Treatment of individuals with cystic fibrosis (CF) has been transformed by small molecule therapies that target select pathogenic variants in the CF transmembrane conductance regulator (CFTR). To expand treatment eligibility, we stably expressed 43 rare missense CFTR variants associated with moderate CF from a single site in the genome of human CF bronchial epithelial (CFBE41o-) cells. The magnitude of drug response was highly correlated with residual CFTR function for the potentiator ivacaftor, the corrector lumacaftor, and ivacaftor-lumacaftor combination therapy. Response of a second set of 16 variants expressed stably in Fischer rat thyroid (FRT) cells showed nearly identical correlations. Subsets of variants were identified that demonstrated statistically significantly higher responses to specific treatments. Furthermore, nearly all variants studied in CFBE cells (40 of 43) and FRT cells (13 of 16) demonstrated greater response to ivacaftor-lumacaftor combination therapy than either modulator alone. Together, these variants represent 87% of individuals in the CFTR2 database with at least 1 missense variant. Thus, our results indicate that most individuals with CF carrying missense variants are (a) likely to respond modestly to currently available modulator therapy, while a small fraction will have pronounced responses, and (b) likely to derive the greatest benefit from combination therapy.

Missense DNA variants have variable effects upon protein function. Consequently, interpreting their pathogenicity is challenging, especially when they are associated with disease variability. To determine the degree to which functional assays inform interpretation, we analyzed 48 CFTR missense variants associated with variable expressivity of cystic fibrosis (CF). We assessed function in a native isogenic context by evaluating CFTR mutants that were stably expressed in the genome of a human airway cell line devoid of endogenous CFTR expression. 21 of 29 variants associated with full expressivity of the CF phenotype generated <10% wild-type CFTR (WT-CFTR) function, a conservative threshold for the development of life-limiting CF lung disease, and five variants had moderately decreased function (10% to ∼25% WT-CFTR). The remaining three variants in this group unexpectedly had >25% WT-CFTR function; two were higher than 75% WT-CFTR. As expected, 14 of 19 variants associated with partial expressivity of CF had >25% WT-CFTR function; however, four had minimal to no effect on CFTR function (>75% WT-CFTR). Thus, 6 of 48 (13%) missense variants believed to be disease causing did not alter CFTR function. Functional studies substantially refined pathogenicity assignment with expert annotation and criteria from the American College of Medical Genetics and Genomics and Association for Molecular Pathology. However, four algorithms (CADD, REVEL, SIFT, and PolyPhen-2) could not differentiate between variants that caused severe, moderate, or minimal reduction in function. In the setting of variable expressivity, these results indicate that functional assays are essential for accurate interpretation of missense variants and that current prediction tools should be used with caution.

Rationale: The advent of precision treatment for cystic fibrosis using small-molecule therapeutics has created a need to estimate potential clinical improvements attributable to increases in cystic fibrosis transmembrane conductance regulator (CFTR) function.Objectives: To derive CFTR function of a variety of CFTR genotypes and correlate with key clinical features (sweat chloride concentration, pancreatic exocrine status, and lung function) to develop benchmarks for assessing response to CFTR modulators.Methods: CFTR function assigned to 226 unique CFTR genotypes was correlated with the clinical data of 54,671 individuals enrolled in the Clinical and Functional Translation of CFTR (CFTR2) project. Crosssectional FEV 1 % predicted measurements were plotted by age at which measurement was obtained. Shifts in sweat chloride concentration and lung function reported in CFTR modulator trials were compared with function-phenotype correlations to assess potential efficacy of therapies.Measurements and Main Results: CFTR genotype function exhibited a logarithmic relationship with each clinical feature. Modest increases in CFTR function related to differing genotypes were associated with clinically relevant improvements in cross-sectional FEV 1 % predicted over a range of ages (6-82 yr). Therapeutic responses to modulators corresponded closely to predictions from the CFTR2-derived relationship between CFTR genotype function and phenotype.Conclusions: Increasing CFTR function in individuals with severe disease will have a proportionally greater effect on outcomes than similar increases in CFTR function in individuals with mild disease and should reverse a substantial fraction of the disease process. This study provides reference standards for clinical outcomes that may be achieved by increasing CFTR function.

