Cystic fibrosis (CF) is a life-shortening disease that is caused by mutations in the CF transmembrane conductance regulator
) gene that lead to the loss or dysfunction of CFTR channel activity [1
]. Clinically, CF can affect multiple organs, including the upper airway, lungs, pancreas, sweat glands, intestine, liver, and vas deferens. The leading cause of morbidity and mortality for CF patients is chronic lung disease [3
]. The incidence of CF and the frequency of specific mutations vary among ethnic populations [5
CFTR is a cAMP/cGMP-regulated chloride (Cl−
) and bicarbonate (HCO3−
) channel that is primarily expressed at the apical (luminal) membrane of epithelial cells lining the airway, gut, and exocrine glands, where it regulates transepithelial fluid secretion and homeostasis [7
]. CFTR is a member of the ATP-binding cassette transporter superfamily, and it consists of 1480 amino acids. CFTR is composed of two membrane-spanning domains (MSD1 and MSD2), two nucleotide binding domains (NBD1 and NBD2), and a regulatory domain (R) [7
]. The CFTR channel can be activated through the phosphorylation of its R domain by various protein kinases (e.g., PKA) and binding and hydrolysis of ATP at its NBDs. CFTR channel activity is determined by the number, open probability, and conductance of channels at cell surface [10
]. Mutations in the CFTR
gene can alter one or more of these parameters, which causes the impairment or loss of the channel activity. Currently, 2103 CFTR mutations have been identified [11
], which were traditionally categorized into six groups based on the nature of defect(s) [12
]. Because some mutations have multiple defects, an expanded classification method was also proposed [14
]. F508del is the most prevalent CFTR mutation; approximately 80–85% of CF patients carry it on at least one allele worldwide [15
]. The classification of CFTR mutations helps to define strategies to restore CFTR channel function that is based on mutation-specific defect(s). The U.S. Food and Drug Administration has approved several CFTR modulators for CF therapy, including Trikafta™, Kalydeco®
, and Symdeko®
(Vertex Pharmaceuticals Inc., Boston, MA, USA) [17
Among these 2103 known CFTR mutations, only a relative few have been studied in detail at both the molecular and phenotypic levels. Therefore, it is critical to study the molecular and clinical characteristics of rare CFTR mutations, particularly of those seen in minority populations, to identify the defect(s) and help develop effective therapies. In this paper, we present two clinical cases of pediatric CF subjects who have a missense mutation F1099L (c.3297C>G). We also characterized the mutation at the molecular level in order to identify the nature of defect(s).
2. Materials and Methods
2.1. Clinical Features
These two subjects received standard care at the University of Tennessee Cystic Fibrosis Research and Care Center at LeBonheur Children’s Hospital (Memphis, TN, USA). Their medical records and CF Foundation Registry data were retrospectively analyzed after Institutional Review Board approval (UTHSC 13-02779-XM). The clinical information was de-identified.
2.2. Antibodies and Reagents
Antibodies: anti-CFTR (clone MM13-4, EMD Millipore Corporation, CA, USA), anti-CFTR (ACL-006, Alomone labs, Jerusalem, Israel), anti-β-actin (Sigma, MO, USA), goat anti-mouse IgG secondary antibody, HRP (Pierce Biotechnology, IL, USA), and goat anti-rabbit IgG Alexa Fluor 488 (Thermo Fisher Scientific, MA, USA). VX-809 (Selleckchem, TX, USA). Other reagents that were used in this study were purchased from Sigma or Fisher Scientific (PA, USA).
2.3. Generation of F1099L-CFTR Mutation (cDNA Name: c.3297C>G)
QuikChange™ Site-Directed Mutagenesis Kit (Stratagene, CA, USA) was used to generate c.3297C>G point mutation on a pcDNA3.1-wild type (WT)-CFTR background. The primers used were:
(TTC > TTG, amino acid F > amino acid L)
We confirmed all of the sequences at the Molecular Resource Center at The University of Tennessee Health Science Center.
2.4. Cell Culture and Plasmids Transfection
WT and CFTR mutant were expressed in HEK-293 cells (ATCC® CRL-1573™, Manassas, VA, USA). The cells were grown in DMEM/F12(1:1) medium (Invitrogen, NY, USA) containing 1% penicillin-streptomycin (Invitrogen) and 10% fetal bovine serum (Invitrogen) supplements, and then incubated at 37 °C with 5% CO2, unless otherwise stated. The cells were transfected using Lipofectamine 2000 (Invitrogen) following the manufacturer’s instruction. At 48 h post-transfection, the cells were used for Western blotting or iodide efflux assay.
