Novel thiazide-sensitive Na–Cl cotransporter mutation in a Chinese patient with Gitelman's syndrome presenting as hypokalaemic paralysis

Nai-Lin Cheng1, Ming-Ching Kao3, Yaw-Don Hsu2 and Shih-Hua Lin1,

1 Division of Nephrology, Department of Medicine and 2 Department of Neurology, Tri-Service General Hospital, National Defense Medical Center, Taipei and 3 Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan

Keywords: Gitelman's syndrome; hypocalciuria; hypokalaemia; mutation; paralysis



   Introduction
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 Introduction
 Case
 Discussion
 References
 
Gitelman's syndrome (GS) is an autosomal recessive renal tubular disorder characterized by hypokalaemic metabolic alkalosis, hypomagnesaemia and hypocalciuria [1]. The electrolyte disturbances resemble those observed in chronic administration of thiazide diuretics. GS usually results from inactivating mutations of the SLC12A3 gene encoding the thiazide-sensitive Na+–Cl- cotransporter (NCCT) on the apical membrane of distal convoluted tubule (DCT) cells [2,3]. Loss of NCCT function leads to decreased Na+ and Cl- reabsorption in the DCT.

The initial clinical presentation of GS includes transient episodes of muscle weakness and tetany, usually in children or young adults [4]. Some patients are completely asymptomatic. Although the neuromuscular manifestations are common and the hypomagnesaemia present in GS can also precipitate neuromuscular symptoms, to the best of our knowledge, profound hypokalaemia complicated by paralysis is rarely seen as the presenting feature in patients with GS [5]. With the paralytic presentation, GS may be misdiagnosed as hypokalaemic periodic paralysis (HPP) such as thyrotoxic periodic paralysis or familial periodic paralysis due to acute shift of K+ into cells, leading to improper management [6]. Furthermore, the documented mutations in the thiazide-sensitive NCCT responsible for GS have been not reported in Chinese patients. In this report, we describe a Chinese male who manifested with acute hypokalaemic paralysis resulting from GS caused by one novel NCCT mutation N426K, and another already known NCCT mutation T163M, respectively.



   Case
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 Introduction
 Case
 Discussion
 References
 
A 21-year-old Chinese male presented to the emergency department with muscular weakness that progressed to paralysis involving all extremities. He denied nausea, vomiting, diarrhoea or the use of diuretics. However, other past clinical signs including nocturnal voiding, polydipsia, carpopedal spasm and tetany had been noted since childhood. His family and past medical history were unremarkable. On physical examination, his blood pressure was 116/72 mm Hg, heart rate 76 beats/min and body weight 56 kg. His thyroid gland was not enlarged. There was a symmetric flaccid paralysis with areflexia in the lower and upper extremities. The remainder of the physical examination was normal.

The biochemical studies are shown in Table 1Go. Hypokalaemia (1.8 mmol/l) and hypomagnesaemia (0.5 mmol/l) were the most striking biochemical abnormalities. Urine K+ concentrations were very low with levels close to 11 mmol/l. At first glance, this may prompt the physician to consider extrarenal K+ wasting or increased transcellular shift of K+. However, urine K+ to urine creatinine ratio (UK+/Cr), fractional excretion rate of potassium (FEK) and transtubular potassium gradient (TTKG), three common and valuable bedside indices of renal K+ excretion, were high (UK+/Cr 2.8 mmol/mmol, FEK 15.3% and TTKG 6), suggesting that renal K+ wasting was responsible for the hypokalaemia (Table 1Go). His ECG revealed a normal sinus rhythm (76 beats/min) with prominent U waves. Abdominal sonography did not reveal renal stones or nephrocalcinosis. KCl replacement therapy (10 mmol/h) was started for his hypokalaemia. I.v. magnesium sulfate (8 g) for his accompanying hypomagnesaemia was also administered over 4 h. His muscle strength increased when plasma K+ concentration reached 2.7 mmol/l 1 day after KCl and magnesium therapy. His persistently elevated urine Na+ and Cl- concentrations in the absence of diuretics suggested a diagnosis of a Bartter's-like syndrome. However, his markedly low urine calcium excretion rate (32 mg/day) and calcium/creatinine ratio (0.04 mmol/mmol) showed that the clinical picture was caused by GS. This patient was treated with oral KCl 64 mmol, spironolactone 100 mg and magnesium oxide 1.0 g/day. His plasma K+ and magnesium (Mg2+) concentrations have been controlled around 3.0 and 0.6 mmo/l, respectively. He has been stable without any neuromuscular symptoms.


