Next-Generation Sequencing for Cystic Fibrosis: Florida Newborn Screening Experience

Cystic fibrosis (CF) is an autosomal recessive genetic condition affecting nearly 1 in 4000 newborns. Early diagnosis and treatment have been shown to improve the care of individuals with CF, which is enhanced through newborn screening (NBS). The state of Florida has been performing CF NBS since 2007, and in 2022, Florida implemented enhanced next generation sequencing (NGS). The goal of this change was to identify individuals from under-represented racial and ethnic groups, who may have rare or de novo variants. NBS screening for CF involved a first tier with immunoreactive trypsinogen (IRT) ≥ 50 or the top 4% of daily specimens, whichever is lower, reflexing to a second tier. As of 2022, the second tier has evolved to an expanded sequence with an Agena 74-variant panel. Single variants would then reflex to the third tier utilizing NGS. NGS is able to confirm what is detected in second-tier testing, adding variants not included in the Agena panel, and refining the TG replications for Poly-T variants to determine pathogenicity of 5T results. When there is a variant of varying clinical consequence between the two databases, the most conservative classification is selected. Individuals with variants would then be referred to one of the contracted CF NBS referral centers for confirmatory sweat chloride testing (sweat). With implementation of NGS, referrals nearly tripled in 2022-2024, with 538 referrals in 2019; 485 in 2020; and 805 in 2021; followed by 1223 referrals made in 2022; 1146 in 2023; and 1294 in 2024. In 2022-2024, 71% of referrals to the contracted NBS CF referral centers were for single variant results, and no cases of CF were identified from these referrals. The number of CF cases remained about the same, ranging from 23 to 40 through the years 2019-2024. The number of CRMS/CFSPID cases, however, tripled going from 10 to 12 in 2019-2022 to over 100 in 2024. The reason for this change seems to be related to complex heterozygous genetic variants as opposed to abnormal sweat. Implementation of NGS for CF in Florida led to a significant increase in the identification of CFTR variants which affected all aspects of the NBS CF process, from an increased workload on the NBS laboratory and follow-up staff, to an increase in referrals to the NBS CF referral centers. The majority of referrals were for single-variant results, which meant the infants had a very low likelihood of having CF. It is recommended that when an algorithm involving NGS is utilized, one should verify that there are appropriate processes for sweat, including the manner in which single-variant CF results are handled, avoiding unnecessary healthcare utilization.