Article Title: Aberrant Splicing Events Associated to CDH23 Noncanonical Splice Site Mutations in a Proband with Atypical Usher Syndrome 1
Figure Lengend Snippet: In vivo and in vitro splicing analysis of the identified CDH23 alleles. ( A ) Analysis of the CDH23 mRNAs of whole blood samples (treated and untreated with cycloheximide, CHX) from patient and control by RT-PCR, showing the presence of aberrantly spliced CDH23 transcripts in addition to the wild-type transcript. Lane 1—patient sample (−CHX), Lane 2—patient sample (+CHX), Lane 3—control sample (−CHX), Lane 4—negative control. The band of 563 bp corresponded to the transcript with an in-frame skipping of exon 46. The band around 760–780 bp corresponded to the wild-type as well as to the aberrant transcripts with additional +7 and +13 nucleotides. Subsequent Sanger sequencing of cloned individual bands confirmed the insertion of 7 bp and 13 bp (774 pb, 780 pb bands) corresponding to each mutant allele. Transcripts bearing premature stop codon due to frameshift were only amplified in cycloheximide-treated blood samples (+CHX). ( B ) Schematic representation of the studied genomic region of CDH23 gene and the midigene construct for in vitro splicing assays (MGC1). The position of each mutation is indicated. The genomic region encompassing exons 45, 46, and 47 was cloned between the splice donor (SD) and acceptor (SA) sites within the pSPL3 vector. ( C ) In vitro splicing assays in HEK293T cells transfected with either the wildtype (WT) or mutant CDH23 midigenes (MGC1-WT, MGC1-15A, and MGC1-9A, respectively) with or without cycloheximide treatment (+CHX). All constructs (MGC1-WT, MGC1-15A, and MGC1-9A) produced skipping of exon 46. CHX treatment in cells transfected with the mutant constructs increased the relative amplification of the aberrantly spliced transcripts. ( D ) Sanger sequence analysis of each transcript band confirmed the wild-type splicing event in cells transfected with MGC1-WT, in contrast to the addition of +13 and +7 nucleotides in exon 46 in cells transfected with MGC1-15A and MGC1-9A, respectively.
Article Snippet: In Vitro Splicing Assays in HEK293T Cells For individual analysis of each of the CDH23 variants, (c.6050-15G > A and c.6050-9G > A), genomic fragments of 3751bp that spanned exons 45, 46, and 47 were subcloned into the HIV-tat intron of the pSPL3 expression vector (Addgene, Watertown, MA, USA).
Techniques: In Vivo, In Vitro, Reverse Transcription Polymerase Chain Reaction, Negative Control, Sequencing, Clone Assay, Mutagenesis, Amplification, Construct, Plasmid Preparation, Transfection, Produced