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

Background Gitelman's syndrome (GS) is a rare salt-losing renal tubular disorder associated with SLC12A3 gene mutations, which encodes the Na-Cl co-transporter (NCCT). GS is characterized by hypokalaemic metabolic alkalosis, hypomagnesemia, hypocalciuria and elevated renin-angiotensin-aldosterone (RAA) level. The variability of phenotypes is likely to be associated with the variety of SLC12A3 mutations. Methods In this study, we reported the clinical features and the genetic analysis of a GS family pedigree. Results We identified novel mutations of SLC12A3 , with c.433 C>T (p.Arg145Cys), c.1077 C>G (p.Asn359Lys), and c.1666 C>T (p.Pro556Ser). The proband exhibited hypokalaemia, hypomagnesemia, metabolic alkalosis, but hypercalcuria and kidney stone. The increased urinary calcium excretion made it confused to Bartter's syndrome (BS). The persistent renal potassium wasting associated renal tubular lesions finally affected urinary calcium reabsorption, leading to the increased calcium excretion. Genetic analysis revealed mutations of SLC12A3 with C433T (Arg145Cys, Het), C1077G (Asn359Lys, Het), and C1666T (Pro556Ser, Het). Those missense mutations led to the predicted amino acid change, caused differences of NCCT protein structures and function. One sister of the proband carried the same mutant sites, however, exhibited milder phenotypes including hypokalemia, hypomagnesemia, RAAS activation, but not elevated urinary calcium excretion. With administration of antisterone, potassium chloride and magnesium supplement, the serum potassium and magnesium were maintained in normal ranges.


Abstract Background
Gitelman's syndrome (GS) is a rare salt-losing renal tubular disorder associated with SLC12A3 gene mutations, which encodes the Na-Cl co-transporter (NCCT). GS is characterized by hypokalaemic metabolic alkalosis, hypomagnesemia, hypocalciuria and elevated renin-angiotensin-aldosterone (RAA) level. The variability of phenotypes is likely to be associated with the variety of SLC12A3 mutations.

Methods
In this study, we reported the clinical features and the genetic analysis of a GS family pedigree.

Results
We identified novel mutations of SLC12A3 , with c.433 C>T (p.Arg145Cys), c.1077 C>G (p.Asn359Lys), and c.1666 C>T (p.Pro556Ser). The proband exhibited hypokalaemia, hypomagnesemia, metabolic alkalosis, but hypercalcuria and kidney stone. The increased urinary calcium excretion made it confused to Bartter's syndrome (BS). The persistent renal potassium wasting associated renal tubular lesions finally affected urinary calcium reabsorption, leading to the increased calcium excretion. Genetic analysis revealed mutations of SLC12A3 with C433T (Arg145Cys, Het), C1077G (Asn359Lys, Het), and C1666T (Pro556Ser, Het). Those missense mutations led to the predicted amino acid change, caused differences of NCCT protein structures and function. One sister of the proband carried the same mutant sites, however, exhibited milder phenotypes including hypokalemia, hypomagnesemia, RAAS activation, but not elevated urinary calcium excretion. With administration of antisterone, potassium chloride and magnesium supplement, the serum potassium and magnesium were maintained in normal ranges.

Conclusions
In this study, we identified the novel mutations of SLC12A3 and the varieties of clinical features.
Further efforts are needed to investigate the diversity in clinical manifestations of GS and its correlation with SLC12A3 mutations. Background Gitelman's syndrome (GS, OMIM#263800) is an autosomal recessive inherited, salt-losing renal tubular disorder, with the clinical features including hypokalemia, renal potassium loss, metabolic alkalosis, hypomagnesemia, hypocalciuria with normal blood pressure [1]. GS is associated with mutations of SLC12A3 (solute carrier family 12 member 3) gene, which locates in chromosome 16q13 and encodes the thiazide-sensitive Na-Cl cotransporter (NCCT) of distal convoluted tubule [2]. Till now, there are more than 400 varieties of SLC12A3 related to GS have been reported [2, 3,4,5].
Among those mutations include nonsense, missense, deletion, insertion, and splice-site, missense mutations account for the most in GS [6]. Most SLC12A3 mutations in GS are simple or complex heterozygous mutations and few of them are homozygous [7].
The clinical symptoms of GS are variable, including muscle weakness, paresthesias, numbness, polyuria, and growth retardation in children [2]. Some patients are asymptomatic or mildly symptomatic, or only exhibit non-specific fatigue, leading to the miss diagnosis or misdiagnosis.
Severe and persistent hypokalemia may lead to glucose intolerance, cardiac and renal dysfunction.
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). GS and Bartter's syndrome (BS) both exhibit hypokalemia, metabolic alkalosis, and normal blood pressure with high plasma renin activity and high aldosterone concentration. They exhibit similar phenotypes, which make it difficult to diagnose. Urinary calcium excretion is considered to be an important clue to distinguish these two diseases [8].
In this study, we identified novel compound hybrid mutations of SLC12A3 with c.433 C>T (p.Arg145Cys), c.1077 C>G (p.Asn359Lys), and c.1666 C>T (p.Pro556Ser) in a family pedigree of GS.
The proband presented hypercalcuria and renal calcification. The further genotype-phenotype correlation analysis is needed to provide deeper insights into GS.

