Screening for the Most Common Mutations of CFTR Gene among Egyptian Children with Difficult-to-Treat Asthma

Abstract

Cystic fibrosis (CF) is panethnic autosomal recessive disease that affects the exocrine glands of pancreas, lungs, and intestine. It is often misdiagnosed in developing countries as difficult-to-treat asthma. We enrolled 150 Egyptian families with one or more probands who were complaining of difficult-to-treat asthma, and 112 cases were studied extensively through history taking including pedigree construction and clinical examination. In addition, spirometry and computed tomography of the chest were done in selected cases. All cases were subjected to quantitative sweat chloride test and molecular screening for the three most common mutations of cystic fibrosis transconductance regulator ( CFTR ) gene ( ΔF508 , G542X , W1282X ) using amplification refractory mutation system (ARMS) technique. Probands of difficult-to-treat asthma comprised 66 males and 46 females; their age range was 1 to 14 years. Sixty-one probands (54.5%) were carriers of one or more of the studied mutations (36 cases and 25 carriers). Six carriers of single mutations had mild respiratory symptoms and negative sweat test. The most common allele was ΔF508 , 60 alleles in 56 individuals (4 were homozygous ΔF508 / ΔF508 ) followed by W1282X in 25 individuals and G542X in 12 individuals. Allele W1282X had an increased risk of recurrent chest infection and bronchiectasis. Moreover, cases with two mutations had more severe symptoms compared with those with a single mutation. CFTR mutations and CF-related syndromes are not rare as thought in Egypt, especially among the high-risk difficult-to-treat asthma. The readily available ARMS technique is recommended for ΔF508 and/or W1282X screening on priority basis among these children.

Introduction

Cystic fibrosis (CF) is transmitted in an autosomal recessive pattern, due to mutation of cystic fibrosis transmembrane conductance regulator ( CFTR ) gene that results in formation of viscid mucous. CFTR is a 230-kb gene located on chromosome 7 encoding a 1,480-amino acid polypeptide, which functions as a chloride channel in the epithelial membrane. ¹

Bronchial asthma is a complex respiratory disorder characterized by marked heterogeneity in disease triggers and response to therapy. ² The interaction of genetic, environmental, and host factors may contribute to the development of childhood asthma and defines its progress, including persistence and severity. ³ Most asthmatic children maintain adequate control of symptoms with low or medium doses of inhaled corticosteroids; however, difficult-to-control asthma is associated with persistent symptoms despite high doses of inhaled or systemic steroid therapy. The most common cause of poorly controlled asthma is the lack of compliance or nonoptimal medical therapy. In addition, conditions that mimic asthma do not respond to the guideline-based therapies. These conditions include structural airway abnormalities, aspiration, gastroesophageal reflux, and CF. ⁴

The diagnosis of CF consensus statements relies on both a clinical presentation as well as evidence of CFTR dysfunction. Therefore, the presence of deleterious mutations in both copies of CFTR gene can satisfy the diagnostic criteria for CF. ⁵ Discovery of the ΔF508 mutation was a landmark in CF research. It was the first and most common disease-causing mutation, and revealed the complexity in polypeptide maturation and translocation to the proper cellular location in apical membranes. ⁶ Since then, more than 2,000 mutations in CFTR gene have been reported, of which two nonsense mutations ( G542X and W1282X ) are relatively common in Egypt and other countries. ^{7 8} However, several cases with positive genetic tests may not fit into the full diagnostic criteria for CF (CFTR-related disease or syndrome) and can present with chronic rhinosinusitis, recurrent wheezes, or cough ⁹ and can be a cause of poor asthma control.

Herein, we aimed to screen for three common CFTR mutations ( ΔF508 , G542X , and W1282X ), using amplification refractory mutation system (ARMS) technique in association with sweat chloride test among cases labeled as difficult-to-treat asthma in North East Egypt to ensure an alternative diagnosis and proper management.

