Next-Generation Molecular Testing of Newborn Dried Blood Spots for Cystic Fibrosis.

Autor:	Lefterova MI; Department of Pathology, Stanford University Medical Center, Stanford, California., Shen P; Stanford Genome Technology Center, Stanford University, Palo Alto, California., Odegaard JI; Department of Pathology, Stanford University Medical Center, Stanford, California., Fung E; Department of Pathology, Stanford University Medical Center, Stanford, California., Chiang T; Department of Pathology, Stanford University Medical Center, Stanford, California., Peng G; Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas., Davis RW; Stanford Genome Technology Center, Stanford University, Palo Alto, California., Wang W; Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas., Kharrazi M; California Department of Public Health, Environmental Health Investigations Branch, Richmond, California., Schrijver I; Department of Pathology, Stanford University Medical Center, Stanford, California; Department of Pediatrics, Stanford University Medical Center, Stanford, California., Scharfe C; Department of Pathology, Stanford University Medical Center, Stanford, California; Stanford Genome Technology Center, Stanford University, Palo Alto, California. Electronic address: curt.scharfe@yale.edu.
Jazyk:	angličtina
Zdroj:	The Journal of molecular diagnostics : JMD [J Mol Diagn] 2016 Mar; Vol. 18 (2), pp. 267-82. Date of Electronic Publication: 2016 Feb 01.
DOI:	10.1016/j.jmoldx.2015.11.005
Abstrakt:	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. (Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
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