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Table 1.  Biochemical studies on admission

 
Genomic DNA was isolated and purified from peripheral blood of the family members and used for PCR amplication of the individual exons of the SLC12A3 gene (NCBI number is NT-024766.4, GI: 14778846). The 26 exons of the complete coding region of the protein were examined by PCR-single strand conformation polymorphism (SSCP) analysis as described previously [3]. Amplification, SSCP screening and direct sequencing of the NCCT gene revealed that the patient was compound heterozygous for point mutations. A thymine to cytosine single-base substitution at nucleotide 494 (C494T, ACG to ATG) and a guanine to cytosine substitution at nucleotide 1284 (C1284G, AAC to AAG) were found in exon 3 and exon 10, respectively. This resulted in a missense mutation from threonine to methionine at codon 163 (T163M) in the first extracellular domain and a missense mutation from asparagine to lysine at codon 426 (N426K) on a glycosylation site in the fourth extracellular domain. These two NCCT gene mutations were confirmed by restriction analysis (Figure 1Go). The PCR fragments of exon 3 digested by the BceAI endonuclease and exon 10 by Hpy8I were separated on a 2% agarose gel. Genetic analysis in the family demonstrated that T163M in exon 3 was transmitted from the patient's father and N426K in exon 10 was from his mother, consistent with autosomal recessive inheritance. These two mutations were not detected in the 50 normal and unrelated healthy subjects. Of note, the T163M mutation has been reported recently [7].



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Fig. 1.  Pedigree of the family and mutation analysis of PCR amplified genomic DNA. Males and females are indicated by squares and circles, respectively. The PCR fragments of exon 3 and exon 10 of NCCT gene were digested with BceAI and Hpy8I endonucleases, respectively. (A) BceAI digestion: DNA of the patient and his father bearing the T163M yields one fragment (205 bp) as the mutation abolishes a BceAI restriction site. (B) Hpy8I digestion: DNA of the individuals bearing the N426K mutation yields three fragments (139, 131 and 122 bp).

 



   Discussion
 Top
 Introduction
 Case
 Discussion
 References
 
Hypokalaemia associated with muscle weakness and even paralysis is not rarely seen in clinical practice. HPP due to an acute shift of K+ into cells without total K+ deficit and non-HPP due to an excessive renal excretion of K+ are two diagnostic entities [6]. Clinical presentations between HPP and non-HPP are almost indistinguishable and their causes are usually not evident from the history. Measurement of urinary K+ excretion and an assessment of the acid–base status may be helpful in the differential diagnosis [6]. A very low rate of excretion of K+ and the absence of a metabolic acid–base disorder suggest HPP, whereas a high rate of excretion of K+ accompanied by either metabolic alkalosis or metabolic acidosis favours non-HPP. This patient with hypokalaemia and paralysis had excessive renal excretion of K+ despite a low urinary K+ concentration, suggesting a non-HPP with renal K+ wasting. Because of his metabolic alkalosis and normal blood pressure, the differential diagnosis included conditions such as profound vomiting, bulimia, use of diuretics and hereditary renal K+ wasting such as BS or GS. Persistent high urine Na+ and Cl- concentrations, extremely low urine calcium excretion rate, and hypomagnesaemia in the absence of diuretics suggested a diagnosis of GS. In this patient, molecular analysis substantiated NCCT mutations causing GS.

Hypokalaemia in GS is usually mild to moderate in degree (>2 mmol/l) [4,5,8]. Profound hypokalaemia (1.8 mmol/l) with muscle paralysis in the absence of extrarenal K+ loss as the presenting feature in GS is uncommon, as evidenced by a review of the literature. As reported by Cruz el al. [5], ~6% of GS patients present with hypokalaemic paralysis. It is still unclear why only some patients with GS have such profound hypokalaemia. Mastroianni et al. [3] have suggested that patients carrying homozygous frameshift mutations have more severe hypokalaemia than patients carrying homozygous or compound heterozygous missense mutations. However, profound hypokalaemia (K 1.6 mmol/l) has been documented in a GS patient with a homozygous missense mutation (Arg655Cys) [6]. Our patient with compound heterozygous missense mutations (T163M and N426K) also had severe hypokalaemia. As the number of GS patients with a specific mutation is increasingly high, the establishment of meaningful genotype/phenotype correlations may answer this question. In addition, the impact of individual mutations on the function of the NCCT needs to be determined. This underscores the necessity of functional analysis of SLC12A3 mutations in an in vitro expression system.

