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Background: PhiC31 integrase facilitates efficient integration of transgenes into human and mouse genomes and is considered for clinical gene therapy. However recent studies have shown that the enzyme can induce various chromosomal abnormalities in primary human embryonic cells and mammalian cell lines. The mechanisms involved are unknown, but it has been proposed that PhiC31 attachment sites in the host genome recombine leading to chromosomal translocations., Results: We have studied possible effects of the PhiC31 integrase expression in human adult fibroblasts by karyotyping. All control cells were cytogenetically normal, whereas cells expressing PhiC31 integrase show chromosomal abnormalities confirming our previous results using primary embryonic fibroblasts. In order to study the early mechanisms involved we measured H2AX phosphorylation - a primary event in the response to DNA double-strand-breaks. Transient transfection with PhiC31 integrase encoding plasmids lead to an elevated number of cells positive for H2AX phosphorylation detected by immunofluorescence. Western blot analysis confirmed the upregulated H2AX phosphorylation, whereas markers for apoptosis as well as p53 and p21 were not induced. Cells transfected with plasmids encoding the Sleeping Beauty transposase remained cytogenetically normal, and in these cells less upregulation of H2AX phosphorylation could be detected., Conclusion: In primary human fibroblasts expression of PhiC31 integrase leads to a DNA damage response and chromosomal aberrations.

Menkes disease is an X-linked disorder of copper metabolism caused by mutations in the ATP7A gene. Whereas most of the patients exhibit a severe classical form, about 9% of the patients exhibit a milder form of Menkes disease. The mildest form is called occipital horn syndrome (OHS). Mutations in the ATP7A gene can be identified in 95-98% of the Menkes disease patients by standard screening techniques. Investigation of RNA isolated from the fibroblasts of eleven patients with no identified mutations was performed, and revealed inclusion of new pseudo-exons into the ATP7A mRNA from three unrelated patients: two patients with OHS and one patient with classical Menkes disease. The pseudo-exons were inserted between exons 10 and 11, between exons 16 and 17 and between exons 14 and 15 in the three patients, as a result of deep intronic mutations. This is the first time the activation of pseudo-exons is demonstrated in the ATP7A gene, and it demonstrates the usefulness of RNA analysis, in terms of revealing disease-causing mutations in noncoding regions. The fact that three different mutations cause disease by the activation of pseudo-exon inclusion also indicates that in Menkes disease this is an important mechanism, which has hitherto been overlooked. [ABSTRACT FROM AUTHOR]

Background: PhiC31 integrase facilitates efficient integration of transgenes into human and mouse genomes and is considered for clinical gene therapy. However recent studies have shown that the enzyme can induce various chromosomal abnormalities in primary human embryonic cells and mammalian cell lines. The mechanisms involved are unknown, but it has been proposed that PhiC31 attachment sites in the host genome recombine leading to chromosomal translocations. Results: We have studied possible effects of the PhiC31 integrase expression in human adult fibroblasts by karyotyping. All control cells were cytogenetically normal, whereas cells expressing PhiC31 integrase show chromosomal abnormalities confirming our previous results using primary embryonic fibroblasts. In order to study the early mechanisms involved we measured H2AX phosphorylation - a primary event in the response to DNA double-strand-breaks. Transient transfection with PhiC31 integrase encoding plasmids lead to an elevated number of cells positive for H2AX phosphorylation detected by immunofluorescence. Western blot analysis confirmed the upregulated H2AX phosphorylation, whereas markers for apoptosis as well as p53 and p21 were not induced. Cells transfected with plasmids encoding the Sleeping Beauty transposase remained cytogenetically normal, and in these cells less upregulation of H2AX phosphorylation could be detected. Conclusion: In primary human fibroblasts expression of PhiC31 integrase leads to a DNA damage response and chromosomal aberrations. [ABSTRACT FROM AUTHOR]