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