Study of USH1 Splicing Variants through Minigenes and Transcript Analysis from Nasal Epithelial Cells
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{"title"=>"Study of USH1 Splicing Variants through Minigenes and Transcript Analysis from Nasal Epithelial Cells", "type"=>"journal", "authors"=>[{"first_name"=>"María José", "last_name"=>"Aparisi", "scopus_author_id"=>"35760804400"}, {"first_name"=>"Gema", "last_name"=>"García-García", "scopus_author_id"=>"35760956400"}, {"first_name"=>"Elena", "last_name"=>"Aller", "scopus_author_id"=>"6603438070"}, {"first_name"=>"María Dolores", "last_name"=>"Sequedo", "scopus_author_id"=>"55568674900"}, {"first_name"=>"Cristina", "last_name"=>"Martínez-Fernández de la Cámara", "scopus_author_id"=>"55607149400"}, {"first_name"=>"Regina", "last_name"=>"Rodrigo", "scopus_author_id"=>"10641302800"}, {"first_name"=>"Miguel", "last_name"=>"Armengot", "scopus_author_id"=>"7006089246"}, {"first_name"=>"Julio", "last_name"=>"Cortijo", "scopus_author_id"=>"7006928415"}, {"first_name"=>"Javier", "last_name"=>"Milara", "scopus_author_id"=>"14016117100"}, {"first_name"=>"Manuel", "last_name"=>"Díaz-LLopis", "scopus_author_id"=>"7003936253"}, {"first_name"=>"Teresa", "last_name"=>"Jaijo", "scopus_author_id"=>"12761027800"}, {"first_name"=>"José María", "last_name"=>"Millán", "scopus_author_id"=>"7202018326"}], "year"=>2013, "source"=>"PLoS ONE", "identifiers"=>{"scopus"=>"2-s2.0-84874386300", "sgr"=>"84874386300", "issn"=>"19326203", "doi"=>"10.1371/journal.pone.0057506", "pmid"=>"23451239", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "pui"=>"368418112"}, "id"=>"7a30a50f-7f62-303c-89c2-ff754c04a5d4", "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.", "link"=>"http://www.mendeley.com/research/study-ush1-splicing-variants-through-minigenes-transcript-analysis-nasal-epithelial-cells", "reader_count"=>34, "reader_count_by_academic_status"=>{"Unspecified"=>5, "Professor > Associate Professor"=>3, "Researcher"=>7, "Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>8, "Student > Master"=>3, "Other"=>3, "Student > Bachelor"=>2, "Lecturer > Senior Lecturer"=>1}, "reader_count_by_user_role"=>{"Unspecified"=>5, "Professor > Associate Professor"=>3, "Researcher"=>7, "Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>8, "Student > Master"=>3, "Other"=>3, "Student > Bachelor"=>2, "Lecturer > Senior Lecturer"=>1}, "reader_count_by_subject_area"=>{"Unspecified"=>5, "Biochemistry, Genetics and Molecular Biology"=>4, "Agricultural and Biological Sciences"=>19, "Medicine and Dentistry"=>4, "Neuroscience"=>1, "Pharmacology, Toxicology and Pharmaceutical Science"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>4}, "Neuroscience"=>{"Neuroscience"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>19}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>4}, "Unspecified"=>{"Unspecified"=>5}, "Pharmacology, Toxicology and Pharmaceutical Science"=>{"Pharmacology, Toxicology and Pharmaceutical Science"=>1}}, "reader_count_by_country"=>{"South Africa"=>1}, "group_count"=>1}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/969966"], "description"=>"<p>Gel electrophoresis shows the different splicing processes for WT minigene and mutants constructions. COS-7 cell transfection experiments were performed in duplicate. <b>A. c.470G>A (p.S157N, </b><b><i>MYO7A</i></b><b>).</b> Band A is the correct transcript of exon 5 (<i>MYO7A</i>). Band B is the skipping of involved exon. <b>B. c.1342_1343delAG (p.S448LfsX2, </b><b><i>MYO7A</i></b><b>).</b> Band A is the correct transcript corresponding to the exon 12 (<i>MYO7A</i>). Band B is the skipping of exon 12. <b>C. c.3652G>A (p.G1218R </b><b><i>MYO7A</i></b><b>).</b> Band A is the correct transcript of exon 29 (<i>MYO7A</i>). The band B is the exon skipping. Band C is the heteroduplex formation from band A and band B. Band D is the aberrant splicing process that include the deletion of 103-bp of 5′end of exon 29. <b>D. c.2289+1G>A (</b><b><i>CDH23</i></b><b>).</b> Band A is the normal transcript of exon 21 (<i>CDH23</i>). Band B is the skipping of exon 21. Band C is the aberrant splicing process that includes the first 149 nucleotides of intron 21. <b>E. c.6049G>A (p.G2017S, </b><b><i>CDH23</i></b><b>).