Novel mutations in thiazide-sensitive Na-Cl cotransporter gene of patients with Gitelman's syndrome

Gitelman's syndrome (GS) is an autosomal recessive disorder characterized by metabolic alkalosis, hypokalemia, hypomagnesemia, and hypocalciuria that has recently been reported to be linked to thiazide-sensitive Na-Cl cotransporter (TSC) gene mutations. In this study, possible mutations in the TSC gene of six Japanese patients clinically diagnosed with GS were investigated. Twenty-six exons encoding TSC were amplified by PCR and then completely sequenced by the direct sequencing method. Patient A showed a missense mutation of Arg 642 to Cys on the paternal allele and a missense mutation of Val 578 to Met and a 2-bp deletion (nucleotide 2543-2544) on the maternal allele. This deletion results in a frameshift that alters codon 837 to encode a stop signal rather than phenylalanine, and it is predicted to lead to loss of the latter half of the intracellular carboxy terminus. In the second family, two affected sisters, patients B and C, had a homozygous missense mutation of Thr 180 to Lys. Both of their parents, who are consanguineously married, have a heterozygous Thr180Lys mutation. Patient D has a homozygous mutation Thr180Lys, which is the same as the second family. Haplotype analysis indicates that patients B and C are not related to patient D. In patients E and F, we could identify only one mutant allele; Ala569Glu and Leu849His, respectively. All of the mutations identified are novel except for the Arg642Cys mutation, which has been found in a Japanese GS patient. Although further in vitro study is required to prove that the mutations are responsible for GS, it is possible that Thr180Lys and Arg642Cys mutations might be common mutations in Japanese GS.     

A novel mutation of the thiazide-sensitive sodium chloride cotransporter gene in a Japanese family with Gitelman syndrome

Gitelman syndrome is an inherited renal disorder characterized by impaired NaCl reabsorption in the distal convoluted tubule leading to hypokalemia, hypomagnesemia and normocalcemic hypocalciuria. It has been shown that this syndrome results from mutations in the gene encoding the thiazide-sensitive sodium chloride cotransporter (TSC). We performed the mutational analysis in the TSC gene of a 30-year-old Japanese woman with Gitelman syndrome and found two mutations at adjacent spots in both alleles. One was a frame shift mutation which generated stop codon at position 671, the other was a single nucleotide mutation, which resulted in an aminoacid substitution at position 672, Met to Ile. Her 52-year-old mother and two daughters had neither hypokalemia nor hypomagnesemia. However, her mother and her 8-year-old daughter had the Met672Ile mutation as heterozygotes. Her 4-year-old daughter had the same frame shift mutation as her mother, a heterozygotic mutation. These results suggest that Gitelman syndrome requires 2 compound heterozygotic mutations and the coexistence of the large deletion in the C-terminal domain with Met672Ile substitution of the TSC could impair the transporter activity underling the hypokalemia and hypomagnesemia in this patient.     

Mimicry of surreptitious diuretic ingestion and the ability to make a genetic diagnosis

Gitelman's syndrome, also known as "hypocalciuric variant of Bartter's syndrome", is a cause of chronic hypokalemia and hypomagnesemia in adults. A specific gene has been found responsible for this disorder, encoding the thiazide-sensitive NaCl coporter (TSC) in the distal convoluted tubule. We describe a psychiatric patient with chronic symptomatic hypokalemia and hypomagnesemia whose electrolyte disturbances were subsequently misdiagnosed as an acute alcohol and benzodiazepine withdrawal syndrome, as chronic diuretic abuse and as a classical Bartter's syndrome. Finally, genetic investigation revealed the presence of mutations in the SLC12A3 gene leading to the proper diagnosis of Gitelman's syndrome. We emphasize that Gitelman's syndrome should be suspected in every hypokalemic patient with biochemical resemblance of diuretic ingestion, especially when repeated toxic screens for diuretics are negative. The ability to make a molecular-genetic diagnosis can be of practical benefit in confusing clinical settings.     

