title: Exon skipping in the ATM gene in normal individuals: the effect of blood sample storage on RT-PCR analysis.
abstract: Mutations in ATM, the gene defective in the human genetic disorder ataxia-telangiectasia (A-T), have been described in A-T patients and in a variety of tumor samples. Most of these arise due to exon skipping. We developed an RT-PCR based protein truncation test (PTT) to screen for ATM mutations in breast cancer patients showing adverse response to radiotherapy. An additional PTT product was evident in the ATM gene in peripheral blood mononuclear cells (PBMCs) from blood samples that were collected 2 days or more prior to RNA extraction. Lymphoblastoid cell lines established from the same blood samples showed no evidence of the additional band. Cloning and sequencing of the additional RT-PCR product revealed an exact deletion of exon 20 (2639 del 200), pointing to exon skipping. RT-PCR analysis of RNA extracted from freshly prepared PBMCs from 3 normal individuals showed no evidence of the additional RT-PCR product but when the blood was stored for 2-3 days prior to RNA extraction the lower molecular weight band was evident in every sample. DNA sequencing confirmed this to be due to loss of exon 20. These data suggest that mRNA-based mutation analysis on ATM should be carried out on unstored blood samples to avoid artificial loss of exons that give rise to apparent mutations.
authors.affiliation: The Queensland Cancer Fund Research Laboratories, Queensland Institute of Medical Research, Brisbane, Australia.
authors.forename: G W
authors.initials: GW
authors.lastname: Birrell
authors.forename: J R
authors.initials: JR
authors.lastname: Ramsay
authors.forename: J J
authors.initials: JJ
authors.lastname: Tung
authors.forename: M F
authors.initials: MF
authors.lastname: Lavin
mesh_terms: D017398:Alternative Splicing; D001260:Ataxia Telangiectasia / Q000235:genetics*; D064007:Ataxia Telangiectasia Mutated Proteins; D001793:Blood Preservation / Q000379:methods*; D001943:Breast Neoplasms / Q000097:blood / Q000235:genetics / Q000532:radiotherapy; D018797:Cell Cycle Proteins; D002461:Cell Line, Transformed; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins; D005091:Exons / Q000235:genetics*; D006801:Humans; D017346:Protein Serine-Threonine Kinases / Q000097:blood / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017384:Sequence Deletion; D014158:Transcription, Genetic; D025521:Tumor Suppressor Proteins
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D025521:Tumor Suppressor Proteins; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases