Identification of compound mutations of SLC12A3 gene in a Chinese pedigree with Gitelman syndrome exhibiting Bartter syndrome-liked phenotypes

Background Gitelman syndrome is a rare salt-losing renal tubular disorder associated with mutation of SLC12A3 gene, which encodes the Na-Cl co-transporter (NCCT). Gitelman syndrome is characterized by hypokalemia, metabolic alkalosis, hypomagnesemia, hypocalciuria, and renin-angiotensin-aldosterone system (RAAS) activation. Different SLC12A3 variants may lead to phenotypic variability and severity. Methods In this study, we reported the clinical features and genetic analysis of a Chinese pedigree diagnosed with Gitelman syndrome. Results The proband exhibited hypokalaemia, hypomagnesemia, metabolic alkalosis, but hypercalciuria and kidney stone formation. The increased urinary calcium excretion made it confused to Bartter syndrome. The persistent renal potassium wasting resulted in renal tubular lesions, and might affect urinary calcium reabsorption and excretion. Genetic analysis revealed mutations of SLC12A3 gene with c.433C > T (p.Arg145Cys), c.1077C > G (p.Asn359Lys), and c.1666C > T (p.Pro556Ser). Potential alterations of structure and function of NCCT protein due to those genetic variations of SLC12A3 are predicted. Interestingly, one sibling of the proband carried the same mutant sites and exhibited similar clinical features with milder phenotypes of hypokalemia and hypomagnesemia, but hypocalciuria rather than hypercalciuria. Family members with at least one wild type copy of SLC12A3 had normal biochemistry. With administration of spironolactone, potassium chloride and magnesium supplement, the serum potassium and magnesium were maintained within normal ranges. Conclusions In this study, we identified compound mutations of SLC12A3 associated with varieties of clinical features. Further efforts are needed to investigate the diversity in clinical manifestations of Gitelman syndrome and its correlation with specific SLC12A3 mutations.


Background
Gitelman syndrome (OMIM#263800) is an autosomal recessive inherited salt-losing renal tubular disorder. Gitelman syndrome is characterized with clinical features including hypokalemia, renal potassium wasting, metabolic alkalosis, hypomagnesemia, hypocalciuria, and RAAS activation with normal blood pressure [1]. Gitelman syndrome is associated with mutations of SLC12A3 (solute carrier family 12 member 3) gene, which locates on chromosome 16q13 and encodes the thiazidesensitive Na-Cl cotransporter (NCCT) of distal convoluted tubule (DCT) [2]. Till now, there are more than 400 varieties of SLC12A3 related to Gitelman syndrome have been reported [2][3][4][5]. Among those mutations, most are missense, deletion, insertion, and splice-site mutations [6]. Most SLC12A3 mutations in Gitelman syndrome are found as simple or complex heterozygous mutations, and few of them are homozygous [7].
The clinical symptoms of Gitelman syndrome are variable, including muscle weakness, paresthesia, numbness, polyuria, and growth retardation in children [2]. Some patients are asymptomatic or mildly symptomatic, or only exhibit non-specific fatigue, leading to the frequent misdiagnosis. Disordered renal reabsorption of sodium and chloride leads to a series of pathophysiological changes and clinical manifestations, including decreased blood volume, and activated renin-angiotensin aldosterone system (RAAS). Severe and persistent hypokalemia may lead to glucose intolerance, cardiac and renal dysfunction. Bartter syndrome is the most important genetic disorder to consider in the differential diagnosis of Gitelman syndrome, since both exhibit hypokalemia, metabolic alkalosis, and increased plasma renin activity and aldosterone levels with normal blood pressure. Such similar phenotypes as those diseases share can make differential diagnose challenging; however, urinary calcium excretion is often considered to be an important clue to distinguish these two disorders [8].
In this study, we reported a Chinese familial Gitelman syndrome, and identified compound mutations of SLC12A3 with c.433C > T (p.Arg145Cys), c.1077C > G (p.Asn359Lys), and c.1666C > T (p.Pro556Ser). The proband presented hypercalciuria and renal calcification, exhibiting Bartter syndrome-like biochemical phenotypes. Further analysis of genotype-phenotype correlation analysis is needed to provide deeper insights into Gitelman syndrome.

Patient recruitment
Participants were recruited from a Chinese pedigree. The diagnosis of Gitelman syndrome was based on clinical symptoms, biochemical parameters and genetic analysis of SLC12A3 mutations. All participants denied a history of laxatives, diuretics, or other agents including insulin, β-receptor activator or Chinese herbal medicine. This study was approved by the ethics committee of the Affiliated Hospital of Qingdao University.