The functional expression of NCCT mutations that were identified previously among patients with GS have been investigated in the Xexopus laevis oocyte expression system [9,10]. The results revealed that misrouting of human mutant NCCT were mainly responsible for the defective NaCl reabsorption in GS. This is similar to two well-known diseases with protein misfolding—the {Delta}F508 mutant of the cystic fibrosis transmembrane conductance regulator and mutants of aquaporin-2 observed in autosomal recessive nephrogenic diabetes insipidus.

The mechanism of decreased NaCl reabsorption by the substitution of methionine for threonine at codon 163 and lysine for asparagine at codon 426 remains unclear. Threonine is an important amino acid in determining the three-dimensional structure of polypeptide, and is usually well-conserved in members of a given protein family [2]. In particular, the asparagine substitution eliminates a glycosylation site in the fourth extracellular domain. Post-translation glycosylation is an important process for proper protein function. The mutation in this site may alarm the ‘quality control’ mechanisms in the Golgi apparatus. Finally, the threonine 163 and asparagine 426 residues might be necessary for proper conformation or binding of another protein important for normal targeting and trafficking of NCCT.

In conclusion, we have identified a novel NCCT mutation, together with another recently reported NCCT mutation, in a Chinese patient with GS presenting with hypokalaemic paralysis. When clinicians encounter muscle weakness or paralysis in any hypokalaemic patient, GS should be considered as a diagnostic possibility. The identification and molecular analysis of GS patients offers the opportunity to study the relationship between structure and function of the NCCT protein.



   Acknowledgments
 
This study was supported by a grant from the National Science Council, Taiwan (NSC 90-2314-B-016-084).

Conflict of interest statement. None declared.



   Notes
 
Correspondence and offprint requests to: Shih-Hua Lin, MD, Division of Nephrology, Department of Medicine, Tri-Service General Hospital, No 325, Section 2, Cheng-Kung Road, Neihu 114, Taipei, Taiwan. Email: l521116{at}ndmctsgh.edu.tw Back



   References
 Top
 Introduction
 Case
 Discussion
 References
 

  1. Gitelman H, Graham J, Welt L. A new familial disorder characterized by hypokalemia and hypomagnesemia. Trans Assoc Am Physicians 1966; 79:92–96
  2. Simon DB, Nelson-Williams C, Bia MJ et al. Gitelman's variant of Bartter's syndrome, inherited hypokalemic alkalosis, is caused by mutations in the thiazide-sensitive Na-Cl cotransporter. Nature Genet 1996; 12:24–30[ISI][Medline]
  3. Mastroianni N, Bettinelli A, Bianchetti M et al. Novel molecular variants of the Na-Cl cotransporter gene are responsible for Gitelman syndrome. Am J Hum Genet 1996; 9:1019–1026
  4. Peters M, Jeck N, Reinalter SS et al. Clinical presentation of genetically defined patients with hypokalemic salt-losing tubulopathies. Am J Med 2002; 112:183–190[CrossRef][ISI][Medline]
  5. Cruz DN, Shaer AJ, Bia MJ, Lifton RP, Simon DB. Gitelman's syndrome revisited: an evaluation of symptoms and health-related quality of life. Kidney Int 2001; 59:710–717[CrossRef][ISI][Medline]
  6. Lin SH, Lin YF, Halperin ML. Hypokalemia and paralysis. Q J Med 2001; 4:133–139
  7. Syren ML, Tedeschi S, Cesareo L et al. Identification of fifteen novel mutations in the SLC12A3 gene encoding the Na-Cl co-transporter in Italian patients with Gitelman syndrome. Hum Mutat 2002; 20:78.
  8. Lemmink HH, Knoers NV, Karolyi L et al. Novel mutations in the thiazide-sensitive NaCl cotransporter gene in patients with Gitelman syndrome with predominant localization to the C-terminal domain. Kidney Int 1998; 54:720–730[CrossRef][ISI][Medline]
  9. Kunchaparty S, Palcso M, Berkman J et al. Defective processing and expression of thiazide-sensitive Na-Cl cotransporter as a cause of Gitelman's syndrome. Am J Physiol 1999; 277:F643–F649[ISI][Medline]
  10. De Jong JC, Van Der Vliet WA, Van Den Heuvel LP et al. Functional expression of mutations in the human NaCl cotransporter: evidence for impaired routing mechanisms in Gitelman's syndrome. J Am Soc Nephrol 2002; 13:1442–1448[Abstract/Free Full Text]
Received for publication: 1.10.02
Accepted in revised form: 19.12.02