In 2017, the US Food and Drug Administration initiated expansion of drug labels for the treatment of cystic fibrosis (CF) to include CF transmembrane conductance regulator (CFTR) gene variants based on in vitro functional studies. This study aims to identify CFTR variants that result in increased chloride (Cl^-) transport function by the CFTR protein after treatment with the CFTR modulator combination elexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA). These data may benefit people with CF (pwCF) who are not currently eligible for modulator therapies. Plasmid DNA encoding 655 CFTR variants and wild-type (WT) CFTR were transfected into Fisher Rat Thyroid cells that do not natively express CFTR. After 24 h of incubation with control or TEZ and ELX, and acute addition of IVA, CFTR function was assessed using the transepithelial current clamp conductance assay. Each variant's forskolin/cAMP-induced baseline Cl^- transport activity, responsiveness to IVA alone, and responsiveness to the TEZ/ELX/IVA combination were measured in three different laboratories. Western blots were conducted to evaluate CFTR protein maturation and complement the functional data. 253 variants not currently approved for CFTR modulator therapy showed low baseline activity (<10 % of normal CFTR Cl^- transport activity). For 152 of these variants, treatment with ELX/TEZ/IVA improved the Cl^- transport activity by ≥10 % of normal CFTR function, which is suggestive of clinical benefit. ELX/TEZ/IVA increased CFTR function by ≥10 percentage points for an additional 140 unapproved variants with ≥10 % but <50 % of normal CFTR function at baseline. These findings significantly expand the number of rare CFTR variants for which ELX/TEZ/IVA treatment should result in clinical benefit.

Newborn screening for cystic fibrosis (CF) has been universal in the US since 2010; however, there is significant variation among newborn screening algorithms. Systematic reviews were used to develop seven recommendations for newborn screening program practices to improve timeliness, sensitivity, and equity in diagnosing infants with CF: (1) The CF Foundation recommends the use of a floating immunoreactive trypsinogen (IRT) cutoff over a fixed IRT cutoff; (2) The CF Foundation recommends using a very high IRT referral strategy in CF newborn screening programs whose variant panel does not include all CF-causing variants in CFTR2 or does not have a variant panel that achieves at least 95% sensitivity in all ancestral groups within the state; (3) The CF Foundation recommends that CF newborn screening algorithms should not limit CFTR variant detection to the F508del variant or variants included in the American College of Medical Genetics-23 panel; (4) The CF Foundation recommends that CF newborn screening programs screen for all CF-causing CFTR variants in CFTR2; (5) The CF Foundation recommends conducting CFTR variant screening twice weekly or more frequently as resources allow; (6) The CF Foundation recommends the inclusion of a CFTR sequencing tier following IRT and CFTR variant panel testing to improve the specificity and positive predictive value of CF newborn screening; (7) The CF Foundation recommends that both the primary care provider and the CF specialist be notified of abnormal newborn screening results. Through implementation, it is anticipated that these recommendations will result in improved sensitivity, equity, and timeliness of CF newborn screening, leading to improved health outcomes for all individuals diagnosed with CF following newborn screening and a decreased burden on families.

Rationale: Lung disease is the major cause of morbidity and mortality in persons with cystic fibrosis (pwCF). Variability in CF lung disease has substantial non-CFTR (CF transmembrane conductance regulator) genetic influence. Identification of genetic modifiers has prognostic and therapeutic importance. Objectives: Identify genetic modifier loci and genes/pathways associated with pulmonary disease severity. Methods: Whole-genome sequencing data on 4,248 unique pwCF with pancreatic insufficiency and lung function measures were combined with imputed genotypes from an additional 3,592 patients with pancreatic insufficiency from the United States, Canada, and France. This report describes association of approximately 15.9 million SNPs using the quantitative Kulich normal residual mortality-adjusted (KNoRMA) lung disease phenotype in 7,840 pwCF using premodulator lung function data. Measurements and Main Results: Testing included common and rare SNPs, transcriptome-wide association, gene-level, and pathway analyses. Pathway analyses identified novel associations with genes that have key roles in organ development, and we hypothesize that these genes may relate to dysanapsis and/or variability in lung repair. Results confirmed and extended previous genome-wide association study findings. These whole-genome sequencing data provide finely mapped genetic information to support mechanistic studies. No novel primary associations with common single variants or rare variants were found. Multilocus effects at chr5p13 (SLC9A3/CEP72) and chr11p13 (EHF/APIP) were identified. Variant effect size estimates at associated loci were consistently ordered across the cohorts, indicating possible age or birth cohort effects. Conclusions: This premodulator genomic, transcriptomic, and pathway association study of 7,840 pwCF will facilitate mechanistic and postmodulator genetic studies and the development of novel therapeutics for CF lung disease.

Background The Clinical and Functional Translation of CFTR project (CFTR2) classified some cystic fibrosis transmembrane conductance regulator (CFTR) gene variants as non-cystic fibrosis (CF)-causing. To evaluate this, the clinical status of children carrying these mutations was examined. Methods We analyzed CF disease-defining variables over 2–6 years in two groups of California CF screen- positive neonates born from 2007 to 2011: (1) children with two CF-causing variants and (2) children with one CF-causing and one non-CF-causing variant, as defined by CFTR2. Results Children carrying non-CF-causing variants had significantly higher birth weight, lower immunoreactive trypsinogen and sweat chloride values, higher first year growth curves, and a lower rate of persistent Pseudomonas aeruginosa colonization compared to children with two CF-causing variants. Conclusions The outcomes in children 2–6 years of age with the L997F, G576A, R1162L, V754M, R668C, R31C, and S1235R variants are consistent with the CFTR2 non-CF-causing classification.

Elucidating the functional consequence of molecular defects underlying genetic diseases enables appropriate design of therapeutic options. Treatment of cystic fibrosis (CF) is an exemplar of this paradigm as the development of CFTR modulator therapies has allowed for targeted and effective treatment of individuals harboring specific genetic variants. However, the mechanism of these drugs limits effectiveness to particular classes of variants that allow production of CFTR protein. Thus, assessment of the molecular mechanism of individual variants is imperative for proper assignment of these precision therapies. This is particularly important when considering variants that affect pre-mRNA splicing, thus limiting success of the existing protein-targeted therapies. Variants affecting splicing can occur throughout exons and introns and the complexity of the process of splicing lends itself to a variety of outcomes, both at the RNA and protein levels, further complicating assessment of disease liability and modulator response. To investigate the scope of this challenge, we evaluated splicing and downstream effects of 52 naturally occurring CFTR variants (exonic = 15, intronic = 37). Expression of constructs containing select CFTR intronic sequences and complete CFTR exonic sequences in cell line models allowed for assessment of RNA and protein-level effects on an allele by allele basis. Characterization of primary nasal epithelial cells obtained from individuals harboring splice variants corroborated in vitro data. Notably, we identified exonic variants that result in complete missplicing and thus a lack of modulator response (e.g. c.2908G>A, c.523A>G), as well as intronic variants that respond to modulators due to the presence of residual normally spliced transcript (e.g. c.4242+2T>C, c.3717+40A>G). Overall, our data reveals diverse molecular outcomes amongst both exonic and intronic variants emphasizing the need to delineate RNA, protein, and functional effects of each variant in order to accurately assign precision therapies. Genetic variants that impact pre-mRNA splicing are a common cause of genetic disease and have varying downstream molecular consequences. As a result, precision therapies that function at the protein level are not always effective for these variants and thus careful assessment is necessary. Here we evaluate RNA-level effects of 52 variants in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and show that study of splicing and its consequences allows for more accurate assignment of precision therapies.