Fig 1.
Nonsense and frameshift mutations at 3’ region that synthesize complex glycosylated truncated protein can respond to CFTR modulators.
(A) Schematic of CFTR-Expression Minigene with full-length introns 25 and 26 (EMG-i25-i26) constructed in pcDNA5FRT plasmid. CFTR expression is driven by a CMV promoter. The location of each studied variant is shown relative to CFTR exons and regions predicted to elicit NMD. (B) Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) showing relative steady state levels of CFTR transcript in HEK293 stable cells expressing wild-type EMG or EMGs with nonsense or frameshift variants, as indicated. Values were normalized to B2M. Mean ± SEM (n = 3) measured in triplicates. P value was determined by one way ANOVA. **** indicates significant difference (P≤0.0001) when compared with CFTR mRNA abundance in cells expressing WT-EMG. (C) Immunoblot (IB) of the steady state amounts of immature core-glycosylated (band B) and the mature complex-glycosylated mature CFTR protein (band C). Lysates were collected from HEK293 cells expressing WT-EMG or EMGs with different PTC-generating variants. Lysates from cells expressing either intronless WT CFTR or F508del served as controls, or empty vector as negative control. 40 μg of total cell lysates were electrophoresed and IB was probed with anti-CFTR antibody (596 # Cystic Fibrosis Foundation Therapeutics). (D) Schematic illustration showing three groups of 3’- nonsense variants based on mRNA stability and protein maturity. (E) Immunoblot of HEK293 stable cells expressing Q1390X or E1418X. The cells were incubated for 48 h with DMSO (.03%) or corrector compounds (lumacaftor and tezacaftor either alone or in combination—3 μM each). CFTR was visualized with anti-CFTR antibody, 596 (CFFT). (F) A representative Ussing chamber tracing of EMG E1418X-expressing CFBE stable cells grown on snap-wells. Short-circuit current (Isc) measurements were recorded in Ussing chambers after treatment of cells with 0.03% DMSO (vehicle) or 3 μM corrector compounds (lumacaftor/tezacaftor or both) for 48 h. Cells were mounted on Ussing chambers to measure CFTR mediated chloride channel activity as a proxy of CFTR function. After stabilization of the basal current, forskolin (10 μM) was added to the basolateral chamber followed by potentiator, ivacaftor (10 μM), and CFTR Inhibitor 172 (10 μM) added to the apical chambers. Inh-172 was added earlier in DMSO no ivacaftor (dashed blue line) treated cells. (G and H) Stacked bar graphs indicate effect of modulator treatment on CFBE (G) and MDCK (H) stable cells expressing different CFTR 3’ nonsense variants. Change in Isc (ΔIsc) was defined as the current inhibited by Inh-172 after sustained Isc responses were achieved upon stimulation with forskolin alone or sequentially with ivacaftor. Mean ± SEM (n = 3–8). WT-CFTR function represents forskolin stimulated Isc without modulator treatment in cells expressing EMG i25-i26. P value was determined by one way ANOVA. **** P ≤0.0001, ** P ≤0.01, * P≤0.05, and n.s. (not significant, P>0.05); when compared with forskolin stimulated CFTR function in DMSO (vehicle) treated cells expressing respective variant.
Fig 2.
Corrector treatment increases CFTR activation response of nonsense variants in exon 22 that result in mature truncated CFTR.
(A) Schematic of CFTR-Expression Minigene with abridged introns 21 and abridged intron 22 (EMG-i21-i22) constructed in pcDNA5FRT plasmid. CFTR exons are shown in boxes and two abridged introns in dashed lines. The location of each studied variant is shown relative to the CFTR exons and regions predicted to elicit NMD. (B) RT-qPCR showing relative steady state levels of CFTR transcript in HEK293 stable cells expressing wild-type EMG or EMGs with truncations at residue position, as indicated on the labels. Values were normalized to B2M. Mean ± SEM (n = 3) measured in triplicates. P value was determined by one way ANOVA. **** indicates significant difference (P≤0.0001) when compared with CFTR mRNA abundance in cells expressing WT-EMG. (C) Steady state levels of CFTR protein from HEK293 cells transiently transfected with wild-type EMG or EMGs with different nonsense variants. 40 μg of total cell lysates were electrophoresed and IB was probed with anti-CFTR antibody-MM13-4 (EMD Millipore). (D) Representative IB showing sensitivity of CFTR to PNGaseF and Endo H. Mature complex glycosylated band is sensitive to PNGase only, whereas immature core glycosylated band is sensitive to both PNGase and EndoH. (E). Schematic illustration of the nonsense variants in the protein context showing their classification into two groups based on mRNA stability and protein maturity. Each nonsense variant truncates CFTR at intracellular loop 6 (ICL6) just before NBD2. (F) A representative Ussing chamber tracing of CFBE cells stably expressing S1196X-EMG. Short-circuit (Isc) measurements were recorded in Ussing chambers after treatment of cells with 0.03% DMSO (vehicle) or 3 μM corrector compounds (lumacaftor/tezacaftor or both) for 48 h. (G and H) Stacked bar graphs indicate effect of modulator treatment on CFBE (G) and MDCK (H) stable cells expressing different CFTR 3’ nonsense variants. Change in Isc (ΔIsc) was defined as the current inhibited by Inh-172 after sustained Isc responses were achieved upon stimulation with forskolin alone or sequentially with ivacaftor. Mean ± SEM (n = 3–8). WT-CFTR function represents forskolin stimulated Isc without modulator treatment in cells expressing EMG i21-i22. P value was determined by one way ANOVA. **** indicates significant difference (P ≤0.0001), n.s. (not significant, P>0.05); when compared with forskolin stimulated CFTR function in DMSO (vehicle) treated cells expressing respective variant.
