Newborn Screening for Severe Primary Immunodeficiency Diseases in Sweden-a 2-Year Pilot TREC and KREC Screening Study

Abstract

Newborn screening for severe primary immunodeficiencies (PID), characterized by T and/or B cell lymphopenia, was carried out in a pilot program in the Stockholm County, Sweden, over a 2-year period, encompassing 58,834 children. T cell receptor excision circles (TREC) and kappa-deleting recombination excision circles (KREC) were measured simultaneously using a quantitative PCR-based method on DNA extracted from dried blood spots (DBS), with beta-actin serving as a quality control for DNA quantity. Diagnostic cutoff levels enabling identification of newborns with milder and reversible T and/or B cell lymphopenia were also evaluated. Sixty-four children were recalled for follow-up due to low TREC and/or KREC levels, and three patients with immunodeficiency (Artemis-SCID, ATM, and an as yet unclassified T cell lymphopenia/hypogammaglobulinemia) were identified. Of the positive samples, 24 were associated with prematurity. Thirteen children born to mothers treated with immunosuppressive agents during pregnancy (azathioprine (n = 9), mercaptopurine (n = 1), azathioprine and tacrolimus (n = 3)) showed low KREC levels at birth, which spontaneously normalized. Twenty-nine newborns had no apparent cause identified for their abnormal results, but normalized with time. Children with trisomy 21 (n = 43) showed a lower median number of both TREC (104 vs. 174 copies/μL blood) and KREC (45 vs. 100 copies/3.2 mm blood spot), but only one, born prematurely, fell below the cutoff level. Two children diagnosed with DiGeorge syndrome were found to have low TREC levels, but these were still above the cutoff level. This is the first large-scale screening study with a simultaneous detection of both TREC and KREC, allowing identification of newborns with both T and B cell defects.

Keywords: KREC; Newborn screening; TREC; primary immunodeficiency diseases; severe combined immunodeficiency.

Fig. 1

Summary of all newborn screening results between 15 November 2013 and 15 November 2015. *Two infants had low TREC levels, and one had low TREC and KREC levels. **See Fig. 2

Fig. 2

Characteristics of the 64 infants with abnormal screening results recalled for repeat testing. *Azathioprine (n = 9), mercaptopurine (n = 1), azathioprine + tacrolimus (n = 3). **One infant who was premature also had trisomy 21

Fig. 3

Comparison of TREC levels (a) in infants born prior to 25 weeks, between 25 and 36 weeks, and at term (≥37 weeks) gestation. Comparison of KREC levels (b) in infants born prior to 27 weeks, between 27 and 36 weeks, and at term (≥37 weeks) gestation. Each point represents one infant and the number of infants in each group is indicated. The horizontal blue line indicates the mean value of all samples. The dashed horizontal red line represents the cutoff value for TREC (<10 copies/3.2 mm blood spot) and KREC (<6 copies/3.2 mm blood spot) levels, respectively. ****p < 0.0001, Mann-Whitney U test

Fig. 4

Correlation between TREC (a) and KREC (b) levels within all 896 twin pairs. Each point represents one twin pair, where the TREC value for twin 1 (x axis) is plotted against the TREC value for twin 2 (y axis) (a). KREC levels for each pair are similarly depicted (b). Overall, there was poor correlation between specimen pairs, with a calculated Spearman correlation coefficient (R _s) of 0.6 for TREC and 0.57 for KREC (p < 0.0001)