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Allele-specific RT-PCR for the rapid detection of recurrent SLC12A3 mutations for Gitelman syndrome.

ABSTRACT: Recurrent mutations in the SLC12A3 gene responsible for autosomal recessive Gitelman syndrome (GS) are frequently reported, but the exact prevalence is unknown. The rapid detection of recurrent SLC12A3 mutations may help in the early diagnosis of GS. This study was aimed to investigate the prevalence of recurrent SLC12A3 mutations in a Taiwan cohort of GS families and develop a simple and rapid method to detect recurrent SLC12A3 mutations. One hundred and thirty independent Taiwan families with genetically confirmed GS were consecutively enrolled to define recurrent SLC12A3 mutations and determine their prevalence. Using TaqMan probe-based real-time polymerase chain reaction, we designed a mutation detection plate with all recurrent mutations. We validated this mutation detection plate and tested its feasibility in newly diagnosed GS patients. A total of 57 mutations in the SLC12A3 gene were identified and 22 including 2 deep intronic mutations were recurrent mutations consisting of 87.1% (242/278, 18 triple) of all allelic mutations. The recurrent mutation-based TaqMan assays were fully validated with excellent sensitivity and specificity in genetically diagnosed GS patients and healthy subjects. In clinical validation, recurrent mutations were recognized in 92.0% of allelic mutations from 12 GS patients within 4 h and all were confirmed by direct sequencing. Recurrent SLC12A3 mutations are very common in Taiwan GS patients and can be rapidly identified by this recurrent mutation-based SLC12A3 mutation plate.

SUBMITTER: Yan MT

PROVIDER: S-EPMC8363728 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundGitelman syndrome (GS) is an autosomal recessive disorder and mild variant of classic Bartter syndrome. The latter is caused by defects in the genes CLCNKB and/or CLCNKA (chloride voltage-gated channel Ka and Kb). Patients with GS usually have loss-of-function mutations in SLC12A3. No patient has been reported with compound heterozygous mutations in these genes. We report a girl with GS with a paternally inherited heterozygous mutation in SLC12A3, and maternally inherited heterozygous variants in both CLCNKB and CLCNKA.Case presentationIn this report, we reported a female patient (8 y and 10 mo) who had growth retardation (111.8 cm, - 1.62 standard deviation height for age) and normal blood pressure, with persistent hypokalemia, hypomagnesemia, hypocalciuria, hypochloremic alkalosis, and elevated levels of plasma renin and aldosterone. Her younger brother, father, and paternal grandmother all had histories of mild low levels of plasma potassium (3.0-3.5 mmol/L), which were rectified by potassium-rich foods. The genomic DNA of the patient, younger brother, parents, and grandparents were screened for gene variations and pedigree analysis using trio whole exome sequencing (WES). The candidate variants were validated by Sanger sequencing. Protein-protein interaction analysis utilized the following databases: Biogrid, MINT, HPRD, STRING, IntAct, iRefIndex, and ppiTrim. The trio WES screening showed that the patient has paternally inherited SLC12A3 p.N359K, and maternally inherited CLCNKB p.L94I. The paternal grandmother and younger brother are both carriers of SLC12A3 p.N359K. According to the STRING database, SLC12A3 and CLCNKB proteins may interact or coexpress with proteins associated with GS.ConclusionsBased on clinical phenotypes, genetic evidence of the pedigree, and previous reported studies, this case of GS indicates a digenetic inheritance of SLC12A3 and CLCNKB that resulted in renal tubular dysfunction perhaps, due to a genetic double-hit mechanism. The putative pathogenicity of the CLCNKB p.L94I variant requires confirmation.

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Allele-specific RT-PCR for the rapid detection of recurrent SLC12A3 mutations for Gitelman syndrome.

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