4992 Molecular Medicine REPORTS 22: 4992-5002, 2020

Research advances in molecular mechanisms underlying

the pathogenesis of cystic fibrosis: From technical
improvement to clinical applications (Review)
TAO WEI1*, HONGSHU SUI1*, YANPING SU1*, WANJING CHENG1,
YUNHUA LIU1, ZILIN HE1, QINGCHAO JI1 and CHANGLONG XU2

1
Department of Histology and Embryology, Shandong First Medical University and
Shandong Academy of Medical Sciences, Tai'an, Shandong 271000; 2Reproductive Medical Center,
Nanning Second People's Hospital, Nanning, Guangxi Zhuang Autonomous Region 530031, P.R. China

Received April 29, 2020; Accepted September 17, 2020

DOI: 10.3892/mmr.2020.11607

Abstract. Cystic fibrosis (CF) is a chronic disease causing Contents

severe impairment to the respiratory system and digestive
tracts. Currently, CF is incurable. As an autosomal recessive 1. Introduction
disorder, the morbidity of CF is significantly higher among 2. Molecular mechanism underlying the CFTR mutation in CF
Caucasians of European descent, whereas it is less pervasive 3. Technical advances and implementations in CF
among African and Asian populations. The disease is caused 4. Molecular regulators in CF
by identical mutations (homozygosity) or different muta- 5. Clinical applications of CF‑associated molecules
tions (heterozygosity) of an autosomal recessive mutation at 6. Challenges and perspectives
position 7q31.2‑q31.1 of chromosome 7. Diagnostic criteria 7. Conclusions
and guidelines work concurrently with laboratory detec-
tion to facilitate precise CF detection. With technological
advances, the understanding of CF pathogenesis has reached 1. Introduction
an unprecedented level, allowing for increasingly precise
carrier screening, more effective early stage CF intervention Cystic fibrosis (CF) is an autosomal recessive disease that can
and improved prognostic outcomes. These advances signifi- be attributed to the disrupted function of the CF transmem-
cantly increase the life quality and expectancy of patients brane conductance regulator (CFTR) gene (1,2). Although CF
with CF. Given the numerous improvements in the field of CF, predominantly affects the lungs, it is a multiorgan disease (3),
the current review summarized the technical advances in the affecting the pancreas, liver, kidneys (4) and intestine (5).
study of the molecular mechanisms underlying CF, as well as CFTR mutation is the cause of the pathogenesis of CF (6)
how these improvements facilitate the clinical outcomes of and CF is generally a result of the deletion of the phenyl-
CF. Furthermore, challenges and obstacles to overcome are alanine at the 508th position of CFTR, which is induced by
discussed. the loss of three nucleotides (7). In vertebrates, CFTR serves
as a membrane protein and participates in the functions of
Cl‑ channels (8,9). Due to its important regulatory functions,
CFTR is ubiquitous throughout the body and is expressed in
epithelial cells in the kidney, pancreas, airway, intestine (4),
Correspondence to: Dr Changlong Xu, Reproductive Medical sweat glands and the male reproductive tract, where it serves a
Center, Nanning Second People's Hospital, 13 Dancun Road, fundamental role in the transepithelial fluid (10). The number
Nanning, Guangxi Zhuang Autonomous Region 530031, P.R. China of identified CF‑associated mutations are increasing, with
E‑mail: [email protected]; [email protected]
~1,700 CFTR mutations being previously recognized to be
CF‑prone (11); however, this number was re‑estimated at 383
*
Contributed equally
in 2019 according to the Clinical and Functional Translation
Abbreviations: CF, cystic fibrosis; CFTR, cystic fibrosis of CFTR website (www.cftr2.org; date of access: 05/08/20).
transmembrane conductance regulator These potential mutations were screened by a specific criteria
that determines the mutations responsible for the onset of CF:
Key words: cystic fibrosis, cystic fibrosis transmembrane Firstly, the mutation could cause changes in the amino acid
conductance regulator, cystic fibrosis transmembrane conductance sequence, affecting both the expression level and functions
regulator mutation, sweat test, gene therapy of CFTR (12); secondly, the mutation introduces premature
signals and exhibits a novel amino acid sequence that is absent
in the normal CFTR gene (13).
 WEI et al: CURRENT ADVANCES IN CF 4993

