The CDKN2A locus on chromosome 9p21 contains the p14 ARF and p16 INK4a genes, and is frequently deleted in human neoplasms, including brain tumors. In this study, we screened 34 primary (de novo) glioblastomas and 16 secondary glioblastomas that had progressed from low-grade diffuse astrocytomas for alterations of the p14 ARF and p16 INK4a genes, including homozygous deletion by differential PCR, promoter hypermethylation by methylationspecific PCR, and protein expression by immunohistochemistry. A total of 29 glioblastomas (58%) had a p14 ARF homozygous deletion or methylation, and 17 (34%) showed p16 INK4a homozygous deletion or methylation. Thirteen glioblastomas showed both p14 ARF and p16 INK4a homozygous deletion, while nine showed only a p14 ARF deletion. Immunohistochemistry revealed loss of p14 ARF expression in the majority of glioblastomas (38/50, 76%), and this correlated with the gene status, i.e. homozygous deletion or promoter hypermethylation. There was no significant difference in the overall frequency of p14 ARF and p16 INK4a alterations between primary and secondary glioblastomas. The analysis of multiple biopsies from the same patients revealed hypermethylation of p14 ARF (5/15 cases) and p16 INK4a (1/15 cases) already at the stage of low-grade diffuse astrocytoma but consistent absence of homozygous deletions. These results suggest that aberrant p14 ARF expression due to homozygous deletion or promoter hypermethylation is associated with the evolution of both primary and secondary glioblastomas, and that p14 ARF promoter methylation is an early event in subset of astrocytomas that undergo malignant progression to secondary glioblastoma.

Maternal use of BZD and/or HBRA may increase the risk for preterm birth and low birth weight and cause neonatal symptoms, but does not appear to have a strong teratogenic potential. The tentative association with pylorostenosis and alimentary tract atresia needs confirmation.

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