Analysis of renal tubular electrolyte transporter genes in seven patients with hypokalemic metabolic alkalosis

BACKGROUND: Disorders that manifest hypokalemic metabolic alkalosis, such as Bartter's syndrome and Gitelman's syndrome, are caused by the malfunction of renal tubular electrolyte transporters. Bartter's syndrome may be linked to dysfunction of Na-K-2Cl cotransporter (NKCC2), renal outer medullary K channel (ROMK), or Cl channel Kb (ClC-Kb), while Gitelman's syndrome may be linked to Na-Cl cotransporter (NCCT) dysfunction. However, previous genetic analyses in these syndromes have included many heterozygotes for each gene and there has been no further analysis of other genes. Thus, to clarify the interaction of these transporter genes, in the present study we investigated all 4 transporter genes in 7 patients with hypokalemic metabolic alkalosis. METHODS: Seven patients from 5 families (patients A-G) were collected, and a mutation analysis of the 4 renal electrolyte transporter genes was performed by direct sequencing. RESULTS: We identified 12 mutations in these 7 patients. Three mutations (del245Y in NKCC2, R1009X in NCCT, V524I in ClC-Kb) have not been reported previously. In NKCC2 gene screening, patient A was homozygous for del245Y. In ClC-Kb gene screening, L27R was detected in patients B, D, and E. V524I was detected in patient C. Both T562M and E578K were observed in patients B and E. In NCCT gene screening, patients B-G shared a common novel mutation, R1009X, and patients D, E, F, and G carried this mutation in both alleles. Patients B and C carried R1009X in one allele, and a 6-amino acid insertion in exon 6 and L849H in another allele, respectively. The 4 other mutations did not result in any amino acid exchange. Despite the NCCT gene mutation, patients C and E showed normomagnesemia. CONCLUSION: Our findings demonstrate that in Bartter's and Gitelman's syndromes, it may not be uncommon to see mutations in several causative transporter genes.     

A novel splice site mutation of the thiazide-sensitive NaCl cotransporter gene in a Japanese patient with Gitelman syndrome

Gitelman syndrome (GS, MIM 263800) is an inherited disorder characterized by metabolic alkalosis with hypokalemia, hypomagnesemia, and hypocalciuria. The genetic abnormalities causing GS are known to lie in the thiazide-sensitive NaCl cotransporter (TSC), which is expressed in the distal tubule of the kidney. The TSC gene, located at chromosome 16, consists of 26 exons and encodes the protein containing 12 putative transmembrane domains with long intracellular amino and carboxy termini. Most of the abnormalities identified in GS were missense mutations, distributed throughout the TSC gene without a hot spot. A 42-year-old Japanese man was introduced for close examination of hypokalemia. In renal clearance studies using furosemide or thiazide, chloride clearance was increased after furosemide but not after thiazide administration. Furthermore, the distal fractional chloride reabsorption was dramatically decreased by furosemide but not thiazide administration, suggesting a defect in the distal tubule. We then analyzed the TSC gene to confirm the diagnosis of GS, and identified a novel G to T mutation at the acceptor splice site preceding exon 14, resulting in disruption of a conventional 3'AG consensus splice site. Abnormal splicing by this mutation is predicted to cause the formation of truncated TSC with a partial deletion of the transmembrane domain, which will loose the function of transporter. In conclusion, we have identified a unique novel splice site mutation of the TSC gene in GS. The predicted structure of this mutant TSC can conceivably cause an impairment of the transporter activity and thereby be responsible for the development of GS in our patient.     

A novel mutation in the chloride channel gene,CLCNKB, as a cause of Gitelman and Bartter syndromes