Subjects and Methods

This cross-sectional study was conducted in the outpatient clinic of the Allergy and Pulmonology Unit in Mansoura University Children's Hospital, from February 2014 to March 2017. The study was reviewed by the ethics committee of Mansoura University Children's Hospital and parents of children included in this study signed the consent form.

Subjects

We recruited 150 Egyptian families with children diagnosed with difficult-to-treat asthma. All probands were assessed through history taking and clinical examination with stress on family history, pedigree construction, main presenting symptom, frequency of hospital admission, pulmonary function tests (spirometry for children aged older than 7 years), and other advanced investigations, for example, high-resolution computed tomography scan of the chest were done in selected cases.

A total of 112 probands were enrolled in our study after fulfilling the criteria of difficult-to-treat asthma based on the Global Initiative of Asthma guidelines, ¹⁰ and 108 individuals were excluded because they did not fulfill inclusion criteria or declined to be enrolled in the study ( Fig. 1 ).

Fig. 1.

Flowchart for the studied families with difficult-to-treat asthma patients.

Inclusion and Exclusion Criteria

Children aged 1 to 14 years were previously misdiagnosed as bronchial asthma and were presenting with difficult-to-treat asthma symptoms.

Inclusion criteria : All difficult-to-treat asthmatic children aged 1 to 14 years of both sexes were as follows:

1. Progressive airway obstruction possibly identified as asthma not responding to controller asthma therapy (high-dose inhaled corticosteroid according to age group, leukotriene receptor antagonist, and long-acting β 2 agonist).

2. Poorly controlled asthma with persistent airway obstructive disease with day time symptoms more than two times per week and/or more night time symptoms more than two times per month for at least 3 months. ¹⁰

3. Recurrent pneumonia, pulmonary complications such as bronchiectasis, chronic sinusitis, or nasal polyposis (symptoms suggestive of atypical CF).

Exclusion criteria : Any child with the following history was excluded:

1. History of atopy (allergic rhinitis, dermatitis, conjunctivitis, food allergy) in cases, parents, and siblings, documented by negative skin prick testing in asthmatic children.

2. Patients noncompliant on therapy: after rechecking the choice of proper steroid inhaler and ensuring proper inhaler technique (through observing technique and using video demonstrations).

3. Documented lung pathology, for example, lung sequestrations, documented gastroesophageal reflux disease (either by history or by pH metry), history of foreign body aspiration, and congenital heart disease.

The diagnosis of CFTR-related disorder has been defined as a mono symptomatic clinical entity (congenital bilateral absence of the vas deferens/pancreatitis/bronchiectasis) associated with CFTR dysfunction that does not fulfill the diagnostic criteria for CF. Individuals with a CFTR-related disorder (generally mono-organ) should be assessed and followed by a CF physician to assure development of any additional symptoms that are typical for CF. ¹¹

Methods

All probands with difficult-to-treat asthma were tested for sweat chloride quantitatively using the Nanoduct Neonatal Sweat Analysis System model 1030 (Wescor Inc., Logan, Utah, United States, Biomedical Products Division). In accordance with CF, the guidelines of Cystic Fibrosis Foundation, sweat test screening is considered as a standard initial screening test for diagnosis of CF. ⁵ All enrolled probands were then screened for the three most common mutations of CFTR gene ( ΔF508 , G542X , and W1282X ) using the easy, cheap, and popular ARMS technique, first introduced in CF at 1991. ¹² Worldwide, molecular testing is the confirmatory test for cases screened with sweat chloride test in cases diagnosed by newborn screening (NBS) program. ¹³ CF NBS is a complex procedure that uses multiple step combinations of tests on dried blood spots. The first tier is always a measurement of immunoreactive trypsinogen; however, NBS has not been yet established in Egypt for CF. Moreover, Sanger sequencing is not widely available in Egypt for routine service, yet we have used it for selected cases based on symptoms, those with severe symptoms but had only one known mutant allele aiming to search for rare/benign variants in the vicinity of known pathogenic alleles. ¹⁴

