title: The effect of the common c.2299delG mutation in USH2A on RNA splicing.
abstract: Recessive variants in the USH2A gene are an important cause of both Usher syndrome and nonsyndromic retinitis pigmentosa. A single base-pair deletion in exon 13 (c.2299delG, p.Glu767Serfs*21) is considered the most frequent mutation of USH2A. It is predicted to generate a premature termination codon and is presumed to lead to nonsense mediated decay. However the effect of this variant on RNA has not been formally investigated. It is not uncommon for exonic sequence alterations to cause aberrant splicing and the aim of the present report is to evaluate the effect of c.2299delG on USH2A transcripts. Nasal cells represent the simplest available tissue to study splicing defects in USH2A. Nasal brushing, RNA extraction from nasal epithelial cells and reverse transcription PCR were performed in five Usher syndrome patients who were homozygous for c.2299delG, two unaffected c.2299delG heterozygotes and seven control individuals. Primers to amplify between exons 12 and 15 and exons 10 and 14 were utilised. Significant variability was observed between different RT-PCR experiments. Importantly, in controls, PCR product of the expected size were amplified on all occasions (13/13 experiments); for patients this was true in only 4/14 experiments (Fisher exact test p = 0.0002). Bioinformatics tools predict the c.2299delG change to disrupt an exonic splicing enhancer and to create an exonic splicing silencer within exon 13. Here, we report an effect of the common c.2299delG mutation on splicing of exons 12 and 13 of USH2A. Future studies are expected to provide important insights into the contribution of this effect on the phenotype.
authors.affiliation: UCL Institute of Ophthalmology, London EC1V 9EL, United Kingdom; Moorfields Eye Hospital, London EC1V 2PD, United Kingdom; Eye Hospital, University Medical Centre, Ljubljana SL 1000, Slovenia.
authors.forename: Eva
authors.initials: E
authors.lastname: Lenassi
authors.affiliation: UCL Institute of Ophthalmology, London EC1V 9EL, United Kingdom; Moorfields Eye Hospital, London EC1V 2PD, United Kingdom.
authors.forename: Zubin
authors.initials: Z
authors.lastname: Saihan
authors.affiliation: UCL Institute of Child Health, London WC1N 1EH, United Kingdom; Great Ormond Street Hospital for Children NHS Foundation Trust, London WC1N 3JH, United Kingdom.
authors.forename: Maria
authors.initials: M
authors.lastname: Bitner-Glindzicz
authors.affiliation: UCL Institute of Ophthalmology, London EC1V 9EL, United Kingdom; Moorfields Eye Hospital, London EC1V 2PD, United Kingdom. Electronic address: andrew.webster@ucl.ac.uk.
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Webster
mesh_terms: D000328:Adult; D000368:Aged; D017931:DNA Primers; D005091:Exons / Q000235:genetics; D016326:Extracellular Matrix Proteins / Q000235:genetics*; D005260:Female; D005451:Fluorescein Angiography; D017353:Gene Deletion; D006801:Humans; D008297:Male; D008875:Middle Aged; D017354:Point Mutation; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D012326:RNA Splicing / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D041623:Tomography, Optical Coherence; D052245:Usher Syndromes / Q000235:genetics*; D055815:Young Adult
chemical_list: D017931:DNA Primers; D016326:Extracellular Matrix Proteins; C112735:USH2A protein, human
keywords: RNA; RT-PCR; USH2A; Usher syndrome; exon skipping; retinitis pigmentosa; splicing