The prevalence of CF varies with ethnicity (14,15). The prognostic outcomes of CF are discussed. Briefly, a PubMed
relatively high incidence of CF among Caucasians may be (pubmed.ncbi.nlm.nih.gov; date of access: 13/08/2020) search
attributed to their increased number (>1,400) of CFTR muta- was conducted using the following key words: ‘Cystic fibrosis’,
tions (16). Furthermore, while 1/3,000 of Caucasians will ‘molecular’, ‘diagnosis’, ‘prognosis’ and ‘therapy’. Examples
develop CF, the incidence is lowered to 1/15,000 among the were chosen as long as they fulfilled one of the following eligi-
African population, further decreasing in Asian populations bility criteria: i) Provided genetic information regarding the
to 1/30,000, compared to the aforementioned two ethnic pivotal role of CFTR in CF; ii) described the latest progresses
groups (15). The ratio of CF incidence between male and in parsing the molecular mechanisms underlying CF using
female is 1:1; however, the mortality rate of CF‑associated novel techniques; iii) demonstrated the association between
lung infections is higher among female patients as they are CF and other bioactive molecule (molecular chaperone) and
subjected to greater deterioration of pulmonary function at the potential clinical implementations, including CF diagnosis
puberty. These gender/age gaps have been proposed to be a and treatment.
result of the elevation in the hormone secretion (including
estrogen) in adults, which may disrupt airway ion transport in 2. Molecular mechanism underlying the CFTR mutation
lungs (17). in CF
There are two major molecular subtypes of CF: Classic CF
and non‑classic CF. Non‑classic CF refers to CF with better Molecular structure of CFTR. The molecular weight and
prognostic outcome, as certain functions of the CFTR protein length of the CFTR protein are 1,480 amino acids and
are preserved, providing advantages for survival. Non‑classic 168,173 Da, respectively (7,12,27). The length of its coding
patients with CF have ≥1 copy of a defect CFTR gene with sequence, which encodes the amino acid sequence for protein
partially conserved CFTR protein functions. Due to the partial products, is 4,443 bp (28). The intron‑free sequence of the
preservation of pancreatic exocrine functions, the symptoms of CFTR transcript is 6,129 bp in length (12,28), whereas the
digestion disorders are less common among patients suffering normal allelic variant for CFTR is ~250,000 bp in length and
from this milder type of CF. In contrast, patients suffering contains 27 exons (12,28). CFTR is comprised of 5 functional
from classic CF have completely lost their functional CFTR domains (12): Two domains (MSD1 and MSD2) controlling
protein. This subtype is characterized by persistent bacterial membrane‑spanning, which constitute the ion channel for
infection in the airways and sinuses, disrupted fat digestion Cl‑ transportation; an R domain, which exerts regulatory roles;
due to the lack of pancreatic exocrine, male dysgenesis due to and two domains (NBD1 and NBD2) that bind and catalyze
obstructive azoospermi and increased sweat Cl‑ levels (18‑20). the hydrolysis of adenosine triphosphate.
The original description of CF can be dated back to
1938 (21). Since then, progress in the understanding of CF Biological functions of CFTR. The CFTR protein is positioned
has been made in a step‑by‑step manner and the following in the cell membrane (29) and is associated with proteins
50 years has witnessed remarkable improvement in life expec- involved in the active transportation of material through the
tancy and life quality among patients with CF (22), which may cell membrane (12,30). Specifically, CFTR regulates the move-
be attributed to technological innovations. In the late 1950s, ment of Cl‑. Therefore, defects in CFTR gene can render the
a stimulated sweat test to diagnose patients with CF through CFTR protein absent or dysfunctional, thereby blocking the
Cl‑ or Na levels was developed (23) based on the recogni- transportation of Cl‑ to the cell surface (29,30). Additionally,
tion of the altered electrolyte composition in sweat (24). The aside from Cl‑, CFTR regulates the epithelial Na channel (31).
preliminary works contributed markedly to the diagnosis of Abnormalities in the CFTR protein disrupt the balance
CF and the understanding of CF was further promoted nearly between Na and Cl‑ ions (30,32), which leads to changes in
30 years later due to the discovery of the CFTR gene, a key mucous constituents and abnormal reabsorption of H2O. This
mediator of CF (9), which enabled the diagnosis of CF by produces a layer of thick, sticky mucus that cannot be removed
directly identifying 2 mutated CF alleles (25). Aside from by cilia, which eventually causes inadequate mucociliary
improved diagnostics, numerous therapies have been applied function and chronic infections (33). This can be fatal. In the
to treat CF, including antibiotics against infections, nutritional lungs, accumulated mucus can become infested with bacteria
supplementation and/or lung transplantation, through which and the chronic inflammation leads to pneumonia, resulting
the life expectancy of patients with CF can be significantly in deterioration with life‑threatening difficulties in breathing.
prolonged (26). Given the molecular mechanisms of the deficiency of CFTR,
Despite prognostic improvements of CF, the median the common symptoms of CF include severe cough and short-
survival of patients with CF is <50 years (22). As described ness of breath; however, CF can also lead to abnormal bowel
above, as the molecular mechanisms in CF are associated with movements, difficulty in gaining weight and infertility (12,29).
ethnical and sex differences in terms of incidence rate, they
can also be used to determine phenotypes of CF. Therefore, Classification of CFTR mutations. Based on the effects on
the innovation of methods for the detection and identification protein translation, cellular processing or channel gating of
of CF at the molecular level will be beneficial to the diagnosis CFTR (28,30), several different classification systems (Fig. 1)
and prognosis of CF. The current review aimed to summa- have been proposed over the years. Generally, the Class 1
rize the recent research advances of CF, including technical mutation results in severe disease, as this mutation prevents the
improvement in the understanding of the molecular mecha- CFTR protein from being generated. Patients with Class 1A
nism of CF. Additionally, the increasing number of molecular mutation do not synthesize any CFTR mRNA. Furthermore,
markers that have the potential to improve diagnostic and patients with Class 1B produce damaged CFTR mRNA,
4994 Molecular Medicine REPORTS 22: 4992-5002, 2020