BACKGROUND: Gitelman syndrome (GS) and Bartter syndrome (BS) are hereditary hypokalemic tubulopathies with distinct phenotypic features. GS has been considered a genetically homogeneous disorder caused by mutation in the gene encoding the NaCl cotransporter (TSC) of the distal convoluted tubule. In contrast, BS is caused by mutations in the genes encoding either the Na-K-2Cl cotransporter (NKCC2), the K+ channel (ROMK) or the Cl- channel (ClC-Kb) of the thick ascending limb. The purpose of this study was to examine the clinical, biochemical and genetic characteristics of a very large inbred Bedouin kindred in Northern Israel with hereditary hypokalemic tubulopathy. METHODS: Twelve family members affected with hypokalemic tubulopathy, as well as 26 close relatives were clinically and biochemically evaluated. All study participants underwent genetic linkage analysis. Mutation analysis was performed in affected individuals. RESULTS: Evaluation of affected family members (age range 3 to 36 years) revealed phenotypic features of both GS and classic Bartter syndrome (CBS). Features typical of GS included late age of presentation (>15 years) in 7 patients (58%), normal growth in 9 (75%), hypomagnesemia (SMg <0.7mmol/L) in 5 (42%), hypermagnesiuria (FEMg>5%) in 6 (50%) and hypocalciuria (urinary calcium/creatinine mmol/mmol <0.15) in 5 (42%). Features typical of CBS included early age of presentation (<1 year) in 3 (25%), polyuria/dehydration in 4 (33%), growth retardation in 3 (25%), hypercalciuria (urinary calcium/creatinine mmol/mmoverline>0.55) in 4 (33%) and nephrolithiasis in 1 (8%). Linkage analysis in affected patients excluded the TSC gene, SLC12A3, as the mutated gene, but demonstrated linkage to the Cl- channel gene, CLCNKB, on chromosome 1p36. Mutation analysis by direct sequencing revealed a novel homozygous missense mutation, arginine 438 to histidine (R438H), in exon 13 of CLCNKB in all patients. A restriction fragment length polymorphism (RFLP) analysis has been developed to aid in genotyping of family members. CONCLUSIONS: Our findings demonstrate intrafamilial heterogeneity, namely the presence of GS and CBS phenotypes, in a kindred with the CLCNKB R438H mutation. We conclude that GS can be caused by a mutation in a gene other than SLC12A3. The exact role of the CLCNKB R438H mutation in the pathogenesis of the electrolyte and mineral abnormalities in GS and CBS remains to be established.     

Genetic analysis of Gitelman syndrome patients from the Czech Republic and Slovakia--three novel mutations found

BACKGROUND: To investigate the genetic cause of inherited hypokalemic metabolic alkalosis associated with Gitelman's syndrome, we searched for mutations in the SLC12A3 gene (thiazide-sensitive NaCl cotransporter) among a set of patients from the Czech Republic and Slovakia. METHODS: We collected blood samples of patients from 16 families with characteristic clinical features. DNA was analyzed for mutation detection with SSCP and subsequent sequencing. Several mutations might be missed when only the SSCP method is applied, therefore direct sequencing of all the 26 exons became an essential tool. RESULTS: Genetic analysis revealed mainly missense mutations. Two novel mutations, c.480dupC (p.Pro160fsX97) and c.238dupCC (p. Pro79fsX35), caused a frameshift and preliminary stop codon appearance. Missense mutation c.790 G --> C (p.Gly264Arg) has never been reported before. Mutation c.1315G --> A (p.Gly439Ser) was frequent among our collection of unrelated patients (5 out of 16). Homozygous Gly439Ser was observed in a patient with chondrocalcinosis. CONCLUSION: We identified 13 different causative mutations in a cohort of Gitelman syndrome patients. Three of those mutations are novel. The occurrence of two mutation detections per individual corresponding to a recessive trait of inheritance was 62.5%. Gly439Ser is the most frequent type of mutation among our patients. Statistic evaluation of genotype/phenotype correlation could not be carried out.     

