Four novel mutations in the thiazide-sensitive Na–Cl co-transporter gene in Japanese patients with Gitelman's syndrome

Nobuki Maki1, Atsushi Komatsuda1, Hideki Wakui1, Hiroshi Ohtani1, Akihiko Kigawa1, Namiko Aiba1, Keiko Hamai2, Mutsuhito Motegi3, Akihiko Yamaguchi3, Hirokazu Imai1,4 and Ken-ichi Sawada1

1 Third Department of Internal Medicine, Akita University School of Medicine, 2 Department of Internal Medicine, Nakadoori General Hospital and 3 Department of Internal Medicine, Senboku General Hospital, Akita, and 4 Department of Nephrology and Rheumatogy, Aichi Medical University, Aichi, Japan

Correspondence and offprint requests to: Atsushi Komatsuda, MD, Third Department of Internal Medicine, Akita University School of Medicine, 1-1-1 Hondo, Akita City, Akita 010-8543, Japan. E-mail: komatsud{at}med.akita-u.ac.jp



   Abstract
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Gitelman's syndrome (GS) is an autosomal recessive disorder resulting from inactivating mutations in the thiazide-sensitive Na–Cl co-transporter (NCCT) gene. To date, almost 90 mutations have been identified. It is possible that there is a population-specific distribution of mutations. In this study, we analysed mutations in the NCCT gene of seven Japanese patients with GS.

Methods. Peripheral blood mononuclear cells were isolated from patients with GS, their family members and healthy control subjects. A mutation analysis of the NCCT gene was performed completely by direct automated sequencing of polymerase chain reaction-amplified DNA products. In patients with a deletion or splice site mutation, we undertook cDNA sequence analysis.

Results. We identified nine mutations. Five of them [c.185C>T (Thr60Met), c.1712C>T (Ala569Val), c.1930C>T (Arg642Cys), c.2552T>A (Leu849His) and c.1932delC] have been reported in Japanese patients, but not in GS patients from other ethnic groups. The remaining four mutations [c.7A>T (Met1Leu), c.1181_1186+20del26, c.1811_1812delAT and IVS16+1G>A] were novel. In cDNA derived from a patient with c.1181_1186+20del26, a deletion of exon 9 and a frameshift at the start of exon 10 were observed. In cDNA derived from patients with IVS16+1G>A, an additional 96 bp insertion between exons 16 and 17 was observed. Six out of seven patients were compound heterozygotes, and the remaining one carried a single heterozygous mutation.

Conclusions. We found four novel mutations in the NCCT gene in seven Japanese patients with GS. Moreover, our study suggests that the distribution of mutations in the NCCT gene in Japanese GS patients potentially differs from that in other populations.

Keywords: Gitelman's syndrome; Japanese; mutation; thiazide-sensitive sodium–chloride co-transporter



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Gitelman's syndrome (GS) is an autosomal recessive disorder characterized by hypokalaemic metabolic alkalosis, hypomagnesaemia, hypocalciuria, elevated levels of plasma renin and aldosterone, and normal blood pressure [1,2]. Patients with GS are usually diagnosed in adulthood during routine investigation, because the patients generally have mild symptoms including cramps and fatigue at presentation. Some patients suffer from severe symptoms such as tetany, paralysis and rhabdomyolysis [3].

Since the electrolyte disturbances in patients with GS are similar to the effects of chronic thiazide administration, the cause of GS was considered to be a functional defect of the thiazide-sensitive Na–Cl co-transporter (NCCT; also known as TSC or SLC12A3) in the distal convoluted tubules. Simon et al. [4] demonstrated complete linkage of GS to the locus encoding NCCT, which is localized to 16q13. They found a wide variety of non-conservative mutations, consistent with loss of function alleles, in patients with GS. The human NCCT gene consists of 26 exons encoding a protein of 1021 amino acids [4]. Its transcript is expressed mainly in the kidney [5]. To date, almost 90 mutations including missense, nonsense, deletion and splice site mutations have been identified [4,620]. Since almost all mutations are private mutations to each family, efforts to establish genotype/phenotype correlations in GS have been limited [18,19]. A recent study by Coto et al. [20] demonstrated a novel common mutation in 20 gypsy patients with GS who had different clinical manifestations. This suggests the lack of correlation between genotype and clinical phenotype in GS.