2.5. Western Blotting
The cells were lysed in a lysis buffer [1× PBS, containing 0.2% Triton-X-100 and protease inhibitors (cOmplete™, Roche, IN, USA)] for 30 min. at 4 °C, and centrifuged at 12,000 rpm for 15 min. at 4 °C. The total protein levels of supernatants were measured by using Bradford protein assay. The supernatants were mixed with 4× sample buffer, denatured, subjected to SDS-PAGE on 5–14% Gel (Bio-Rad, CA, USA), and then transferred to PVDF membranes (Pierce Biotechnology). The membranes were blocked with blocking buffer (5% milk in 0.1% PBS-T) and then probed with respective antibody against CFTR (MM13-4, 1:1,000 dilution) or β-actin (1:5000 dilution). The protein bands were visualized while using ECL™ Western blot detection reagents (GE Healthcare, Buckinghamshire, UK) and quantified using ImageJ software (US National Institutes of Health). The Supplemental Materials
provided the whole blots and densitometry readings and analysis of the bands of interest (Figures S1–S3
2.6. Real-Time PCR to Measure CFTR mRNA Levels
The total RNA was isolated from HEK-293 cells transfected with WT- or F1099L-CFTR using Purelink RNA Mini Kit (Thermo Fisher Scientific). One microgram (1 μg) of total RNA was converted to cDNA using SuperScript III Reverse Transcriptase (Invitrogen). Real-time PCR was performed using LightCycler 480 (Roche, Indianapolis IN, USA). The primers for human CFTR were:
The primers for human GAPDH were:
The parameters for PCR thermocycling were: 95 °C for 5 min., 95 °C for 10 s (40 cycles), and 60 °C for 30 s. All of the samples were run in triplicate. The levels of CTFR mRNAs were normalized to GAPDH.
2.7. Iodide (I−) Efflux Assay
Cells were grown on poly-lysine-coated 60-cm culture dishes and then transfected with WT-CFTR, F1099L-CFTR, or empty vector using Lipofectamine 2000. At 48 h post transfection, the culture medium was removed, and the cells were loaded for 60 min. at room temperature with an I--containing loading buffer (136 mM NaI, 137 mM NaCl, 4.5 mM KH2PO4, 1 mM CaCl2, 1 mM MgCl2, 10 mM glucose, and 5 mM HEPES, pH 7.2). After removing the loading buffer, the cells were washed five times with an efflux buffer (136 mM NaNO3 replacing 136 mM NaI in the loading buffer) to remove the extracellular NaI. One milliliter (1 mL) of the efflux buffer was then added to each culture dish and the samples were collected after 1 min. The procedure was repeated another three times (note: these four samples were used to establish a stable baseline I− efflux for each culture dish). Subsequently, a cocktail of PKA activating agonists (final concentrations: 10 µM forskolin, 200 µM CPT-cAMP, and 100 µM IBMX) were added to the efflux buffer to activate the CFTR channel and another six samples were collected from each dish to measure the CFTR-mediated I- efflux. The I− concentrations in the collected samples were measured using a combination iodide electrode (9653BNWP, Orion Research Inc., Chelmsford, MA, USA). The I- efflux rates were reported as nano-moles (nmol)/min. Upon the completion of sample collection, the cells were lysed and the total protein concentrations were measured using Bradford protein assay. The maximal I- efflux rates of samples were normalized to their total protein concentrations of cell lysates.
2.8. Immunofluorescence Labeling and Imaging
HEK-293 cells were grown on poly-lysine-coated microscope cover glasses and then transfected with F1009L-CFTR, WT-CFTR, or vector plasmids. After 24 h, VX-809 (5 µM) or DMSO was added and cells were cultured for another 24 h. The cells were washed twice with PBS, fixed with 4% formaldehyde for 10 min., permeabilized with 0.1% Triton-X-100 for 5 min., and then blocked with goat serum (1% in PBS) for 30 min. at room temperature. The cells were incubated with a CFTR polyclonal antibody (ACL-006: 1:50) at 4 °C overnight, washed five times with PBS, and incubated with the secondary anti-rabbit Alexa Fluor 488 (1:2000) for 1 h. The cover glasses were mounted with DAPI Fluoromount-G (SouthernBiotech, AL, USA). Fluorescence images were taken on a Zeiss 710 LSM microscope while using a 60× objective.
2.9. Statistical Analysis
The data were reported as Mean ± S.E.M (standard error of the mean). Student’s t-test was used for statistical analysis and p values < 0.05 were considered to be significant.
Newborn screening programs and full-length CFTR sequencing have facilitated the identification of rare or unique CFTR mutations, especially in minority populations where classical CF phenotypes are uncommon [5
]. Many of these rare mutations have not been studied at the molecular level, and the nature of the defects has not been identified. The lack of this knowledge hinders the development of effective and personalized therapies in minority populations.
