A review of cystic fibrosis: Basic and clinical aspects

Abstract Cystic fibrosis is an autosomal recessive disease caused by mutations of the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). Here we summarize, at the basic descriptive level, clinical and genetic characteristics of cystic fibrosis gene mutations, while emphasizing differences between CF mutations found in Chinese pediatric CF patients compared to those found in Caucasian CF patients. In addition, we describe animal models used to study human cystic fibrosis disease and highlight unique features of each model that mimic specific human CF‐associated signs and symptoms. At the clinical level, we summarize CF clinical manifestations and diagnostic, treatment, and prognostic methods to provide clinicians with information toward reducing CF misdiagnosis and missed diagnosis rates.

Fibrosis Foundation 2018 Registry Report. However, epidemiological data on CF prevalence in China have not yet been reported, aside from observations that the genotypic spectrum of Chinese CF varies widely among resident subpopulations based on their geographical and ethnic origins.
Numerous animal CF models have been established based on specific types of human CFTR mutations, but models differ in their effectiveness in mirroring features of human CF-specific disease.
For example, the mouse CF model differs markedly from human CF at the pathological level, while at the molecular level CFTR genes of pig and human are highly homologous, but their corresponding CFTR protein structures and functions are vastly different. At present, ferret and rabbit CF models hold promise as human CF models, but additional models based on other species should also be evaluated.
Meanwhile, the introduction of human CFTR genes harboring CFTR mutations into genomes of animals holds promise as a strategy for creating better animal models for human CF. Nevertheless, current animal models each have their own unique features that are useful for studying particular aspects of human CF disease, as described below.

| Characteristics of the human cystic fibrosis gene and encoded CFTR protein
Cystic fibrosis is caused by pathogenic mutations in a single large gene located on human chromosome 7 that encodes the cystic fibrosis transmembrane conductance regulator (CFTR) protein. [1][2][3] CFTR belongs to the ABC (ATP-binding cassette) family of proteins, a large group of related proteins that share transmembrane transport functions. The CFTR gene comprises 250 kilobases of genomic sequence that encodes an epithelial cell protein that is composed of 1480 amino acids in its mature state. The CFTR protein forms a cell membrane-spanning chloride channel whose function is regulated by phosphorylation mediated by cAMP- The structure of normal CFTR protein contains two groups of six membrane-spanning structural motifs, two intracellular nucleotidebinding folds (NBFs), and a highly charged 'R domain' containing multiple phosphorylation sites. Activation of the chloride channel requires phosphokinase A-mediated phosphorylation of the R domain and sustained ATP levels within the NBFs. 4,5

| Genetic mutation types in CFTR
CFTR mutations are currently categorized according to cause of dysfunction, including dysfunctional protein translation, cell processing, or CFTR channel gating. Missense (single amino acid substitution) mutations account for 38.74% of CFTR mutants, frameshift (insertion or deletion) mutations account for 16.25%, splicing (incorrect intron splicing) mutations account for 10.93%, and nonsense (early termination codon) mutations account for 8.41% of all known CFTR mutations detected worldwide. 6 Mutations of the CFTR gene fall into six different classes that roughly correspond to specific types of CFTR dysfunction. 7,8 In general, mutations in classes I to III cause more severe disease than those in classes IV to VI. 8,9  and NBO2), whereby some mutants retain varying degrees of sensitivity to nucleotide binding. The mutation giving rise to CFTR substitution G551D, which abolishes ATP binding, is the most common class III mutation in Caucasian populations. Meanwhile, other CFTR mutations within the region encoding the CFTR R domain may also fall into this category. 10 Class IV mutations: Defective conduction. CTFR protein is produced and transported correctly to the cell surface. However, the rate of ion flow and the duration of channel opening are reduced as compared to normal CFTR protein even though chloride currents are generated in response to cAMP stimulation. A mutation that induces a CFTR protein amino acid substitution (R117H) is the most common class IV mutation in Caucasian populations. 10 Class V mutations: Reduced amounts of functional CFTR protein. This class is not included in some classification schemes. It includes several mutations that alter mRNA stability and other types of mutations that alter stability of the mature CFTR protein (with the latter sometimes classified separately into an additional class, class VI). 8,11 Class VI mutations: Decreased CFTR stability. This class causes substantial plasma membrane instability and includes Phe508del when rescued by most correctors (rPhe508del). 8