Sweat Chloride Testing

The device was suitable for all ages. Sweat was induced by pilocarpine iontophoresis, and the results were interpreted by continuous flow analysis on the analyzer display. The flexor aspect of the forearm, approximately halfway between the wrist and elbow, was used for the anodic (positive) skin site. The area was cleaned with alcohol. Reference ranges were determined based on value of sweat chloride level: normal (0–<40 mmol/L), intermediate (40–<60 mmol/L), and abnormal (60 mmol/L) according to the user's manual. The minimum reading value was 3 mmol/L. Tests were conducted at room temperature (22–25°C) with a relative humidity of ∼60.

Molecular testing : From all cases and their parents, 3 mL of peripheral blood were collected from each in a separate test tube containing EDTA solution, pH 8 as an anticoagulant. DNA was extracted, purified, amplified, and analyzed using the following steps.

DNA extraction and purification : The DNA extraction kit (Gentra Systems, United States) uses two reagents: a DNA purification solution and a DNA elution solution. In the kit, a sample is applied directly to the purification matrix that contained a spin column.

Agarose gel electrophoresis : To verify the presence of DNA in good quantity, agarose gel electrophoresis was done using 0.8 to 1% agarose gel to resolve DNA fragments according to their molecular weight where smaller fragments migrate faster than larger ones. Therefore, the size of the fragment can be determined by calibrating the gel, using known size standards, and comparing the distance of the unknown complex DNA.

Polymerase chain reaction (PCR) of the extracted DNA : AmpliTaq Gold PCR master mix contained everything required for PCR amplification over wide range of DNA templates. DNA polymerase is the main ingredient of the PCR master mix, with gold buffer added for optimal enzymatic reactions. AmpliTaq Gold DNA polymerase is chemically modified and remains inactive through the thermal cycling preventing the extension of mis-annealed primers. When the reaction attains the optimal annealing temperature, the enzyme is activated which increase the yield of specific product because the reactants were not wasted in the formation of nonspecific products.

PCR for CFTR reaction : A reaction contains 4 μL DNA, 3 μL of each primer forward and reverse, and 10 μL of master mix. PCR was performed under the following conditions: 94°C denaturation for 5 minutes, 35 cycles as 94°C denaturation for 2 minutes, 60°C annealing for 2 minutes, and 72°C extension for 2 minutes, the procedure was completed by the continuation of the last 72°C for 10 minutes.

ARMS Technique

This is a simple, rapid, and reliable method for the detection of any mutation including single base changes or small deletions. The principle idea behind this method is that oligonucleotides, which are complementary to the gene sequence except for a mismatch at their 3′ OH residue, do not have function as primers in the PCR under adequate conditions. ¹⁵ ARMS technique is composed of two reactions: (1) normal reaction which contains a primer specific for the normal DNA sequence and (2) mutant reaction which contains a mutant-specific primer.

• (Allele 1) dTTATATTGTTGTGTCAGGACCAGCATTACA

• (Allele 2) dTTATATTGTTGTGTCAGGACCAGCATTACG. ¹²

Statistical Methods

Data were explored and analyzed using SPSS (Statistical Package for Social Sciences; SPSS Inc., Chicago, Illinois, United States) version 15.

Results

This is a cross-sectional study that had been conducted in the period from February 2014 to March 2017, it comprised 112 probands with difficult-to-treat asthma: 66 males and 46 females; age range 1 to 14 years with the mean of 3.2 ± 1.2 years at the time of enrollment. All were analyzed for CFTR mutations using ARMS.

