NGS-Based Test for CF Promises Faster, Cheaper, More Thorough Analysis

Testing for the cystic fibrosis (CF) helped to fuel the first wave of clinical molecular diagnostics. Now, an assay developed by researchers at the Stanford University taps the power of multiplex next-generation sequencing (NGS) to enable early detection and management of CF in ways that could consolidate sample types and technologies.

The highly sensitive, specific, rapid, and potentially cost-effective new test is described in a paper published in the March issue of the Journalof Molecular Diagnostics.

Cystic fibrosis is the most common fatal genetic disease in the United States. Newborns in every U.S. state have been screened for CF since2010, but the current tests vary widely and have limitations.

"The assays in use are time-consuming and don't test the entire cystic fibrosis gene," says the study's senior author, Curt Scharfe, M.D., Ph.D.,now at Yale University. "They don't tell the whole story."

Most CF tests now in use begin with immunoreactive trypsinogen testing of dried blood spots (DBSs) taken from newborn heel sticks. Since this assay is sensitive but prone to false positives, newborn screening programs rely on tiered strategies, which reflex hypertrypsinogenemic specimens to methods that interrogate a relatively small number of common CF mutations. If one of the common mutations is identified, the infant's entire CF gene is sequenced to try to confirm whether the baby has a second, less common CF mutation. The process takes up to two weeks and can miss infants who carry two rareCF mutations, particularly in ethnically diverse populations.

The new assay, known as CFseq, reliably sequences the entire CFTR gene from a single 3.2-millimeter newborn DBS punch sample. This is the first time scientists have found a way to reliably use DBSs for this type of sequencing for CF, which typically requires much more DNA. Moreover, the one-step method costs less andtakes about half the time of current testing methods.

"In our new assay, we are reading every letter in the book of the CF gene," says study co-author Iris Schrijver, M.D., from Stanford. "Whatever mutations pop up,the technique should be able to identify. It's a very flexible approach."

In addition to NBS, the test could be used for carrier and diagnostic testing andbroadened to screen for other genetic diseases, not just CF.

Stanford's Molecular Pathology Laboratory has a contract with California for the state's CF newborn testing, so the immediate next steps are staff training and clinical validation of the new assay. California newborn screening officials will then have the opportunity to decide whether they want the new test to replace the currentmethod. Schrijver expects the process will take less than a year.

Takeaway: NGS using DBS provides a more thorough, time efficient, and lesscostly way to screen newborns for CF.