Figure 1. Different types of CFTR mutations. Generally, intact CFTR mRNA can be generated from the cell nucleus and following correct folding, sufficient
amount of normal CFTR protein is transported to the cell membrane to serve as a Cl‑ channel. In contrast, different malfunctions in this multi‑step process
lead to different CFTR defects. CFTR, cystic fibrosis transmembrane conductance regulator; Cl‑, chloride ion.

which cannot be converted into protein (28). In Class 2 muta- F508del mutation in CFTR (36). In the following decades,
tions, the CFTR protein is produced; however, it is misfolded. expression of CFTR had been verified by various models by
The misfolded protein will be prevented from migrating to the reverse transcription‑quantitative PCR (RT‑qPCR). Certain
cell membrane. In Class 3 mutations, channels in the CFTR implementations of RT‑qPCR in CF include the following:
protein are not properly opened due to gate defect (29,32,34). i) In CF cell IB3‑1 transduced with CFTR vectors, CFTR
For the Class 4 mutation, while the CFTR protein is responsive mRNA expression was detected using RT‑qPCR, whereby the
to cell signaling, it is misshapen, resulting in a limited flow of efficiency of transduction was measured (37); and ii) multiplex
Cl‑ ions. Furthermore, in Class 5 mutations, insufficient CFTR fluorescent RT‑qPCR was used for scanning the exons to
protein is produced, leading to a reduction in the number of detect large CFTR rearrangements (38).
CFTR protein channels at the cell membrane (35). In class 6 Aside from the aforementioned utilizations in CFTR
mutations, less stable protein is prematurely degraded after detection, PCR is frequently performed to examine bacte-
it reaches the cell surface. Relatively, this mutation is less rial infection in CF; for instance, PCR was used to detect
severe compared with the other mutations and, therefore, is a Aspergillus fumigatus DNA, which commonly infects the
milder subtype. Generally, the Class 1, 2 and 3 mutations are airways of patients with CF (39), in samples collected from
more common and responsible for insufficiency in the organs patients with CF. Preimplantation genetic testing (PGT) is an
suffering from CF. important method to detect CF before or at pre‑embryonic
stages (40). The updated version of the PGT guidelines
3. Technical advances and implementations in CF regarding CF proposed that PCR analysis should be performed
to detect the causative mutation(s), along with associated poly-
PCR analysis of CF. PCR is a widely used laboratory tech- morphisms within or near to the CFTR gene (41). Nevertheless,
nique that allows for the semi‑quantification of mRNAs. As PCR has its own limitations. For instance, during unequal
early as 1992, allelic specific‑PCR was used for detecting allelic PCR amplification, allele dropout can hamper the
 WEI et al: CURRENT ADVANCES IN CF 4995

detection of CFTR mutations, as the annealing of a primer to 4. Molecular regulators in CF

