title: Next-Generation Molecular Testing of Newborn Dried Blood Spots for Cystic Fibrosis.
abstract: 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.
authors: Department of Pathology, Stanford University Medical Center, Stanford, California.
authors: Martina I
authors: MI
authors: Lefterova
authors: Stanford Genome Technology Center, Stanford University, Palo Alto, California.
authors: Peidong
authors: P
authors: Shen
authors: Department of Pathology, Stanford University Medical Center, Stanford, California.
authors: Justin I
authors: JI
authors: Odegaard
authors: Department of Pathology, Stanford University Medical Center, Stanford, California.
authors: Eula
authors: E
authors: Fung
authors: Department of Pathology, Stanford University Medical Center, Stanford, California.
authors: Tsoyu
authors: T
authors: Chiang
authors: Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas.
authors: Gang
authors: G
authors: Peng
authors: Stanford Genome Technology Center, Stanford University, Palo Alto, California.
authors: Ronald W
authors: RW
authors: Davis
authors: Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas.
authors: Wenyi
authors: W
authors: Wang
authors: California Department of Public Health, Environmental Health Investigations Branch, Richmond, California.
authors: Martin
authors: M
authors: Kharrazi
authors: Department of Pathology, Stanford University Medical Center, Stanford, California; Department of Pediatrics, Stanford University Medical Center, Stanford, California.
authors: Iris
authors: I
authors: Schrijver
authors: Department of Pathology, Stanford University Medical Center, Stanford, California; Stanford Genome Technology Center, Stanford University, Palo Alto, California. Electronic address: curt.scharfe@yale.edu.
authors: Curt
authors: C
authors: Scharfe
mesh_terms: D003365:Costs and Cost Analysis; D003550:Cystic Fibrosis / Q000175:diagnosis / Q000235:genetics*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D056915:DNA Copy Number Variations; D017931:DNA Primers; D059788:Dried Blood Spot Testing / Q000379:methods*; D005820:Genetic Testing / Q000379:methods; D059014:High-Throughput Nucleotide Sequencing / Q000191:economics / Q000379:methods*; D006801:Humans; D007231:Infant, Newborn; D060885:Multiplex Polymerase Chain Reaction / Q000191:economics / Q000379:methods; D015997:Neonatal Screening / Q000191:economics / Q000379:methods*; D011786:Quality Control; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: C505032:CFTR protein, human; D017931:DNA Primers; D019005:Cystic Fibrosis Transmembrane Conductance Regulator