.

Results of complete blood count, blood gas analysis, biochemistry, spot urine and 24‐h urine collection from the index case (IC) and her son.

Parameters	Results IC	Unaffected son	Reference intervals	Units
Haemoglobin	13.8	15.1	12.5–16	g/dL
Haematocrit	39.9	44.3	37–47	%
pH	7.47	—	7.33–7.43
pCO2	41	—	38–48	mmHg
HCO3a	29.8	—	22–28	mmol/L
Serum sodium (Na+)	142	140	136–146	mEq/L
Serum potassium (K+)	3	3.7	3.5–5.1	mEq/L
Serum magnesium (Mg2+)	1.3	2.1	1.9–2.5	mg/dL
Serum calcium (Ca2+)	10	9.9	8.8–10.6	mg/dL
Serum phosphorus (P−)	3.2	3.6	2.5–4.5	mg/dL
Serum creatinine	0.65	0.85	0.51–0.95	mg/dL
CDK‐EPI	> 90	> 90		ml/min/1.73 m2
Urine spot Ca2+	2.4	25.3		mg/dL
24 h urinary Ca2+ excretion	42.8	265.6	100–300	mg/24 h
Urine spot creatinine	69	197	22–328	mg/dL
24 h urinary creatinine excretion	1242	2068.5	600–1800	mg/24 h
Urine spot P−	40.6	83.2		mg/dL
24 h urinary P− excretion	731	873.6	400–1300	mg/24 h
Urine spot uric acid	28.5	70.9		mg/dL
24 h urinary uric acid excretion	513	744.4	250–750	mg/24 h
Urine spot Mg2+	5.2	12.4	4.1–13.8	mg/dL
24 h urinary Mg2+ excretion	93.6	130.2	73–122	mg/24 h
Urine spot Na+	100	234		mEq/L
24 h urinary Na+ excretion	180	245.7	40–220	mEq/24 h
Urine spot K+	46.7	27.9		mmol/L
24 h urinary K+ excretion	84.1	29.3	25–125	mEq/24 h
Urine osmolality	315	913	500–800	mOsmol/Kg
Serum osmolality	284	286	280–300	mOsmol/Kg
Renin	1.824	1.181	0.28–2.84	ng/dL
Aldosterone	26.1	6.67	2.52–39.2	ng/dL
Aldosterone/Renin ratio	14.3	5.6

Abbreviations: Ca, calcium; CDK‐EPI, equation to estimate renal function; K, potassium; Mg, magnesium; Na, sodium; P, phosphorus.

The analytical results showed mild metabolic alkalosis accompanied by hypokalemia, hypomagnesemia, and hypocalciuria. Therefore, considering the patient's history, the differential diagnoses considered were: eating disorders, long‐term laxative abuse, diuretic abuse, Gitelman Syndrome (GS), and Bartter Syndrome (BS). The first three were ruled out during the medical history, and the 24‐h hypomagnesemia and hypocalciuria favoured the diagnosis of GS over BS. Consequently, a genetic study was requested to confirm the results.

The administered treatment included oral K⁺ supplementation, and the patient was advised to take supplements and follow a diet rich in K⁺ and Mg²⁺.

2.1. Genetic Study

2.1.1. Materials and Methods

Before conducting the genetic tests, informed consent was obtained from the patients to publish the case details.

Complete blood samples were collected from the woman as the index case (IC) and later from her son. The patient had no siblings, and her parents were deceased.

The methodology employed for the genetic study was next‐generation sequencing (NGS) using capture and enrichment of the exonic regions and flanking intronic zones of the genes included in the Custom Constitutional Panel Mb (CCP17) sequencing panel, utilising SureSelect Agilent technology, followed by sequencing on the NextSeq platform (Illumina).

The genes included in the panel were: ATP6V1B1, BSND, CA2, CASR, CLCNKA, CLCNKB, CLDN16, CLDN19, FXYD2, HSD11B2, KCNJ1, KCNJ10, KLHL3, MAGED2, NR3C2, SCNN1A, SCNN1B, SCNN1G, SLC12A1, SLC12A2, SLC12A3, SLC4A1, SLC4A4, WNK1, WNK4.

Bioinformatic analysis to assess the impact of variants on protein structure, functionality, and conservation was conducted using the CADD score. For variants potentially affecting splicing, predictors available in Alamut (MaxEnt, SSF, GeneSplicer, and NNSplice) were utilised. The average read depth obtained was 152.10 > 20% in 95.81% of the analysed regions.

The methodology used for the genetic study in the son was similar to that in the IC. It involved DNA extraction, subsequent amplification of the region of interest in the SLC12A3 gene, library preparation using SureSelect Agilent technology, and sequencing on the NextSeq platform (Illumina).

