Recurrent Deep Intronic Mutations in the SLC12A3 Gene Responsible for Gitelman's Syndrome

Abstract

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.

Clinical Severity of Gitelman Syndrome Determined by Serum Magnesium

“…We detected 30 SLC12A3 gene mutations in 32 GS patients, including 23 missense mutations, 4 frameshift mutations, 1 nonsense mutation, and 2 already known disease-causing intronic mutations [21]. All of the mutations found in normomagnesemic GS patients are missense mutations except for one intronic mutation.…”

Identification of Gene Mutations in Autosomal Dominant Polycystic Kidney Disease through Targeted Resequencing

“…Notably, this dataset provides an important filter for common intronic variants, which will be useful to pinpoint rare, potential splice-changing deep intronic variants. [55][56][57] Because of the genomic duplication, these data are not available for the entire duplicated portion of PKD1 from the 1000 Genomes Project and other sequencing projects.…”

Exonic Mutations in the SLC12A3 Gene Cause Exon Skipping and Premature Termination in Gitelman Syndrome

“…25 Hence, to rule out the possibility that deep intronic mutations affected the splicing events, we verified by genomic sequencing that there were no specific single nucleotide polymorphisms in the flanking introns of exon 16 in our patient (data not shown). This suggests that the exon skipping in this patient is exclusively due to the c.2016G.A single nucleotide mutation.…”