CFTR modulators have revolutionized the treatment of individuals with cystic fibrosis (CF) by improving the function of existing protein. Unfortunately, almost half of the disease-causing variants in CFTR are predicted to introduce premature termination codons (PTC) thereby causing absence of full-length CFTR protein. We hypothesized that a subset of nonsense and frameshift variants in CFTR allow expression of truncated protein that might respond to FDA-approved CFTR modulators. To address this concept, we selected 26 PTC-generating variants from four regions of CFTR and determined their consequences on CFTR mRNA, protein and function using intron-containing minigenes expressed in 3 cell lines (HEK293, MDCK and CFBE41o-) and patient-derived conditionally reprogrammed primary nasal epithelial cells. The PTC-generating variants fell into five groups based on RNA and protein effects. Group A (reduced mRNA, immature (core glycosylated) protein, function <1% (n = 5)) and Group B (normal mRNA, immature protein, function <1% (n = 10)) variants were unresponsive to modulator treatment. However, Group C (normal mRNA, mature (fully glycosylated) protein, function >1% (n = 5)), Group D (reduced mRNA, mature protein, function >1% (n = 5)) and Group E (aberrant RNA splicing, mature protein, function > 1% (n = 1)) variants responded to modulators. Increasing mRNA level by inhibition of NMD led to a significant amplification of modulator effect upon a Group D variant while response of a Group A variant was unaltered. Our work shows that PTC-generating variants should not be generalized as genetic ‘nulls’ as some may allow generation of protein that can be targeted to achieve clinical benefit.

Background: Cell-based studies have shown that W1282X generates a truncated protein that can be functionally augmented by modulators. However, modulator treatment of primary cells from individuals who carry two copies of W1282X generates no functional CFTR. To understand the lack of response to modulators, we investigated the effect of W1282X on CFTR RNA transcript levels. Methods: qRT-PCR and RNA-seq were performed on primary nasal epithelial (NE) cells of a previously studied individual who is homozygous for W1282X, her carrier parents and control individuals without nonsense variants in CFTR. Results: CFTR RNA bearing W1282X in NE cells shows a steady-state level of 4.2 ± 0.9% of wild-type (WT) CFTR RNA in the mother and 12.4 ± 1.3% in the father. NMDI14, an inhibitor of nonsense-mediated mRNA decay (NMD), restored W1282X mRNA to almost 50% of WT levels in the parental NE cells. RNA-seq of the NE cells homozygous for W1282X showed that CFTR transcript level was reduced to 1.7% of WT (p-value: 4.6e-3). Negligible truncated CFTR protein was generated by Flp-In 293 cells stably expressing the W1282X EMG even though CFTR transcript was well above levels observed in the parents and proband. Finally, we demonstrated that NMD inhibition improved the stability and response to correctors of W1282X-CFTR protein expressed in the Flp-In-293 cells. Conclusion:These results show that W1282X can cause substantial degradation of CFTR mRNA that has to be addressed before efforts aimed at augmenting CFTR protein function can be effective.

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.

Chromosomal structural variation can cause severe neurodevelopmental and neuropsychiatric phenotypes. Here we present a nonverbal female adolescent with severe stereotypic movement disorder with severe problem behavior (e.g., self-injurious behavior, aggression, and disruptive and destructive behaviors), autism spectrum disorder, severe intellectual disability, attention deficit hyperactivity disorder, and global developmental delay. Previous cytogenetic analysis revealed balanced translocations present in the patient's apparently normal mother. We hypothesized the presence of unbalanced translocations in the patient due to maternal history of spontaneous abortions. Whole-genome sequencing and whole-genome optical mapping, complementary next-generation genomic technologies capable of the accurate and robust detection of structural variants, identified t(3;10), t(10;14), and t(3;14) three-way balanced translocations in the mother and der(10)t(3;14;10) and der(14)t(3;14;10) translocations in the patient. Instead of a t(3;10), she inherited a normal maternal copy of Chromosome 3, resulting in an unbalanced state of a 3q28qter duplication and 10q26.2qter deletion. Copy-imbalanced genes in one or both of these regions, such as DLG1, DOCK1, and EBF3, may contribute to the patient's phenotype that spans neurodevelopmental, musculoskeletal, and psychiatric domains, with the possible contribution of a maternally inherited 15q13.2q13.3 deletion.

No abstract