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

Abstract

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) gamma 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 gamma gene were selected at different sites in the TCR gamma gene so that the size of PCR products could define which V subgroup was involved in rearranged TCR gamma genes; gamma genes involving different V and J subgroups could be determined directly by PCR. Mutant T-lymphocytes with rearranged TCR gamma 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 gamma genes. Rearrangements among all subgroups of the V and J gene segments of the TCR gamma gene could be detected. VgammaI and Jgamma1/2 subgroups were involved in 69 and 71% of rearranged TCR gamma genes, respectively. This PCR-based analysis of TCR gamma 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.