Effectiveness of capillary electrophoresis using fluorescent-labeled primers in detecting T-cell receptor gamma gene rearrangements

Abstract

We describe the use of fluorescent-labeled primers to analyze T-cell receptor gamma gene rearrangements (TCR gamma GR) using capillary electrophoresis in the ABI Prism 310 Genetic Analyzer. We also compare the performance with denaturing gradient gel electrophoresis (DGGE). In a single multiplex polymerase chain reaction (PCR) we amplified TCR gamma GR with primers for all known groups of variable region genes, and joining region genes described in lymphoid neoplasms. Ten reactive samples, followed by five cell lines and 25 tumor samples with 41 individual TCR gamma GR (due to many biallelic rearrangements) previously identified by DGGE, were analyzed to validate the technique. The capillary electrophoresis protocol has 92% concordance for both TCR clonal status (23 of 25) and 95% concordance in the number of individual TCR gamma GR (38 of 41) identified by DGGE. The reproducible sensitivity for detecting TCR gamma GR diluted in reactive lymphoid DNA is 2% in clinical applications. Discrimination of predominant rearrangements requires a minimum ratio of two times the height of the normal distribution of polyclonal peaks. Capillary electrophoresis can provide results within 60 minutes for each specimen after PCR is complete. Capillary electrophoresis provides a faster result than sequence-based separation methods and gives an archival electronic record. Fluorescent labeling allows the identification of both the variable and joining gene segments used in a TCR gamma GR. The effectiveness of capillary electrophoresis is similar to DGGE.

Figure 1.

Schematic of gene segments in the T-cell receptor γ gene. V, Variable region; J, Joining region; GP, Group; C1, Constant region.

Figure 2.

The height (h2) of the peak must exceed the height (h1) of the polyclonal background by a ratio of more than 2.0 (RPB = h2/h1) to define a clonal result. Evaluation of bialellic TCRγGR in cell lines where one peak (h3) is outside the normal distribution of polyclonal T cells suggests that the ratio of such peaks (h3) are better assessed by adding h3 to h1 to calculate h2. Otherwise one peak will be called positive and one peak will be called negative.

Figure 3.

A: Polyclonal TCRγ gene rearrangements are demonstrated by the normal distribution pattern obtained from peripheral blood lymphocytes. Labeling of the TCRγGRs is as follows: the common joining region genes Jγ1/Jγ2-black, JγP-blue, and JγP1/JγP2-green. B: Molt4 cell line with biallelic Vγ2-JγP1/JγP2, Vγ10-JγP1/JγP2 TCRγGR. Molt4 DNA was diluted 50:50 in peripheral blood lymphocyte DNA before PCR. The green peaks identify that the JγP1/JγP2 joining segment is used in both TCRγGR. GR = gene rearrangement. Peak height of a clonal peak should be a minimum of two times the height of the polyclonal background. C: Fluorescent analysis of dual TCRγGR (Vγ2-JγP and Vγ9-JγP1/JγP2) in a peripheral T-cell lymphoma, demonstrating the capability of identifying different joining region genes used in each rearrangement. JγP-blue; JγP1/JγP2-green. D: Sensitivity study by serial dilution of TCRγGR (Vγ3-Jγ1/Jγ2; Vγ4-Jγ1/Jγ2) CEM in peripheral blood lymphocytes. A detection sensitivity of at least 0.5% was obtained with the CEM cell line DNA serially diluted in peripheral blood lymphocyte DNA. Below 0.5%, the ratio (RPB) of the peak height of the TCRγGRs will fall below two times of the polyclonal background. E: Discriminating a clonal peak (right arrow) from the background polyclonal T-cells in this case is difficult. Neither of the two peak heights (left arrows) is greater than two times the maximum height (at 1200) of the main polyclonal background. This illustrates the potential problem that can occur with identifying the significance of oligoclonal peaks. F: Polyclonal TCRγ gene rearrangements are demonstrated in peripheral blood lymphocytes using variable region primers labeled as follows: the common variable region genes of Vγ2–8 and Vγ3-black. The lesser common genes are Vγ9-green; Vγ10-red; Vγ11-blue. G: Fluorescent analysis identifies the variable gene used in dual TCRγGR (Vγ2 and Vγ10). in a peripheral T-cell lymphoma., Vγ2-black; Vγ10-red. x axis, nucleotide length; y axis, intensity of signal; Rox-labeled size standard at 200 nt.