the matched allelic sequence is predominated as compared to
its mismatched counterpart (42). Non‑coding RNAs (ncRNAs) in CF. Although CF is monogenic,
the phenotypes of patients with CF are heterogeneous, which
Implementation of next‑generation sequencing (NGS) in may be attributed to multiple regulators that contribute to CF
CF. The revolutionary innovation of sequencing technolo- pathogenesis (53). Non‑coding (nc)RNAs are a type of RNA
gies, including NGS platforms, allows for the detection of molecule that do not translate into a protein. Instead, ncRNAs
a broader spectrum of potential mutations in CF, particu- exerts regulatory roles in multiple biological processes, such
larly single‑nucleotide polymorphism (43), which are as translation (54), RNA splicing (55) and gene regulation (56),
hypothesized to be a cause of CF (44). NGS outperformed among which microRNA (miRNA or miR), long non‑coding
whole genome sequencing in terms of cost‑efficiency and RNA (lncRNA) and circular RNA (circRNA) have been exten-
its accuracy is guaranteed by stringent thresholds during sively studied. The current review discusses several ncRNAs
data processing (43). Therefore, NGS has been widely used that participate in CF pathogenesis.
in carrier (at‑risk individual) screening, including CFTR
mutation screening, to improve genetic counseling and miRNA and CF. Previous studies have demonstrated that
reduce the incidence of CF among carriers' (at‑risk‑couples) miR‑145, miR‑494 and miR‑101 directly or indirectly target
descendants (43). In another study of methodology estab- and regulate CFTR (53,57‑59). The inhibition of miR‑145
lishment, NGS‑based expanded carrier screening, which through peptide nucleic acids was reported to promote the
determines variants through hybridization capture gene expression of CFTR, as miR‑145 binds to the 3'‑untranslated
enrichment, identified several genetic alterations, including region (3'UTR) of the CFTR gene (57). Additionally, the
copy‑number variants in the CFTR gene. The combination interaction of miR‑494/miR‑101 and CFTR was verified and
of NGS and variant interpretation achieved higher accuracy confirmed through luciferase reporter assays (58). Considering
in identifying CF‑associated phenotypes compared with the evidence that has demonstrated the inhibitory effects of these
traditional method (23 variants screening) (45). Furthermore, miRNAs on CFTR, the exacerbated pulmonary condition
by retrospectively performing NGS assays on patients with caused by air pollution or cigarette smoke was studied and
single CF mutated screened by sweat tests, all CFTR muta- attributed to elevated miR‑101 and miR‑144 (60). Although
tions were correctly detected, indicating that NGS assays most miRNAs that directly target CFTR serve as suppressors,
were completely concordant with traditional methods (46). certain miRNAs exert indirect regulatory roles on CFTR to
These reports demonstrated the effective implementation promote its expression (Fig. 2). For instance, in primary epithe-
of NGS in CF detection, particularly at the early stage of lial cells derived from CF bronchia (CFTR defect phenotype),
the disease. miR‑138 was reported to downregulate the expression of SIN3
transcription regulator family member A (SIN3A), a negative
Gene editing for CF. Clustered regularly interspaced short transcriptional regulator of CFTR (61). In an indirect manner,
palindromic repeats (CRISPR)/CRISPR‑associated protein 9 miR‑138 promoted the expression of CFTR through alleviating
(Cas9) is an emerging technology by which Cas9 proteins the repression that SIN3A imposed on CFTR.
work in conjunction with guide RNA molecules and locate Furthermore, miRNAs participate in other biological
the site of target DNA sequence prior to cutting it out (47), processes. In CF lung epithelial cells, miR‑155 promoted
following which the gap can be filled with the corrected inflammation through the inositol 5'‑phosphatase 1‑PI3K/Akt
gene sequence through the endogenous cellular regeneration. cascade. Moreover, as chronic bacterial lung infection is a
Therefore, this technology can be implemented in different major cause of CF morbidity, exhaled breath condensate was
single‑gene‑driven heritable deficiencies. With the advances used for miRNA profile analysis of patients with CF with
in such gene editing technology, a promising future has been microbial infection. The results demonstrated that 6 miRNAs
demonstrated in regard to the replacement of defective CFTR (has‑miRNA‑432‑5p, hsa‑miRNA‑3170, hsa‑miR449c,
gene at the DNA level, through which normal CFTR function hsa‑miR‑1276, hsa‑miR‑1247 and hsa‑miR‑548) were identi-
could be fundamentally restored (48). Although gene editing fied as potential biomarkers for patients with CF and chronic
is still at its infant stage due to the relatively high off‑target Pseudomonas infection (62). These reports indicated that
rate (49), it has demonstrated greater potential when compared miRNAs serve crucial functions in the pathogenesis of CF and
to traditional CF therapies targeting (instead of editing) DNA, are key diagnosis and prognosis markers for CF.
RNA or proteins. For instance, the functional repair of CFTR
has been successfully performed in an in vitro model derived lncRNA and CF. Dysregulation of lncRNAs have been reported
from stem cells of patients with CF, namely intestinal organ- to be associated with chronic pulmonary infection, adaptive
oids (50). Another approach, Zinc finger nucleases‑mediated immune responses and inflammation in patients with CF (63).
gene editing, was used to correct defective CFTR in induced By working concurrently with several proteins, lncRNA BGas
pluripotent stem cells (51). Notably, a mutation which could (a novel long noncoding RNA located in the intron 11 of the
cause β thalassemia was corrected in human embryos using CFTR gene) regulated CFTR by regulating its local chromatin
CRISPR/Cas9, indicating the capability of embryonic gene and DNA structure (53). Microarray profiling of lncRNAs
editing with this technique (52). Thus, we hypothesized revealed one upregulated (X‑inactive specific transcript) and
that CRISPR/Cas9‑induced gene editing in embryos threat- several downregulated (HOX Transcript Antisense RNA,
ened with potential mutations is a promising for the future Metastasis Associated Lung Adenocarcinoma Transcript
treatment of CF. 1 and Toll Like Receptor 8 Antisense RNA 1) lncRNAs in
4996 Molecular Medicine REPORTS 22: 4992-5002, 2020

