Newborn screening for severe combined immunodeficiency using a novel and simplified method to measure T-cell excision circles (TREC)
Introduction
SCID is a group of > 20 disorders caused by different genetic defects [1]. The incidence has been calculated at approx. 1:58,000 live births [2]. All types of SCID have in common the lack of functional T-cells, leading to a combined cellular and humoral immunodeficiency. Without allogeneic hematopoietic stem cell transplantation (HSCT), children die in the first years of life because of severe infections. The only way to prevent death is early diagnosis followed by HSCT, before infections occur [3]. Indeed, studies have shown that the most important prognostic value for the primary outcome and the long-term survival rate is the clinical status of the patient (patients with active infection at the time of transplantation have a survival rate of 50%, whereas those with no infection 82–90%) [4], [5].
Because of the value of early pre-symptomatic diagnosis, SCID fulfills all the criteria for a disease to be targeted by newborn screening and was recommended being added to the panel of NBS illnesses in 2011 [6]. While a method to screen neonates for SCID in a high throughput format in dried blood spots has not been available until recently, the development of a practicable test by Chan and Puck in 2005 has been a breakthrough [7].
Recently, a number of studies in the US and in Europe proved the importance and the validity of a SCID newborn screening [2], [3].
Efforts have been made in several research laboratories in order to establish an optimal screening test combining good sensitivity and specificity at costs affordable in a massive-scale format [8].
Nevertheless, no agreement on the method and the panel of diseases to be screened has been achieved so far. The most promising method is the quantification of T-cell Receptor Excision Circles (TRECs) on dried blood spots (DBS) by real-time quantitative PCR (qPCR) [9]. TRECs are small episomal pieces of DNA which are generated in the thymus during the VDJ-T-cell receptor gene rearrangement and are therefore good markers for naïve T-cells [10].
Several efforts have been made to implement methods for the early diagnosis of SCID [5], [11], [12], [13], [14], [15], [16], [17]. We established and tested a robust method to detect SCID in newborns through the quantification of TRECs by qPCR. Our method showed a very high sensitivity and specificity and compared favorably to existing methods in terms of time and costs, which plays an important role in the perspective of a population screening on large numbers.
Section snippets
Screened samples
DBS specimens were obtained from Guthrie cards collected by the Newborn Screening Laboratory of Heidelberg University in the period between October and December 2012. After completing the other routine screening tests, DBSs were de-identified and punched for the TREC assay.
After having obtained a written consent from the parents, anonymized DBS specimens of newborns and children with different types of T-cell deficiency were used as positive controls (Supplemental Table 1).
Ethical approval
This pilot study was
Results
We established and tested a new high performance assay for SCID screening based on a qPCR assay for the quantification of TRECs. A very fast DNA extraction phase requiring only few minutes was followed by the qPCR analysis. Calibration curves for TREC quantification were obtained by serially diluting a plasmid coding for the TREC sequence (Fig. 1a–b). β-actin served as semi quantitative internal control. The method was highly reproducible and quantitative over a range from 10 to 100,000 copies
Test quality
All of the positive controls were found to have TREC numbers below the cutoff (estimated sensitivity 1.0). The specificity was estimated based on the proportion of unselected newborns with a TREC number above the cutoff to be 0.990 ± 0.003, CI 99%. The re-test rate of 1.2% and the recall rate of 0.05% lie within the range of other existing tests for SCID and below the range generally accepted in newborn screening [11], [12].
As our study was retrospective and anonymous, we cannot comment on the
Conclusions
In sum, this anonymized pilot phase presents and evaluates a new reliable, rapid and cost-effective method of newborn screening for SCID. We currently plan a prospective non-anonymous screening phase of all newborns that is meant to prepare for a general newborn screening for SCID in Germany.
The following is the supplementary data related to this article.
Acknowledgements
We would like to thank Alessandra Sottini and Luisa Imberti for providing us a TREC-plasmid, as well as all the colleagues of the Pediatric Centers of Ulm and Freiburg who contributed with sending positive control-samples.
We are finally grateful to the Dietmar Hopp Stiftung, St. Leon-Rot, Germany, for the generous funding of this project.
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