Fig 3.
NMD inhibition has a synergistic effect on corrector-potentiator combination response in stable cells expressing nonsense variants in exon 22 that produce mature truncated CFTR.
(A) Relative expression of the alternate CFTR allele in the primary nasal cells of CF individuals carrying exon 22 nonsense variant. Pyrosequencing assay was designed such that exon 22 with upstream and downstream flanking exons was amplified from the corresponding cDNA preparations. Sequencing primer yielded relative abundances of alternate alleles at the respective loci where nucleotide change occurred. (B) CFTR mRNA decay in HEK293 cells stably expressing wild type EMG or EMG harboring nonsense variants R1158X or S1196X. Actinomycin D (3 μg/ml) was added at time 0 to induce transcriptional shut-down. Cells were collected at the indicated time points. Levels of the CFTR mRNAs were assessed by RT-qPCR, normalized to B2M mRNA and displayed as a percentage of the levels at t = 0. Mean ± SEM (n = 3) (C) Efficiency of siRNA mediated knock down of UPF1 detected on IB of whole cell lysates collected from HEK293 cells stably expressing either R1158X or S1196X. GAPDH siRNA and non-target (NT) siRNA were used as positive and negative controls respectively. Beta-Actin was used as loading control. (D) Effect of direct NMD inhibition on the level of CFTR transcript by siRNA mediated knock down of UPF1 in HEK293 cells stably expressing either R1158X or S1196X. Levels of the CFTR mRNAs were assessed by RT-qPCR and normalized to B2M mRNA. Mean ± SEM, n = 3 independent biological triplicates, P value was determined by two way ANOVA. ** (P≤0.01) and *** (P≤0.001) indicate significant difference when compared with CFTR mRNA abundance in untreated cells (E) Short-circuit (Isc) tracings of CFBE-S1196X stable cells recorded in Ussing chambers after direct inhibition of NMD by UPF1. Cells were transfected with Upf1 siRNA at 50% confluency for 4 days before being mounted on Ussing chambers. GAPDH and non-targeted (NT) siRNA transfections were used as controls. Cells were incubated with lumacaftor (3 μM) or DMSO (0.03%) during last 48h of siRNA transfections. (F) Stacked bar graphs indicate effect of UPF1 siRNA in combination with CFTR modulators. Change in Isc (ΔIsc) was defined as the current inhibited by Inh-172 after sustained Isc responses were achieved upon stimulation with forskolin alone or sequentially with ivacaftor. Mean ± SEM (n = 3). P value was determined by one way ANOVA. ** (P≤0.01) indicate significant difference when compared with forskolin stimulated CFTR function in NT siRNA transfected cells and **** (P≤0.0001) indicate significant difference when compared with ivacaftor activated CFTR function in NT siRNA transfected cells incubated with or without lumacaftor. WT-CFTR function represents forskolin stimulated ΔIsc without modulator treatment in cells expressing EMG i21-i22.
Fig 4.
CFTR nonsense variant with splicing defect has residual function that benefits from modulator treatments.