Epigenetic modifications in CF. Epigenetics is a mechanism

that alters gene expression without changing the fundamental
DNA sequence. The two mostly known mechanisms under-
lying epigenetic modifications include histone modifications
and DNA methylation (69), both of which are involved in
regulation of CFTR. It had been hypothesized that aside from
ncRNAs, epigenetics is a contributing factor in the disease vari-
ability of CF (70). Additionally, epigenetic mechanisms have
Figure 2. miRNA regulation of CFTR. The majority of studies have been proposed to be an activator of host defenses that induce a
focused on the direction of miRNAs in the regulation of CFTR, whereby robust immune response (71). The association between immu-
miRNAs lead to silence or degradation of CFTR mRNA by binding to its
nity and epigenetics has demonstrated that DNA methylation
3'UTR. Additionally, miRNAs inhibit the expression of certain CFTR
suppressors, through which they promote the expression of CFTR mRNA. at numerous gene loci in lung macrophages was responsible
miRNA, microRNA; CFTR, cystic fibrosis transmembrane conductance for the malfunction of innate immune cells in lungs with
regulator; UTR, untranslated region. CF (72). Additionally, differentially altered DNA methylation
at CpG sites was associated with lung function and their over-
expression was demonstrated in numerous regulatory genes
the patients with CF compared with the matched non‑CF responsible for cell adhesion (for example, ETS homologous
controls (64). Although the current evidence regarding the factor) and inflammatory responses (for example, baculoviral
association between lncRNAs and CF is relatively limited, it IAP repeat‑containing protein 1) (73) in nasal epithelial samples
is notable that in a comparative study, 636 and 1,974 differen- from patients with CF (74). Furthermore, acetylation has been
tially expressed lncRNAs were found in two groups of patients proposed to be associated with CF. For instance, the inhibition
suffering from CF airway epithelium or CF lung parenchyma of histone deacetylase (HDAC)7 was demonstrated to restore
(compared with the matched non‑CF counterparts), respec- the function of F508del (75) and HDAC2 was reported to
tively. By analyzing these lncRNAs in a comprehensive be responsive to defective CFTR function (76). A previous
manner, the antisense lncRNA RN7SK Pseudogene 237 was study demonstrated that microtubule deacetylase regulated
found to be significantly altered in CF airway tissues, whereas cholesterol accumulation and NF‑κ B activation in CF cells
the downregulation of two intergenic lncRNAs, long intergenic through the HDAC6‑Ac‑tub cascade, which corroborated with
non‑protein coding RNA (LINC)01023 and LINC00176 were the findings that HDAC6 may be a therapeutic site for various
confirmed in CF parenchyma tissues (65). Additionally, in silico CF phenotypes (77). Collectively, therapeutic approaches for
analysis of RNA‑seq data demonstrated that Pseudomonas CF that target epigenetic mechanisms have been considered
aeruginosa infections lead to 108 altered lncRNAs expression promising, as epigenetic alterations are dynamic and revers-
between respiratory epithelial cells derived from patients with ible. However, epigenetic therapy of CF disease is still at its
CF and non‑CF donors. Among these lncRNAs, LINC00862 infant stage (78).
and CTD‑2619J13 were significantly altered at different time
points throughout the process (0 h prior to infection and 2, 4 5. Clinical applications of CF‑associated molecules
and 6 h following infection) (63). These studies indicated that
lncRNAs exerted important regulatory roles in CF, which still Molecular diagnosis of CF. The association between clinical
remain to be fully elucidated. presentations and residual CFTR function has been estab-
lished (79). Congenital absence of the vas deferens is established
circRNA and CF. In the field of RNA, circRNAs are endoge- when the proportion of normal CFTR function is <10%, as this
nous ncRNAs. These RNAs have been identified in organisms, number decreases (<5%), positive sweat test results could be
including eukaryotes, archaea, bacteria and viruses, and act supported and patients might suffer from pulmonary infec-
as a sponge for certain miRNAs in pulmonary diseases (66). tion when CFTR function further drops to <4.5%. The worst
Currently, reports regarding the potential roles of circRNAs cases (CFTR function <1%) lead to pancreatic insufficiency,
in CF are relatively limited. However, due to their close regu- aside from the aforementioned symptoms (80). As varying
latory association with miRNAs, it is likely that circRNAs molecular subtypes are associated with different phenotypes,
participate in the progression of CF. Specifically, in bladder experts from the ‘Cystic Fibrosis Foundation’ convened a
cancer, circ‑solute carrier family 8 member A1 was reported panel of criteria for diagnosing CF in 1996 (81,82). Several
to be a sponge of miR‑494 (67), whereas direct interac- tests, including the sweat test (83,84), nasal potential differ-
tions between circ‑baculoviral IAP repeat containing 6 and ence (NPD) (85‑87), DNA screening (88,89) and a ciliary test,
miR‑145 were indicated in embryonic stem cells (68). Reports were recommended. The current review discussed traditional
concerning miR‑494 and miR‑145 and their involvement in (regular) approaches (sweat test and NPD measurement) and
CFTR were revealed (57,58), indicating that these regula- novel methods.
tory circRNAs may regulate CFTR through these miRNAs. The sweat test is an effective method for detecting CF,
Analogously, it has been hypothesized that numerous miRNAs covering all age ranges (83,84). However, the application of
regulating CFTR may be targets for various circRNAs, which creams and lotions within 1 day prior to sweat collection can
might be involved in CF. This is a novel research avenue to disrupt the precision of diagnosis. The criteria for determining
consider in the future since the elucidation of the mechanisms CF varies based on different ages. In infants up to 6 months
driven by cirRNAs will provide a further understanding on the of age, a CF diagnosis is very unlikely if the level of Cl‑ is
pathogenesis of CF and its complications. not >29 mmol/l. However, the possibility of establishing a
 WEI et al: CURRENT ADVANCES IN CF 4997

