Characteristics of T cell receptor beta gene rearrangements and its role in minimal residual disease detection in childhood T-cell acute lymphoblastic leukemia.
- Author:
Jie-yu LIU
1
;
Zhi-gang LI
;
Chao GAO
;
Lei CUI
;
Min-yuan WU
Author Information
- Publication Type:Journal Article
- MeSH: Adolescent; Base Sequence; Child; Child, Preschool; Female; Gene Rearrangement, beta-Chain T-Cell Antigen Receptor; genetics; Humans; Infant; Male; Molecular Sequence Data; Neoplasm, Residual; diagnosis; genetics; Polymerase Chain Reaction; methods; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; diagnosis; genetics; Sensitivity and Specificity; Sequence Analysis, DNA
- From: Chinese Journal of Pediatrics 2008;46(7):487-492
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo explore the characteristics of T cell receptor beta (TCRbeta) gene rearrangements in children with T-cell acute lymphoblastic leukemia (T-ALL) and establish a system of quantitative detection of MRD with real-time quantitative (RQ-PCR) targeted at TCRbeta gene rearrangement.
METHODSMultiplex polymerase chain reaction (PCR) designed by BIOMED-2 was used to detect TCRbeta gene rearrangements in the bone marrow samples of 26 children with T-ALL. Sequence of junction region were then compared and analyzed in IMGT database. Allele specific oligonucleotide (ASO) upstream primers were designed complementary to the V-D-J or D-J junctional region of TCRbeta gene rearrangements. Samples at diagnosis were serially diluted in DNA obtained from mononuclear cells (MNC) from a pool of 20 healthy donors to generate the patient specific standard curves. Subsequently, a TCRbeta RQ-PCR assay to quantify MRD with germline Jbeta primer/probe combinations was applied in six patients. To check the quantity and quality of DNA, the investigators used RQ-PCR analysis for the N-ras gene.
RESULTSClonal rearrangements were identified in 92.3% childhood T-ALL (Vbeta-Dbeta-Jbeta rearrangements in 84.6%, Dbeta-Jbeta rearrangements in 50%). Comparative sequence analysis of 42 TCRbeta recombinations revealed two downstream Vbeta families (BV5, BV6) were preferentially used. The segment Jbeta2.7 in childhood T-ALL was preferentially used. Jbeta1.3, Jbeta2.4, and Jbeta2.6 were not found to be used. The slope of the standard curves was from -3.54 to -3.37 and the intercepts were from 19.35 to 20.51. The correlation coefficients of all 6 standard curves were excellent (> or = 0.98). All the RQ-PCR quantitative range reached 10(-4). MRD analysis of follow up samples showed that MRD increased before relapse.
CONCLUSIONRQ-PCR analysis of TCRbeta gene rearrangements was highly sensitive and specific, it will be of high value for future T-ALL MRD studies. And quantitative and serial study of MRD may be of prognostic importance.