(A) A schematic illustration of CFTR-Expression Minigene with introns 14–18 (introns 14 and 16 are full-length, and 15, 17, & 18 are abridged). Arrow indicates location of E831X (top). CFTR mRNA splicing patterns of the total RNA extracted from HEK293 cells transiently transfected with E831X-EMG. (B) Steady state amounts of different isoforms of CFTR produced from E831X-EMG-i14-i18 expressed transiently in HEK293 cells. Lysates from cells expressing WT-EMG i14-i18, intronless WT CFTR or F508del served as positive controls, and empty vector as negative control. Immunoblot (IB) was probed with anti-CFTR antibody, 596 (CFFT). Horizontal arrows indicate to isoforms corresponding to (i) a normal codon (E831) substituted with a stop codon, (ii) deletion of complete exon 16, and (iii) deletion of a single amino acid E831. Beta-Actin was used as loading control. (C) Short-circuit (Isc) tracing of CFTR function observed in CFBE-stable cells expressing E831X mounted on Ussing chamber. Cells were treated for 48 h with correctors (lumacaftor/tezacaftor or both, 3 μM each) and acutely with potentiator (ivacaftor, 10 μM). Change in Isc (ΔIsc) was defined as the current inhibited by Inh-172 after sustained Isc responses achieved upon stimulation with forskolin alone or sequentially with ivacaftor. Data are presented as mean±SEM (n = 3). P value was determined by one way ANOVA. **** indicates significant difference (P ≤0.0001) when compared with forskolin stimulated CFTR function in DMSO (vehicle) treated cells. WT-CFTR function represents forskolin stimulated Isc without modulator treatment in cells expressing EMG i14-i18. (D) A tracing of CFTR function observed in primary nasal epithelial cells of an individual harboring E831X/F508del. CF-Human nasal epithelial (HNE) cells were treated for 24 h with lumacaftor and tezacaftor, 3 μM each, and acutely with Ivacaftor (10 μM). Stacked bar graph is a comparison of improvement in CFTR function of E831X/F508del vs F508del/Indel. Alternate Indel alleles were either 2184insA, 2183delAA>G, or 3659del C. P value was determined by one way ANOVA. ** indicates significant difference (P ≤0.01) when compared with forskolin stimulated CFTR function in DMSO (vehicle) treated E831X/F508del HNEs. * (P ≤0.05) when compared with CFTR function in modulator treated F508del/Indel HNEs.
Fig 5.
5’ nonsense variants that do not undergo NMD are the potential targets of read-through agents.
(A) Left panel—Sanger sequencing, and right panel—fragment analysis. Total RNA was extracted from the conditionally reprogrammed nasal epithelial cells of CF individual with F508del/L88X genotype. RT-PCRs were performed using CFTR-specific primers to amplify L88X and F508del regions. Area under the peak was used to determine expression of L88X transcript compared to F508del. (B) RNA-seq of the primary human nasal epithelial cells of healthy and L88X/F508del individuals. Density profile of all expressed genes (top), and relative transcript counts of L88X compared to F508del (bottom). (C) Schematic of CFTR-Expression Minigene with abridged introns 1, 2, 3, 4 and 5 constructed in pcDNA5FRT plasmid. CFTR exons are shown in boxes and abridged introns in dashed lines. The location of each studied variant is shown relative to the CFTR exons and regions predicted to elicit NMD. (D) Graph shows relative steady state levels of CFTR transcript in HEK293 stable cells expressing wild-type EMG or EMGs with truncations at residue position, as indicated on the labels. Values were normalized to B2M. Mean ± SEM (n = 3) measured in triplicates. P value was determined by one way ANOVA. No significant difference (n.s.) (P>0.01) when compared with CFTR mRNA abundance in cells expressing WT-EMG. (E) Immunoblot of the naturally occurring 5’-truncations on the steady state amounts of CFTR protein expressed in HEK293 cells. CFTR was visualized with anti-CFTR antibody-596 (CFFT), and anti-Na+K+ATPase served as control. (F) CFTR function measured in CFBE stable expressing L88X. Cells were incubated for 24 h with readthrough compound (G418, 5 μM and 25 μM)/ corrector (lumacaftor, 3 μM) or both. Short-circuit (Isc) tracing of CFTR function observed in CFBE-stable cells expressing L88X mounted on Ussing chamber. Change in Isc (ΔIsc) was defined as the current inhibited by Inh-172 after sustained Isc responses achieved upon stimulation with forskolin alone or sequentially with ivacaftor. Data are presented as mean ± SEM (n = 3). P value was determined by one way ANOVA. **** (P ≤0.0001), and *** (P ≤0.001) indicate significant difference when compared with forskolin stimulated CFTR function in DMSO (vehicle) treated cells. WT-CFTR function represents forskolin stimulated Isc without modulator treatment in cells expressing EMG i1-i5.
Table 1.
Heterogeneous effects of PTC generating CFTR variants.