Identification of variants of interest located in exonic and intronic regions up to ±10 nucleotides from the studied gene, relative to the reference genome (hg19), was performed after enrichment using DRAGEN Enrichment, Version 3.8.4, and filtering according to specific quality criteria: call quality > 20, coverage > 1×, Genotype Quality > 20, and Allele fraction > 15. Annotation and variant filtering were done through RefLabDB (database and proprietary pipeline). Variant analysis was conducted using annotations and support from Alamut Visual (Interactive Biosoftware).

Bioinformatic analysis to assess the impact of variants on protein structure and functionality was performed using the CADD score. For variants potentially affecting splicing, the SpliceAI metapredictor was used. The average read depth obtained was 152.10 > 20% in 100% of the analysed regions.

2.2. Results

2.2.1. Index Case (IC)

In the IC, two heterozygous variants were found in SLC12A3 (NM_000339.3): c.1315G>A, p.(Gly439Ser), and c.704C>A, p.(Thr235Lys), which have been classified as variants of uncertain significance (VUS) as no previous literature or record of these mutations was found in the Single Nucleotide Polymorphism Database (dbSNP) or Genome Aggregation Database (gnomAD). However, two changes (c.704C>G and c.704C>T) at the same position were previously described in GS.

Genetic analysis of the patient and her son demonstrated the causality of the genotype formed by the combination of the heterozygous and in trans variants:

c.1315G>A, chr16‐56879207 G>A, p.(Gly439Ser) in the SLC12A3 gene (NM_001126108.2), described with the dbSNP code rs759377924. Catalogued according to the ACMG pathogenicity criteria PM3, PS3, PM1, PP2, PM2, PM5, and PP3. Listed in specific databases as a pathogen and associated with familial hypokalemia‐hypomagnesemia by ClinVar. ClinVar IDs: 586601. Nineteen presentations as a pathogen, the last two presentations in 2025: SCV005892576, RCV000713326. By UniProt VAR_039505. Its population frequencies, detailed in the latest version of gnomAD v4.1, show its distribution with a heterozygosity of 0.008%; although it has not yet been identified in many populations, the highest frequency is found in Central European populations. The positional information of codon p.439 of the SLC12A3 protein (GENCODE: ENST00000438926.2, RefSeq: NM_000339.2, NM_001126107.1, NM_001126108.1, UniProt: P55017‐2), cDNA: c.1315‐1317 GGC, calculates a dn/ds change tolerance score of 0.29, within the intolerance range. This region is part of the PF00324 domain. No splicing alteration is predicted from this change c.704C>A, chr16‐56870198 C>A, p.(Thr235Lys) in the SLC12A3 gene (NM_001126108.2), has not yet been described in databases (last access: 05/20/2025; https://franklin.genoox.com/clinical‐db/variant/snp/chr16‐56870198‐C‐A‐hg38?app=acmg‐classification), nor published or detected in other populations. In silico predictors assign the ACMG pathogenicity criteria PM1, PP2, PM2, and PM5. Positional information of codon p.235 of the SLC12A3 protein (GENCODE: ENST00000438926.2, RefSeq: NM_000339.2, NM_001126107.1, NM_001126108.1, UniProt: P55017‐2), cDNA: c.703‐705 ACG, with a dn/ds score of 0.65, slightly intolerant. The position is part of the protein domain PF00324. No ClinVar SNVs were found at this position. This change does not predict a splicing alteration. The first variant was recorded as pathogenic in public databases such as the Human Gene Mutation Database (HGMD), and the CADD bioinformatic predictor estimated a pathogenic effect (26.6).

Additionally, the patient carried benign heterozygous variants in the genes KCNJ10 (NM_002241.4): c.811C>T; p.(Arg271Cys) (ƒ = 4.67000000 gnomAD), CA2 (NM_000067.2): c.34+59_34+65delTCCCCGA (ƒ = 4.92000000 gnomAD), SLC12A3 (NM_000339.2): c.1825+9C>A (ƒ = 3.41000000 gnomAD), SLC12A3 (NM_000339.2): c.1884G>A; p.(Ser628Ser) (ƒ = 8.49000000 gnomAD).

Pathogenic variants in the SLC12A3 gene are associated with GS, which has an autosomal recessive inheritance pattern. In such diseases, two pathogenic variants in trans configuration (one on each allele) are necessary for diagnostic confirmation, or the identification of a single pathogenic variant confirms the status of an asymptomatic carrier. For this reason, the decision was made to study the son.

In the case of the son, the heterozygous presence of the variant c.704C>A p.(Thr235Lys) in the SLC12A3 gene was identified. The presence of only this variant supported the suspicion that both variants in the IC are in trans configuration, increasing evidence of the potential causality of the identified VUS (Figure 1). However, the clinical implication of an unconfirmed pathogenic variant must be assessed by the specialist in the context of the patient's family and clinical situation.

