Detection of Copy-Number Variants in Expanded Carrier Screening Maximizes Identification of Cystic Fibrosis Carriers

Cystic fibrosis is one of the most common autosomal recessive conditions. Medical-society guidelines recommend routine carrier screening for cystic fibrosis via targeted genotyping of 23 frequent single-nucleotide variants (SNVs) and short insertions or deletions (indels) in the CFTR gene. Screening for copy-number variants (CNVs) is recommended only when a reproductive partner is a known carrier. Here we assess the performance and clinical impact of routinely screening for SNVs, indels, and CNVs in a next-generation sequencing (NGS)-based expanded carrier screen (ECS). Pathogenic variants in CFTR from 103,718 patients were discovered via a validated NGS-based ECS. A custom algorithm identified CNVs via relative deviations in NGS read depth. Approximate CNV breakpoints were inferred from the NGS-depth profile. Positive CNVs were orthogonally assessed via multiplex ligation-dependent probe amplification (MLPA). CFTR CNV sensitivity was explored across a range of length scales via in silico simulations. Among carriers of cystic fibrosis, 98.7% had pathogenic SNVs or indels in the CFTR gene (79% had one of the 23 common variants), and the remaining 1.3% harbored a pathogenic CNV spanning at least one exon. In total, we observed 25 unique CNVs with a diversity of breakpoints, suggesting that algorithms must be configured to detect novel CNVs. Further, single-exon deletions were observed for seven different CFTR exons; analysis of confidence scores for these empirical deletions—coupled with extensive simulations—demonstrated that the bioinformatics pipeline was both accurate and robust, even for short CNVs. All CNVs identified via NGS were confirmed via MLPA. CNV detection maximizes identification of cystic fibrosis carriers and can be applied to all patients undergoing an NGS-based ECS test validated to detect these complicated variants. Clinical guidelines recommending screening of only the 23 most frequent variants miss critical identification of carriers and should be revisited.