Establishing an in-house real-time polymerase chain reaction assay to quantify T-cell receptor excision circles and kappa-deleting recombination excision circles for use in screening newborn babies for inborn errors of immunity

Abstract

Inborn errors of immunity (IEI) are genetic disorders that hinder immune responses and are underdiagnosed, particularly in low-to-middle income countries. These disorders occur in one in every 500 live births. Early detection is crucial for preventing health complications. The need for early detection of IEI has led to the development of newborn screening (NBS) programmes; however, many countries lack routine NBS due to financial and technological challenges. The present study aimed to develop an in-house screening technique for T-cell receptor excision circles (TREC) and kappa-deleting recombination excision circles (KREC) quantification using real-time polymerase chain reaction (PCR) to screen for IEI in newborn babies.

We developed an in-house real-time PCR technique to detect and quantify TREC and KREC in human immunodeficiency virus (HIV)-exposed uninfected (HEU) and HIV-unexposed uninfected (HUU) neonates, using dried blood spot (DBS) samples collected and preserved as part of the Siyakhula study cohort. Deoxyribonucleic acid (DNA) was extracted from DBS samples, and TREC and KREC genes were simultaneously detected using a newly developed real-time PCR technique.

Results revealed that HUU infants had statistically significant higher TREC concentrations (P-value = 0.006) than HEU infants, however, no significant difference in KREC concentrations were noted between the two cohorts. Statistical significance was determined by a P-value <0.05. These results indicate reduced TREC levels in immunologically vulnerable babies (HEU).

The TREC and KREC assays showed significant potential as biomarkers for IEI screening. Importantly, the simultaneous detection and quantification of TREC/KREC is a cost-effective method for IEI screening.