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

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 agammaglobulin...

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Autores principales: Gutierrez-Mateo, Cristina, Timonen, Anne, Vaahtera, Katja, Jaakkola, Markku, Hougaard, David M, Bybjerg-Grauholm, Jonas, Baekvad-Hansen, Marie, Adamsen, Dea, Filippov, Galina, Dallaire, Stephanie, Goldfarb, David, Schoener, Daniel, Wu, Rongcong
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7510252/
https://www.ncbi.nlm.nih.gov/pubmed/33072998
http://dx.doi.org/10.3390/ijns5040039
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author Gutierrez-Mateo, Cristina
Timonen, Anne
Vaahtera, Katja
Jaakkola, Markku
Hougaard, David M
Bybjerg-Grauholm, Jonas
Baekvad-Hansen, Marie
Adamsen, Dea
Filippov, Galina
Dallaire, Stephanie
Goldfarb, David
Schoener, Daniel
Wu, Rongcong
author_facet Gutierrez-Mateo, Cristina
Timonen, Anne
Vaahtera, Katja
Jaakkola, Markku
Hougaard, David M
Bybjerg-Grauholm, Jonas
Baekvad-Hansen, Marie
Adamsen, Dea
Filippov, Galina
Dallaire, Stephanie
Goldfarb, David
Schoener, Daniel
Wu, Rongcong
author_sort Gutierrez-Mateo, Cristina
collection PubMed
description 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.
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spelling pubmed-75102522020-10-15 Development of a Multiplex Real-Time PCR Assay for the Newborn Screening of SCID, SMA, and XLA Gutierrez-Mateo, Cristina Timonen, Anne Vaahtera, Katja Jaakkola, Markku Hougaard, David M Bybjerg-Grauholm, Jonas Baekvad-Hansen, Marie Adamsen, Dea Filippov, Galina Dallaire, Stephanie Goldfarb, David Schoener, Daniel Wu, Rongcong Int J Neonatal Screen Article 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. MDPI 2019-11-02 /pmc/articles/PMC7510252/ /pubmed/33072998 http://dx.doi.org/10.3390/ijns5040039 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Gutierrez-Mateo, Cristina
Timonen, Anne
Vaahtera, Katja
Jaakkola, Markku
Hougaard, David M
Bybjerg-Grauholm, Jonas
Baekvad-Hansen, Marie
Adamsen, Dea
Filippov, Galina
Dallaire, Stephanie
Goldfarb, David
Schoener, Daniel
Wu, Rongcong
Development of a Multiplex Real-Time PCR Assay for the Newborn Screening of SCID, SMA, and XLA
title Development of a Multiplex Real-Time PCR Assay for the Newborn Screening of SCID, SMA, and XLA
title_full Development of a Multiplex Real-Time PCR Assay for the Newborn Screening of SCID, SMA, and XLA
title_fullStr Development of a Multiplex Real-Time PCR Assay for the Newborn Screening of SCID, SMA, and XLA
title_full_unstemmed Development of a Multiplex Real-Time PCR Assay for the Newborn Screening of SCID, SMA, and XLA
title_short Development of a Multiplex Real-Time PCR Assay for the Newborn Screening of SCID, SMA, and XLA
title_sort development of a multiplex real-time pcr assay for the newborn screening of scid, sma, and xla
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7510252/
https://www.ncbi.nlm.nih.gov/pubmed/33072998
http://dx.doi.org/10.3390/ijns5040039
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