title: Study of USH1 splicing variants through minigenes and transcript analysis from nasal epithelial cells.
abstract: Usher syndrome type I (USH1) is an autosomal recessive disorder characterized by congenital profound deafness, vestibular areflexia and prepubertal retinitis pigmentosa. The first purpose of this study was to determine the pathologic nature of eighteen USH1 putative splicing variants found in our series and their effect in the splicing process by minigene assays. These variants were selected according to bioinformatic analysis. The second aim was to analyze the USH1 transcripts, obtained from nasal epithelial cells samples of our patients, in order to corroborate the observed effect of mutations by minigenes in patient's tissues. The last objective was to evaluate the nasal ciliary beat frequency in patients with USH1 and compare it with control subjects. In silico analysis were performed using four bioinformatic programs: NNSplice, Human Splicing Finder, NetGene2 and Spliceview. Afterward, minigenes based on the pSPL3 vector were used to investigate the implication of selected changes in the mRNA processing. To observe the effect of mutations in the patient's tissues, RNA was extracted from nasal epithelial cells and RT-PCR analyses were performed. Four MYO7A (c.470G>A, c.1342_1343delAG, c.5856G>A and c.3652G>A), three CDH23 (c.2289+1G>A, c.6049G>A and c.8722+1delG) and one PCDH15 (c.3717+2dupTT) variants were observed to affect the splicing process by minigene assays and/or transcripts analysis obtained from nasal cells. Based on our results, minigenes are a good approach to determine the implication of identified variants in the mRNA processing, and the analysis of RNA obtained from nasal epithelial cells is an alternative method to discriminate neutral Usher variants from those with a pathogenic effect on the splicing process. In addition, we could observe that the nasal ciliated epithelium of USH1 patients shows a lower ciliary beat frequency than control subjects.
authors: Research Group on Sensorineural Diseases, Instituto de Investigación Sanitaria-La Fe, Valencia, Spain.
authors: María José
authors: MJ
authors: Aparisi
authors: Gema
authors: G
authors: García-García
authors: Elena
authors: E
authors: Aller
authors: María Dolores
authors: MD
authors: Sequedo
authors: Cristina
authors: C
authors: Martínez-Fernández de la Cámara
authors: Regina
authors: R
authors: Rodrigo
authors: Miguel
authors: M
authors: Armengot
authors: Julio
authors: J
authors: Cortijo
authors: Javier
authors: J
authors: Milara
authors: Manuel
authors: M
authors: Díaz-LLopis
authors: Teresa
authors: T
authors: Jaijo
authors: José María
authors: JM
authors: Millán
mesh_terms: D000070585:Cadherin Related Proteins; D015820:Cadherins / Q000235:genetics; D016022:Case-Control Studies; D002923:Cilia / Q000502:physiology; D019295:Computational Biology / Q000379:methods; D004847:Epithelial Cells / Q000378:metabolism / Q000502:physiology*; D014644:Genetic Variation; D006801:Humans; D009154:Mutation; D000081412:Myosin VIIa; D009218:Myosins / Q000235:genetics; D009666:Nose / Q000166:cytology*; D012326:RNA Splicing / Q000235:genetics*; D052245:Usher Syndromes / Q000235:genetics*
chemical_list: C442480:CDH23 protein, human; C498835:CDHR15, human; D000070585:Cadherin Related Proteins; D015820:Cadherins; C000631455:MYO7A protein, human; D000081412:Myosin VIIa; D009218:Myosins