A novel splice variant of the DNA-PKcs gene is associated with clinical and cellular radiosensitivity in a patient with xeroderma pigmentosum.
BACKGROUND
Radiotherapy-induced DNA double-strand breaks (DSBs) are critical cytotoxic lesions. Inherited defects in DNA DSB 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 DSB repair.


OBJECTIVE
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 gamma-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 and 3904) encompassing exon 31 of the gene.


CONCLUSIONS
We provide evidence of a novel splice variant of the DNA-PKcs gene associated with radiosensitivity in a patient with xeroderma pigmentosum and report the first double mutant in distinct DNA repair pathways being consistent with viability.
Brunel Institute of Cancer Genetics and Pharmacogenomics, Division of Biosciences, School of Health Sciences and Social Care, Brunel University, Uxbridge UB8 3PH, Middlesex, UK.
Fatemeh
F
Abbaszadeh
Peter H
PH
Clingen
Colin F
CF
Arlett
Piers N
PN
Plowman
Emma C
EC
Bourton
Matthew
M
Themis
Evgeny M
EM
Makarov
Robert F
RF
Newbold
Michael H L
MH
Green
Christopher N
CN
Parris
D017398:Alternative Splicing / Q000502:physiology; D000595:Amino Acid Sequence; D002470:Cell Survival / Q000235:genetics / Q000528:radiation effects; D002478:Cells, Cultured; D051747:DNA-Activated Protein Kinase / Q000235:genetics / Q000502:physiology*; D006258:Head and Neck Neoplasms / Q000235:genetics / Q000473:pathology / Q000532:radiotherapy*; D006394:Hemangiosarcoma / Q000235:genetics / Q000473:pathology / Q000532:radiotherapy*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000502:physiology; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000502:physiology*; D020033:Protein Isoforms / Q000235:genetics / Q000502:physiology; D011832:Radiation Injuries / Q000235:genetics; D011836:Radiation Tolerance / Q000235:genetics*; D012535:Scalp; D017386:Sequence Homology, Amino Acid; D012878:Skin Neoplasms / Q000235:genetics / Q000473:pathology / Q000532:radiotherapy*; D014983:Xeroderma Pigmentosum / Q000235:genetics* / Q000473:pathology
D007527:Isoenzymes; D009687:Nuclear Proteins; D020033:Protein Isoforms; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human