Patients' recruitment
Seven participants (three men and four women) from a Chinese family were recruited. The diagnosis of GS depends on clinical symptoms, biochemical parameters and genetic analysis. All participants denied a history of laxatives, diuretics, or other drugs including insulin, β-receptor activator or Chinese medicinal herbs. This study was approved by the ethics committee of the Affiliated Hospital of Qingdao University.

Genetic analysis
Genomic DNA was extracted from peripheral blood sample using the QIAamp Blood DNA Mini Kit

Clinical presentation
A 42-year-old male was presented to our hospital with fatigue, repeated muscle weakness and quadriplegia for ten years. Laboratory investigation exhibited hypokalemia, hypomagnesemia, increased urinary potassium excretion, activated renal-aldosterone system, hypercalcaemia and hypercalcuria ( Table 1). The fraction excretion rate of potassium (FEK%) was significantly increased to 30.5-49.2% (normal range 8-12%), suggesting that hypokalemia resulted from renal potassium wasting. Thyroid function, cortical and adreno-corticotropic hormone (ACTH) were normal. Other possible causes of hypokalaemia such as thyrotoxic periodic paralysis, renal tubule acidosis, Cushing's syndrome were excluded. Serum calcium slightly increased, and urinary calcium excretion was also increased (FECa 2.66%, urinary calcium to creatine ratio 0.70) ( Table 1)  The patient was supplied with potassium chloride sustained release tables, antisterone and magnesium. During the one-year fellow-up, serum potassium and magnesium was sustained in normal range.
Biochemistry profiles of the other family pedigree One of his sisters (II-4, Fig. 2A) also showed hypokalemia, hypomagnesaemia, elevated reninaldosterone level, normal blood pressure, but with normocalcemia and hypocalciuria ( Table 1). The sister was administrated with potassium chloride, and the serum potassium level was corrected into normal range, without symptoms such as fatigue, muscle weakness, tetany, or paresthesia during the treatment. In addition, the levels of serum potassium, sodium, calcium, magnesium, urinary potassium and calcium were unremarkable in the other family members of the pedigree (Table 2).

Genetic analysis
The amplification and sequencing of SLC12A3 gene were performed on the family pedigree. The  Three-dimensional structure prediction of NCCT and the potential dysfunction The SLC12A3 encoded NCCT contains 12 transmembrane segments and N-and C-terminal domains.
We identified the alteration of NCCT structure induced by the variants of SLC12A3 (C433T, Arg145Cys; C1077G, Asn359Lys; and C1666T, Pro556Ser) (Fig. 3), using the SWISS-MODEL workspace (http://swiss-model.expasy.org). Those missense mutations led to the predicted amino acid change, caused differences of NCCT protein structures and function, finally leading to the electrolyte disturbance.
In this study, we reported a family pedigree of GS with novel heterozygous mutations of SLC12A3,

