title: Rapid screen for truncating ATM mutations by PTT-ELISA.
abstract: Mutations in the ataxia-telangiectasia mutated (ATM) gene are responsible for the autosomal recessive genetic disorder, ataxia-telangiectasia (A-T). Approximately 80% of ATM mutations found in A-T patients results in truncations, which can be detected by Protein Truncation Test (PTT). Conventional PTT uses SDS-PAGE electrophoresis to detect mobility of radiolabeled truncated protein fragments. In this study, we developed a non-radioactive Protein Truncation Test which utilizes an enzyme-linked immunosorbent assay (PTT-ELISA) to detect ATM mutations in eight overlapping fragments. N- and C-terminal epitopes (c-myc and V5, respectively) were introduced into transcription/translation products, which could then be detected by Sandwich ELISA. Using this assay, we screened 9 newly diagnosed A-T patients consecutively. Of the 18 expected mutations, 14 truncating mutations were independently identified by cDNA direct sequencing and/or DNA dHPLC analysis. PTT-ELISA detected all of these 14. Four mutations were novel. The PTT-ELISA provides a rapid method for detecting truncating mutations in large genes and should be considered prior to using more laborious or costly methods, such as direct sequencing.
authors.affiliation: Department of Pathology and Laboratory Medicine, The David Geffen School of Medicine at UCLA, CA 90095, United States.
authors.forename: Liutao
authors.initials: L
authors.lastname: Du
authors.forename: Chih-Hung
authors.initials: CH
authors.lastname: Lai
authors.forename: Patrick
authors.initials: P
authors.lastname: Concannon
authors.forename: Richard A
authors.initials: RA
authors.lastname: Gatti
mesh_terms: D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000235:genetics*; D002478:Cells, Cultured; D004252:DNA Mutational Analysis / Q000379:methods*; D004268:DNA-Binding Proteins / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D012680:Sensitivity and Specificity; D025521:Tumor Suppressor Proteins / Q000235:genetics*
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