Basic demographic and clinical data of all probands are shown in Table 1 ; the main clinical presentation is shortness of breath among all studied probands (90.2 and 78.4%) in CFTR positive and CFTR negative probands, respectively, and recurrent wheezing among individuals tested positive for CFTR mutations (98.4%) which is statistically significant compared with the whole cohort ( p  = 0.02). Positive consanguinity and family history of chronic lung diseases are statistically significantly higher among carriers of gene mutation compared with the whole probands (70.5 and 78.7%, respectively with p  < 0.05 for both).

Table 1. Demographic and clinical data of probands of difficult-to-treat asthma and CFTR mutation positive cases .

Characteristic	CFTR positive probands (61) N (%)	CFTR negative probands (51) N (%)
Gender
Male	40 (65.6%)	29(56.8%)
Female	21 (34.4%)	22(43.1%)
Consanguinity	43 (70.5%) a	0
Failure to thrive	15 (24.6%)	0
Age of onset of first episode of asthma in y (mean ± SD)	2.9 ± 1.1 a	4.3 ± 1.4
Shortness of breath	55 (90.2%)	40 (78.4%)
Chronic cough	50 (82%)	24 (47.05%)
Wheezes	60 (98.3%) a	15 (29.4%)
Nasal polyp	7 (11.5%)	3 (5.8%)
Sinusitis	14 (22.9%)	6 (11.7%)
Family history of chronic lung disease	48 (78.7%)	0
Previous hospital admission	48 (78.7%)	22 (43.13%)
Clubbing	12 (19.7%)	0
CT of the chest
Indicated in 27
Normal	6/61 (9.8%)	6/51 (11.7%)
Consolidation	9/61 (14.7%)
Bronchiectasis	12/61 (19.7%)
Intermediate positive sweat test	55 (90.1%) a	31 (60.7%)
Result of sputum cultures (21)
Pseudomonas aeruginosa	12/21 (57.1%)	Not done
Streptococci	9/21 (42.8%)

Abbreviations: CFTR, cystic fibrosis transconductance regulator; CT, computed tomography; SD, standard deviation.

^a Statistically significant chi-square test, p  < 0.05.

Out of the 112 probands of difficult-to-treat, 61 individuals carried one or more of the studied three mutations (54.5%): 36 individuals with two mutations (considered CF after sweat chloride testing for confirmation) and 25 individuals with single mutations. The most common allele was ΔF508 , 60 alleles in 56 individuals (four cases were homozygous ΔF508 ) followed by W1282X in 25 and G542X in 12 individuals. Suspected cases were genotyped either homozygous ΔF508 / ΔF508 (4 cases), compound heterozygous ΔF508 / W1282X , or ΔF508 / G542X (in 20 and 12 cases, respectively). Twenty carriers were ΔF508 and the remaining five were W1282X carriers ( Table 1 ). Among the 20 probands with difficult-to-treat asthma presenting with chronic sinusitis, 14 tested positive for CFTR mutation(s) (70%).

Subjects with single W1282X mutation had an increased risk for recurrent chest infection and bronchiectasis. Moreover, probands with two mutations (36 cases, 59%) were at increased risk for severe symptoms, earlier onset of symptoms, higher incidence of complications, and increased frequency of hospital admissions (data were not shown in the tables).

We compared the results of sweat chloride testing versus ARMS among all probands. Out of 86 suspected cases with intermediate positive sweat test, only 55 (63.9%) yielded positive results with ARMS, and the remaining 31 (36.04%) cases showed rare unidentified mutant variants (CF-related disorders). Probands with severe symptoms falling among this last group were sequenced and 5 of 31 (16.3%) showed rare mutant variants. Six carriers with only one allele who had mild symptoms were negative for sweat chloride testing ( Fig. 2 ).

Fig. 2.

Distribution of the CFTR genotypes among the 61 difficult-to-treat asthma probands. CFTR, cystic fibrosis transconductance regulator.

CT of the chest was done for 27 cases with chronic symptoms (chronic wet cough, hemoptysis, or restrictive pattern in pulmonary function testing); 6 cases were free, 9 showed consolidation areas, and 12 showed peribronchial thickening, mild bronchiectatic changes with ground glass opacities ( Table 1 ). Significant number of cases with CT changes (15 of 21, 71.4%) were positive for CFTR mutations ( p  = 0.012): 9 (60%) had double mutations and 6 (40%) had single mutation; the five individuals with single W1282X allele had positive CT changes (data were not shown in the tables). Sputum culture was done for 21 cases with positive CT changes and revealed Pseudomonas aeruginosa in 12 (57.1%) patients and streptococci in 9 (42.8%) patients.

Discussion

CF is the most common life-threatening autosomal recessive disease in the United States, occurring in ∼1 in 4,000 newborns. ¹⁶ There has been a surprising degree of difficulty encountered worldwide in establishing the diagnosis of CF in a minority of cases and because of this, health care providers continue to be faced with uncertain cases and challenging diagnostic dilemmas. ¹⁷

An important advance since the publication of the last U.S. Cystic Fibrosis Foundation diagnostic consensus criteria is a better definition of CFTR-related disorders, which is simply defined as a clinical entity associated with CFTR dysfunction that does not fulfill diagnostic criteria for CF. Clinicians should avoid the use of terms such as classic/nonclassic CF, typical/atypical CF, or delayed CF because these terms have no harmonized definition and could be confusing for families or caregivers. ⁵ However, The European CF Society Diagnostic Network Working Group has maintained a nonclassic or atypical CF label. ¹⁸ Previously, individuals with atypical CF usually have one severe mutation and one less common mutation or abnormality of trinucleotide repeats on their other CFTR gene. In addition, their respiratory symptoms include recurrent pneumonia, progressive obstruction possibly identified as asthma, chronic sinusitis, or nasal polyposis. The identification of this disease status is essential because of the discrepancy in prognosis and treatment strategies. ¹⁹

CF is not a rare disease in Egypt; it had been diagnosed in 4.6% of the studied cases with clinical picture suggestive of disease with their median age 4 years at the time of diagnosis; this age was higher than that reported in other studies due to the delay in diagnosis. ²⁰ In the current study, 112 children with difficult-to-treat asthma were enrolled, they were subjected to CFTR screening using ARMS technique to have some clinical correlations; a high incidence of CFTR mutations (61 of 112, 54.5%) had been found. In a previous study, only 9.1% of the 1,091 newly diagnosed CF patients were identified through NBS and prenatal diagnosis represented 3.9%. The residual cases were diagnosed based on clinical manifestations; 50% of them were pulmonary disease. Also, in our work, there were no cases with a positive family history of CF, yet 78.7% of individuals with CFTR mutations had family histories suggestive of chronic pulmonary symptoms. These results indicate the importance of CFTR mutation screening among suspected cases with chronic and/or recurrent pulmonary symptoms even in the absence of malnutrition or failure to thrive. ²¹

Arab studies on CF consider the presence of many patients who are undiagnosed taking in consideration the high kindred rate between 25 and 60%. Al-Mahroos found out an 80% rate of consanguineous marriage in Bahrain. ²² On the contrary, our current study showed consanguineous marriage of 38.4% for the whole studied population and 70.5% for the CFTR mutation carriers, although homozygous mutant is present in only four cases. An interesting observation in our study was that among the 20 probands with difficult-to-treat asthma presented with chronic sinusitis, at least 14 individuals (70%) carried one or more CFTR mutation ( Table 2 ); a finding that should draw attention to the necessity of molecular testing of CFTR in chronic refractory asthmatics who have chronic sinusitis as well.

Table 2. Distribution of CFTR alleles and genotypes among probands with difficult-to-treat asthma .

Genotype	Individuals	Alleles
ΔF508 /–	20	20
ΔF508 / ΔF508	4	8
ΔF508 / W1282X	20	40
ΔF508 / G542X	12	24
W1282X /–	5	5
G542X /–	–	–
Total	61	97

Abbreviation: CFTR, cystic fibrosis transconductance regulator.