In Japanese patients with GS, missense mutations resulting in amino acid substitutions and deletion mutations have been reported [4,7,11,13,1517]. In these mutations, Thr60Met seems to be the most common mutation in Japanese patients with GS [16], which has not been described in GS patients from other ethnic groups [18,19].

In the present study, we investigated possible mutations in the NCCT gene of seven Japanese patients with GS and their family members. We identified nine mutations, including four novel mutations. The remaining five mutations have been reported only in Japanese GS patients. Our results suggest that there is a population-specific distribution of mutations in the NCCT gene.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Patients
Seven Japanese patients with hypokalaemic metabolic alkalosis, hypomagnesaemia and hypocalciuria were examined in Akita University Hospital and its affiliated hospitals in Akita, Japan. As shown in Table 1, five patients have some symptoms associated with hypokalaemia or hypomagnesaemia. Patient 5 had proteinuria. Since the level of urinary N-acetyl-ß-D-glucosaminidase was elevated to 14.7 U/l (normal: 0–7.4) in this patient, proteinuria might be caused by renal tubular injury due to hypokalaemia. The biochemical data of the seven patients are shown in Table 2. All patients fulfilled the diagnostic criteria for GS reported by Bettinelli et al. [2].


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Table 1. Clinical characteristics of GS patients

 

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Table 2. Biochemical data of GS patients

 
The protocol of this study was approved by the ethics committee of the institution involved, and informed consent for genetic studies was obtained from the patients and their family members and from 50 healthy Japanese control subjects.

Molecular analysis of the NCCT gene
Genomic DNA was prepared from peripheral lymphocytes of the patients, the family members and the 50 healthy subjects using a DNA isolation kit (GenTLE; Takara Bio Inc., Ohtsu, Japan). Twenty-six exons of the NCCT gene were amplified by polymerase chain reaction (PCR) using primers as described by Simon et al. [4]. We designed three sets of PCR primers for 5' splice sites of exon 1 and exon 26 (Table 3) to observe splice site mutations.


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Table 3. PCR primers for genomic DNA amplification in NCCT

 
To confirm deletion or splice site mutations, we performed cDNA sequence analysis. Total RNA was also isolated from peripheral lymphocytes of the patients, the family members and the 50 healthy subjects using an RNA isolation kit (RNeasy Mini Kit; Qiagen GmbH, Hilden, Germany). Reverse transcription was performed using a kit with an oligo(dT) primer (First-Strand cDNA Synthesis Kit; Amersham Pharmacia Biotech UK Ltd, Buckinghamshire, UK). The cDNA was amplified by PCR using several sets of primers, which were constructed to cover the regions of the boundary between two exons or intra-exons (Table 4).


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Table 4. PCR primers for cDNA amplification in NCCT

 
When heterozygous mutations were suspected by direct sequence data of PCR products or two bands of PCR products were observed on polyacrylamide gel electrophoresis, the PCR products were then inserted into the pCR®2.1 vector of a cloning kit (TA Cloning® Kit; Invitrogen Corp., Carlsbad, CA). The sequences comprising mutations were reamplified from 20 independent positive clones. These PCR products were sequenced directly using a DNA sequencing kit (BigDyeTM Terminator Cycle Sequencing Kit; Applied Biosystems Japan Ltd, Tokyo, Japan) on an automated DNA sequencer (ABI 377; Perkin-Elmer Applied Biosystems, Foster City, CA). Sequence analysis and mutation identification were performed using Sequencing Analysis software (Perkin-Elmer Applied Biosystems), as previously described elsewhere [21].

When only one mutant in the NCCT gene was identified, we also analysed 19 exons of the CLCNKB gene, as described by Simon et al. [22].



   Results
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 Subjects and methods
 Results
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 References
 
Mutations detected in seven Japanese patients with GS and their family members are summarized in Table 5. Nine mutations including four novel mutations in patients 1, 3, 4, 5 and 6 were found.