| Characteristics of mutations in Chinese pediatric CF patients
In order to collect information pertaining to CFTR mutations associ- also revealed the value of neonatal screening for achieving early CF detection and treatment in some countries. Moreover, the results also revealed that a CF diagnosis can be confirmed in some cases using sweat chloride-based tests, while in other cases genetic testing is needed to confirm a CF diagnosis.

| CLINI C AL FE ATURE S OF C YS TI C FIB ROS IS
CF is caused by dysfunctional transport of chloride and/or other ions (such as sodium and bicarbonate) that leads to generation of thick, viscous secretions (eg mucus) in the lungs, pancreas, liver, intestine, and reproductive tract and increased salt content in sweat gland secretions.
Ultimately, progressive lung disease is the main cause of CF complications and patient mortality. 8 The course of disease varies greatly and can begin from a few months after birth to decades after birth, with many patients exhibiting mild or atypical symptoms. Therefore, clinicians should take care to avoid excluding CF as a possible diagnosis in cases where patients exhibit only a few typical CF signs and symptoms. anaerobes that may be identified using next-generation sequencing technology. 32

| Sinus disease
The majority of CF patients develop sinus disease. 34   With regard to other CF-associated digestive system disorders, 10% to 20% of newborns with CF present with meconium ileus characterized by obstruction of the bowel by meconium, which is a risk factor for poor CF prognosis. 38 Rectal prolapse, which previously was rarely detected in children with CF, has been detected frequently in recent years and appears to be associated with constipation and/or malnutrition. Focal biliary cirrhosis caused by inspissated bile is present in many patients and may cause elevated serum alkaline phosphatase and lobular hepatomegaly. A minority of CF patients develop periportal fibrosis, cirrhosis, symptomatic portal hypertension, and variceal bleeding that are associated with progressive liver disease. 37

| Reproductive system diseases
More than 95% of men with CF are infertile because of defects in sperm transport, although spermatogenesis is not affected.
Intriguingly, nearly one-half of all men with congenital bilateral absence of the vas deferens and normal lung function possess two CFTR mutations. 39 Meanwhile, females with CF are less fertile than normal healthy women, due to malnutrition and the production of abnormally tenacious cervical mucus. Nonetheless, females with CF may become pregnant and those who do should be counselled accordingly about contraception and childbearing decisions. 40 Indeed, comprehensive genetic counselling is essential for prospective parents with CF.

| Diagnostic criteria of cystic fibrosis
Both of the following criteria must be met to diagnose CF 43

| Sweat chloride
The sweat chloride test remains the primary test used for CF diagnosis.
If the concentration of chlorine is greater than 60 mmol/L, the diagnosis of CF is confirmed, while a high concentration of 40-60 mmol/L is suspicious, and a concentration <40 mmol/L is normal (excluding adrenal insufficiency). However, new clinical guidelines 43

| ANIMAL MODEL S OF C YS TI C FIB ROS IS
To date, many animal models of CF have been established that vary according to type of CFTR mutation. Phenotypes of human and animal models of cystic fibrosis are listed in Table 3. belongs to the P2-type ATPase family and shares sequence homologies with both the gastric H,K-ATPase (ATP4A) and the Na,K-ATPase (ATP1A). ATP12A mediates the electroneutral exchange of H + for potassium (K + ) but may also function in a Na + /K + exchange mode. 48 In humans lacking CFTR, unchecked H + secretion by the nongastric H + /K + adenosine triphosphatase (ATP12A) acidified airway surface liquid, which impaired airway host defenses. However, the expression of ATP12A is low in murine airways, which may partly explain the very mild pulmonary phenotype in murine models of CF. 49 In any case, mouse models are not helpful for studying the long-term pathology of human CF disease, due to the short lifespan of mice.

