The Alu insertion in the CLCN5 gene of a patient with Dent’s disease leads to exon 11 skipping

Abstract

Alu sequences are short, interspersed elements that have generated more than one million copies in the human genome. They propagate by transcription followed by reverse transcription and integration, causing mutations, recombination, and changes in pre-mRNA splicing. We have recently identified a 345-bp long Alu Ya5 element inserted in codon 650 within exon 11 of the chloride channel ClC-5 gene (CLCN5) of a patient with Dent's disease. A microsatellite pedigree analysis indicated that the insertion occurred in the germline of the maternal grandfather. Dent's disease is an X-linked renal tubular disorder characterized by low-molecularweight proteinuria, hypercalciuria, nephrolithiasis, and nephrocalcinosis. Here, we found, by RT-PCR amplification of RNA extracted from the patient's blood and subsequent DNA sequencing, that the Alu insertion led to an aberrant splicing of the CLCN5 pre-mRNA that skipped exon 11. Using the ESE finder and RESCUE-ESE Web interfaces, we identified two high-score exonic splicing enhancer (ESE) sequences in the site of insertion. The functional significance of these ESE motifs is suggested by our observation that these sequences are highly conserved among mammal CLCN5 genes. Therefore, we suggest that the Alu insertion causes exon skipping by interfering with splicing regulatory elements.The altered splicing would predict a truncated ClC-5 protein that lacks critical domains for sorting and chloride channel function.

A missense mutation in the chloride/proton ClC-5 antiporter gene results in increased expression of an alternative mRNA form that lacks exons 10 and 11. Identification of seven new CLCN5 mutations in patients with Dent’s disease

“…Poly-A mRNA (200 ng) was reverse transcribed as described above. Amplifications were carried out using previously described conditions (Claverie-Martin et al 2005). To study the coding region in the splicing variant, a primer (5¢-ATTCCAAGCAATCGCCTAAG-3¢) was designed to specifically detect skipping of exons 10 and 11.…”

“…To study the coding region in the splicing variant, a primer (5¢-ATTCCAAGCAATCGCCTAAG-3¢) was designed to specifically detect skipping of exons 10 and 11. After testing the specificity of the primer, it was used along with primer CLCN5cDN 2F (5¢-GAC-TTCTTGGAGGAGCCAATC-3¢) (Claverie-Martin et al 2005) to amplify the complete coding region (starting in exon 2) in all tissues. The program for this amplification was 94°C for 2 min, followed by 40 cycles each consisting of three steps, 94°C for 30 s, 60°C for 45 s, and 72°C for 60 s, with a final extension at 72°C for 5 min.…”

An Evolutionary Biology Approach to Understanding Neurological Disorders

“…Indeed, one in 50 individuals will carry a de novo L1 insertion, and one in 20 individuals a de novo Alu insertion (Collier & Largaespada, 2007). The active nature of many human retrotransposons is therefore linked to disease-causing somatic and germline mutations (Collier & Largaespada, 2007;Wallace et al, 1991;Oldridge et al, 1999;Claverie-Martin et al, 2003). Repeats may also contribute to DNA secondary structures that are more prone to breakage (Yatsenko et al, 2009).…”

Splicing defects caused by exonic mutations inPKD1as a new mechanism of pathogenesis in autosomal dominant polycystic kidney disease

“…1B). 10,[29][30][31] Defective mRNAs produced by these events may include exons or intron sequences with premature stop codons. It is not easy to predict the cytoplasmic fate of these aberrant messages, but some of them are probably degraded by nonsensemediated decay (NMD).…”