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Amplification and Expression of Splice Variants of the Gene Encoding the P450 Cytochrome 25-Hydroxyvitamin D₃ 1,-Hydroxylase (CYP 27B1) in Human Malignant Glioma

Institut für Humangenetik, Theoretische Medizin [R. M. M., K. R., B. D., U. F., E. M.], Neurochirurgische Universitätsklinik [W-I. S.], and Abteilung für Neuropathologie [W. F.], Universität des Saarlandes, 66421 Homburg/Saar, Germany

Purpose: Recently, we reported the isolation of six novel genes termed glioma-amplified sequences (GASs) from the glioblastoma cell line TX3868 using microdissected mediated cDNA capture (U. Fischer et al., Hum. Mol. Genet., 5: 595–600, 1996). The aim of this study was to further characterize the gene GAS89.

Experimental Design: To determine the amplification frequency, we performed comparative PCR studies and Southern blot hybridization experiments. To identify full-length clones of GAS89 we screened a HybriZAP library. Reverse transcription-PCR was performed to isolate splice variants and to determine expression levels.

Results: We identified for the gene GAS89 an amplification frequency of 25% in 28 examined glioblastoma multiforme samples. Screening a HybriZAP library, we isolated an incomplete gene sequence showing identity with the gene for 25-hydroxyvitamin D₃ 1,-hydroxylase. Different full-length clones were then isolated using PCR primers chosen from the 3'- and 5'-untranslated regions. As determined by sequencing, the clones represent various splice variants of the 25-hydroxyvitamin D₃ 1,-hydroxylase gene. The clones encode truncated proteins but also one potentially functional enzyme variant. Reverse transcription-PCR studies revealed overexpression of several variants in glioblastoma samples with GAS89 amplification in comparison with normal brain RNA and glioblastoma without GAS89 amplification.

Conclusions: This is the first report of gene amplification for 25-hydroxyvitamin D₃ 1,-hydroxylase and the appearance of mRNA splice variants in glioblastoma multiforme. The endogenous expression of the 25-hydroxyvitamin D₃ 1,-hydroxylase gene and the appearance of alternative splice variants reveal a new feature of the molecular pathogenesis of glioblastoma and may represent a new target for glioma therapy.

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