</b> Band A is the correct transcript of exon 46 (<i>CDH23</i>). Band B is the skipping of exon 46. Band C is the heteroduplex formation from the band A and B. <b>F. c.8722+1delG (</b><b><i>CDH23</i></b><b>).</b> Band A is the correct splicing process of exon 60 (<i>CDH23</i>). Band B is the abnormal splicing process of exon 60 that shows a deletion of the last nucleotide (G) of the involved exon. <b>G. c.3717+2dupTT (</b><b><i>PCDH15</i></b><b>).</b> Band A is the correct transcript of exon 27 (<i>PCDH15</i>). Band B is the skipping of the involved exon. Band C is the transcript corresponding to the new donor splice site from exon 27 plus the first 52 nucleotides of the intron 27. Band D is the heteroduplex formation from the band B and the band D.</p>", "links"=>[], "tags"=>["vitro", "splicing", "assays", "mutations", "ush1"], "article_id"=>639329, "categories"=>["Molecular Biology", "Neuroscience", "Genetics"], "users"=>["María José Aparisi", "Gema García-García", "Elena Aller", "María Dolores Sequedo", "Cristina Martínez-Fernández de la Cámara", "Regina Rodrigo", "Miguel Armengot", "Julio Cortijo", "Javier Milara", "Manuel Díaz-LLopis", "Teresa Jaijo", "José María Millán"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0057506.g001", "stats"=>{"downloads"=>2, "page_views"=>38, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_In_vitro_splicing_assays_for_the_seven_splicing_mutations_identified_in_the_USH1_genes_/639329", "title"=>"In vitro splicing assays for the seven splicing mutations identified in the USH1 genes.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-26 09:43:49"}
  • {"files"=>["https://ndownloader.figshare.com/files/969971"], "description"=>"<p>Gel electrophoresis shows the RT-PCR products obtained for USH1 patients and control samples. Electropherograms of the transcripts obtained show the molecular characterization of the effect of the studied variants. All experiments were performed in duplicate. <b>A. c.5856G>A (p.K1952K, </b><b><i>MYO7A</i></b><b>).</b> Band A is the correct splicing process of the exons 38–45 of the <i>MYO7A</i> gene. Band B is the skipping of exon 42. RP-280 replicate 1 shows two transcripts corresponding to the WT allele (band A) and mutant allele (band B). RP-280 replicate 2 shows only the band B corresponding to the aberrant transcript. <b>B. c.2289+1G>A (</b><b><i>CDH23</i></b><b>).</b> Band A is the transcript corresponding to the normal RNA processing of the exons 18–24 of the <i>CDH23</i> gene. Band B is the aberrant transcript that includes the first 149 nucleotides and the last 54 nucleotides of intron 21<b>. C. c.6049G>A (p.G2017S, </b><b><i>CDH23</i></b><b>).</b> RP-1534 shows only the WT allele. <b>D. c.8722+1delG (</b><b><i>CDH23</i></b><b>).</b> RP-928 shows the heterozygous transcript from WT allele and the mutant allele. It presents a deletion of the last nucleotide (G) of exon 60 of <i>CDH23</i> gene.</p>", "links"=>[], "tags"=>["ush1", "variants", "nasal", "epithelial"], "article_id"=>639334, "categories"=>["Molecular Biology", "Neuroscience", "Genetics"], "users"=>["María José Aparisi", "Gema García-García", "Elena Aller", "María Dolores Sequedo", "Cristina Martínez-Fernández de la Cámara", "Regina Rodrigo", "Miguel Armengot", "Julio Cortijo", "Javier Milara", "Manuel Díaz-LLopis", "Teresa Jaijo", "José María Millán"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0057506.g002", "stats"=>{"downloads"=>1, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Transcript_analysis_of_USH1_variants_in_nasal_epithelial_cells_/639334", "title"=>"Transcript analysis of USH1 variants in nasal epithelial cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-26 09:44:53"}