CF diagnosis increases when Cl‑ levels range 30‑59 mmol/l. the CFTR gene was used in an attempt to restore CFTR
Generally, the diagnosis of CF can be confirmed when this function (1,94‑97). The rationale behind this method was to
level is >60 mmol/l. The criteria vary slightly in patients aged restore the dysfunctional gene or to supplement the patient
>6 months. CF cannot be diagnosed when the Cl‑ concentra- with the corrected version of the protein prior to irrevers-
tion is <39 mmol/l. When levels range 40‑59 mmol/l, a higher ible damage (95,98,99). For this technique, the DNA has to
probability of CF is expected. The diagnosis of CF can be penetrate the nucleus to be transcribed, which is the major
established if the Cl‑ levels are >60 mmol/l. Collection of a barrier in gene therapy. In practice, gene therapy entails inha-
sufficient volume of sweat is required for laboratory assays, lation of a spray which delivers therapeutic DNA to the lungs.
through which Na and Cl‑ concentrations are determined. During the therapeutic process, either viral vectors (including
Incorrect results occur due to contamination, evaporation, adenovirus, lentivirus and herpes virus) or non‑viral vectors
insufficient sample and technical errors (83,84). (such as plasmids) were used. The best therapeutic outcome
NPD measurement is used to follow‑up patients with would be the successful replacement of the defective gene
CF (85‑87). NPD is generally used to evaluate the voltage in the lungs to cure CF fundamentally. In other outcomes,
between the reference electrode and the exploring electrode, CF symptoms are alleviated by decelerated disease progres-
which is sensitive and specific (85‑87). In vivo, NPD provides sion; specifically, to clear aberrant and excessive secretions,
data about incorrect ion transport due to CFTR protein combat pulmonary infections and to prevent intestinal
dysfunction in the nasal epithelial cells of patients. Ancillary obstruction (99). Additionally, gene therapy is the first and
test is used to verify the phenotype of patients and identify ion most advanced vector system using recombinant retroviruses
channel abnormality. However, specific skills are required to ex vivo. In vivo gene therapy uses vectors based on the recom-
perform the test and interpret the results. binant form of adenovirus. The recent virus‑based system is an
DNA screening can detect severe mutations, including adeno‑associated system and numerous vector systems have
F508del and minor mutations such as the 5T variant, and is been validated in clinical trials involving human participants.
particularly useful to detect CF in patients who are unable Among them, adenoviruses and adeno‑related viruses have
to perform the sweat test (88,89). This method can provide been widely used (37,100). Aside from virus vectors, cationic
a general idea associated with the severity of the illness and lipids‑based vectors are also popular (99).
can detect less severe CF variants, including azoospermia and Transcript supplementation therapy using the correct version
congenital bilateral absence of the vas deferens (90). Previously, of CFTR mRNA transfected or transduced into the respective
children suffering from CFTR‑associated metabolic disor- target cells has been documented (95). In this therapy, mRNA
ders were classified into non‑typical or moderate type of CF. is actively producing CFTR in the cytoplasm, thereby circum-
However, these indistinct categories lack stringent criteria. venting the nuclear membrane. However, protein delivery is