CYP17A1基因的复合杂合突变导致17α-羟化酶／17，20-碳链裂解酶缺陷症

目的通过1例17α-羟化酶／17,20-碳链裂解酶缺陷症（17-OHD）患者的临床特点和基因突变分析,探讨17-OHD的基因分子机制及突变对P450 c17蛋白功能的影响。方法收集患者临床资料,提取患者外周血白细胞DNA,PCR扩增CYP17A1基因的8个外显子及内含子边界,测序确定CYP17A1基因的突变位点,用突变位点保守性分析和计算机模拟P450 c17蛋白三维结构,分析突变对P450 c17蛋白功能的影响。结果患者临床表现及内分泌功能检查完全符合17-OHD特点,PCR产物和克隆测序发现,CYP17A1基因的一个等位基因的第2外显子125位密码子发生了CGA→CAA的突变,导致Arg125Gin错义突变,另一个等位基因的第6外显子的第360和361密码子缺失G核苷酸及C转换为A核苷酸,导致Leu361Phe错义突变和以后的移码突变,形成一个只包含417个氨基酸的截短蛋白,该蛋白缺乏17α-羟化酶和17,20-碳链裂解酶催化活性部位。通过P450c17蛋白分子三维建模分析表明,121位色氨酸与血红素和125位精氨酸分别形成氢键,当125位精氨酸突变为谷氨酰胺时,121位色氨酸不能再与125位谷氨酰胺形成氢键,从而使P450c17蛋白分子结构的稳定性受到影响,影响了其功能。结论通过CYP17A1基因突变分析,进一步从分子遗传学方面证实了患者的临床诊断,CYP17A1突变导致的P450c17蛋白的结构改变是该17-OHD患者临床表现的基因分子基础。     

Compound heterozygous mutations in the SRD5A2 gene exon 4 in a male pseudohermaphrodite patient of Chinese origin

The goal of this study was to perform 5-alpha-reductase type 2 gene (SRD5A2) analysis in a male pseudohermaphrodite (MPH) patient with normal testosterone (T) production and normal androgen receptor (AR) gene coding sequences. A patient of Chinese origin with ambiguous genitalia at 14 months, a 46,XY karyotype, and normal T secretion under human chorionic gonadotropin (hCG) stimulation underwent a gonadectomy at 20 months. Exons 1-8 of the AR gene and exons 1-5 of the SRD5A2 gene were sequenced from peripheral blood DNA. AR gene coding sequences were normal. SRD5A2 gene analysis revealed 2 consecutive mutations in exon 4, each located in a different allele: 1) a T nucleotide deletion, which predicts a frameshift mutation from codon 219, and 2) a missense mutation at codon 227, where the substitution of guanine (CGA) by adenine (CAA) predicts a glutamine replacement of arginine (R227Q). Testes located in the inguinal canal showed a normal morphology for age. The patient was a compound heterozygote for SRD5A2 mutations, carrying 2 mutations in exon 4. The patient showed an R227Q mutation that has been described in an Asian population and MPH patients, along with a novel frameshift mutation, Tdel219. Testis morphology showed that, during early infancy, the 5-alpha-reductase enzyme deficiency may not have affected interstitial or tubular development.     

Spectrum of mutations in Gitelman syndrome

Gitelman's syndrome (GS) is a rare, autosomal recessive, salt-losing tubulopathy caused by mutations in the SLC12A3 gene, which encodes the thiazide-sensitive NaCl cotransporter (NCC). Because 18 to 40% of suspected GS patients carry only one SLC12A3 mutant allele, large genomic rearrangements may account for unidentified mutations. Here, we directly sequenced genomic DNA from a large cohort of 448 unrelated patients suspected of having GS. We found 172 distinct mutations, of which 100 were unreported previously. In 315 patients (70%), we identified two mutations; in 81 patients (18%), we identified one; and in 52 patients (12%), we did not detect a mutation. In 88 patients, we performed a search for large rearrangements by multiplex ligation-dependent probe amplification (MLPA) and found nine deletions and two duplications in 24 of the 51 heterozygous patients. A second technique confirmed each rearrangement. Based on the breakpoints of seven deletions, nonallelic homologous recombination by Alu sequences and nonhomologous end-joining probably favor these intragenic deletions. In summary, missense mutations account for approximately 59% of the mutations in Gitelman's syndrome, and there is a predisposition to large rearrangements (6% of our cases) caused by the presence of repeated sequences within the SLC12A3 gene.     

