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Updated: Aug 15, 2026
Detection of Alternative Splicing During Epithelial-Mesenchymal Transition
Published on: October 9, 2014
Detecting tissue-specific alternative splicing and disease-associated aberrant splicing of the PTCH gene with exon
Kazuaki Nagao1, Naoyuki Togawa, Katsunori Fujii
1Department of Genetics, National Research Institute for Child Health and Development, Tokyo, Japan.
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.
Insights
Researchers discovered novel PTCH gene splicing variations, including a brain-specific exon, using oligonucleotide microarrays. This method also identified disease-associated splicing defects in nevoid basal cell carcinoma syndrome (NBCCS) patients.
Area of Science:
- Genetics
- Molecular Biology
- Bioinformatics
Background:
- Mutations in the patched (PTCH) gene are linked to nevoid basal cell carcinoma syndrome (NBCCS), a disorder associated with developmental anomalies and increased cancer risk.
- Alternative splicing (AS) of PTCH mRNA can generate diverse isoforms, but comprehensive analysis and disease relevance are not fully understood.
Purpose of the Study:
- To identify and characterize novel PTCH mRNA isoforms generated by alternative splicing.
- To investigate the tissue-specific regulation of PTCH alternative splicing.
- To develop and validate a microarray-based method for detecting aberrant PTCH splicing in genetic disorders like NBCCS.
Main Methods:
- Design and utilization of custom oligonucleotide microarrays with exon, exon-exon junction, and intron probes for comprehensive PTCH splicing analysis.
- Normalization of probe intensities based on total PTCH expression and probe sensitivity.
- Validation of microarray findings using reverse transcription polymerase chain reaction (RT-PCR).
Main Results:
- Identification of numerous PTCH mRNA isoforms, including a novel exon (12b), primarily generated through alternative splicing.
- Exon 12b demonstrated specific expression in brain and heart tissues, particularly the cerebellum.
- The developed microarray technique successfully detected disease-associated aberrant splicing events in NBCCS patients, linked to mutations disrupting splice donor sites.
Conclusions:
- Oligonucleotide microarrays with exon junction probes are effective for studying tissue-specific alternative splicing.
- This approach provides a powerful tool for investigating aberrant splicing in genetic disorders, aiding in the diagnosis and understanding of diseases like NBCCS.
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