Spectrum of mutations in Gitelman syndrome

Abstract

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.

Figure 1.

Frequency and distribution of the 172 detected mutations in 711 alleles. On the horizontal axis, each bar represents one mutation (there is no relation with the actual position in the exon). Dotted line corresponds to an allele frequency >2%.

Figure 2.

MLPA and QMPSF electropherograms for SLC12A3 from two patients. For MLPA, each peak represents one exon of the SLC12A3 gene and 13 control probes. For QMPSF, each peak represents one analyzed exon and the HMBS internal control. Control samples are shown in red and patients' samples in blue. (A1) The MLPA half doses for exons 19 to 23 in patient GT004 and peak height ratio showing the deleted exons with ratio <0.7. (A2) MLPA duplication of exons 1 to 3 in patient GT137 and peak height ratio showing the duplicated exons with ratio >1.3. (B1) QMPSF half doses for exons 19 to 23 in patient GT004. (B2) QMPSF duplication of exons 1 to 3 in patient GT137.

Figure 3.

Pattern of mutations by type including genomic rearrangements at the SLC12A3 gene.

Figure 4.

Mapping and characterization of breakpoints of SLC12A3 heterozygous deletions. (A) Schematic representation of the genomic organization of the 26 exons of the SLC12A3 gene and the location of breakpoints of 7 deletions: (A1) E2_E3del: 3342 bp deletion (c.282 + 667_c.506–205del). (A2) E2_E3del: 2720 bp deletion (c.283–273_c.506–213del). (A3) E4_E6del: 1508 bp deletion (c.506–315_852 + 185del). (A4) E14del: 1183 bp deletion (c.1169 + 773_c.1825 + 247del). (A5) E18del: 1355 bp deletion (c.2178 + 269_c.2285 + 685del). (A6) E24_E25del: 10711bp deletion (c.2748–324_c.2952–505). (A7) E26del: 2416bp deletion (c.2952–1593_*677delins25). The LCRs on or near the breakpoints are indicated. (B) Sequence analysis showing breakpoints of deletions 1 and 4. Breakpoints of deletion 1 are inside Alu repeats, suggesting a nonallelic homologous recombination. Breakpoints of deletion 4 are inside nonhomologous LCRs. (C) Sequence alignments at the breakpoints of two SLC12A3 heterozygous deletions probably originated from nonhomologous end-joining and one complex rearrangement. For deletions 2 and 4, boxes indicate the nucleotide microhomology. Brackets depict short motifs that might have facilitated the rearrangements. For deletion 7, boxes indicate the repetitive motifs; the read sequence is the 18-bp repetition that is present in inserted sequence and in the 3′UTR breakpoint.

Figure 5.

Characterization of SLC12A3 E1_E7del for patient BT213. (A) MLPA electropherograms for the proband and his mother. Each peak represents one exon of the SLC12A3A gene and 13 control probes. (Aa) The electropherogram of the proband shows abnormal height peaks (in blue) in seven exons (numbered) compared with a normal control (in red). The peak ratio graph shows the 7 deleted exons for which the ratio was <0.7. (Ab) The electropherogram of the mother showing normal height peaks for all of the exons (blue) compared with a normal control (red). Normal peak ratios revealed an equivalent number of copies. (B1) Pedigree showing the constructed haplotypes resulting from biallelic SNP genotyping. The proband is shown in black. The SNPs covering the approximately 16-kb region upstream from the SLC12A3 gene are shown, including the two SNPs mapped to intron 8 of the SLC12A3 gene. The two deletion-flanking SNPs, covering a region of 89.9 kb, are underlined. Chromosome 16 showing both SLC12A3 and NUP93 (on the right). Cen, centromere; Tel, telomere. (B2) Results of molecular karyotyping. Array-comparative genomic hybridization log 2 ratio plot of chromosome 16 of the proband from the Agilent 244k, showing a 13-kb deletion from the probe lying within the 5′UTR of the SLC12A3 gene (56,899,150 Mb; hg19) to the probe that is specific to exon 9 (56,912,240 Mb; hg19). The deleted probes are in green, and unaltered probes (gain or loss) are in black.