Combination of TREC Measurement and Next-Generation Sequencing in Newborn Screening for Severe Combined Immunodeficiency: A Pilot Program in Japan

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 <30 copies/μL and beta-actin copy number of ≥50 copies/μL. In patients with TREC negative results, genomic DNA was subjected to DNA capture designed using SureDesign (Agilent, Santa Clara, USA), covering a total of 349 genes associated with PIDs, inherited bone marrow failure syndromes, and the 22q11.2 region. Target capture, enrichment, and indexing were performed according to the manufacturer's instructions. Generated libraries were sequenced using a HiSeq 2500 platform (Illumina, San Diego, USA). This study was approved by the ethical committees of the Nagoya University Graduate School of Medicine and Fujita Health University.

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 (<1500 /μL). These infants avoided live vaccines and received appropriate interventions to prevent infection. Using target sequencing analyses, we identified four patients with PIDs, including 22q11.2 deletion syndrome (n = 2), Wiskott‒Aldrich syndrome (n = 1), and combined immunodeficiency with an unknown causative gene (n = 1).

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.