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

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 cost-effective and fast approach for a combined screening of three diseases that benefit from nucleic-acid based methods in high-throughput NBS laboratories.

Current Status of Newborn Bloodspot Screening Worldwide 2024: A Comprehensive Review of Recent Activities (2020–2023)

“…Recognizing the need to address the transition from pediatric to adult medicine as the screened population ages, a guideline for transition has also been developed [ 807 ]. A 2022 report provides pilot testing data for a multi-analyte MS/MS procedure that simultaneously detects SCD, BIO, and TYR-I [ 808 ], and a 2023 report provides information on a combination multiplex qPCR and MS/MS procedure for simultaneously detecting SCD, SMA, and SCID [ 809 ].…”

Multiplex Real-Time PCR-Based Newborn Screening for Severe Primary Immunodeficiency and Spinal Muscular Atrophy in Osaka, Japan: Our Results after 3 Years

“…Therefore, we were able to implement SMA-NBS without having to charge any additional fees. Tesorero R et al also reported on the scalability and cost-effectiveness of multiplex qPCR assays by integrating screening for sickle cell disease into a pre-existing duplex NBS assay for SCID and SMA [ 38 ]. In addition, as the basis for optional NBS in Osaka had been established by the preceding PID-NBS, the obstetricians in the region had gained a deeper understanding of other treatable target diseases for NBS, including SMA, that should be diagnosed in the neonatal period.…”

High Throughput Newborn Screening for Sickle Cell Disease – Application of Two-Tiered Testing with a qPCR-Based Primary screen

“…More detailed descriptions about the methods, including measures for quality control, are provided in the supplemental file (section “Method descriptions”) and elsewhere 13 16 .…”