Inactivation mutations of the luminal thiazide-sensitive NaCl cotransporter (NCC) in the distal convoluted tubules or the basolateral chloride channel (CLCNKB) in the distal nephron are the most common genetic mutations in Gitelman's syndrome (GS) or Bartter's syndrome (BS). We conducted clinical and molecular studies in Chinese patients with GS or BS. Twenty patients with chronic hypokalemia (15 males and five females, age 25 +/- 7 yr) from 15 unrelated Chinese families were investigated. All had renal K+ wasting, metabolic alkalosis, and normotension. The urinary calcium excretion rate was used to distinguish between BS or GS on clinical grounds. Clinical symptoms and biochemical studies were recorded. Molecular analysis included PCR single-strand confirmational polymorphism, direct sequencing of both the NCC and CLCNKB genes, and restriction fragment length polymorphism. Sixteen patients had a clinical diagnosis of GS with hypocalciuria and four BS without hypocalciuria. Four of these 20 patients did not have hypomagnesemia. The males had severe hypokalemia [1.9 +/- 0.4 mEq/liter (mmol/liter)] with paralytic episodes, whereas females had moderate hypokalemia [2.6 +/- 0.2 mEq/liter (mmol/liter)] and less severe symptoms. There were no mutations detected in CLCNKB. Twelve NCC mutations, including six novel mutations and nine recurrent ones, were identified. Allele frequency of the detected NCC mutations was 3% in 100 healthy subjects. Some GS patients with NCC mutations may have normocalciuria and/or normomagnesemia. Gender differences may account for phenotype variability. Screening of these identified NCC mutations remains the gold standard for the diagnosis of GS.

SummaryBackground and objectives Gitelman's syndrome (GS) is an autosomal recessive renal tubular disorder caused by mutations in the SLC12A3 gene encoding the thiazide-sensitive Na ϩ -Cl Ϫ cotransporter (NCC). Despite meticulous sequencing of genomic DNA, approximately one-third of GS patients are negative or heterozygotes for the known mutations.Design, Setting, Participants, & Measurements Because blood leukocytes express NCC mRNA, we evaluate whether deep intronic mutations contribute to GS patients with uniallelic or undetectable SLC12A3 mutations. Twenty-nine patients with GS (men/women ϭ 16/13), including eight negative and 21 uniallelic SLC12A3 mutations from 19 unrelated families, and normal controls were enrolled in an academic medical center. Analysis of cDNA from blood leukocytes, sequencing of the corresponding introns of genomic DNA for abnormal transcript, and analysis of NCC protein expression from renal biopsy were performed. Results We identified nine Taiwan aboriginal patients carrying c.1670 -191C3 T mutations in intron 13and 10 nonaboriginal patients carrying c.2548ϩ253C3 T mutations in intron 21 from 14 families (14/ 19). These two mutations undetected in 100 healthy subjects created pseudoexons containing new premature termination codons. Haplotype analysis with markers flanking SLC12A3 revealed that both mutations did not have founder effects. Apical NCC expression in the DCT of renal tissue was markedly diminished in two patients carrying deep intronic mutations.Conclusions Deep intronic mutations in SLC12A3 causing defective NCC expression can be identified with the RNA-based approach in patients with GS. c.1670 -191C3 T and c.2548ϩ253C3 T are hot spot mutations that can be screened in GS patients with uniallelic or negative SLC12A3 mutations.