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Background Severe combined immune deficiency (SCID) is a potentially fatal primary immunodeficiency due to the absence of T and B lymphocyte function. Early intervention for patients with SCID results in a higher survival rate. From 2017, we launched the first optional newborn screening (NBS) for SCID in Japan based on the detection of T-cell receptor excision circles (TREC). However, NBS for severe B-cell lymphopenia, such as X-linked agammaglobulinemia (XLA), has not been a standard screening test because of a high false-positive rate of Kappa-deleting recombination excision circles (KREC), which reflects the replication of B cells. XLA is characterized by severe B-cell lymphopenia and marked reduction of all classes of serum immunoglobulins. Patients with XLA require early diagnosis and immunoglobulin replacement therapy to prevent the development of bronchiectasis caused by recurrent infections. This study aimed to analyze the results of NBS for SCID and elucidate the utility of NBS for SCID and XLA using the TREC/KREC assay. Patients and Methods We enrolled infants who received NBS for SCID (n = 29,447) between April 2017 and June 2018. Using the EnLiteTM TREC kit, we measured TRECA and β-actin, which are used as controls for monitoring sample amplification. Samples with less than 30 copies/µL and adequate β-actin were defined as positive TRECA. All infants with positive TRECA were followed up for at least 12 months. We measured TRECB and KREC using the EnLiteTM TREC/KREC kit in these infants. As positive controls, we used TRECB and KREC in patients with SCID and XLA, respectively. Furthermore, all infants with positive TRECA were evaluated using flow cytometric analysis and target capture-based next-generation sequencing (NGS) analysis covering 349 primary immunodeficiency- and bone marrow failure-related genes to evaluate CD4+CD45RA+ T-cell counts and identify diagnostic variants. This study was approved by the institutional review board of Nagoya University Graduate School of Medicine. Results Of the infants who underwent NBS for SCID, 43 (0.15%) infants showed positive TRECA. All 43 infants were followed up in Nagoya University for at least 12 months. Of these, we identified one case with DiGeorge syndrome showing severe lymphopenia but did not identify typical SCID. TRECB and KREC were measured in 43 infants with positive TRECA. To determine which kit is more useful to detect T-cell lymphopenia, we compared TRECA with TRECB in 1454 infants with normal TRECA and 43 with positive TRECA. All healthy infants with normal TRECA showed TRECB with more than 30 copies/µL but nine patients with SCID showed extremely low TRECB (median [range], 0 [0-3] copies/µL). Only 6 of 43 (14%) infants showed TRECB with less than 30 copies/µL. Moreover, we analyzed the correlation between CD4+CD45RA+ T-cell counts and TRECB. Compared with 37 infants with normal TRECB, 6 infants with positive TRECB demonstrated significantly lower CD4+CD45RA+ T-cell counts (P = 0.026). However, target capture-based NGS did not identify any diagnostic variants among them. This finding suggested that using this kit, false-positive rates might be decreased from 0.15% (43/29,447) to 0.02% (6/29,447). Using this kit, we assessed KREC in 1454 infants with normal TRECA and 43 with positive TRECA. Of these, we identified one case with less than 30 copies/µL KREC who was diagnosed with congenital asplenia. As positive controls, all six patients with XLA showed quite low KREC (0 [0-9] copies/µL). Compared with previously reported KREC assay, this kit may result in lower false-positive rates. Furthermore, to demonstrate whether KREC reflects the replication of B cells, we analyzed the correlation with CD19+ B-cell counts and KREC. Among 43 infants with positive TRECA, infants with less than 500/µL CD19+ B cells showed significantly lower KREC than those with more than 500/µL CD19+ B cells (P = 0.014). Conclusion We conducted the first large-scale study to evaluate the utility of the newly released EnLiteTM TREC/KREC kit. This kit may be more useful than the current TREC kit to identify infants with T-cell lymphopenia and to avoid unnecessary follow up. Compared with previous NBS for XLA, the false-positive rate of this assay was within an acceptable range. Furthermore, TRECB and KREC were assessed with almost the same screening cost and labor. Therefore, we are considering switching from the current TREC kit to this TREC/KREC kit. Disclosures No relevant conflicts of interest to declare.

INTRODUCTION Severe combined immunodeficiency disease (SCID) is the most severe form of primary immunodeficiency disorders (PIDs). Impaired cellular and humoral immunity renders the affected infants susceptible to various infections and results in death within the first 2 years of life. Affected infants are asymptomatic at birth, untreated disease leads to death, and prompt treatment (i.e., hematopoietic stem cell transplantation, gene therapy, or enzyme replacement therapy) is linked to significant improvement in outcome. Thus, SCID meets the disease criteria for newborn screening (NBS). The T-cell receptor excision circle (TREC) is an excellent marker of recently formed T cells, and quantitative PCR-based measurement of TREC is an excellent tool in population-based NBS for SCID. Recent progress in next-generation sequencing (NGS) has enabled the simultaneous sequencing of numerous nucleic acids, detecting single nucleotide changes as well as copy number variants. We launched a pilot newborn optional screening program for SCID, combining the measurement of TREC and NGS in Japan. PATIENTS AND METHODS We measured TREC copy number using the Enlite™ Neonatal TREC assay (Perkin Elmer, Turku, Finland), which utilizes the duplex amplification of TREC and beta-actin in the same reaction for each specimen. We used TREC negative cutoffs as follows: TREC copy number of RESULTS From April 2017 to March 2018, we screened a total of 22,865 newborns, covering 57% of the total number of births in the Aichi prefecture, Japan. We identified 48 (0.21%) newborns with TREC negative results. These newborns were referred to the Nagoya University Hospital or Fujita Health University Hospital and received thorough immunological examination, including target capture sequencing. Among them, 12 (25%) newborns had background diseases, including Down syndrome (n = 4), gastrointestinal defects (n = 3), congenital diaphragmatic hernia (n = 2), congenital chylothorax (n = 2), and severe congenital heart anomaly (n = 1). Immunological assessment identified 11 (23%) infants with lymphocytopenia ( CONCLUSION We successfully launched a pilot newborn optional screening program for SCID, combining the measurement of TREC and NGS in Japan. We did not identify typical SCID patients probably because of the relatively small sample size. However, this newborn screening program, incorporating an NGS assay as a second test, achieved early accurate diagnoses of patients with other PIDs with TREC negative results. Consequently, this program may facilitate patient management and optimize treatment outcomes. Disclosures No relevant conflicts of interest to declare.

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