Slow progressive FSGS associated with an F392L<Emphasis Type="Italic"> WT1</Emphasis> mutation

Constitutional missense mutations in the WT1 gene are usually associated with the Denys-Drash syndrome, characterized by a rapid progressive nephropathy, male pseudohermaphroditism, and an increased risk for Wilms tumor. We report here a patient with scrotal hypospadias and a slow progressive nephropathy due to focal and segmental glomerulosclerosis. WT1 mutation analysis revealed a constitutional missense mutation in exon 9 resulting in an exchange F392L. This mutation has previously been reported by others in a patient with a similar mild course of nephropathy. In contrast, a mutation in the corresponding codon of exon 8 (F364L) was previously found by us in a patient with a very rapid progression to end-stage renal disease. Whether the position of a mutation may influence the course of the nephropathy must be evaluated in a larger patient cohort. The individual tumor risk for this alteration cannot be given at present because neither of the two patients has shown evidence of a Wilms tumor or a gonadoblastoma to date.Petras Kaltenis and Valérie Schumacher contributed equally to the work     

Functional expression of mutations in the human NaCl cotransporter: evidence for impaired routing mechanisms in Gitelman's syndrome

Gitelman's syndrome is an autosomal recessive renal tubular disorder characterized by hypokalemic metabolic alkalosis, hypomagnesemia, and hypocalciuria. This disorder results from mutations in the thiazide-sensitive NaCl cotransporter (NCC). To elucidate the functional implications of mutations associated with this disorder, metolazone-sensitive (22)Na(+) uptake, subcellular localization, and glycosidase-sensitive glycosylation of human NCC (hNCC) were determined in Xenopus laevis oocytes expressing FLAG-tagged wild-type or mutant hNCC. Injection of 10 ng of FLAG-tagged hNCC cRNA resulted in metolazone-sensitive (22)Na(+) uptake of 3.4 +/- 0.2 nmol Na(+)/oocyte per 2 h. Immunocytochemical analysis revealed sharp immunopositive staining at the plasma membrane. In agreement with this finding, a broad endoglycosidase H-insensitive band of 130 to 140 kD was present in Western blots of total membranes. The plasma membrane localization of this complex-glycosylated protein was confirmed by immunoblotting of purified plasma membranes. The mutants could be divided into two distinct classes. Class I mutants (G439S, T649R, and G741R) exhibited no significant metolazone-sensitive (22)Na(+) uptake. Immunopositive staining was present in a diffuse band just below the plasma membrane. This endoplasmic reticulum and/or pre-Golgi complex localization was further suggested by the complete absence of the endoglycosidase H-insensitive band. Class II mutants (L215P, F536L, R955Q, G980R, and C985Y) demonstrated significant metolazone-sensitive (22)Na(+) uptake, although uptake was significantly lower than that obtained with wild-type hNCC. The latter mutants could be detected at and below the oocyte plasma membrane, and immunoblotting revealed the characteristic complex-glycosylated bands. In conclusion, this study substantiates NCC processing defects as the underlying pathogenic mechanism in Gitelman's syndrome.     

Linkage of Gitelman syndrome to the thiazide-sensitive sodium-chloride cotransporter gene with identification of mutations in Dutch families

Gitelman syndrome is a mostly autosomal recessive disorder affecting the renal tubular function associated with hypokalemia and hypomagnesemia. Functional studies point to a defect in the distal renal tubule in the thiazide-sensitive, electroneutral sodium-chloride cotransporter (TSC). Based upon the localization of a 2.6 cDNA encoding the human TSC to chromosome 16q13, polymorphic markers spanning the region from 16p12 to 16q21 were tested for linkage to the Gitelman syndrome locus in three Dutch families with autosomal recessive inheritance of this disorder. Using two-point linkage analysis, a maximum LOD score (Z_max of 4.49 (at Θ = 0.00) was found for the marker D16S408. One crucial recombination event places the Gitelman syndrome locus distal to D16S419 at 16q12-13. Subsequently we have tested our group of Gitelman patients for mutations in the human TSC gene. Two mutations were identified in three Gitelman families. Our study confirms that the human TSC gene is involved in Gitelman syndrome. Patients from three Gitelman families reveal two identical human TSC mutations, suggesting these families share a common ancestor. Received April 19, 1996; received in revised form and accepted May 24, 1996     

