Multiplex polymerase chain reaction-based analysis of T-cell receptor γ gene rearrangements for the determination of T-lymphocyte clonality

• Published in: Environmental and molecular mutagenesis 2000, Vol.35 (1), p.1-8
• Main Authors: Ma, Hongbao, Smith, Darlene H., Hsie, Abraham W., Ward Jr, Jonathan B.
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Electrophoresis, Polyacrylamide Gel; Gene Rearrangement, gamma-Chain T-Cell Antigen Receptor; General aspects. Methods; hprt gene; Humans; Hypoxanthine Phosphoribosyltransferase - genetics; Medical sciences; multiplex PCR; Mutation; mutational spectrum; polyacrylamide gel electrophoresis (PAGE); Polymerase Chain Reaction - methods; T-cell receptor gamma (TCR γ) gene; T-lymphocyte clonality; T-Lymphocytes - enzymology; TCR ^g gene; Toxicology
• ISSN: 0893-6692; 1098-2280
• DOI: 10.1002/(SICI)1098-2280(2000)35:1<1::AID-EM1>3.0.CO;2-C

Determination of the frequency of mutations at hprt or other loci in human lymphocytes provides a useful biomarker for human exposure to mutagens. One problem, however, is distinguishing between unique mutants and sibling mutants arising as progeny of an earlier mutant cell. We have developed a multiplex polymerase chain reaction (PCR)–based method to analyze T‐cell receptor (TCR) γ gene rearrangements for determination of T‐cell clonality in mutational spectrum analysis. PCR primers for different subgroups of the V gene segment of the TCR γ gene were selected at different sites in the TCR γ gene so that the size of PCR products could define which V subgroup was involved in rearranged TCR γ genes; γ genes involving different V and J subgroups could be determined directly by PCR. Mutant T‐lymphocytes with rearranged TCR γ genes containing the same V and J subgroups were analyzed using PCR‐based denaturing polyacrylamide gel electrophoresis. All of the 161 hprt mutant clones analyzed contained rearranged TCR γ genes. Rearrangements among all subgroups of the V and J gene segments of the TCR γ gene could be detected. VγI and Jγ1/2 subgroups were involved in 69 and 71% of rearranged TCR γ genes, respectively. This PCR‐based analysis of TCR γ gene rearrangements provides a simple and comprehensive method for identifying the clonality of mutant T‐lymphocytes in human hprt mutant lymphocyte assay and mutational spectrum analysis. Environ. Mol. Mutagen. 35:1–8, 2000 © 2000 Wiley‐Liss, Inc.