Carrier Screening for Cystic Fibrosis among IVF Patients Utilizing Next Generation DNA Sequencing Detects Common, Rare, and Otherwise Undetectable Mutations

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Carrier screening for cystic fibrosis (CF) is recommended by the American College of Medical Genetics, the American Congress of Obstetricians and Gynecologists and societies representing the Ashkenazi Jewish population. Due to cost considerations and restrictive technologies, traditional CF carrier screening assays are designed to look for only the most common CF mutations. Next generation DNA sequencing (NGS) allows for comprehensive determination of CF carrier status through its ability to detect a much larger set of mutations, thereby increasing detection of CF mutations in Caucasians, as well as in patients of other or mixed ethnicities. NGS is expected to increase the sensitivity of preconception screening for CF carriers over traditional assays by increasing dramatically the number of CF mutations detected (≥550 vs. ∼100). Our objective was to evaluate the clinical effectiveness of NGS for detecting not only common but also rare CF mutations that would go undetected using traditional carrier screening assays. A high-throughput and proprietary methodology (comprised of multiplex gene capture, NGS and computational analysis), which looks for over 550 known pathogenic CF mutations, was applied to over 4,000 patient DNA samples, as of this writing. Clinical reports were issued on the presence or absence of disease-causing CF mutations. Among 4,000 patients screened for CF utilizing NGS, we were able to detect 114 carriers, a frequency consistent with that reported in the literature given the ethnic mix tested. Several of these mutations – including S1455X (c.4364C>G), P67L (c.200C>T), Q1476X (c.4426C>T) - were not detected by traditional screening assays. Had traditional screening assays been used, these carriers would have been missed, thereby increasing the risk of having a child with CF. Additionally, during test validation, a DNA sample from a CF patient was found to have not only a known mutation (R334/c.100C>T) from one parent, but also a previously uncharacterized mutation (c.3368-2A>T) from the other parent. If the parent carrying c.3368-2A>T had been tested with a traditional, more limited carrier screening assay, this at-risk couple would have been missed. NGS has significant advantages over traditional carrier screening assays. Due to the vast number of pathogenic mutations detectable, NGS enables comprehensive examination of carrier status for CF in persons of all ethnicities, and is, therefore, expected to yield higher detection rates, resulting in fewer missed carriers.