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Table 5. NCCT gene mutations in Japanese GS patients and their family members

 
Patient 1 had two heterozygous mutations. One of the mutations was a C to T substitution at nucleotide 185 in exon 1 (c.185C>T), resulting in an alteration of threonine to methionine at codon 60 (Thr60Met). Another mutation was a novel 26 bp deletion from the end of exon 9 (nucleotides 1181–1186) to intron 9 (c.1181_1186+20del26). Analysis of cDNA from exon 8 to exon 10 in the patient revealed that the deletion causes a heterozygous defect of exon 9 and a frameshift at the start of exon 10, resulting in the predictable termination at amino acid 423 (Figure 1). The first mutation was observed in the patient's son. The second mutation was found in the patient's sister. However, the son and the sister had no electrolyte disorders (data not shown).



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Fig. 1. Nucleotide sequence of the NCCT cDNA from exon 8 to exon 10 in patient 1. Direct sequence analysis shows heterozygous transitions of cDNA from exon 8 to exon 10 (left). The results of TA cloning of these PCR products reveal a normal sequence clone and an abnormal clone, which showed a defect of exon 9 and a frameshift at exon 10. This finding shows that the patient is a heterozygote for these alterations.

 
Patient 2 had one heterozygous mutation, which was a C to T substitution at nucleotide 1712 in exon 14 (c.1712C>T). This missense mutation produces an alanine to valine substitution at amino acid 569 (Ala569Val). This mutation was not observed in the patient's son. No mutations in the CLCNKB gene were found in this patient.

Patients 3 and 4 (brothers) had two heterozygous mutations. One of them was the same mutation of a novel deletion at nucleotides 1811–1812 (AT) in exon 14 (c.1811_1812delAT). This deletion causes a frameshift, resulting in the predictable termination at amino acid 649. An additional mutation observed in patient 3 was a T to A substitution at nucleotide 2552 in exon 22 (c.2552T>A), resulting in alteration of leucine to histidine at codon 849 (Leu849His). Another mutation observed in patient 4 was a C to T substitution at nucleotide 1930 in exon 15 (c.1930C>T), resulting in alteration of arginine to cysteine at amino acid 642 (Arg642Cys). The patients’ father was a compound heterozygote (Arg642Cys and Leu849His), and had hypomagnesaemia (1.5 mg/dl) and hypocalciuria (urinary calcium/creatinine g ratio: 0.07). His serum potassium was 3.6 mmol/l. The patient's mother was heterozygous for c.1811_1812delAT, but had no electrolyte disorders (data not shown).

Patients 5 and 6 (brother and sister) had the same heterozygous mutations. One of the mutations was a novel A to T substitution at nucleotide 7 in exon 1 (c.7A>T), resulting in alteration of methionine to leucine at the start codon (Met1Leu). Another mutation was a novel G to A substitution at a 5' splice site of intron 16 (IVS16+1G>A). Sequence analysis of cDNA from exon 13 to exon 17 in the patients revealed that this mutation produced 32 additional amino acids between exon 16 and exon 17 (Figure 2). Their father had a heterozygous IVS16+1G>A, and their mother had a heterozygous c.7A>T. However, the parents had no electrolyte abnormalities (data not shown).



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Fig. 2. Nucleotide sequence of the NCCT cDNA from exon 13 to exon 17 in patient 5. The results of TA cloning of these PCR products reveal an abnormal sequence clone with 96 additional nucleotides and a normal sequence. This finding shows that the patient has a 96 bp insertion between exon 16 and exon 17. The asterisk indicates the mutated nucleotide.

 
Patient 7 had two heterozygous mutations. One of the mutations was a deletion of C at nucleotide 1932 in exon 16 (c.1932delC). It causes a frameshift, resulting in the predictable termination at amino acid 671. Another mutation was a T to A substitution at nucleotide 2552 in exon 22 (c.2552T>A), resulting in alteration of leucine to histidine at amino acid 849 (Leu849His). His mother and sister had a heterozygous c.2552T>A, but had no electrolyte disorders (data not shown).

