Molecular Analysis of Patients With Type III Bartter Syndrome: Picking Up Large Heterozygous Deletions With Semiquantitative PCR

Abstract

Type III Bartter syndrome (BS) (OMIM607364) is caused by mutations in the basolateral chloride channel ClC-Kb gene (CLCNKB). The CLCNKB gene is sometimes reported as having a large deletion mutation, but all cases reported previously were large homozygous deletions and a large heterozygous deletion is impossible to detect by direct sequencing. This report concerns a genetic analysis of five Japanese patients with type III BS. To identify the mutations, we used polymerase chain reaction (PCR) and direct sequencing. To detect large heterozygous deletion mutations of the CLCNKB gene, we conducted semiquantitative PCR amplification using capillary electrophoresis. The result was that four mutations were identified, comprising one novel 2-bp deletion mutation, an entire heterozygous deletion, and a heterozygous deletion mutation of exons 1 and 2. The nonsense mutation W610X was detected in all patients, and this mutation is likely to constitute a founder effect in Japan. Capillary electrophoresis is a new method and extremely useful for detecting large heterozygous deletions, and should be used to examine type III BS cases in whom only a heterozygous mutation has been detected by direct sequencing. This is the first report to identify large heterozygous deletion mutations in the CLCNKB gene in patients with type III BS. (Pediatr Res 62: 364-369, 2007)

Differential diagnosis of Bartter syndrome, Gitelman syndrome, and pseudo–Bartter/Gitelman syndrome based on clinical characteristics

“…10,17 Patients suspected to have BS/GS with no CLCNKB or SLC12A3 mutations were diagnosed as having p-BS/GS. If patients suspected to have BS/GS carried only one CLCNKB or SLC12A3 mutant allele, we performed additional semiquantitative polymerase chain reaction 10,18 or multiplex ligation-dependent probe amplification using the SALSA P266-CLCNKB or P136-SLC12A3 multiplex ligation-dependent probe amplification assays (MRC-Holland, Amsterdam, The Netherlands) to detect large heterozygous deletions. Total RNA from leukocytes and/or urine sediment was isolated as previously described 19,20 and analyzed to detect splicing abnormalities.…”

HNF1B alterations associated with congenital anomalies of the kidney and urinary tract

“…To detect HNF1B gene deletions, we performed semiquantitative polymerase chain reaction (PCR) amplification using capillary electrophoresis (Agilent 2100 Bioanalyzer with DNA 1000 Lab Chips; Agilent Technologies, Palo Alto, CA, USA), as previously described [14]. We applied this method to exons 2, 4, and 9 of the HNF1B gene.…”

A Deep Intronic Mutation in the SLC12A3 Gene Leads to Gitelman Syndrome

“…Genomic DNA was isolated from peripheral blood leukocytes of the patient as well as from normal control subjects with the Qiagen kit (Qiagen Inc., Chatsworth, CA), according to the manufacturer's instructions. Primer pairs for the SLC12A3 gene and the CLCNKB gene were generated following previous reports (8,9). Polymerase chain reaction (PCR) was performed and the resultant products were analyzed, including every intron-exon boundary, by direct sequencing with a DNA sequencer (PerkinElmer-ABI, Foster City, CA).…”