Novel nonsense mutation in the Na+/HCO3- cotransporter gene (SLC4A4) in a patient with permanent isolated proximal renal tubular acidosis and bilateral glaucoma

Permanent isolated proximal renal tubular acidosis (pRTA) with ocular abnormalities is a systemic disease involving short stature, isolated pRTA, mental retardation, and ocular abnormalities. Kidney Na+/HCO3- cotransporter (kNBC1) cDNA from peripheral lymphocytes from a patient with permanent isolated pRTA and bilateral glaucoma was screened, and a novel homozygous mutation, namely a cytosine-to-thymine transition at nucleotide 234, which resulted in the formation of a stop codon at codon 29, was identified. This homozygous mutation, Q29X, was identified in the unique 5'-end of the kNBC1 gene (SLC4A4) of the patient. Cosegregation of this Q29X mutation with the disease and heterozygosity in the parents of the affected patient were observed. The absence of this mutation in 156 alleles from 78 Japanese individuals indicates that this mutation is directly related to the disease and is not a common DNA sequence polymorphism. This nonsense mutation predicts a truncated kNBC1 protein that lacks the 1007 amino acids of the carboxyl-terminus, and the effect on kNBC1 cotransport activity is likely to be a loss of function. In contrast, the pancreatic Na+/HCO3- cotransporter of the patient is not likely to be affected by this nonsense mutation. These results have implications for understanding the role of kNBC1 in the pathophysiologic processes of pRTA associated with ocular abnormalities and mental retardation.     

Prophylactic thyroidectomy in multiple endocrine neoplasia: the impact of molecular mechanisms of<Emphasis Type="Italic"> RET</Emphasis> proto-oncogene

BACKGROUND: Multiple endocrine neoplasia (MEN) type 2, a cancer syndrome inherited in the dominant fashion, is defined by the occurrence of medullary thyroid carcinoma (MTC), either as a singular lesion (familial medullary thyroid carcinoma, FMTC) or with the variable expression of pheochromocytoma, hyperparathyroidism (MEN 2A), ganglioneuromas, buccal neuromas and Marfanoid-like phenotype (MEN 2B). DISCUSSION: Germline mutations of the RET proto-oncogene, localized on chromosome 10q11.2, have been identified as the underlying genetic cause of the disorder. In the majority of patients with MEN 2A/FMTC missense mutations at exon 10 or exon 11 are identifiable. Cysteine to arginine exchange at codon 634 is the mutation most frequently found. In MEN 2B approximately 95% of patients present with a mutation at codon 918 (exon 16). Additionally, less frequent mutations in other codons have been found in both syndromes. The DNA-based genotype analysis enables the identification of gene carriers at risk of developing MTC and offer them prophylactic thyroidectomy prior to development of any thyroid pathologies. Prophylactic surgery is generally recommended for MEN 2A/FMTC gene carriers at the age of 4-6 years. Due to the aggressiveness of the MEN 2B syndrome gene carriers should be operated by the age of 1 year. Presumably some less virulent mutations allow postponement of the prophylactic treatment to the second to fourth decade of life. CONCLUSIONS: Compared to standard presymptomatic biochemical screening, genetic testing and consecutive prophylactic treatment contribute to better outcome of individuals at risk for MTC.     

Recessive missense mutations in LAMB2 expand the clinical spectrum of LAMB2-associated disorders

Congenital nephrotic syndrome is clinically and genetically heterogeneous. The majority of cases can be attributed to mutations in the genes NPHS1, NPHS2, and WT1. By homozygosity mapping in a consanguineous family with isolated congenital nephrotic syndrome, we identified a potential candidate region on chromosome 3p. The LAMB2 gene, which was recently reported as mutated in Pierson syndrome (microcoria-congenital nephrosis syndrome; OMIM #609049), was located in the linkage interval. Sequencing of all coding exons of LAMB2 revealed a novel homozygous missense mutation (R246Q) in both affected children. A different mutation at this codon (R246W), which is highly conserved through evolution, has recently been reported as causing Pierson syndrome. Subsequent LAMB2 mutational screening in six additional families with congenital nephrotic syndrome revealed compound heterozygosity for two novel missense mutations in one family with additional nonspecific ocular anomalies. These findings demonstrate that the spectrum of LAMB2-associated disorders is broader than previously anticipated and includes congenital nephrotic syndrome without eye anomalies or with minor ocular changes different from those observed in Pierson syndrome. This phenotypic variability likely reflects specific genotypes. We conclude that mutational analysis in LAMB2 should be considered in congenital nephrotic syndrome, if no mutations are found in NPHS1, NPHS2, or WT1.     