FIGURE 1.

Identification of the c.704C>A, p.(Thr235Lys), variant in the SLC12A3 gene by next‐generation sequencing (NGS) in the patient and her son. The figure shows two panels: In panel A, corresponding to the mother, a deep sequencing of 47 reads was obtained, with a heterozygosity frequency of the A allele of 37%. In panel B, corresponding to segregation analysis in her son, coverage increase to 10 356 and 49%‐heterozygosity frequency of the A allele corroborated the existence of the variant. The results were consistent with a heterozygous state in both patients and in trans location in the mother (affected by GS).

3. Discussion

GS also known as familial hypokalemia‐hypomagnesemia, has long been considered a benign tubulopathy [7]. It is estimated to have an incidence of around 1:40.000 individuals [6].

As mentioned earlier, this condition can be asymptomatic or associated with mild or nonspecific symptoms. However, this perception has been challenged due to the phenotypic variability and severity of the disease [1]. Severe manifestations such as growth delay, chondrocalcinosis, tetany, rhabdomyolysis, seizures, and ventricular arrhythmia have also been described [9, 10, 11]. Interfamilial and intrafamilial phenotypic variability has been documented in genetically confirmed GS patients, even in those with identical mutations in SLC12A3. A combination of genotype, gender, modifier genes, compensatory mechanisms, as well as environmental factors or dietary habits, could be involved in such variability [7, 12].

The detection of biallelic inactivating mutations in the SLC12A3 gene is crucial for GS diagnosis. To date, over 500 different mutations of the SLC12A3 gene have been reported in affected patients [13]. Most patients are compound heterozygotes for mutations, but a significant number of GS patients carry a single SLC12A3 mutation [1], including nonsense, frameshift, and splice site mutations distributed throughout the protein [6].

Loss‐of‐function mutations in the Na‐Cl cotransporter (NCC) lead to deficient Na⁺ absorption. Consequently, more Na⁺ reaches the collecting ducts, and the excess Na⁺ and water loss in the urine causes mild volume contraction. This activates the renin‐angiotensin‐aldosterone mechanism, leading to increased renin and aldosterone production. Aldosterone acts on cortical collecting ducts to enhance Na⁺ reabsorption through Na⁺ channels, thereby maintaining salt homeostasis at the expense of increased excretion of K⁺ and H⁺. This results in hypokalemia and metabolic alkalosis [14]. Hypomagnesemia is likely due to defects in the transient receptor potential channel subfamily M, member 6 (TRPM6), responsible for Mg²⁺ reabsorption in the distal convoluted tubule (DCT) [15]. There are two possible mechanisms for hypocalciuria in GS: mild volume contraction may cause increased Na⁺ and Ca²⁺ reabsorption in the proximal convoluted tubule, or inhibition of NCC in the DCT leads to increased Na⁺ uptake in exchange for enhanced Ca²⁺ extrusion through the Na⁺–Ca²⁺ exchanger 1 (NCX1) at the basolateral membrane. This results in increased Ca²⁺ reabsorption through the transient receptor potential cation channel subfamily V, member 5 (TRPV5) [16].

The analytical sensitivity and specificity of genetic tests for GS are 90%–100% and 100%, respectively. However, because some GS patients have only one mutated allele in SLC12A3, the clinical sensitivity and specificity turn out to respectively be 80% and 100%, depending on the genetic methods used [17].

It is important to note that with the increasing availability of genetic data based on new populations and functional studies that have identified more variables, the classification of variants may change: the pathogenicity of genetic variants previously associated with the disease could be questioned, and vice versa, variants of uncertain significance (VUS) could be confirmed as pathogenic [18, 19, 20].

4. Conclusion

In conclusion, the diagnosis of GS is based on clinical symptoms and biochemical abnormalities, which should subsequently be confirmed through genetic testing. In these cases, the use of an NGS‐based gene panel to parallelly sequence all relevant genes in the differential diagnosis of GS is recommended.

In our case, genetic studies revealed two heterozygous mutations in the SLC12A3 gene, including a novel mutation not reported to date. The identification of this mutation in trans increased the evidence of the VUS pathogenicity.

Consent

Informed consent was obtained from the patient, as well as verbal and written assent from the child.

Conflicts of Interest

The authors declare no conflicts of interest.

Tomás‐Simó P., Sierra‐Rivera A., Checa‐Ros A., Rodríguez‐López R., Galán‐Serrano A., and D'Marco L., “Novel Pathogenic Genotype in SLC12A3 Associated to Gitelman Syndrome: A Case Report,” Nephrology 30, no. 9 (2025): e70127, 10.1111/nep.70127.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.