Biochemistry tests
The peripheral blood sample and urine sample were collected. The blood and urine electrolytes were measured with an automatic biochemical analyzer. Plasma renin activity, plasma angiotensin, and plasma aldosterone were measured using a radioimmunoassay.

Clinical manifestations of the proband
A 42-year-old male was presented to hospital with chief complaint of fatigue, repeated muscle weakness and paralysis over ten-year period. Laboratory investigation exhibited hypokalemia, hypomagnesemia, increased urinary potassium excretion, activated RAAS, hypercalcemia and hypercalciuria ( Table 1). The fractional excretion rate of potassium (FE K %) was significantly increased to 30.5-49.2% (normal range 8-12%), suggesting that hypokalemia is resulted from renal potassium loss. Thyroid function, cortical and adreno-corticotropic hormone (ACTH) were normal. Other possible causes of hypokalemia such as thyrotoxic periodic paralysis, renal tubular acidosis and hypercortisolism were excluded. Serum calcium was slightly increased, with elevated urinary calcium excretion (FE Ca 2.66%, urinary calcium to creatine ratio 0.70) ( Table  1). The glucose stimulated insulin secretion (GSIS) test showed the delayed insulin release and insulin resistance. Renal calcification was detected by computed tomography (CT) (Fig. 1A-D). Kidney biopsy revealed the large vacuolar degeneration or atrophy in renal tubular cells ( Fig.  1E-H), suggesting that renal tubular lesions might be associated with persistent renal potassium wasting.
The patient was supplied with potassium chloride sustained release tables, spironolactone and magnesium. During the fellow-up, serum potassium and magnesium levels were maintained within normal range.

Biochemistry profiles of the other pedigree relatives
Plasma biochemical results showed that one of proband's sister (II-4, Fig. 2A) showed Gitelman syndromephenotypes with hypokalemia, hypomagnesaemia, elevated renin-aldosterone level, and normal blood pressure, but with normocalcemia and hypocalciuria ( Table  1). The sister was therefore administrated with potassium chloride, and the serum potassium level was corrected into normal range, without symptoms of fatigue, muscle weakness, tetany, or paresthesia during the treatment. The levels of serum potassium, sodium, calcium, magnesium, urinary potassium and calcium were unremarkable in the other relatives of the pedigree (Table 1).

Genetic analysis
Sequencing of SLC12A3 gene was performed on the familial relatives of the proband. Genetic analysis revealed that the proband and sibling II-4 carried same  Tables 1 and 2). These results suggested that c.433C > T and c.1666C > T located on the same chromosome (in cis), while c.1077C > G representing the trans variant led to the compound heterozygosity in the proband and sibling (II-4). The frequency of these variants was examined in the reference database. The variant c.433C > T was not identified in 1000 Genomes, but was examined in gnomAD (0.000012) and Esp6500 (0.000077) at very low frequency. The variant c.1077C > G was identified in 1000 Genomes (0.0001997). The variant c.1666C > T was not identified in 1000 Genomes, but was examined in gnomAD at low frequency (0.00002). The potential pathogenicity of those three variants were studied using prediction bioinformatics ( Table 2). In addition, no mutations were detected in other 40 genes reported to be associated with Gitelman and Bartter syndrome, including CLCNKA/CLCNKB (encodes the chloride channel ClC-Kb) and BSND (encodes chloride channel accessory subunit), KCNJ1 (encodes the thick ascending limb potassium channel), and CASR (encodes Calcium-sensing receptor) were not detected.
RAAS (standing condition) Three-dimensional structure prediction of NCCT and the potential dysfunction The SLC12A3-encoded NCCT protein contains 12 transmembrane segments as well as N-and C-terminal domains. We identified the alteration of NCCT structure induced by the compound mutations of SLC12A3 (C433T, Arg145Cys; C1077G, Asn359Lys; and C1666T, Pro556Ser) (Fig. 3), using the SWISS-MODEL workspace (http://swiss-model.expasy.org). Results indicate that amino acid change in NCCT protein due to the missense mutations of SLC12A3 might lead to alteration of NCCT protein structures that can affect function, resulting in the electrolyte disturbance.

