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Complexity of the 5'UTR region of the CLCN5 gene: eleven 5'UTR ends are differentially expressed in the human kidney.

ABSTRACT: Dent disease 1 represents a hereditary disorder of renal tubular epithelial function associated with mutations in the CLCN5 gene that encoded the ClC-5 Cl-/H+ antiporter. All of the reported disease-causing mutations are localized in the coding region except for one recently identified in the 5'UTR region of a single patient. This finding highlighted the possible role for genetic variability in this region in the pathogenesis of Dent disease 1.The structural complexity of the CLCN5 5'UTR region has not yet been fully characterized. To date 6 different 5' alternatively used exons--1a, 1b, 1b1 and I-IV with an alternatively spliced exon II (IIa, IIb)--have been described, but their significance and differential expression in the human kidney have not been investigated. Therefore our aim was to better characterize the CLCN5 5'UTR region in the human kidney and other tissues.To clone more of the 5' end portion of the human CLCN5 cDNA, total human kidney RNA was utilized as template and RNA ligase-mediated rapid amplification of cDNA 5' ends was applied.The expression of the different CLCN5 isoforms was studied in the kidney, leucocytes and in different tissues by quantitative comparative RT/PCR and Real--Time RT/PCR.Eleven transcripts initiating at 3 different nucleotide positions having 3 distinct promoters of varying strength were identified. Previously identified 5'UTR isoforms were confirmed, but their ends were extended. Six additional 5'UTR ends characterized by the presence of new untranslated exons (c, V and VI) were also identified. Exon c originates exon c.1 by alternative splicing. The kidney uniquely expresses all isoforms, and the isoform containing exon c appears kidney specific. The most abundant isoforms contain exon 1a, exon IIa and exons 1b1 and c. ORF analysis predicts that all isoforms except 3 encode for the canonical 746 amino acid ClC-5 protein.Our results confirm the structural complexity of the CLCN5 5'UTR region. Characterization of this crucial region could allow a clear genetic classification of a greater number of Dent disease patients, but also provide the basis for highlighting some as yet unexplored functions of the ClC-5 proton exchanger.

SUBMITTER: Tosetto E

PROVIDER: S-EPMC4105828 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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Complexity of the 5'UTR region of the CLCN5 gene: eleven 5'UTR ends are differentially expressed in the human kidney.

Tosetto Enrica E Casarin Alberto A Salviati Leonardo L Familiari Alessandra A Lieske John C JC Anglani Franca F

BMC medical genomics 20140707

<h4>Background</h4>Dent disease 1 represents a hereditary disorder of renal tubular epithelial function associated with mutations in the CLCN5 gene that encoded the ClC-5 Cl-/H+ antiporter. All of the reported disease-causing mutations are localized in the coding region except for one recently identified in the 5'UTR region of a single patient. This finding highlighted the possible role for genetic variability in this region in the pathogenesis of Dent disease 1.The structural complexity of the ...[more]

PMID: 25001568

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Project description:We performed RNA sequencing (RNAseq) on peripheral blood mononuclear cells (PBMCs) to identify differentially expressed gene transcripts (DEGs) after kidney transplantation and after the start of immunosuppressive drugs. RNAseq is superior to microarray to determine DEGs because it's not limited to available probes, has increased sensitivity, and detects alternative and previously unknown transcripts. DEGs were determined in 32 adult kidney recipients, without clinical acute rejection (AR), treated with antibody induction, calcineurin inhibitor, mycophenolate, with and without steroids. Blood was obtained pre-transplant (baseline), week 1, months 3 and 6 post-transplant. PBMCs were isolated, RNA extracted and gene expression measured using RNAseq. Principal components (PCs) were computed using a surrogate variable approach. DEGs post-transplant were identified by controlling false discovery rate (FDR) at < 0.01 with at least a 2 fold change in expression from pre-transplant. The top 5 DEGs with higher levels of transcripts in blood at week 1 were TOMM40L, TMEM205, OLFM4, MMP8, and OSBPL9 compared to baseline. The top 5 DEGs with lower levels at week 1 post-transplant were IL7R, KLRC3, CD3E, CD3D, and KLRC2 (Striking Image) compared to baseline. The top pathways from genes with lower levels at 1 week post-transplant compared to baseline, were T cell receptor signaling and iCOS-iCOSL signaling while the top pathways from genes with higher levels than baseline were axonal guidance signaling and LXR/RXR activation. Gene expression signatures at month 3 were similar to week 1. DEGs at 6 months post-transplant create a different gene signature than week 1 or month 3 post-transplant. RNAseq analysis identified more DEGs with lower than higher levels in blood compared to baseline at week 1 and month 3. The number of DEGs decreased with time post-transplant. Further investigations to determine the specific lymphocyte(s) responsible for differential gene expression may be important in selecting and personalizing immune suppressant drugs and may lead to targeted therapies.

Dataset Information

Complexity of the 5'UTR region of the CLCN5 gene: eleven 5'UTR ends are differentially expressed in the human kidney.

Publications

Complexity of the 5'UTR region of the CLCN5 gene: eleven 5'UTR ends are differentially expressed in the human kidney.

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