Introduction
The purpose of neonatal screening is the early recognition of treatable, mostly genetically determined diseases that manifest with a high rate of morbidity and mortality. Mass screening of newborn infants with dried blood spots (DBS) started in the early 1960s based on a method developed by Guthrie and Susi for diagnosing phenylketonuria [
1]. Peripheral blood from a heel stick was blotted onto filter paper (referred to as “Guthrie card”) at 3 to 5 days after birth, dried, and sent by mail to centralized laboratories for analysis.
The US congress decided in 2006 to include 29 core and 25 secondary disorders in its newborn screening program based on a priority list of disorders. In recent years, a number of additional disorders, among them severe combined immunodeficiency (SCID), have been included in this list, based on the well-recognized Wilson and Jungner criteria [
2]. In Sweden, neonatal screening with DBS was initiated in 1965 and today, a total of 24 disorders are included in the program.
Genetically determined disorders of immunity are commonly referred to as primary immunodeficiencies (PID) and were first recognized clinically over 60 years ago with the identification of X-linked agammaglobulinemia (XLA) [
3]. Today, the group of PID includes more than 250 distinct entities, which are divided into T cell deficiencies, B cell deficiencies (the predominant group), combined T and B cell deficiencies, complement defects, and granulocyte defects [
4].
Primary immunodeficiencies as a group are not rare diseases, but should be considered in all patients presenting with severe, atypical, or recurrent infections. The overall prevalence is unknown, but varies greatly (from 1:600 for IgA deficiency, 1:20,000 for common variable immunodeficiency, 1:50,000 for SCID, and 1:100,000 for XLA) [
4] and references therein [
5]. The estimated incidence of severe forms of PID, which require immediate attention, is variable in different populations, but would be expected to be in the order of 5–10 per 100,000 live births.
In 2005, Chan and Puck published a seminal paper, describing the T-cell receptor excision circle (TREC) assay as a tool for large-scale screening for SCID and other T cell lymphopenias [
6]. T cell receptor genes are normally edited during T cell differentiation, and the deleted fragments are circularized and do not undergo further replication in dividing cells. Thus, TRECs are a marker of recently formed T cells. The TREC copy number can be determined using a quantitative PCR-based method using DNA extracted from routinely collected DBS. The TREC assay was implemented in the state of Wisconsin in 2008 [
7] and its inclusion in the newborn screening programs in the USA was recommended in 2010. Currently, 38 states are screening for SCID using the TREC assay, with the remaining 12 states due to commence in 2017 (Jeffrey Modell Foundation,
http://www.info4pi.org/). More than three million newborns have been tested to date [
8]. The results show a frequency of SCID of 1/58,000 children (overall 1/7300 with clinically relevant forms of T cell lymphopenia) and a high survival rate (92 %) after treatment.
Congenital B cell lymphopenia can be identified by screening for kappa-deleting recombination excision circles (KREC), the circular by-product of B cell immunoglobulin kappa gene rearrangement [
9]. Patients with severe forms of B cell deficiency such as XLA are easily identified by the KREC assay [
10] and are treated with regular gammaglobulin infusions, thereby allowing a near to normal life. Furthermore, it has been shown that delayed-onset forms of SCID and other severe forms of PID that present with normal T cell numbers and TREC levels above cutoff in newborn screening programs may be identified using a combined TREC and KREC screening approach [
11].
We thus initiated a screening program for all newborns in the county of Stockholm in Sweden using a combined TREC and KREC assay [
12]. We previously analyzed 10,058 samples as part of a feasibility study, and one XLA patient was identified (Bruton tyrosine kinase gene mutation, c.1480C>T, p.Gln494Ter), thus demonstrating proof of principle of the combined screening approach [
12]. Although a small cohort of Spanish newborns have been screened using the combined TREC/KREC assay [
13], this represents the first large-scale prospective study where both T and B cell defects (lymphopenia) can be identified and the results of the first 2 years of the screening program are reported here.
Discussion
Since 2008, TREC analysis has been the method of choice for screening of newborns for severe forms of primary T cell lymphopenia [
6,
7,
16], a method that will capture most, but not all, children with SCID (exceptions being patients with mutations in
ZAP70 [
17,
18],
MHC class II [
19‐
21], and
ADA (delayed-onset disease [
11]). We subsequently developed a triplex method that also includes KREC analysis to assess for potential B cell lymphopenia in DBS samples. This method can successfully identify patients with XLA [
12], selected patients with delayed-onset adenosine deaminase deficiency (ADA) [
11], Nijmegen-breakage syndrome (NBS) [
12], and purine nucleoside phosphorylase (PNP) deficiency [
22]. The combined assay has recently also been used to screen patients with SCID [
23,
24], ATM [
12,
25], Wiskott-Aldrich syndrome (WAS) [
26], DiGeorge syndrome [
27‐
29], and trisomy 21 [
30], and it has been suggested to be included in routine screening of newborns for primary immunodeficiency [
31‐
34]. KREC levels have also been used to monitor immune reconstitution after bone marrow/stem cell transplantation [
35‐
37].