A young female with Spitzer-Weinstein syndrome diagnosed by thiazide test

Spitzer-Weinstein syndrome is a rare disorder characterized by thiazide responsive hyperkalemia and normal anion gap metabolic acidosis, similar to Gordon syndrome. The hyperfunction of thiazide-sensitive Na+-Cl- cotransporter (TSC) is the main pathophysiological mechanism. We presented a 19-year-old female with short stature, normal blood pressure, persistently elevated serum potassium, and metabolic acidosis. The diagnosis of Spitzer-Weinstein Syndrome was made by clinical pictures and thiazide test. After taking 4 mg hydrochlorothiazide, her daily urine potassium excretion increased from 18.1 mmol to 41.8 mmol, urine pH decreased from 7.32 to 6.50, and urinary net charge decreased from 65.7 to 54.4. This patient then took hydrochlorothiazide 4 mg daily. The persistent hyperkalemia and metabolic acidosis were corrected. Thiazide, a powerful inhibitor of TSC, proved to be a useful tool for the diagnosis and treatment of Spitzer-Weinstein syndrome.     

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

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.     

A Young Female with Spitzer-Weinstein Syndrome Diagnosed by Thiazide Test

Spitzer-Weinstein syndrome is a rare disorder characterized by thiazide responsive hyperkalemia and normal anion gap metabolic acidosis, similar to Gordon syndrome. The hyperfunction of thiazide-sensitive Na⁺-Cl^? cotransporter (TSC) is the main pathophysiological mechanism. We presented a 19-year-old female with short stature, normal blood pressure, persistently elevated serum potassium, and metabolic acidosis. The diagnosis of Spitzer-Weinstein Syndrome was made by clinical pictures and thiazide test. After taking 4 mg hydrochlorothiazide, her daily urine potassium excretion increased from 18.1 mmol to 41.8 mmol, urine pH decreased from 7.32 to 6.50, and urinary net charge decreased from 65.7 to 54.4. This patient then took hydrochlorothiazide 4 mg daily. The persistent hyperkalemia and metabolic acidosis were corrected. Thiazide, a powerful inhibitor of TSC, proved to be a useful tool for the diagnosis and treatment of Spitzer-Weinstein syndrome.     

Malignant pancreatic endocrine tumor in a child with tuberous sclerosis

Tuberous sclerosis complex (TSC) is an autosomal dominant condition whose signs and symptoms may vary from a few hypopigmented skin spots to epilepsy, severe mental retardation, and renal failure. The disease is caused by mutations in either TSC1 or TSC2 gene, at chromosome 9q34 and 16p13.3. Inactivation of both alleles at TSC1 or TSC2 loci is associated with the development of hamartomas in different organs, and only rarely with malignant neoplasms. In this study we present a 6-year-old boy with TSC and with a malignant islet cell tumor of the pancreas. Mutation analysis of DNA extracted from peripheral blood cells of the patient identified an R1459X de novo mutation in exon 33 of the TSC2 gene. Immunohistochemical analysis with anti-tuberin antibodies on paraffin-embedded tissue sections showed loss of tuberin immunostaining in tumor cells but normal expression in residual normal pancreas. DNA analysis of tumor and normal cells showed chromosome 16p13 loss of heterozygosity in malignant pancreatic islet cell tumor but not in normal pancreas. These findings suggest a role for tuberin, the TSC2 gene product, in the pathogenesis of malignant pancreatic endocrine tumor.