Therefore, this resulted in ambiguities in subtype stratifica- often ineffective and it is difficult to include natural posttran-
tion. Currently, DNA screening is widely used in newborn scriptional protein modifications. Additionally, RNA antisense
screening for improved stratification of the different subtypes therapy is taken into account in CF treatment. The hypothesis
of CF. Nevertheless, regular evaluation remains important (42). is to use inhaled RNA antisense to produce functional CFTR
The most significant benefit of newborn screening and early protein by inducing RNA to work more efficiently (53). Notably,
diagnosis of CF is the possibility to treat disease‑prone patients nanotechnology using package miRNAs to treat CF was proven
prior to the occurrence of serious symptoms (91). to be safe and effective. However, more research is required
One of the consequences of developing CF is the chronic before applying this model to other diseases (66).
pulmonary infection caused by colonized bacteria at an Another alternative for CFTR treatment includes modu-
early age. Phenotypic features associated with CF diagnosis lator therapies (101), which can be categorized into two
provides information about chronic sinopulmonary disease groups: Potentiators and correctors. The potentiators act on the
manifestation due to many microorganism, including CFTR ion channels. Therefore, these modulators are geared
Staphylococcus aureus, nontypeable Haemophilus influ‑ toward the class III subject group (gate defect), among which
enzae, mucoid/non‑mucoid Stenotrophomonas maltophilia, ivacaftor prolonged the opening of the CFTR channel, thereby
Pseudomonas aeruginosa and Burkholderia cepacian (92). facilitating Cl‑ ion flow (102). In January 2012, the U.S. Food
These pathogenic bacteria can provoke gastrointestinal and Drug Association approved ivacaftor use and, currently,
dysfunction responsible for intestinal, pancreatic, hepatic ivacaftor is the only licensed CFTR potentiator (103).
and nutritional troubles. Identification of the microorganism Observational data based on clinical and in vitro studies have
in patients with CF guides the path for subsequent antibiotic indicated that ivacaftor is efficient for several mutations within
therapy (93). As mentioned in previous sections, microorganism classes III, IV and V in rat thyroid cell lines (102‑104).
detection can be performed by analyzing the expression profile The correctors serve a key role in the transportation of
of miRNAs (62) or other novel biomarkers (63). Therefore, nascent proteins (104). For instance, corrector lumacaftor is
traditional PCR analysis, microarray methods and NGS are considered a stabilizer that increases the stability of mutated
capable of biomarkers profiling. CFTR proteins, through which these proteins could be trans-
ported to the cell membrane more effectively and remain there
Molecular therapy for CF. Molecular therapy serves a crucial for an extended period of time (105). These stable substrates
role in CF treatment. The current review discussed several could be further enhanced by potentiators (106). Monotherapy
alternatives, which are summarized in Fig. 3. In 1993, a gene with lumacaftor, as a corrector, failed to demonstrate signifi-
therapy clinical trial was performed. The first trial focused cant results in homozygous patients (106,107). Furthermore,
on the nasal epithelium and adenovirus vectors containing another type of corrector, tezacaftor, demonstrated great
4998 Molecular Medicine REPORTS 22: 4992-5002, 2020

