RESUMEN
Detection of clonal T cell receptor gamma (TCRG) gene rearrangements by PCR is widely used in both the diagnostic assessment of lymphoproliferative disorders and the follow-up of acute lymphoblastic leukaemia (ALL), when residual positivity in excess of 10(-3) at morphological complete remission is increasingly recognised to be an independent marker of poor prognosis. This is largely based on specific detection of V-J rearrangements from childhood cases. We describe rapid, multifluorescent Vgamma and Jgamma PCR typing of multiplex amplified diagnostic samples, as applied to 46 T-ALL. These strategies allow selected analysis of appropriate cases, immediate identification of Vgamma and Jgamma segments in over 95% of alleles, improved resolution and precision sizing and a sensitivity of detection at the 10(-2)-10(-3) level. We demonstrate preferential V-J combinations but no difference in V-J usage between children and adults, nor between SIL-TAL1-negative and -positive cases. A combination of fluorescent multiplex and Vgamma-Jgamma-specific monoplex follow-up, as described here, will allow detection of both significant clonal evolution and of the diagnostic clone at a level of prognostic significance, by techniques which can readily be applied to large-scale prospective studies for which real-time analysis is required.
Asunto(s)
Leucemia-Linfoma de Células T del Adulto/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Adolescente , Secuencia de Bases , Niño , Clonación Molecular , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Fluorescencia , Humanos , Región de Unión de la Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/inmunología , Reacción en Cadena de la Polimerasa , Receptores de Antígenos de Linfocitos T gamma-delta/genéticaRESUMEN
The selection of various T-cell receptor (TCR) gene families and complex rearrangements during intra-thymic differentiation provide the basis for the expression of antigen specificity by mature T cells. TCR beta variable (TCRBV) transcripts can be identified by RT-PCR, but multiple reactions are required to detect all genes of the TCRBV subfamilies. We describe here a multiplex PCR method that amplifies 46 functional genes comparing 23 TCRBV families in 5 reactions where each reaction contains 4 to 7 specific primers together with a single fluorescence-tagged TCR beta constant region primer. Between 8 and 10 distinct subtypes within each of the 23 TCRBV families can be identified by analysis of the CDR3 length. Multiplex PCR products isolated from agarose gels can be subjected to direct sequencing for confirmation and definitive clonotyping if necessary. The data illustrated here show that the multiplex PCR technique is useful for screening TCRBV usage and can be easily adapted for analysis of clonal composition in T-cell populations.
Asunto(s)
Reacción en Cadena de la Polimerasa/métodos , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Línea Celular , Cartilla de ADN , Citometría de Flujo , Humanos , Linfocitos TRESUMEN
The field of medical, molecular diagnostics has grown rapidly over the last few years, becoming increasingly informative to both clinician and patient. As genes associated with specific diseases have been discovered and sequenced, many genotype-phenotype relationships have been defined. For those genetic diseases with associated, defined, gene mutations, sophisticated DNA diagnostic tests are being developed. As an example, the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, is associated with Cystic Fibrosis (CF). We have developed a new molecular diagnostic technology, PCR/OLA/SCS, and applied it first to the diagnosis of CF. Test design in the field of molecular diagnostics must consider such characteristics as specificity, sensitivity, ease and speed of protocol, multiplex capacity, and cost. PCR/OLA/SCS addresses these requirements. Polymerase Chain Reaction (PCR) is widely used in both diagnostics and research. We have combined well established PCR technology with Oligonucleotide Ligation Assay (OLA) and Sequence-Coded Separation (SCS), two relatively new technologies.