Quantification of κ-deleting recombination excision circles in Guthrie cards for the identification of early B-cell maturation defects

Department of Pediatrics, National Defense Medical College, Saitama, Japan.
The Journal of allergy and clinical immunology (Impact Factor: 11.48). 03/2011; 128(1):223-225.e2. DOI: 10.1016/j.jaci.2011.01.052
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    • "KREC measurement has been proposed as a potential tool for the identification of these two diseases, because B-cell maturation defects occur before IGKDEL events, and therefore KRECs should be not produced/detected in these patients. Indeed, a study performed by analyzing KRECs derived from dried blood spots revealed that no KRECs were detected in 30 XLA and 5 non-XLA patients [40]. We employed the combined TREC/KREC assay to measure the extent of T- and B-cell neoproduction in CVID adult patients with no acute infections and undergoing Ig passive immunotherapy. "
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    ABSTRACT: T-cell receptor excision circles (TRECs) and kappa-deleting recombination excision circles (KRECs) are circular DNA segments generated in T and B cells during their maturation in the thymus and bone marrow. These circularized DNA elements persist in the cells, are unable to replicate, and are diluted as a result of cell division, thus are considered markers of new lymphocyte output. The quantification of TRECs and KRECs, which can be reliably performed using singleplex or duplex real-time quantitative PCR, provides novel information in the management of T- and B-cell immunity-related diseases. In primary immunodeficiencies, when combined with flow cytometric analysis of T- and B-cell subpopulations, the measure of TRECs and KRECs has contributed to an improved characterization of the diseases, to the identification of patients' subgroups, and to the monitoring of stem cell transplantation and enzyme replacement therapy. For the same diseases, the TREC and KREC assays, introduced in the newborn screening program, allow early disease identification and may lead to discovery of new genetic defects. TREC and KREC levels can also been used as a surrogate marker of lymphocyte output in acquired immunodeficiencies. The low number of TRECs, which has in fact been extensively documented in untreated HIV-infected subjects, has been shown to increase following antiretroviral therapy. Differently, KREC number, which is in the normal range in these patients, has been shown to decrease following long-lasting therapy. Whether changes of KREC levels have relevance in the biology and in the clinical aspects of primary and acquired immunodeficiencies remains to be firmly established.
    Full-text · Article · May 2013 · Journal of Translational Medicine
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    • "Analogous to SCID, efforts have been initiated to set-up KREC-based screening for B-cell maturation defects (Nakagawa et al., 2011). These initial results revealed the presence of >200 KREC copies/μg DNA in healthy children, whereas no KRECs were detected in 30 XLA patients and 5 non-BTK agammaglobulinemia patients. "
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    ABSTRACT: The vast majority of patients suffering from a primary immunodeficiency (PID) have defects in their T- and/or B-cell compartments. Despite advances in molecular diagnostics, in many patients no underlying genetic defect has been identified. B- and T-lymphocytes are unique in their ability to create a receptor by genomic rearrangement of their antigen receptor genes via V(D)J recombination. During this process, stable circular excision products are formed that do not replicate when the cell proliferates. Excision circles can be reliably quantified using real-time quantitative (RQ-)PCR techniques. Frequently occurring δREC-ψJα T-cell receptor excision circles (TRECs) have been used to assess thymic output and intronRSS-Kde recombination excision circles (KREC) to quantify B-cell replication history. In this perspective, we describe how TRECs and KRECs are formed during precursor - T- and B-cell differentiation, respectively. Furthermore, we discuss new insights obtained with TRECs and KRECs and specifically how these excision circles can be applied to support therapy monitoring, patient classification and newborn screening of PID.
    Full-text · Article · May 2011 · Frontiers in Immunology
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    ABSTRACT: Severe combined immunodeficiency (SCID) and X-linked agammaglobulinemia (XLA) are inborn errors of immune function that require prompt diagnosis and treatment to prevent life-threatening infections. The lack of functional T or B lymphocytes in these diseases serves as a diagnostic criterion and can be applied to neonatal screening. A robust triplex PCR method for quantitation of T-cell receptor excision circles (TRECs) and κ-deleting recombination excision circles (KRECs), using a single Guthrie card punch, was developed and validated in a cohort of 2560 anonymized newborn screening cards and in 49 original stored Guthrie cards from patients diagnosed with SCID, XLA, ataxia-telangiectasia, Nijmegen-breakage-syndrome, common variable immunodeficiency, immunoglobulin A deficiency, or X-linked hyper-IgM syndrome. Simultaneous measurement of TREC and KREC copy numbers in Guthrie card samples readily identified patients with SCID, XLA, ataxia-telangiectasia and Nijmegen-breakage-syndrome and thus facilitates effective newborn screening for severe immunodeficiency syndromes characterized by the absence of T or B cells.
    Full-text · Article · Nov 2011 · Blood
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