Figure 3. Schematic of several alternatives of molecular therapy. Comparisons were made between prior to and post‑treatment. In supplement therapy, the
corrected version of the CFTR protein (protein supplementation) or mRNA (RNA antisense therapy) were delivered to the cell. These exogenous molecules
exerted their regulatory roles mainly in the cytoplasm. In gene therapy, packaged lentiviruses correcting the CFTR gene are directly inserted into the cell
nucleus, thereby facilitating the normal transcription of CFTR mRNA. Furthermore, in modulator therapy, potentiators enhance the gating properties of
malfunctioned CFTR, correctors induce correct CFTR protein folding/trafficking and amplifiers increase the production of immature CFTR protein, providing
sufficient substrate for the corrector and potentiator. CFTR, cystic fibrosis transmembrane conductance regulator.
 WEI et al: CURRENT ADVANCES IN CF 4999

result, improving the processing and trafficking of mutated corresponding molecular regulators and their clinical imple-
CFTR, and promoting chloride transportation in bronchial mentations. Emerging technology, including NGS analysis
epithelial cells derived from F508del/F508del donors, which and gene therapy, will improve the understanding of the
were achieved without the problems associated with luma- underlying molecular mechanisms. Increasing numbers of
caftor (for example, pulmonary exacerbation and increment novel molecular regulators, such as miRNAs and lncRNAs,
in weight) (108). The underlying mechanism and propriety have been reported, some of which displayed potential to
of tezacaftor are very close to those of lumacaftor (107), as be biomarkers of CF. CF diagnosis was improved by carrier
tezacaftor therapy increases Cl‑ transport. When combined screening, while newborn screening facilitates the prog-
with ivacaftor, tezacaftor is efficient in transporting the CFTR nostic outcome via the timely intervention of CF at the early
protein to its correct position on cell surfaces (109‑111). stage. The developed understanding of molecular variants
Therefore, potentiators and correctors are often combined to (genotypes) of CF defects have enabled the development of
treat patients with CF. Specifically, CFTR potentiators increase increasingly precise and customized CF treatments, which
the activity of CFTR on epithelial surfaces, whereas CFTR significantly prolonged the survival of patients with CF
correctors promote processing and trafficking of mutated with novel therapies, including gene, supplementation and
protein. In order to restore the availability and functionality of modulator therapies. These have demonstrated promising
CFTR protein in the epithelium, CFTR modulator drugs are future for CF treatment. Although rapid progresses have
taken orally (106). been reported in the understanding and treatment of CF,
Furthermore, the third type of modulator, which is still improvements are required and challenges remain to be
in development, is termed the amplifier. These modulators overcome.
selectively promote cellular immature CFTR protein produc-
tion, supplying correctors and potentiator with sufficient Acknowledgements
substrate (112). For instance, patients with CF and F508del
mutations received gentamicin nasal drops for 14 days, which Not applicable.
led to a 22% increase in their wild‑type CFTR function (113).
Additionally, curcumin was used to treat CF by potentiating Funding
the activation of CFTR (114). Currently, triple combination
therapy (elexacaftor, tezacaftor and ivacaftor) outperformed This work was supported by the National Natural Science
dual combination therapy (elexacaftor and tezacaftor) as it Foundation of China (grant no. 81670004), the Guangxi Natural
promoted the Cl‑ and fluid transportation, thereby further Science Fund Project (grant nos. 2017GXNSFAA198163,
increasing the beat frequency of cilia, as manifested by in vitro 2017GXNSFAA198149 and 2020GXNSFAA159099), the
efficacy in F508Del/F508Del human bronchial epithelial Nanning Scientific Research and Technology Development
cells (115). Project (grant nos. 20153124, 20163138 and 20153011), the
Youth Science Foundation of Guangxi Medical University
6. Challenges and perspectives (grant no. GXMUYSF201307), the Scientific Research Project
of Health Committee of Guangxi (grant nos. Z2014456
Despite the fact that considerable data have been obtained in and Z2015197), the Guangxi Key Laboratory of Bio‑targeting
regard to the molecular mechanisms of CF, challenges still of Theranostics Fund (grant no. GXSWBX2020001), the
remain. Further research is required concerning the following Key R&D plan of Qingxiu District Science and Technology
aspects: i) Although 3‑base‑pair deletion and >100 related Planning Project (grant no. 2020025) and the Academic
variants have been reported to account for CF pathogenesis, Promotion Programme of Shandong First Medical University
phenotypes of other variants, particularly those with single (grant no. 2019QL013).
amino acid alterations, remain to be elucidated (41); ii) inter-
pretation regarding molecular and genetic results of CFTR Availability of data and materials
(whether specific variation should be defined as ‘disease‑prone’
or ‘neutral’) has remained controversial, mainly due to the Not applicable.
one‑to‑many association between CF genotypes to pheno-
types (116), which result in difficulties in associating genetic Authors' contributions
information with clinical traits; iii) while gene therapies (gene
editing) exhibit potential in CF treatment, the efficiency is TW, HS and YS wrote and revised the manuscript. WC,
decreased by high off‑target effects (117); iv) another defect YL, ZH and QJ contributed in drafting the manuscript. CX
due to technical restriction is that prior to being intracellularly designed the work. All authors read and approved the final
de‑packaged, the transferred gene can be severely damaged manuscript.
by multiple natural barriers, including mucus and the immune
response (118); and v) the spectrum of treatable mutations Ethics approval and consent to participate
should be extended (119).
Not applicable.
7. Conclusions
Patient consent for publication
The current review summarizes the advances in the under-
standing of the molecular mechanisms underlying CF, the Not applicable.
5000 Molecular Medicine REPORTS 22: 4992-5002, 2020

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