  • {"files"=>["https://ndownloader.figshare.com/files/1010895"], "description"=>"<p>The nucleotides that have been modified are indicated in bold.</p><p>The positions of deleted nucleotides are represented with (−).</p>", "links"=>[], "tags"=>["site-directed"], "article_id"=>671302, "categories"=>["Molecular Biology", "Neuroscience", "Genetics"], "users"=>["María José Aparisi", "Gema García-García", "Elena Aller", "María Dolores Sequedo", "Cristina Martínez-Fernández de la Cámara", "Regina Rodrigo", "Miguel Armengot", "Julio Cortijo", "Javier Milara", "Manuel Díaz-LLopis", "Teresa Jaijo", "José María Millán"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0057506.t002", "stats"=>{"downloads"=>3, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Primers_used_for_the_site_directed_mutagenesis_/671302", "title"=>"Primers used for the site-directed mutagenesis.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2013-02-25 00:21:42"}
  • {"files"=>["https://ndownloader.figshare.com/files/1010741"], "description"=>"<p>Not performed in this study are indicated with (−).</p><p>NMD: Nonsense mediated decay.</p>*<p>c.5856G>A (p.K1952K, <i>MYO7A</i>) was previously analyzed by Jaijo et al. <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057506#pone.0057506-Jaijo5\" target=\"_blank\">[26]</a>.</p>", "links"=>[], "tags"=>["ush1", "variants"], "article_id"=>671143, "categories"=>["Molecular Biology", "Neuroscience", "Genetics"], "users"=>["María José Aparisi", "Gema García-García", "Elena Aller", "María Dolores Sequedo", "Cristina Martínez-Fernández de la Cámara", "Regina Rodrigo", "Miguel Armengot", "Julio Cortijo", "Javier Milara", "Manuel Díaz-LLopis", "Teresa Jaijo", "José María Millán"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0057506.t005", "stats"=>{"downloads"=>1, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Effects_of_USH1_variants_on_splicing_/671143", "title"=>"Effects of USH1 variants on splicing<b>.</b>", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2013-02-25 00:19:03"}
  • {"files"=>["https://ndownloader.figshare.com/files/969973"], "description"=>"<p><b>A</b>. Comparison between USH1 group and Control group. The nasal ciliary beat frequency was significantly different between these groups (Mann Whitney Test, p  = 0.031). <b>B</b>. Comparison between the <i>MYO7A</i> group and <i>CDH23</i> group, <i>MYO7A</i> and Control group and <i>CDH23</i> group and Control group. The nasal ciliary beat frequency was significantly different between <i>CDH23</i> group and Control group (P<0.05).</p>", "links"=>[], "tags"=>["ciliary"], "article_id"=>639336, "categories"=>["Molecular Biology", "Neuroscience", "Genetics"], "users"=>["María José Aparisi", "Gema García-García", "Elena Aller", "María Dolores Sequedo", "Cristina Martínez-Fernández de la Cámara", "Regina Rodrigo", "Miguel Armengot", "Julio Cortijo", "Javier Milara", "Manuel Díaz-LLopis", "Teresa Jaijo", "José María Millán"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0057506.g003", "stats"=>{"downloads"=>1, "page_views"=>15, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Nasal_ciliary_beat_frequency_in_five_MYO7A_and_three_CDH23_patients_and_thirty_controls_/639336", "title"=>"Nasal ciliary beat frequency in five <i>MYO7A</i> and three <i>CDH23</i> patients, and thirty controls.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-26 09:45:09"}
  • {"files"=>["https://ndownloader.figshare.com/files/1010871"], "description"=>"*<p>The primer MYO_EXP_25-33-EXT-R was designed by Vaché et al. <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057506#pone.0057506-Vach1\" target=\"_blank\">[11]</a>.</p>", "links"=>[], "tags"=>["fragments", "cdnas", "nasal"], "article_id"=>671276, "categories"=>["Molecular Biology", "Neuroscience", "Genetics"], "users"=>["María José Aparisi", "Gema García-García", "Elena Aller", "María Dolores Sequedo", "Cristina Martínez-Fernández de la Cámara", "Regina Rodrigo", "Miguel Armengot", "Julio Cortijo", "Javier Milara", "Manuel Díaz-LLopis", "Teresa Jaijo", "José María Millán"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0057506.t003", "stats"=>{"downloads"=>1, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Primers_used_to_amplify_fragments_of_MYO7A_and_CDH23_cDNAs_from_nasal_cells_/671276", "title"=>"Primers used to amplify fragments of <i>MYO7A</i> and <i>CDH23</i> cDNAs from nasal cells.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2013-02-25 00:21:16"}