These mutations were all absent from 100 Japanese control chromosomes.



   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
In the present study, we found nine mutations (five missense mutations, three deletion mutations generating frameshifts, and one splice site mutation) in the NCCT gene in seven Japanese patients with GS. To the best of our knowledge, four of these mutations [c.7A>T (Met1Lue), c.1181_1186+20del26, c.1811_ 1812delAT and IVS16+1G>A] are novel.

Dramatic structural alterations of the mutated proteins, resulting in loss of normal NCCT function, can be expected in these novel mutations. First, mutations of the translation initiator ATG (Met1) have rarely been observed in inherited diseases. Huang et al. [23] and Sperandeo et al. [24] suggested the use of alternative ATGs sharing homology with Kozak's consensus sequence of GCC[G/A]CCATGG [25] for translation initiation in their cases. In our case, a significant homology with the Kozak consensus sequence is found in the 7th in-frame ATG (Met279, GCTGGCATGG). If we used this ATG, the mutated protein would lack the first 278 amino acid residues. Secondly, in the deletion mutations c.1181_1186+ 20del26 and c.1811–1812delAT, the predictable terminations are amino acids 423 and 649, respectively. These mutated proteins would lack the C-terminal portion. Finally, in IVS16+1G>A, the mutated protein would contain an additional 32 amino acids.

Another five mutations found in our study [c.185C>T (Thr60Met), c.1712C>T (Ala569Val), c.1930C>T (Arg642Cys), c.2552T>A (Leu849His) and c.1932delC] have been described in Japanese patients with GS [11,13,1517], but not in GS patients from other populations [18,19]. (Note that in reference [13], Ala569Val is incorrectly termed as Ala569Glu.) In particular, Thr60Met found in patient 1 has been reported in several Japanese patients with GS. Kitagawa et al. [16] investigated mutations in the NCCT gene of 12 Japanese patients with GS, and found that three of them had a heterozygous mutation of Thr60Met. This mutation is the most common in their study. Matsuki et al. [15] also reported that one of three Japanese patients with GS had this mutation. Therefore, Thr60Met is likely to be the common mutation in Japanese GS patients.

In the present study, we found compound heterozygous mutations in six out of seven patients. This finding is compatible with an autosomal recessive pattern of inheritance of GS. However, only one mutant allele was identified in the remaining patient (patient 2). Monkawa et al. [13] suggested several explanations for why only one mutant allele is identified in some patients with GS. First, there may be mutations in the gene-regulating fragments in the promoter region. Secondly, large heterozygous deletions may not be found by standard mutation detection methods. Thirdly, there may be a heterozygous mutation in a gene other than the NCCT gene. We analysed mutations in the CLCNKB gene in patient 2, because a mutation in the gene recently has been shown to cause GS [26]. However, we could not detect abnormalities in the CLCNKB gene.

In family 3 (Table 5), the father had compound mutations [c.1930C>T (Arg642Cys) and c.2552T>A (Leu849His)], and the mother was a carrier for c.1811_1812delAT. Both patients 3 and 4 inherited heterozygous mutations from their parents. The mutant alleles derived from the father were different in patients 3 and 4. The father's GS phenotype was milder than that of his children. These clinical discrepancies cannot be clarified at the moment. If only clinical and biochemical data were available in the family 3 members, autosomal dominant transmission would be considered, as in the GS families reported by Bettinelli et al. [27]. Genetic analyses of the family members are necessary in these cases to clarify the exact genetic mode of inheritance.

In summary, we found nine mutations in the NCCT gene in seven Japanese patients with GS. Among them, four mutations were novel. The remaining five mutations have been reported in only Japanese patients. Our study therefore suggests that the distribution of mutations in the NCCT gene in Japanese GS patients potentially differs from that in other populations.



   Acknowledgments
 
We thank Dr K. Yahata, Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, for providing TSC primers. This study was supported in part by the 21st Century COE Programme from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Conflict of interest statement. None declared.



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 4.12.03
Accepted in revised form: 25. 2.04