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. 2019 Nov 2;5(4):39.
doi: 10.3390/ijns5040039. eCollection 2019 Dec.

Development of a Multiplex Real-Time PCR Assay for the Newborn Screening of SCID, SMA, and XLA

Cristina Gutierrez-Mateo  1 , Anne Timonen  2 , Katja Vaahtera  2 , Markku Jaakkola  2 , David M Hougaard  3 , Jonas Bybjerg-Grauholm  3 , Marie Baekvad-Hansen  3 , Dea Adamsen  3 , Galina Filippov  1 , Stephanie Dallaire  1 , David Goldfarb  1 , Daniel Schoener  1 , Rongcong Wu  1
Affiliations

Development of a Multiplex Real-Time PCR Assay for the Newborn Screening of SCID, SMA, and XLA

Cristina Gutierrez-Mateo et al. Int J Neonatal Screen. .

Abstract

Numerous studies have shown evidence supporting the benefits of universal newborn screening for primary immunodeficiencies (PID) and for Spinal Muscular Atrophy (SMA). We have developed a four-plex, real-time PCR assay to screen for Severe Combined Immune Deficiencies (SCID), X-linked agammaglobulinemia (XLA), and SMA in DNA extracted from a single 3.2 mm punch of a dried blood spot (DBS). A simple, high-throughput, semi-automated DNA extraction method was developed for a Janus liquid handler that can process 384 DBS punches in four 96-well plates in just over one hour with sample tracking capability. The PCR assay identifies the absence of exon 7 in the SMN1 gene, while simultaneously evaluating the copy number of T-cell receptor excision circles (TREC) and Kappa-deleting recombination excision circles (KREC) molecules. Additionally, the amplification of a reference gene, RPP30, was included in the assay as a quality/quantity indicator of DNA isolated from the DBS. The assay performance was demonstrated on over 3000 DNA samples isolated from punches of putative normal newborn DBS. The reliability and analytical accuracy were further evaluated using DBS controls, and contrived and confirmed positive samples. The results from this study demonstrate the potential of future molecular DBS assays, and highlight how a multiplex assay could benefit newborn screening programs.

Keywords: DBS; KREC; Newborn Screening; SCID; SMA; SMN1; TREC; XLA; real-time PCR.

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Conflict of interest statement

Conflicts of InterestC.G-M., A.T., K.V., M.J., G.F., S.D., D.G., D.S. and R.W. are PerkinElmer employees. Authors representing PerkinElmer had a role in designing the study, in collection, analyses, and interpretation of the data, in the writing of the manuscript, and in the decision to publish the results. The other authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Histogram of RPP30, SMN1, TREC, and KREC Cts.
Figure 2
Figure 2
Histogram of TREC and KREC copy numbers/105 cells.
Figure 3
Figure 3
Amplification plots for: (a) A reference SMA positive sample with three copies of the SMN2 gene showing no amplification of the SMN1 locus; (b) an individual over 55 years old (SCID-like) showing no amplification for the TREC locus and normal amplification for the other three targets; (c) a contrived SMA, SCID, and XLA positive sample showing amplification only for RPP30. (d) A reference SMA carrier sample showing robust amplification of the SMN1 locus.
See this image and copyright information in PMC

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