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. 2025 Nov 8;45(1):154.
doi: 10.1007/s10875-025-01927-6.

Next-generation Sequencing and Other Second Tier Tests in Newborn Screening for (X-linked) Agammaglobulinemia

Maartje Blom  1 , Annelotte J Duintjer  2 , Ingrid Pico-Knijnenburg  2 , Sandra Imholz  3 , Sahila Balkassmi  4 , Hermine A van Duyvenvoorde  4 , Mirjam van der Burg  5
Affiliations

Next-generation Sequencing and Other Second Tier Tests in Newborn Screening for (X-linked) Agammaglobulinemia

Maartje Blom et al. J Clin Immunol. .

Abstract

Purpose: Patients with X-linked agammaglobulinemia (XLA) suffer from severe, recurrent infections potentially leading to life-threatening complications. Early diagnosis and timely treatment can prevent infections and secondary complications, emphasizing a role for newborn screening (NBS). NBS for XLA is based on quantification of kappa-deleting recombination excision circles (KRECs). KREC-based screening could result in a large number of false-positive referrals associated with high impact for parents and health care systems, indicating the need for a second tier test.

Methods: KRECs were measured in NBS cards (N = 110,491) with a multiplex TREC/KREC qPCR assay. As second tier test options, an alternative qPCR multiplex assay, epigenetic immune cell counting for relative B-cell quantification and targeted next-generation sequencing with B-cell deficiency gene panel including 73 genes were performed on NBS cards of newborns with low KRECs.

Results: In total, 136/110,491 newborns had KRECs below cut-off. With the alternative qPCR multiplex assay, 16/110 of these newborns (14.5%) had KRECs above cut-off and would not have been referred. With epigenetic immune cell counting, 16.5% (17/103) had relative B-cell counts in the range of healthy controls. Targeted NGS showed promising results as 87 out of 103 (84%) newborns with low KRECs did not show any pathogenic/likely pathogenic variants and would not have been referred for follow-up diagnostics.

Conclusion: Several second tier tests can potentially reduce the number of false-positive referrals in NBS for XLA. NGS seems to be the most effective technique in NBS for XLA and other forms of agammaglobulinemia. Our results show promising first steps towards the implementation of NBS for XLA.

Keywords: B-cell deficiency; BTK; Bruton tyrosine kinase; Genetics; KREC; Kappa-deleting recombination excision circles; NBS; NGS; Newborn screening; Next generation sequencing; X-linked agammaglobulinemia; XLA.

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Conflict of interest statement

Declarations. Ethics Approval: This is an observational study using leftover material from the NBS program. No ethical approval is required, the use of NBS cards was approved by the National Institute for Public Health and the Environment (RIVM; no 2019–3). Competing Interests: The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
KREC copies analysed with a second qPCR assay. A. Correlation plot of KREC copies analysed with both assays (SPOT-it, ImmunoIVD and NeoMDx, PerkinElmer/Revvity) in healthy controls (N = 80), Pearson R correlation was 0.83 (P < 0.01). B. KREC levels in copies/105 cells of healthy newborns (N = 80), newborns with low KREC levels (N = 110) and confirmed XLA patients (N = 3) measured with the NeoMDx assay (PerkinElmer/Revvity). The red dotted line is the cutoff of the manufacturer at KREC ≤ 459 copies/105 cells. The black line shows mean KREC copies/105 cells
Fig. 2
Fig. 2
Epigenetic immune cell counting of B lymphocytes. A. Relative (epi) BLC counts as a percentage of total leukocytes of healthy newborns (N = 311), newborns with low KRECs (N = 103) and XLA patients (N = 4) measured with epigenetic qPCR (Epimune GmbH). The mean is depicted with a black line. B. Log-transformed epigenetic BLC/GAPDH copies with a 99.9% confidence interval (red ellipse). Healthy newborns (N = 311) are depicted in red and newborns with low KREC levels are depicted in green (N = 103)
Fig. 3
Fig. 3
Scores of evident artefacts compared to suspected indel variants
Fig. 4
Fig. 4
Overview of variant filtering and interpretation of the workflow of NGS as a second tier in NBS for B-cell deficiencies
Fig. 5
Fig. 5
Venn-diagram of samples of newborns with low KRECS who that were included in all three second-tier tests (N=94) and would have been referred after initial KREC analysis based on the three different second tier test options; next generation sequencing (NGS), epigenetic immune cell counting and analysis with a second qPCR with different KREC primers
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Supplementary concepts