Discussion
In this study, we reported a Chinese pedigree of Gitelman syndrome with heterozygous compound mutations of SLC12A3, exhibiting hypokalemia and hypomagnesemia. However, the proband exhibited hypercalciuria and renal calcification, which made it difficult to We identified variants of SLC12A3, c.433C > T (p.Arg145Cys), c.1077C > G (p.Asn359Lys), and c.1666C > T (p.Pro556Ser), but no mutations found in CLCNKA/ CLCNKB, BSND, KCNJ1, CASR that are known causing Batter syndrome. Therefore, clinical diagnosis of Gitelman syndrome was made, and the identified SLC12A3 variations were predicted to contribute clinical features of Gitelman syndrome. With administration of aldosterone antagonist spironolactone, potassium and magnesium supplement, the serum potassium and magnesium was maintained in nearly normal range during fellow-up. The phenotype variability may be associated with the pathogenic variabilities of SLC12A3 mutations. Genetic analysis is a useful tool for the diagnosis and differential diagnosis of such similarlypresenting diseases as Gitelman syndrome and Bartter syndrome. Further investigation is needed to provide better understanding of genotype-phenotype association of NCCT dysfunction in Gitelman syndrome. Gitelman syndrome is a salt-losing tubulopathy with the clinical features of hypokalemic alkalosis, hypomagnesemia and hypocalciuria. Chronic hypokalemia leads to symptoms of weakness, fatigue, thirst, and paralysis. Severe cases can cause rhabdomyolysis, ventricular arrhythmias, or even sudden cardiac arrest [9]. Gitelman syndrome is associated with dysfunction of NCCT protein encoded by SLC12A3 gene in the renal DCT. The decreased reabsorption of Na + and Cl − leads to compensatory excessive exchange through Na + /K + and Na + /H + pumps, resulting in excessive K + and H + excretion and hypokalemic alkalosis. In a small minority of Gitelman syndrome patients, mutations in the CLCNKB gene encoding the chloride channel ClC-Kb have been identified [10].
We identified compound mutations of SLC12A3, c.433C > T (p.Arg145Cys), c.1077C > G (p.Asn359Lys), and c.1666C > T (p.Pro556Ser). The proband and his affected sister carried three compound heterozygous mutations, suggesting that c.433 C > T and c.1666 C > T probability occurred in cis on one allele, and c.1077C > G occurred in trans. Interestingly, the other relatives with only a single affected chromosome show normal biochemistry. The phenotypes are more severe in patients with more than one mutated allele, with lower serum potassium level, which were more difficult to be corrected with potassium supplements [6,11].
Bartter syndrome (especially type III) is the most important renal salt-wasting disease which should be considered as the differential diagnosis of Gitelman syndrome. Bartter syndrome is also characterized by hypokalemia, metabolic alkalosis, polyuria, increased renin activity and aldosterone levels, but without hypertension or edema. It exhibits the increased urinary calcium excretion, but rarely leads to nephrocalcinosis. Bartter syndrome could be caused by mutations of NKCC2 (Na + -K + -2Cl − cotransporter) expressed in the thick ascending limb (TAL) of Henle loop (Type 1 Bartter Syndrome) [12], ROMK (outwardly rectifying potassium channel) (Type 2 Bartter Syndrome), or CLCNKB (chloride channel) (Type 3 Bartter Syndrome) which is a regulator of NKCC2. Type 4 Bartter Syndrome is induced by mutations of both the kidney-specific chloride channel ClC-Ka and ClC-Kb, leading to dysfunction of Cl − reabsorption. Activating mutations of calciumsensing receptor (CaSR) suppresses the NKCC2 and ROMK expression to induce type 5 Bartter syndrome [13]. The site of defect in Bartter syndrome is at the TAL of the Henle loop, whereas in Gitelman syndrome  Fig. 3 The model structure of Na-Cl cotransporter (NCCT) protein with variants induced by novel mutations of SLC12A3 to show potential influence. The differences of modeled structure compared to wild type (A) were indicated in circles. The visible differences of protein structure was induced by (B) co-existence of c.433 C > T (p.Arg145Cys) and c.1666 C > T (p.Pro556Ser), or (C) c.1077 C > G (p.Asn359Lys) lead to differences from wild type protein structure. It may induce the alteration of the function of NCCT is at the renal DCT [14]. Gitelman syndrome used to be thought as a mild type of Bartter syndrome. However, the pathogenesis and clinical characteristics are different. Bartter syndrome typically presents in infancy or early childhood, with more severe clinical manifestations and complications, such as severe electrolyte derangements, short stature, polyuria, and hypercalciuria induced nephrocalcinosis [15]. Gitelman syndrome usually shows hypomagnesaemia with increased urinary magnesium excretion (FE Mg > 4%), but lower urinary calcium excretion (uCa/uCr < 0.2) [8]. A diuretic loading test using furosemide and hydrochlorothiazide can be helpful in differentiating Gitelman syndrome from Bartter syndrome [16]. Hypocalciuria in Gitelman syndrome is generally a result of the increased calcium reabsorption in the proximal tubule and distal renal unit, which is caused by NCCT dysfunction [17]. In this report, the proband exhibited hypokalemia, hypomagnesaemia, metabolic alkalosis, but with hypercalciuria, similar to the features of Bartter syndrome, which makes it confused for differential diagnosis. It is contradicted with the features of hypocalciuria in classic Gitelman syndrome. Chronic renal potassium loss can cause renal tubular epithelial cell injury or vacuolar deformation to reduce the reabsorption of calcium [18]. The persistent renal potassium wasting might result in renal tubular lesions, and affect urinary calcium reabsorption and excretion, leading to persistent hypercalcuria. Additionally, loss-offunction of NCCT up-regulates the expression of intestinal calcium transporter, and increases calcium uptake in digestive tract [19]. Hypercalcemia inhibits PTH release via negative feedback; conversely, the suppressed PTH level reduces the calcium reabsorption by the renal tubule, and increases urinary calcium excretion. The patient described here also has diabetes mellitus. Hyperglycemia causes osmotic diuresis leading to urinary calcium excretion. Finally, increased urinary calcium excretion and chronic hypomagnesaemia are the causes of renal calcification. The relationship between mutated gene sites and urinary calcium levels has not been reported. It is unclear whether hypercalcuria is associated with three variants of SLC12A3.
Patients with Gitelman syndrome have a tendency of glucose intolerance and impaired insulin secretion [20]. Potassium plays an important role in the regulation of insulin release. Reduced extracellular potassium ion concentration could suppress the insulin secretion and release via ATP-sensitive potassium channel on beta-cells. Long-term low potassium and magnesium level is one of the factors for diabetes development. In addition, hyperaldosteronism was also reported to promote insulin resistance [21]. Studies have indicated that Gitelman syndrome can be combined with autoimmune diseases such as Graves' disease, Hashimoto's thyroiditis, IgA nephropathy, Sjogren's syndrome, or latent autoimmune diabetes in adults (LADA) [22,23].
The therapeutic strategy for Gitelman syndrome focuses on the correction of electrolyte disturbance, especially potassium and magnesium replacement. The level of serum magnesium may affect the severity and effect of potassium supplement [6,24]. Other therapeutic options include the inhibitors of the secondary elevated RAAS, such as using non-selective or selective aldosterone antagonist spironolactone or eplerenone, or NaCl transporter blockers such as potassium-sparing diuretic aminophenidine. The lossof-function mutations in the SLC12A3 gene result in Gitelman syndrome. Thus, the thiazide diuretics should be avoided for Gitelman syndrome patients as long-term treatment option [25]. Non-steroidal antiinflammatory drugs (NASIDs) such as indomethacin can suppress renin secretion by inhibiting renal prostaglandin E2 (PGE 2 ) synthesis, and ameliorate the upregulation of aldosterone level induced by potassium supplement. It also could increase potassium level without worsening sodium and volume depletion in Gitelman syndrome patients [26]. However, the gastrointestinal side effect and interstitial renal damage make the application to be limited.

Conclusions
In this study, we reported a pedigree of Gitelman syndrome and identified compound mutations of SLC12A3. Combined with clinical features, biochemistry profiles and genetic analysis, the diagnose could be made. The proband exhibited persistent hypercalcuria, which is contrary to the typical biochemical alteration in Gitelman syndrome. Diagonotic procedures combined clinical features and biochemistry profiles with genetic counselling are necessary for correctly differentiate Gitelman syndrome from Bartter syndrome. Further investigation will explore the correlation between genotype and phenotype are needed for provide better understanding of Gitelman syndrome.
Additional file 1. The variants of SLC12A3 identified in this pedigree of Gitelman syndrome. A. The mutant sequence of SLC12A3 mRNA and amino acid. Red characters to show the mutant nucleotide or amino acid. B. The model of Na-Cl cotransporter (NCCT) and affected amino acid site. NCCT is a 12 times transmembrane structure with 1030 amino acids. Star and red to show the mutant amino acids and sites, Arg145Cys, Asn359Lys, and Pro556Ser located in the respective spots.