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.

Novel compound heterozygous mutation of SLC12A3 in Gitelman syndrome co-existent with hyperthyroidism: A case report and literature review

“…To date, all the identified mutations are widely distributed along 26 exons and some introns of SLC12A3. Combined heterozygosity with different mutations in each allele is the most frequent variant, with missense mutation being the most common type[ 26 - 28 ]. Notably, compound heterozygosity is a frequent occurrence in patients with GS coexisting with AITD (Table 3 ).…”

“…In the present case, triple mutations, including two missense mutations and one frameshift duplication, were identified. Triple mutations, with one mutation in one allele, have increasingly been reported in several studies[ 26 , 28 , 29 ]. Interestingly, the mutations p.Thr163Met and p.Arg871His are consistent with our findings on the same allele in unrelated Chinese-Taiwanese GS patients with triple mutations implying linkage disequilibrium, suggesting that there may be many hot spot mutations in SLC12A3 [ 26 ] (Figure 2 ) .…”

“…Triple mutations, with one mutation in one allele, have increasingly been reported in several studies[ 26 , 28 , 29 ]. Interestingly, the mutations p.Thr163Met and p.Arg871His are consistent with our findings on the same allele in unrelated Chinese-Taiwanese GS patients with triple mutations implying linkage disequilibrium, suggesting that there may be many hot spot mutations in SLC12A3 [ 26 ] (Figure 2 ) . In contrast, the recurrent mutations are very different in Japanese and European GS patients[ 30 , 31 ] .…”

Establishment of a Rapid Typing Method for Coronavirus Disease 2019 Mutant Strains Based on PARMS Technology

“…At the same time, this presents new challenges to the detection of SARS-COV-2. Some studies on genotyping are helpful to SARS-CoV-2 screening [14][15][16]. Zhenrui Xue et al developed Taqman-MGB nanoPCR system to detect clinical single-base mutation [14].…”