A high-throughput newborn screening approach for SCID, SMA, and SCD combining multiplex qPCR and tandem mass
Rafael Tesorero1, Joachim Janda1, Friederike Hörster1
1Department 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.
Insights
A new multiplex assay enables rapid, cost-effective newborn screening for severe combined immunodeficiency (SCID), spinal muscular atrophy (SMA), and sickle cell disease (SCD). This approach allows for early diagnosis and treatment, improving health outcomes for newborns.
Area of Science:
- Biochemistry
- Genetics
- Public Health
Background:
- Early diagnosis of SCID, SMA, and SCD is crucial for timely treatment and improved patient outcomes.
- High-throughput nucleic acid-based methods offer a fast and cost-effective solution for newborn screening (NBS).
- Germany's NBS program now includes SCD screening, necessitating advanced analytical platforms.
Purpose of the Study:
- To develop and validate a combined, high-throughput assay for simultaneous screening of SCID, SMA, and SCD.
- To implement a two-tier screening strategy for SCD using qPCR and MS/MS.
- To assess the efficiency and cost-effectiveness of the multiplex assay in a real-world NBS setting.
Main Methods:
- A multiplex quantitative real-time PCR (qPCR) assay was developed for simultaneous detection of SCID, SMA, and first-tier SCD screening.
- DNA was extracted from dried blood spots for quantification of T-cell receptor excision circles (SCID), SMN1 deletion (SMA), and a housekeeping gene.
- A two-tier SCD screening involved qPCR for HBB: c.20A>T allele and tandem mass spectrometry (MS/MS) for carrier/patient differentiation.
Main Results:
- The assay screened 96,015 newborns between July 2021 and March 2022.
- Two SCID cases and 14 SMA cases were identified.
- First-tier SCD screening detected 431 samples with the HBB: c.20A>T allele, leading to the identification of 17 HbS/S, five HbS/C, and two HbS/β thalassemia patients.
Conclusions:
- The developed quadruplex qPCR assay provides a cost-effective and rapid method for combined screening of SCID, SMA, and SCD.
- This approach is suitable for high-throughput NBS laboratories utilizing nucleic acid-based methods.
- Early detection through this multiplex assay can significantly improve health outcomes for affected newborns.
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.
