TY - JOUR
T1 - Next-Generation Molecular Testing of Newborn Dried Blood Spots for Cystic Fibrosis
AU - Lefterova, Martina I.
AU - Shen, Peidong
AU - Odegaard, Justin I.
AU - Fung, Eula
AU - Chiang, Tsoyu
AU - Peng, Gang
AU - Davis, Ronald W.
AU - Wang, Wenyi
AU - Kharrazi, Martin
AU - Schrijver, Iris
AU - Scharfe, Curt
N1 - Publisher Copyright:
© 2016 American Society for Investigative Pathology and the Association for Molecular Pathology.
PY - 2016/3/1
Y1 - 2016/3/1
N2 - Newborn screening for cystic fibrosis enables early detection and management of this debilitating genetic disease. Implementing comprehensive CFTR analysis using Sanger sequencing as a component of confirmatory testing of all screen-positive newborns has remained impractical due to relatively lengthy turnaround times and high cost. Here, we describe CFseq, a highly sensitive, specific, rapid (<3 days), and cost-effective assay for comprehensive CFTR gene analysis from dried blood spots, the common newborn screening specimen. The unique design of CFseq integrates optimized dried blood spot sample processing, a novel multiplex amplification method from as little as 1 ng of genomic DNA, and multiplex next-generation sequencing of 96 samples in a single run to detect all relevant CFTR mutation types. Sequence data analysis utilizes publicly available software supplemented by an expert-curated compendium of >2000 CFTR variants. Validation studies across 190 dried blood spots demonstrated 100% sensitivity and a positive predictive value of 100% for single-nucleotide variants and insertions and deletions and complete concordance across the polymorphic poly-TG and consecutive poly-T tracts. Additionally, we accurately detected both a known exon 2,3 deletion and a previously undetected exon 22,23 deletion. CFseq is thus able to replace all existing CFTR molecular assays with a single robust, definitive assay at significant cost and time savings and could be adapted to high-throughput screening of other inherited conditions.
AB - Newborn screening for cystic fibrosis enables early detection and management of this debilitating genetic disease. Implementing comprehensive CFTR analysis using Sanger sequencing as a component of confirmatory testing of all screen-positive newborns has remained impractical due to relatively lengthy turnaround times and high cost. Here, we describe CFseq, a highly sensitive, specific, rapid (<3 days), and cost-effective assay for comprehensive CFTR gene analysis from dried blood spots, the common newborn screening specimen. The unique design of CFseq integrates optimized dried blood spot sample processing, a novel multiplex amplification method from as little as 1 ng of genomic DNA, and multiplex next-generation sequencing of 96 samples in a single run to detect all relevant CFTR mutation types. Sequence data analysis utilizes publicly available software supplemented by an expert-curated compendium of >2000 CFTR variants. Validation studies across 190 dried blood spots demonstrated 100% sensitivity and a positive predictive value of 100% for single-nucleotide variants and insertions and deletions and complete concordance across the polymorphic poly-TG and consecutive poly-T tracts. Additionally, we accurately detected both a known exon 2,3 deletion and a previously undetected exon 22,23 deletion. CFseq is thus able to replace all existing CFTR molecular assays with a single robust, definitive assay at significant cost and time savings and could be adapted to high-throughput screening of other inherited conditions.
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U2 - 10.1016/j.jmoldx.2015.11.005
DO - 10.1016/j.jmoldx.2015.11.005
M3 - Article
C2 - 26847993
AN - SCOPUS:84960811674
SN - 1525-1578
VL - 18
SP - 267
EP - 282
JO - Journal of Molecular Diagnostics
JF - Journal of Molecular Diagnostics
IS - 2
ER -