A high-throughput newborn screening approach for SCID, SMA, and SCD combining multiplex qPCR and tandem mass spectrometry

PLOS ONE RESEARCH ARTICLE A high-throughput newborn screening approach for SCID, SMA, and SCD combining multiplex qPCR and tandem mass spectrometry Rafael Tesorero ID1☯*, Joachim Janda ID1☯*, Friederike Hörster1, Patrik Feyh1, Ulrike Mütze ID1, Jana Hauke1, Kathrin Schwarz1, Joachim B. Kunz2, Georg F. Hoffmann1, Jürgen G. Okun1 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Tesorero R, Janda J, Hörster F, Feyh P, Mütze U, Hauke J, et al. (2023) A high-throughput newborn screening approach for SCID, SMA, and SCD combining multiplex qPCR and tandem mass spectrometry. PLoS ONE 18(3): e0283024. https:// doi.org/10.1371/journal.pone.0283024 Editor: Elsayed Abdelkreem, Sohag University Faculty of Medicine, EGYPT Received: November 22, 2022 Accepted: February 28, 2023 Published: March 10, 2023 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0283024 Copyright: © 2023 Tesorero et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. 1 Department of General Pediatrics, Division for Neuropediatrics and Metabolic Medicine, Center for Child and Adolescent Medicine and Dietmar Hopp Metabolic Center, University Hospital Heidelberg, Heidelberg, Germany, 2 Department of Pediatric Oncology, Hematology, Oncology and Immunology, University of Heidelberg, Heidelberg, Germany ☯ These authors contributed equally to this work. * (RT); (JJ) Abstract Early diagnosis of severe combined immunodeficiency (SCID), spinal muscular atrophy (SMA), and sickle cell disease (SCD) improves health outcomes by providing a specific treatment before the onset of symptoms. A high-throughput nucleic acid-based method in newborn screening (NBS) has been shown to be fast and cost-effective in the early detection of these diseases. Screening for SCD has been included in Germany’s NBS Program since Fall 2021 and typically requires high-throughput NBS laboratories to adopt analytical platforms that are demanding in terms of instrumentation and personnel. Thus, we developed a combined approach applying a multiplexed quantitative real-time PCR (qPCR) assay for simultaneous SCID, SMA, and 1st-tier SCD screening, followed by a tandem mass spectrometry (MS/MS) assay for 2nd-tier SCD screening. DNA is extracted from a 3.2-mm dried blood spot from which we simultaneously quantify T-cell receptor excision circles for SCID screening, identify the homozygous SMN1 exon 7 deletion for SMA screening, and determine the integrity of the DNA extraction through the quantification of a housekeeping gene. In our two-tier SCD screening strategy, our multiplex qPCR identifies samples carrying the HBB: c.20A>T allele that is coding for sickle cell hemoglobin (HbS). Subsequently, the 2nd tier MS/MS assay is used to distinguish heterozygous HbS/A carriers from samples of patients with homozygous or compound heterozygous SCD. Between July 2021 and March 2022, 96,015 samples were screened by applying the newly implemented assay. The screening revealed two positive SCID cases, while 14 newborns with SMA were detected. Concurrently, the qPCR assay registered HbS in 431 samples which were submitted to 2nd-tier SCD screening, resulting in 17 HbS/S, five HbS/C, and two HbS/β thalassemia patients. The results of our quadruplex qPCR assay demonstrate a costeffective and fast approach for a combined screening of three diseases that benefit from nucleic-acid based methods in high-throughput NBS laboratories. PLOS ONE | https://doi.org/10.1371/journal.pone.0283024 March 10, 2023 1 / 16 PLOS ONE Funding: The newborn screening pilot study (“Expansion of Newborn Screening by an additional 28 target diseases”) is generously supported by the Dietmar Hopp Foundation, St. Leon- Rot, Germany (2311220 and 1DH1911376 to G.F.H.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Multiplex newborn screening for SCID, SMA and SCD Introduction The first quantitative real-time PCR (qPCR)-based newborn screening (NBS) in Germany was implemented in August 2019 to detect severe combined immunodeficiency (SCID), a group of inherited primary immunodeficiencies with an incidence estimated to be between 1:30,000– 1:50,000 and which are characterized by the absence or extremely low numbers of naïve Tcells [1]. Infants born with SCID typically are asymptomatic at birth, but if not diagnosed and treated early, the diseases turn fatal within the first year of life due to opportunistic infections. Curative human stem cell transplantation (HSCT), enzyme replacement or gene therapy within the first months of life substantially increase the survival rates of SCID patients [2]. Because SCID is characterized by low to undetectable levels of T-cells, it can be detected early by measuring thymic function through the quantification of T-cell receptors excision circles (TRECs) from peripheral blood [3]. TRECs are stable, non-replicative, extrachromosomal circular DNA byproducts generated during the T-cell receptor rearrangement that occur in about 70% of newly matured T-cells. They are elevated in healthy newborns, and decline with increasing age due to decreased thymic activity [4]. TRECs can be effectively and rapidly quantified by a qPCR assay from infant dried blood spots (DBS) used for NBS programs. The TREC assay was first implemented in 2008 in Wisconsin, USA as a method for SCID screening [5]. Since then, several countries have nationally or regionally established SCID screening in their NBS programs by means of qPCR, opening up the possibilities for genetic screening of other diseases without a biomarker. Another disease recently added to the German NBS panel that benefits from early detection from nucleic-based methods is spinal muscular atrophy (SMA), an autosomal recessively inherited disorder characterized by the degeneration of alpha motor neurons in the spinal cord, which results in progressive proximal muscle weakness and atrophy [6]. With an incidence of 1:6,000 to 1:10,000, SMA is the most common inherited neurodegenerative disease and was the leading genetic cause of death in early childhood [7,8]. The severity of symptoms depends on the SMA type classification, which is based on age of onset and achieved motor function [9]. The survival motor neuron (SMN) protein is encoded by two genes, the main functiona (...truncated)