Analysis of T-cell receptor-γ gene rearrangements using heteroduplex analysis by high-resolution microcapillary electrophoresis.
Determination of T-cell clonality has an important additional value for diagnosis of T-cell lymphomas. Various molecular methods have been developed, including polymerase chain reaction (PCR) of T-cell receptor γ (TCRγ). The detection of PCR products usually relies commonly on either GeneScan (GS) analysis or heteroduplex (HD) analysis by polyacrylamide gel electrophoresis (PAGE). These techniques have their disadvantages, being relatively time-consuming and laborious or requiring expensive equipment. Here, we propose an alternative method that combines multiplex PCR and HD analysis by microcapillary electrophoresis (ME) on the Agilent 2100 Bioanalyzer. The sensitivity of the method was determined with clonal PEER T-cell line DNA dilution in polyclonal DNA and was evaluated as 1-5%. Fifty-three samples from patients with T-cell lymphoproliferative disorders were analyzed by HD analysis using ME and GS analyses. Comparison of the two techniques showed them to be highly concordant (93% similarity). The rate of clonality detection by GS analysis was higher than HD analysis by ME, but none of the discordant patients (n=5) has yet developed lymphoma. HD analysis by ME to reveal TCRγ gene rearrangements in clinical specimens was consistent with clinical data and the outcome of patients. Detection of T-cell clonality by HD analysis with ME is sensitive, practical, safe and represents a potential alternative to PAGE and GS analysis.
Département de Biochimie et Biologie Moléculaire, Assistance Publique des Hôpitaux de Marseille, Marseille, France. Nathalie.Beaufils@mail.ap-hm.fr
Nathalie
N
Beaufils
Amin
A
Ben Lassoued
Arnaud
A
Essaydi
Jean-Philippe
JP
Dales
Christine
C
Formisano-Tréziny
Nathalie
N
Bonnet
Jean-Jacques
JJ
Grob
Jean
J
Gabert
D002999:Clone Cells / Q000378:metabolism; D004526:Efficiency; D019075:Electrophoresis, Capillary / Q000379:methods; D015334:Gene Rearrangement, gamma-Chain T-Cell Antigen Receptor; D019675:Genes, T-Cell Receptor gamma / Q000235:genetics*; D020180:Heteroduplex Analysis / Q000379:methods*; D006801:Humans; D008232:Lymphoproliferative Disorders / Q000175:diagnosis / Q000235:genetics; D008832:Microchemistry / Q000379:methods; D012189:Retrospective Studies; D012680:Sensitivity and Specificity