Newborn Screening and early diagnosis of Severe T-cell and B-cell Lymphopenia using TREC/KREC Assay.

Objective: Primary immunodeficiency disorders (PID) are a heterogeneous group of genetic disorders resulting from quantitative or functional defects in immune cells. Recently, T-cell receptor excision circles (TRECs) and κ-deleting recombination excision circles (KRECs) have been used for the detection of T and B cell lymphopenia in newborn screening programs based on region-specific cutoff levels. Here, we report the newborn screening program for obtaining the cutoff levels for TREC and KREC copy numbers that may be applied in our newborn screening program and as a diagnostic tool in infants with a family history of PID in Iran. Materials and methods: DNA was extracted from a single 3.2mm punch from dried blood spots collected from 2160 anonymized newborns, which were obtained from two major referral health centers for newborn screening between 2014 and 2016. Samples from thirty patients with a definite diagnosis of severe combined immunodeficiency, X-linked agammaglobulinaemia (XLA), ataxia telangiectasia (AT) and twenty-five healthy controls were used for determining the cutoffs. Samples from other patients including those with IgA deficiency (IgAD), Wiskott-Aldrich syndrome, Hyper-IgM syndromes (HIgM) and Dedicator of cytokinesis 2 (DOCK2) deficiency were considered as controls. TREC, KREC and beta-actin (ACTB) copy numbers were analyzed in all samples using triplex-quantitative real-time PCR. The diagnostic efficiency of this method was evaluated using ROC curve analysis. Results: Subsequent to statistical analysis, the cutoff values were calculated and obtained with a high sensitivity and specificity. Among the thirty cases with abnormal results (1.4%), twelve cases (0.6%) were retested and were subsequently shown to be in the normal range. Moreover, all of the positive, disease control patients were correctly identified. Conclusion: Determining cutoff levels with a high sensitivity and specificity for both TREC and KREC is essential for correctly identifying children with severe forms of PID in newborn screening programs, whilst minimising false positive results. Early diagnosis of patients with PID enables early implementation of appropriate preventative measures and definitive therapies such as hematopoietic stem cell transplantation. [ABSTRACT FROM AUTHOR]