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Detection of PTEN nonsense mutation and ψPTEN expression in central nervous system high-grade astrocytic tumors by a yeast-based stop codon assay

Oncogene volume 19, pages 4346–4353 (2000)Cite this article

Abstract

We have developed a new yeast-based assay for the detection of PTEN nonsense mutation, and applied it to a total of 42 astrocytic tumors. The assay utilizes homologous recombination of PCR-amplified PTEN cDNA samples to a yeast vector which expresses an in-frame PTEN::ADE2 chimera protein. An allele of nonsense mutation in the sample PTEN mRNA gives a truncated chimera protein in a yeast cell, resulting in the formation of a red colony. The assay and subsequent sequence analysis demonstrated nonsense mutations as red colonies of more than 10% in one of 10 anaplastic astrocytomas and six of 18 glioblastomas, but none in six pilocytic astrocytomas or in eight astrocytomas. Sequence analysis of white colonies showed one missense mutation in a glioblastoma. Interestingly, four of seven nonsense mutations were frame-shifts due to exon skipping. In addition, pink colonies were found in one of six pilocytic astrocytomas, three of eight astrocytomas, two of 10 anaplastic astrocytomas, and 10 of 18 glioblastomas. Sequence analysis of the pink colonies revealed a sequence similar to those reported as ψPTEN/PTH2. By testing mRNA and genomic DNA, it was found to be a processed pseudogene which was transcribed. The ψPTEN expression was complementary to PTEN mutation, for 14 of 18 glioblastomas showed either PTEN mutation or ψPTEN expression and only one case showed both PTEN mutation and ψPTEN expression (P<0.046), suggesting a pathological role of ψPTEN expression as an alternative to PTEN mutation in glioblastomas.

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Acknowledgements

We thank Drs Yutaka Sawamura, Kenji Mitsumori and Hisatoshi Saito for providing clinical samples, Ms Yumiko Shinohe for her excellent technical assistance and Ms Masako Yanome for her help in preparation of the manuscript.

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Authors and Affiliations

  1. Department of Neuropathophysiology, Section of Neurosurgery, Hokkaido University Graduate School of Medicine, Sapporo, 060-8638, Japan

    C-L Zhang, H Kobayashi, M Nozaki & H Abe

  2. Division of Cell Biology, Cancer Institute, Hokkaido University Graduate School of Medicine, Sapporo, 060-8638, Japan

    M Tada & T Moriuchi

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Zhang, CL., Tada, M., Kobayashi, H. et al. Detection of PTEN nonsense mutation and ψPTEN expression in central nervous system high-grade astrocytic tumors by a yeast-based stop codon assay. Oncogene 19, 4346–4353 (2000). https://doi.org/10.1038/sj.onc.1203795

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