We identified a missense mutation F1099L in two pediatric CF patients. We characterized this mutation at the molecular level while using a heterologous cell expression model, and found that (1) F1099L-CFTR has a defect in protein maturation. When compared to WT-CFTR, the total protein level and maturation efficiency of F1099L mutant were significantly decreased; (2) F1099L-CFTR has an impaired channel function (23% of WT-CFTR); (3) F1099L-CFTR is a temperature-sensitive mutant; lowering the temperature promoted the protein maturation; and, (4) F1099L-CFTR responded very well to VX-809 treatment. The total CFTR protein level and the channel function were markedly increased (3.7- and three-fold, respectively) with the use of VX-809, to levels almost comparable to WT-CFTR.
Our molecular characterization of F1099-CFTR was conducted in HEK-293 cells. Although HEK-293 is one of the most commonly used cell lines for studying CFTR protein expression and channel function, caution should be exercised when trying to associate these data with clinical findings. For the purpose of developing personalized medicine for these two or other patients with a F1099L mutation, a better approach is to harvest the rectal or trachea/bronchial biopsies and test their responses to CFTR modulators in a Ussing chamber, or to convert these biopsies into organoids and use the forskolin-induced swelling assay to test their responses [18
]. The CFTR modulators to be tested should include the active components of Trikafta™: VX-661 (Tezacaftor), VX-445 (Elexacaftor), and VX-770. Currently, there are seven patients with F1099L in the CFTR2 database. F1099L was listed as a mutation with varying clinical consequences [19
]. By using extensive sequencing of CFTR
gene, McGinniss and colleagues identified F1099L in a pediatric patient who has the genotype of F508del/F1099L and showed positive newborn IRT and borderline sweat chloride levels (48, 52 mmol/L) [20
]. Degrugillier et al. reported a clinical case of a patient with F508del and F1099L who had severe chronic rhinosinusitis. The authors found that F1099L-CFTR matured less fully than WT-CFTR and that VX-809 alone or in combination with VX-770 promoted the maturation of F1099L-CFTR, to a level that is comparable to WT-CFTR. They did not find significant difference in the correction efficiency between VX-809 alone and a combination of VX-809 and VX-770 [21
]. Recently, Dr. Cutting’s group assessed the function of 48 CFTR
missense variants in CF bronchial epithelial cells (CFBE41o-). They found that F1099L-CFTR reduced the chloride conductance to 15.1 ± 6.4% of WT-CFTR [22
]. Our data are consistent with these findings. The independent and corroborative results from different research groups consolidate our understanding of F1099L mutation and help to develop effective therapies for patients with this mutation.
It has been reported that, in addition to F508del, VX-809 also worked on other CFTR mutations, although with varying efficacies [23
]. In a previous study, we found that VX-809 could partially rescue the expression and function of a G1208D mutation [24
]. VX-809 was more efficacious for rescuing F1099L than for G1208D. F1099L-CFTR is located in the 4th cytosolic loop (CL4) between the transmembrane domain TM10 and TM11 based on the Swiss-Prot numbering system or the original Science paper number [25
]. A cluster of mutations have been found in CL4 that cause misfolding of the CFTR protein; examples included R1070W, R1070Q, F1074L, A1067T, and R1066H [16
]. CL4 has been shown to interact with NBD1, and this interface is important for CFTR folding [28
]. Studies on the three-dimensional structures of WT, F508del, and these other responding mutations could help to unveil the mechanisms of action of VX-809 and other newer correctors (e.g., Tezacaftor and Elexacaftor), and develop more effective CFTR correctors.
It is known that the pulmonary disease phenotypes do not correlate well with CFTR genotypes, and that other genetic (e.g., modifier genes) and environment factors (e.g., socioeconomic status) affect the disease severity [29
]. Our finding that F1099L-CFTR has a residual CFTR function (23% of normal CFTR) seems to correlate with the mild disease phenotypes that were shown in these two subjects, with both having pancreatic sufficiency and normal chest radiograph. This finding also correlates with the sweat chloride levels in these two subjects: when paired with G551D in patient #1, the sweat chloride levels were high (84–116 mmol/L); when paired with 3849+10kbC->T (which usually associates with mild disease presentation) in patient #2, the sweat chloride levels were low in the range of 24–43 mmol/L.
Although our patients currently showed mild CF disease phenotypes, we cannot, as of yet, predict the effect of F1099L mutation on disease progression. It is known that patients with residual CFTR channel activity can still develop bronchiectasis with chronic sino-pulmonary infections and other CF-related diseases when they get older [12
]. Given that F1099L was very responsive to VX-809 correction, we speculate that VX-809 and similar CFTR modulators (e.g., Tezacaftor and Elexacaftor) could be beneficial for patients with this mutation. For subject #1, who has G551D and F1099L mutations, it might be interesting to test whether Orkambi®
, or Trikafta™ might be a better option for therapy than Kalydeco®
alone by using a personalized medicine approach if his clinical status worsens on ivacaftor alone.