Analysis of T-cell receptor-gamma gene rearrangements using oligonucleotide microchip: a novel approach for the determination of T-cell clonality.
T-cell clonality estimation is important for the differential diagnosis between malignant and nonmalignant T-cell proliferation. Routinely used methods include polymerase chain reaction (PCR) analysis of T-cell receptor-gamma (TCR-gamma) gene rearrangements followed by Genescan analysis, polyacrylamide gel electrophoresis, or heteroduplex analysis to visualize amplification products. Here, we present a new method for the analysis after PCR of TCR-gamma rearrangements using hybridization on oligonucleotide microchip. A microchip was designed to contain specific probes for all functional variable (V) and joining (J) gene segments involved in rearrangements of the TCR-gamma locus. Fluorescently labeled fragments of rearranged gamma-chain from patients and donors were obtained in a multiplex nested PCR and hybridized with a microchip. The results were detected using a portable microchip analyzer. Samples from 49 patients with T-cell lymphomas or leukemias and 47 donors were analyzed for T-cell clonality by microchip and single-strand conformation polymorphism analysis, which served as a standard reference method. Comparison of two techniques showed full concordance of the results. The microchip-based approach also allowed the identification of V and J gene segments involved in the particular TCR-gamma rearrangement. The sensitivity of the method is sufficient to determine 10% of clonal cells in the sample.
Gra
Olga A
OA
Department of Biological Microchips, Engelhardt Institute of Molecular Biology, Moscow, Russia.
Sidorova
Julia V
JV
Nikitin
Eugene A
EA
Turygin
Alexander Y
AY
Surzhikov
Sergey A
SA
Melikyan
Anait L
AL
Sudarikov
Andrey B
AB
Zasedatelev
Alexander S
AS
Nasedkina
Tatyana V
TV
D000328:Adult; D000368:Aged; D002999:Clone Cells / Q000166:cytology / Q000378:metabolism; D005260:Female; D005787:Gene Frequency; D015329:Gene Rearrangement, T-Lymphocyte / Q000235:genetics*; D019675:Genes, T-Cell Receptor gamma / Q000235:genetics*; D006801:Humans; D008297:Male; D008875:Middle Aged; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012680:Sensitivity and Specificity; D013601:T-Lymphocytes / Q000166:cytology* / Q000276:immunology / Q000378:metabolism*