In vivo and in vitro splicing assay of SLC12A1 in an antenatal salt-losing tubulopathy patient with an intronic mutation

Abstract

Type I Bartter syndrome (BS), an inherited salt-losing tubulopathy, is caused by mutations of the SLC12A1 gene. While several intronic nucleotide changes in this gene have been detected, transcriptional analysis had not been conducted because mRNA analysis is possible only when renal biopsy specimens can be obtained or occasionally when mRNA is expressed in the leukocytes. This report concerns a type I BS patient due to compound heterozygosity for the SLC12A1 gene. Genomic DNA sequencing disclosed the presence of two novel heterozygous mutations of c.724 + 4A > G in intron 5 and c.2095delG in intron 16, but it remains to be determined whether the former would be likely to influence the transcription. In this report, we conducted both in vivo assay of RT-PCR analysis using RNA extracted from the proband's urinary sediments and in vitro functional splicing study by minigene construction, and obtained evidence that this intronic mutation leads to complete exon 5 skipping. To the best of our knowledge, this is the first study to use non-invasive methods for both an in vivo assay and an in vitro functional splicing assay of inherited kidney disease. These analytical assays could be adapted for all inherited kidney diseases.

Somatic mosaicism and variant frequency detected by next-generation sequencing in X-linked Alport syndrome

“…16,17 The present study compared the allele frequencies between DNA extracted from these two sources and obtained very similar findings (Table 2). Therefore, we compared the variant frequency of either kidney biopsies or urinary sediments with the phenotype in our analysis.…”

Differential diagnosis of Bartter syndrome, Gitelman syndrome, and pseudo–Bartter/Gitelman syndrome based on clinical characteristics

“…If patients suspected to have BS/GS carried only one CLCNKB or SLC12A3 mutant allele, we performed additional semiquantitative polymerase chain reaction 10,18 or multiplex ligation-dependent probe amplification using the SALSA P266-CLCNKB or P136-SLC12A3 multiplex ligation-dependent probe amplification assays (MRC-Holland, Amsterdam, The Netherlands) to detect large heterozygous deletions. Total RNA from leukocytes and/or urine sediment was isolated as previously described 19,20 and analyzed to detect splicing abnormalities. Patients with type III BS and GS with homozygous or compound heterozygous mutations in CLCNKB or SLC12A3 were included in this study, whereas patients with only heterozygous mutations were excluded.…”

Aplicação da biologia molecular na abordagem da síndrome de Bartter: relato de caso

“…2,5,6,9,12 The recent study by Brochard et al,2 who investigated mutations in 42 children with BS, is worth mentioning. Most of those children had heterozygous mutations of the KCNJ1 gene (45%).…”

“…Transient neonatal hyperkalemia, which is generally little diagnosed, was detected in 63% of the children with a mutation of the KCNJ1 gene, not being observed in children with mutations of other genes. 2 Nozu et al 12 showed that analysis of the genetic material of urinary cells may obviate the need of more invasive procedures, such as blood sampling and renal biopsy. The method proposed by those authors allowed detection of mutations in the SLC12A1 gene of BS type I.…”

“…The method proposed by those authors allowed detection of mutations in the SLC12A1 gene of BS type I. 12 In Brazil, even in reference centers, the main difficulty in the diagnosis of BS lies in the precise identification of the mutation involved. Accordingly, the use of molecular biology techniques in the approach to BS diagnosis may speed diagnosis and treatment, allowing genetic counseling to be provided.…”