Discussion
GS is a salt-losing tubulopathy with the clinical features of hypokalemic alkalosis, hypomagnesaemia and hypocalciuria. Chronic low potassium leads to symptoms of weakness, fatigue, thirst and the abnormal heart palpitation. Severe case can cause rhabdomyolysis, ventricular arrhythmias, or sudden cardiac arrest [9]. GS is associated with dysfunction of NCCT encoded by SLC12A3 gene in the renal distal convoluted tubule (DCT). The decreased reabsorption of Na + and Cl − leads to compensatory excessive exchange through Na + /K + and Na + /H + pump, and results in excessive K + and H + excretion and hypokalemic alkalosis. In a small minority of GS patients, mutations in the CLCNKB gene encoding the chloride channel ClC-Kb have been identified [10].
To our knowledge, this is the first time to report the novel c.1077 C > G (p.Asn359Lys) and c.1666 C > T (p.Pro556Ser) mutations of SLC12A3 and its relation with phenotypes. The phenotypes are more severe in patients with more than one mutated alleles, with lower serum potassium level, and more difficult to be corrected with potassium supplement [6,11].
BS (especially type III) is the most important renal salt-wasting disease which should be considered as the differential diagnosis of GS. BS is also characterized by hypokalemia, metabolic alkalosis, polyuria, increased renin activity and aldosterone level, but without hypertension or edema. It exhibits the increased urinary calcium excretion, but rarely leads to nephrocalcinosis. BS is caused by mutations of NKCC2 (Na + -K + -2Cl − cotransporter) in the thick ascending limb (TAL) of Henle loop (Type 1 BS) [12], ROMK (outwardly rectifying potassium channel) (Type 2 BS), or CLCNKB (chloride channel) (Type 3 BS) which is a regulator of NKCC2. A minority of GS patients has been shown to have mutations of CLCNKB gene [10]. Type 4 BS 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 calcium-sensing receptor (CaSR) suppresses the NKCC2 and ROMK expression to induce type 5 GS [13]. The site of defect in BS is at the TAL of the Henle loop, whereas in GS is at the renal DCT [14].
GS used to be thought as a mild type of BS. However, the pathogenesis and clinical characteristics are different. BS 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]. GS usually shows hypomagnesaemia with increased urinary manganese excretion (FEMg > 4%), but lower urinary calcium excretion (uCa/uCr < 0.2) [8].
Diuretic loading test using furosemide and hydrochlorothiazide is helpful to differ GS from BS [16].
Usually, hypocalciuria in GS is related to the increased calcium reabsorption in the proximal tubule and distal renal unit, which is caused by NCCT dysfunction [17]. In this case, the proband exhibited hypokalemia, hypomagnesaemia, metabolic alkalosis, but with hypercalcuria, similar to the features of BS, which makes it confused for differential diagnosis. It is contradicted with the features of hypocalcuria in classic GS. Chronic renal potassium loss can cause renal tubular epithelial cell injury or vacuolar deformation, to reduce the reabsorption of calcium [18]. In addition, loss-of-function of NCCT up-regulates the expression of intestinal calcium transporter, and increases calcium uptake in gut tract [19]. Hypercalcemia inhibits PTH release by negative feedback. In reverse, the suppressed PTH level reduces the calcium reabsorption by the renal tubule, and increases urinary calcium excretion. This patient also has diabetes mellitus. Hyperglycemia causes osmotic diuresis to increase urinary calcium excretion. 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 mutations of SLC12A3.
It is reported that GS patient 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 of diabetes development. In addition, hyperaldosteronism was also reported to promote insulin resistance [21].
Studies have shown that GS 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 of GS 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 options include the inhibitors for the secondary elevated renin-aldosterone system (RAAS), such as non-selective or selective aldosterone antagonist antisterone or eplerenone, or NaCl transporter blockers such as aminophenidine [25]. Non-steroidal anti-inflammatory drugs (NASID) such as indomethacin can suppress renin secretion by inhibiting renal prostaglandin E2 (PGE2) synthesis, and ameliorate the up-regulation of aldosterone level induced by potassium supplement. It also could increase potassium level without worsening sodium and volume depletion in GS patients [26]. However, the gastrointestinal side effect and interstitial renal damage make the application to be limited.

Conclusions
In this study, we identified novel mutations of SLC12A3 and reviewed the advances in genetic analysis, diagnosis, differential diagnosis and management of GS. Combined with clinical features, biochemistry profiles and genetic analysis, the diagnose could be made. Further studies on the correlation between genotype and phenotype are needed to provide better understanding of GS. Declarations Ethic approval and consent to participate:

List Of Abbreviations
All procedures performed in this study involving human participants were in accordance with the ethical standards of the Affiliated Hospital of Qingdao University with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. We obtained the written informed consent from all the participants.

Consent to publish:
The clinical data and images were obtained from the proband and his family members. All the participants gave their written consent for the information to be published.

Availability of data and materials:
The data generated and analyzed in this study are not publicly available due to protection of privacy, but are available from the corresponding author on reasonable request.

Competing interests:
All authors declare that they have no competing of interest. for clinical data collection, including biochemistry profiles and genetic analysis.

Authors' contributions:
DB and CY collected and interpreted the patient and family pedigree' data, analyzed the clinical data and genetic sequencing, summarized and wrote the paper. LX is the major contributors in writing the manuscript. WY provided key guidance of intellectual content and important discussion to this study.
WF, ZY, and SX provided important discussion and suggestions. ZW summarized all results and controlled the whole study. All authors read and approved the final manuscript.
authors have no conflict of interest related to this work.   Table 3. Summary of the variants of SLC12A3 in the GS pedigree.

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