Shawky et al stated that in high-risk group of children with chronic lung disease, the ΔF508 mutation comprised 25% of all CFTR mutations. ²³ Recently, in another Egyptian study done on 32 CF patients, 61% were ΔF508 mutation positive, 22.2% were homozygous, and 38.8% were heterozygous. ²⁴ Analysis of frequency of the three studied mutations in our cases revealed that the most common allele was ΔF508 ; 60 alleles in 56 individuals followed by W1282X in 25 and G542X in 12 individuals. Probands with two alleles were genotyped either homozygous ΔF508 / ΔF508 (4 cases), compound heterozygous ΔF508 / W1282X , or ΔF508 / G542X (in 20 and 12 cases, respectively). Hence, ΔF508 allele frequency in our work is 60 out of 97 (61.9%) which is close to the Caucasian frequency of 70%. ⁶ In another Egyptian study, ²⁵ 14 patients (38.9%) were heterozygous for ΔF508 and 10 patients (27.8%) were negative which is quite different from ours (52.5 and 8.2%, respectively). This discrepancy could be explained by our larger sample size, the different selection criteria, or the use of allele-specific primers in ARMS technique applied in our study. These patients had the typical phenotypic features of CF, thus if a child with difficult-to-treat asthma was genotyped negative for ΔF508 , further survey for other mutations should be done using the commercially available panels in Egypt; otherwise, sequencing for rare mutations becomes mandatory. The reported outcomes of CF are still indecisive; our work as well as some studies had concluded a positive relation of ΔF508 mutation with asthma resistance. ²⁶ However, earlier studies had reported a protective association. ²⁷ No significant relationship has been found between ΔF508 mutation and asthma-like symptoms in many European studies. ^{28 29}

The next most common CFTR mutation found in our study was W1282X (25 allele out of 97, 25.8%) followed by G542X (found in 12 cases, 12.4%). Both are nonsense mutations due to premature stop codon. In another study, a significant repetition of G542X mutation had been observed among cases when compared with controls; nevertheless, only G551D was mainly related to asthma risk, but G542X showed no association with asthma. ¹ Another interesting point in our work is the clear link between individuals with single W1282X mutation and the increased risk of recurrent chest infection and bronchiectasis; moreover, probands with double mutations (36 cases, 59%) were at increased risk for severe symptoms, earlier onset of symptoms, higher incidence of complications, and increased frequency of hospital admissions. This observation can be considered as specific pulmonary phenotype of CFTR-related disorders. However, the clinical implication of the W1282X mutation was denied among studied Indian subjects. ¹

In our study, comparing the results of sweat chloride testing versus ARMS among all probands; out of 86 cases with intermediate positive sweat test, only 55 (63.9%) were yielded positive results with ARMS and the remaining 31 (36.04%) cases included rare unidentified mutant variants. Probands with severe symptoms falling among this last group were sequenced; 5 of 31 (16.3%) showed rare mutant variants. Six carriers with only one allele who had mild symptoms were negative for sweat chloride testing (CFTR-related disorders). Recently, a 12-year-old Egyptian child who had been treated for difficult asthma was finally diagnosed as CF, and the authors highlighted the importance of considering CF in all patients with such clinical state regardless of their ethnicity. ³⁰ So, difficult-to-treat asthmatic children should be screened for CFTR mutations with top priority to ΔF508 followed by W1282X mutation especially if sequencing is not available.

In conclusion, asthma presentations are variable which could be due to environmental, ethnic, or variable gene expression caused by allelic heterogeneity or modifier genes; a phenomenon that is confirmed in many research reports and refuted in others. Moreover, lack of neonatal screening for CF in Egypt and other developing countries results in misdiagnosis of many cases of CF as bronchial asthma. This conflicting conclusion may necessitate the implementation of further multicenter multiethnic observational study.