Recurrent deep intronic mutations in the SLC12A3 gene responsible for Gitelman's syndrome

Abstract

Background 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(+)-Cl(-) 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 = 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.1670-191C→T mutations in intron 13 and 10 nonaboriginal patients carrying c.2548+253C→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.1670-191C→T and c.2548+253C→T are hot spot mutations that can be screened in GS patients with uniallelic or negative SLC12A3 mutations.

Figure 1.

The identification of deep intronic mutation c.1670–191C→T in intron 13 of SLC12A3. (A) RT-PCR analysis using exon 11 to 15-specific primers in patient 1 shows a comparatively large band (729 bp) in contrast to a relatively small band (491 bp) in normal control. Sequencing of this larger mRNA transcript demonstrated a 238-bp insertion of cryptic exon between exons 13 and 14. (B) Direct sequencing of intron 13 demonstrates a homozygous single base substitution of C to T at the position −191 just before 1670 coding DNA (c.1670–191C→T).

Figure 2.

The identification of a deep intronic mutation c.2854+253C→T in the intron 21. (A) Electrophoresis of the RT-PCR products by using exon 20 to 24-specific primers in patient 10 shows the incorporation of a 90-bp cryptic exon following exon 21. (B) Direct sequencing of intron 21 revealed a heterozygous single base substitution of C to T at position 253 just after 2548 coding DNA (c.2548+253C→T).

Figure 3.

Cryptic exons created by two deep intron mutations causing translation frameshift with introduction of premature termination codons TGA after 10 amino acids (A) and TAA after 4 amino acid (B), respectively, as shown with underlining. Constitutive and cryptic exons are represented by light gray and dark boxes, respectively. Mutated nucleotides are indicated by arrows.

Figure 4.

Pedigree of family K. Men are indicated by squares, and women are indicated by circles. T163M (exon 3) and R871H (exon 22) were transmitted from the father, and (c.2548+253C→T) in intron 21 was transmitted from the mother.

Figure 5.

Immunofluorescence of NCC and calbindin-D28k in renal biopsy tissue. (A) The position of each mutation in the predicted topology of human NCC is represented by arrows. An antibody recognized amino-terminal (amino acids 75 to 90) of NCC was applied to detect NCC protein expression. (B) Representative immunofluorescence micrographs of NCC (green, left panels), calbindin-D28k (red, middle panels), and merged (right panels) in renal tissue from normal control, disease control, and GS patients with homozygous c.1670–191C→T and heterozygous c.2548+253C→T mutations. The scale bars indicate 50 μm. aa, amino acids.