Ours is the largest study to date using a combined TREC/KREC assay for newborn screening. One SCID patient was identified (and successfully transplanted) in our cohort of 58,000 infants, comparable with the data published by Kwan et al. on a very large US cohort [
8]. One patient with ATM was also identified. This disorder is very rare, with an estimated incidence of 1 in 200,000 according to recent figures from the USA [
38]. It will be interesting to determine if this finding is due to serendipity or if the disease is more frequent in Scandinavia. Our third patient showed a T cell lymphopenia of unknown cause. Such cases have been previously described in studies in the USA with incidences varying between 3 and 4 per 100,000 children in different states and with a predicted incidence of 1 in 160,000 in a British study (Professor Bobby Gaspar, Great Ormond Street Hospital, personal communication). Recently, during the third year of screening, yet another SCID patient (ADA deficiency) and a patient with severe T cell lymphopenia have been identified.
The number of referrals for flow cytometric analysis in our cohort was low (approximately 1:20,000), markedly lower than that reported in the US study of Kwan et al. [
8] (ranging from 1/735 to 1/7500 in the different states). This is a reflection of the strict resampling strategy that was applied to children with low, but not absent TREC or KREC levels. These results also indicate that the addition of KREC testing to the TREC assay in a newborn screening program does not critically drive the demand for second tier testing nor does it increase the fiscal impact of the test itself due to the minimal additional cost of multiplexing.
Prematurity has been considered a predisposing factor for low TREC numbers in studies in American infants [
8]. The TREC and KREC levels in our study did show a downward trend with decreasing gestational age, although in the vast majority, the levels were above the cutoff, even in markedly premature infants. This notion was also recently supported [
13] in a small Spanish cohort of newborns.
Twins and triplets were markedly overrepresented among the positive samples (n = 11/64), potentially reflecting prematurity in the majority of cases (n = 8/11). There was a notable difference in TREC and KREC levels within twin pairs returning normal screening results. Although information regarding zygosity was not available, it is expected that one in four pairs is monozygotic and therefore genetically identical, and this observation will thus be further investigated in the future studies.
Thirteen children with low KREC levels were born to 12 mothers who had been treated with azathioprine during pregnancy, three of whom were concomitantly receiving tacrolimus. Two children born to the same azathioprine-treated mother, 2 years apart, both had low KREC levels. Low KREC levels were also recently noted by de Felipe et al. [
13] in a small cohort of Spanish children born to mothers treated with azathioprine. As azathioprine may cross the placenta (and is mutagenic), it is generally contraindicated during pregnancy unless used for treatment of severe disease. As B lymphocytes are markedly more sensitive to drug-induced apoptosis than T lymphocytes [
39], the selectivity for a reduction in KREC copies alone is not surprising.
We anticipated that some infants with trisomy 21 would screen positive since they may have low B cell and/or T cell production [
30]. However, only one of the 43 infants screened during the 2-year period had a KREC level below cutoff, although infants with trisomy 21 had generally lower levels than those in the general newborn population. In Sweden, the estimated frequency of chromosome 22q deletions is around 1/4000, suggesting that some 14–15 children in the screened cohort would suffer from this form of PID. However, less than 10 % of chromosome 22q deletion patients had pronounced T cell lymphopenia (measured as low TREC levels) and an additional 5–10 % had levels close to the cutoff. Thus, we would have expected only one to two children to be identified in our tested cohort, which is not statistically different from the present result (no child identified).
Most of the children in our study were recalled due to low KREC levels. This was expected, as we did not know what the “true” cutoff level for children with XLA would be and thus applied a fairly large safety margin. Ongoing studies in our laboratory, analyzing KREC levels in a large cohort of children with mutation proven XLA, may allow further adjustment of the recall level of KREC, thus reducing the efforts and costs associated with “false” positive results.
Newborn screening for metabolic diseases has undergone major changes during the past decades with inclusion of numerous very rare diseases in the program. We anticipate a similar development in the PID screening field where the KREC may ultimately be added to the TREC assay currently used in several national screening programs. The decision regarding implementation of TREC vs. combined TREC/KREC screening can be guided by considering the advantages and disadvantages of each screening approach. The advantages for adding KREC screening include the identification of children with XLA, late onset ADA, some patients with NBS, and other selected forms of PID whose disease may be undetected by TREC screening alone [
8,
23,
40]. In addition, it may assist in distinguishing patients with SCID caused by deficiency in the production of T as well as B cells or only T cells, thus aiding in the diagnostic process. On the negative side, increased costs associated with the additional KREC assay may be considered a disadvantage of the combined screening strategy. With the possibility to multiplex PCR reactions, the individual cost for additional screening markers such as KREC in an existing TREC screening system is negligible (<€0.10 per newborn), but associated costs incurred in follow-up and further testing for infants with abnormal screening results should also be considered. However, it should be noted that the recall rate using the combined TREC/KREC assay is similar to that of TREC screening alone [
8], with a lower rate of follow-up FACS studies than in other published studies to date.
As markers for other genetic diseases have been proposed which can be multiplexed with the TREC/KREC system, future efforts may be focused on the extension of existing newborn screening tests for PID [
26]. This may potentially include complement deficiencies (using DBS eluates as described by Janzi et al. [
41] for C3 deficiencies and Hamsten et al. [
42] for C2 deficiencies) and granulocyte defects, thus allowing coverage of a majority of the primary immunodeficiency diseases. Targeted exome sequencing or even whole genome sequencing of newborns may ultimately be considered, which could lead to identification of additional defects of immunity and a large number of other inherited, potentially fatal diseases.