  • {"files"=>["https://ndownloader.figshare.com/files/1010846"], "description"=>"<p>Tails added at the beginning of the primer are indicated in bold.</p><p>Enzyme restriction sites used in this study are indicated in italics.</p>", "links"=>[], "tags"=>["genetics and genomics", "molecular biology", "neuroscience"], "article_id"=>671249, "categories"=>["Molecular Biology", "Neuroscience", "Genetics"], "users"=>["María José Aparisi", "Gema García-García", "Elena Aller", "María Dolores Sequedo", "Cristina Martínez-Fernández de la Cámara", "Regina Rodrigo", "Miguel Armengot", "Julio Cortijo", "Javier Milara", "Manuel Díaz-LLopis", "Teresa Jaijo", "José María Millán"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0057506.t001", "stats"=>{"downloads"=>1, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Primers_used_to_amplify_the_specific_insert_/671249", "title"=>"Primers used to amplify the specific insert.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2013-02-25 00:20:49"}
  • {"files"=>["https://ndownloader.figshare.com/files/1010767"], "description"=>"<p>Results from four different bioinformatic programs used to predict the effect on the splicing process.</p>", "links"=>[], "tags"=>["bioinformatic", "programs", "splicing"], "article_id"=>671169, "categories"=>["Molecular Biology", "Neuroscience", "Genetics"], "users"=>["María José Aparisi", "Gema García-García", "Elena Aller", "María Dolores Sequedo", "Cristina Martínez-Fernández de la Cámara", "Regina Rodrigo", "Miguel Armengot", "Julio Cortijo", "Javier Milara", "Manuel Díaz-LLopis", "Teresa Jaijo", "José María Millán"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0057506.t004", "stats"=>{"downloads"=>0, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Results_from_four_different_bioinformatic_programs_used_to_predict_the_effect_on_the_splicing_process_/671169", "title"=>"Results from four different bioinformatic programs used to predict the effect on the splicing process.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2013-02-25 00:19:29"}
  • {"files"=>["https://ndownloader.figshare.com/files/1010818"], "description"=>"<p>Family healthy carriers are indicated with an asterisk (*).</p>", "links"=>[], "tags"=>["ush1", "patients", "carriers", "mutations", "presented"], "article_id"=>671222, "categories"=>["Molecular Biology", "Neuroscience", "Genetics"], "users"=>["María José Aparisi", "Gema García-García", "Elena Aller", "María Dolores Sequedo", "Cristina Martínez-Fernández de la Cámara", "Regina Rodrigo", "Miguel Armengot", "Julio Cortijo", "Javier Milara", "Manuel Díaz-LLopis", "Teresa Jaijo", "José María Millán"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0057506.t006", "stats"=>{"downloads"=>0, "page_views"=>7, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Genotypes_of_the_five_USH1_patients_and_the_two_family_healthy_carriers_of_USH1_mutations_presented_in_this_study_/671222", "title"=>"Genotypes of the five USH1 patients and the two family healthy carriers of USH1 mutations presented in this study.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2013-02-25 00:20:22"}
  • {"files"=>["https://ndownloader.figshare.com/files/1010791"], "description"=>"<p>Nasal ciliary beat frequency of USH1 patients and controls.</p>", "links"=>[], "tags"=>["ciliary", "ush1", "patients"], "article_id"=>671195, "categories"=>["Molecular Biology", "Neuroscience", "Genetics"], "users"=>["María José Aparisi", "Gema García-García", "Elena Aller", "María Dolores Sequedo", "Cristina Martínez-Fernández de la Cámara", "Regina Rodrigo", "Miguel Armengot", "Julio Cortijo", "Javier Milara", "Manuel Díaz-LLopis", "Teresa Jaijo", "José María Millán"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0057506.t007", "stats"=>{"downloads"=>1, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Nasal_ciliary_beat_frequency_of_USH1_patients_and_controls_/671195", "title"=>"Nasal ciliary beat frequency of USH1 patients and controls.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2013-02-25 00:19:55"}

PMC Usage Stats | Further Information

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Relative Metric

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