title: Detecting tissue-specific alternative splicing and disease-associated aberrant splicing of the PTCH gene with exon junction microarrays.
abstract: Mutations in the human ortholog of Drosophila patched (PTCH) have been identified in patients with autosomal dominant nevoid basal cell carcinoma syndrome (NBCCS), characterized by minor developmental anomalies and an increased incidence of cancers such as medulloblastoma and basal cell carcinoma. We identified many isoforms of PTCH mRNA involving exons 1-5, exon 10 and a novel exon, 12b, generated by alternative splicing (AS), most of which have not been deposited in GenBank nor discussed earlier. To monitor splicing events of the PTCH gene, we designed oligonucleotide arrays on which exon probes and exon-exon junction probes as well as a couple of intron probes for the PTCH gene were placed in duplicate. Probe intensities were normalized on the basis of the total expression of PTCH and probe sensitivity. Tissue-specific regulation of AS identified with the microarrays closely correlated with the results obtained by RT-PCR. Of note, the novel exon, exon 12b, was specifically expressed in the brain and heart, especially in the cerebellum. Additionally, using these microarrays, we were able to detect disease-associated aberrant splicings of the PTCH gene in two patients with NBCCS. In both cases, cryptic splice donor sites located either in an exon or in an intron were activated because of the partial disruption of the consensus sequence for the authentic splice donor sites due to point mutations. Taken together, oligonucleotide microarrays containing exon junction probes are demonstrated to be a powerful tool to investigate tissue-specific regulation of AS and aberrant splicing taking place in genetic disorders.
authors.affiliation: Department of Genetics, National Research Institute for Child Health and Development, Tokyo, Japan.
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Nagao
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Togawa
authors.forename: Katsunori
authors.initials: K
authors.lastname: Fujii
authors.forename: Hideki
authors.initials: H
authors.lastname: Uchikawa
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Kohno
authors.forename: Masao
authors.initials: M
authors.lastname: Yamada
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Miyashita
mesh_terms: D017398:Alternative Splicing; D001478:Basal Cell Nevus Syndrome / Q000235:genetics*; D001932:Brain Neoplasms / Q000235:genetics; D005091:Exons / Q000235:genetics*; D006338:Heart Neoplasms / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D009928:Organ Specificity / Q000235:genetics; D000072079:Patched Receptors; D000072081:Patched-1 Receptor; D020033:Protein Isoforms / Q000235:genetics; D011956:Receptors, Cell Surface / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: C000606236:PTCH1 protein, human; D000072079:Patched Receptors; D000072081:Patched-1 Receptor; D020033:Protein Isoforms; D011956:Receptors, Cell Surface