| CF rat models
Compared with mice, rats are appreciably bigger and provide better tissue specimens and blood samples for analysis. Compared with larger animals, rats have a shorter gestation and earlier sexual maturity. Like humans, rats have extensive submucosal glands, which are implicated in the development of CF airway disease. 50 To date, several CF rat models have been generated with interesting phenotypes. The first CF rat model was a CFTR-knockout rat strain. 51 Recently, two CF rat models of KO and F508del CFTR using CRISPR/Cas9 gene editing have showed encouraging results. 52,53 They revealed CF manifestations including reduced survival, intestinal obstruction, bioelectric defects in the nasal epithelium, histopathological changes, and male reproductive abnormalities.
Moreover, they represent a novel resource to advance the development of CF therapeutics.

| Porcine CF models
Pigs have a large number of offspring, mature rapidly, and have a long lifespan, enabling researchers to study long-term pathology and prognosis of CF. In addition, pig anatomy and physiology mimic corresponding human characteristics and porcine CFTR is 92% homolo-

| The ferret CF model
The ferret CF model shares many CF pathological characteristics with human CF, especially in newborns. However, considerable effort is needed to produce enough CFTR−/− ferrets to ensure that

| The rabbit CF model
Recently, CRISPR/CAS9 has been used to generate CFTR knockout and F508del genomic mutations to create CF rabbit models. 58 Rabbits are considered to be an ideal species for simulating human CF lung disease, as their airway anatomy and inflammatory responses resemble corresponding human characteristics. Preliminary findings of experiments using CF rabbits to model human CF indicate that CF rabbits will likely be useful for modelling human CF disease.

| Prospective animal models for CF research
With the development of rapid and accurate gene editing technologies such as CRISPR/CAS9, better animal models of human CF can now be created by introducing specific CFTR genes and mutations into animal genomes. Development of animal models that accurately mimic human CF will facilitate development of experimental pulmonary therapies, identification of new therapeutic targets, and enable clarification of complex mechanisms underlying initiation and progression of pulmonary CF. 59 Although no perfect CF animal model exists, each animal model has its own unique advantages for use in studying specific CF-related pathogenic mechanisms.  Trikafta (tezacaftor plus elexacaftor and ivacaftor) is the third drug approved by FDA that rescues defects caused by F508del, which is superior to its predecessors. Trikafta is also effective in CF patients with one copy of F508del-CFTR mutation. It demonstrates safety and sustained efficacy for 24 weeks or longer in people with CF and one or more F508del alleles. 64 To prevent long-term infection and inflammation that eventually cause irreversible bronchiectasis and respiratory failure, lung transplantation is feasible for end-stage patient treatment depending on the health of the particular patient.

| Management of cystic fibrosis
In addition to timely diagnosis and treatment, long-term follow-up and monitoring of CF patient status are also very important. For that can adversely affect treatment compliance and long-term prognosis. Therefore, psychological counselling is also very important.

| PROG NOS IS OF C YS TI C FIB ROS IS
Although cystic fibrosis is currently incurable and greatly reduces life expectancy, the average CF survival age has increased significantly over the past 50 years and now exceeds 40 years of age. Thus, CF is no longer viewed solely as a childhood disease, but now is recognized as a disease of children and adults. Currently more than half of CF patients are adults as old as 60 years of age, indicating that active treatment can improve prognosis, increase quality of life, and prolong lifespan. Time to diagnosis and treatment, severity of lung disease, nutritional and general conditions, and mental state are key factors that influence prognosis. 44 With regard to pediatric CF patients, attention should be paid to improving awareness and compliance of family members to prevent infection, actively treat acute exacerbations, and comply with recommended care instructions to maximize quality of life and long-term survival.

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interest.