A novel splice variant of the DNA-PKcs gene is associated with clinical and cellular radiosensitvity in a xeroderma pigmentosum patient

Abstract

Background: Radiotherapy-induced DNA double strand breaks (DSB) are critical cytotoxic lesions. Inherited defects in DSB DNA repair pathways lead to hypersensitivity to ionising radiation, immunodeficiency and increased cancer incidence. A patient with xeroderma pigmentosum complementation group C, with a scalp angiosarcoma exhibited dramatic clinical radiosensitivity following radiotherapy, resulting in death. A fibroblast cell line from non-affected skin (XP14BRneo17) was hypersensitive to ionising radiation and defective in DNA double strand break repair.

Aim: To determine the genetic defect causing cellular radiation hypersensitivity in XP14BRneo17 cells.

Methods: Functional genetic complementation whereby copies of human chromosomes containing genes involved in DNA DSB repair (chromosomes 2, 5, 8 10, 13 and 22) were individually transferred to XP14BRneo17 cells in an attempt to correct the radiation hypersensitivity. Clonogenic survival assays and γ-H2AX immunofluorescence were conducted to measure radiation sensitivity and repair of DNA DSBs. DNA sequencing of defective DNA repair genes was performed.

Results: Transfer of chromosome 8 (location of DNA-PKcs gene), and transfection of a mammalian expression construct containing the DNA-PKcs cDNA restored normal ionising radiation sensitivity and repair of DNA DSBs in XP14BRneo17 cells. DNA sequencing of the DNA-PKcs coding region revealed a 249 bp deletion (between base pairs 3656-3904) encompassing exon 31 of the gene.

Conclusion: We provide evidence of a novel splice variant of the DNA-PKcs gene associated with radiosensitivity in a xeroderma pigmentosum patient and report the first double mutant in distinct DNA repair pathways being consistent with viability.