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1

V gene replacement in T and B lymphocytes: illicit or regimented rearrangement?

100%
Golub R.
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issue 4
255-262
EN
Lymphocyte can undergo novel types of secondary genetic rearrangements that alter the specificity of a pre-existing B cell receptor (BCR) or T cell receptor (TCR). V gene replacement is one of them and it replaces an already rearranged V gene segment with an upstream germline V gene segment. This review focuses on the molecular aspect of rearrangement. The role of this mechanism in the immune system is debated.
2

Implementation of the standard strategy for identification of Ig/TCR targets for minimal residual disease diagnostics in B-cell precursor ALL pediatric patients: Polish experience

100%
Dawidowska M., Jolkowska J., Szczepanski T., Derwich K., Wachowiak J., Witt M.
Archivum Immunologiae et Therapiae Experimentalis
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2008
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vol. 56
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issue 6
409-418
EN
Introduction: Minimal residual disease (MRD), detected based on immunoglobulin and T-cell receptor (Ig/TCR) gene rearrangements as markers of residual leukemic cells, is currently the most reliable prognostic factor in acute lymphoblastic leukemia (ALL). A feasibility study is presented of the standard strategy for the identification of Ig/TCR targets for MRD diagnostics in Polish ALL patients by identifying Ig/TCR gene rearrangement pattern using standard primer sets and protocols. Materials and Methods: The PCR-heteroduplex approach based on BIOMED-1 and BIOMED-2 protocols (recommended as the European standard) was used to detect IGH, IGK-Kde, TCRD, TCRG, and TCRB rearrangements in 58 Polish B-cell precursor ALL patients. Sequencing and homology analysis between the obtained and germline Ig/TCR sequences enabled identification of the rearrangements.The U-Gauss test was used for statistical analysis of the Ig/TCR rearrangement pattern in Polish patients compared with relevant data on other nationalities. Results: The following pattern was identified: IGH: 83% (VH-JH: 74%, DH-JH: 9%), IGK-Kde: 41%, TCRD: 78% (incomplete TCRD: 55%, Vdelta2-Ddelta3: 45%, Ddelta2-Ddelta3: 21%, Vdelta2-Jalpha: 35%), TCRG: 50%, and TCRB: 13%. Considerable convergence of the Ig/TCR pattern in Polish patients and those of other nationalities (mainly West Europeans) was demonstrated. Statistically relevant differences were only found between the incidence of DH-JH in Polish (9%) and Dutch patients (24%; p<0.05) and Polish and Italian patients (19%; p<0.05), VH-JH in Polish (74%) and Chilean patients (100%; p<0.05), and TCRG in Polish (50%) and Brazilian patients (69%; p<0.05). Conclusions: The convergence of Ig/TCR patterns in Polish and European patients indicates that the strategy for Ig/TCR target identification based on standard primers and protocols might be directly used for the construction of Polish standards and recommendations for MRD diagnostics.
3

Novel competitive PCR methods for quantitation of T-cell receptor delta (TCRD) gene rearrangements

100%
Masternak M. M., Przybylski G. K., Smoczkiewicz P., Plotek W., Kowalczyk D. W., Nowak J. S.
Journal of Applied Genetics
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2002
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vol. 43
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issue 2
235-244
EN
Since the invention of the polymerase chain reaction (PCR) several quantitative PCR-based approaches have been described. Recently, the real-time PCR method became a standard in quantitative PCR, although high costs of the necessary equipment and reagents make it unaffordable for many laboratories. In this paper we describe two novel competitive PCR techniques, which were used to determine the frequency of T-cell receptor delta gene (TCRD) rearrangements in peripheral blood leukocytes. In the reference gene competitive PCR (rgc-PCR) the rearranged TCRD gene competes with the reference gene (RAG1) for common reagents (dNTPs and Taq polymerase). The intensity ratio of amplification products, TCRD/RAG1, corresponds to the portion of cells containing a rearrangement. A series of reactions was performed, in which RAG1 primers were added to the PCR after different numbers of cycles. On the basis of the number of cycles needed to obtain equal band intensity, the frequency of cells containing a rearrangement was calculated. In the common primer competitive PCR (cpc-PCR), two gene rearrangements, Vdelta1-Jdelta1 and Vdelta2-Jdelta1, compete for the common Jd1 primer. The competing genes are amplified from the same genomic DNA template; therefore unlike in the method using the internal competitor, the results are not affected by the quantity or quality of the analysed sample. We showed that the rgc-PCR and cpc-PCR are reliable and give reproducible results. The methods do not require any expensive equipment or reagents, and can be used to determine the frequency of gene rearrangements.
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