The cystic fibrosis transmembrane regulator (CFTR) gene codes for the CFTR protein; a chloride channel protein that helps in the transportation of chloride ions and water molecules across the cell membranes of lungs, liver, pancreas, and skin. CFTR is a member of the ATP-binding cassette family of membrane transport proteins, but appears to be unique within this family by functioning as an ion channel rather than an active transporter protein. CFTR binds to ATP in order to open the channel for chloride ion transport across the membrane. This transport of chloride ions helps in controlling the movement of water in tissues and thereby, maintains the fluidity of mucus and other secretions. The CFTR protein contains twelve tranmembrane alpha-helices that are presumed to form the pore region by which chloride ions cross the membrane. Functional evidence implicates tranmembranes 1 and 6 as playing key roles in forming the pore and interacting with chloride ions to determine the functional permeation properties.
Mutations in the gene that disrupt the proper folding of the protein lead to loss of chloride ion transport. This, consequently results in a disruption of the chloride and water balance required to maintain the thin mucus in the airway and digestive tract. In addition, non-functional CFTR gene also lead to defects in the sodium ion channel, ENaC, leading to further loss of water in the upper airway. The mucus, therefore, gets thickened and sticky, leading to the characteristic signs and symptoms of Cystic Fibrosis (CF). CFTR gene mutations have also been implicated in the disorder Congenital Bilateral Absence of the Vas Deferens (CBAVD), characterized by an absence of vas deferens since birth. Interestingly, studies have indicated that mutations in the CFTR gene could also protect carriers against Salmonella typhi, the bacteria causing typhoid fever.
The CFTR gene, located on chromosome 7q31.2, is approximately 250 Kb in length, and codes for an mRNA with 27 exons. The CFTR protein consists of 1480 amino acids, and weighs 168 kDa. Expression of the genes is normally seen in the endothelial cells of the umbilical vein, lung microvasculature, RBCs, pancreas, lung epithelia, sweat glands, colon, parotid glands, and the liver.
Over 1000 mutations have been identified in the CFTR gene. However, CF causing mutations are mainly clustered in the nucleotide binding domains of the protein. In fact, more than 70% of patients with cystic fibrosis show a single mutation that involves the deletion of three nucleotides of exon 10, within the first nucleotide binding domain, resulting in the deletion of phenylalanine at position 508 in the protein product. This mutation is commonly referred to as delta F508 (c.3882_3885delTATT). The mutated protein is unable to fold in a proper fashion, and is destroyed by the cellular degradation pathway. Most of the other mutations causing cystic fibrosis are rare and are called "private mutations". The delta F508 is also the most common mutation seen among patients with CBAVD. However, some other mutations have been identified to be specific for CBAVD, with the exclusion of CF. These include the splice site variant IVS8-5T.
Eskandarani (2002) undertook a study to identify the CFTR gene mutations existing in the Bahraini population. The study group included 19 Bahraini children (12 males, 7 females; mean age: 5.4 years) belonging to 13 unrelated families. The rate of consanguinity among these families was 77%. Genetic screening for 15 CTFR mutations common in the Arab population was performed on all patients using RFLP and/or ARMS-PCR. Eight mutations were detected in these patients, most common of which were: 2043delG (31%), 548A-T (19%), 4041C-G (8%), and delta F508 (c.3882_3885delTATT; 8%). Both 2043delG and 548A-T mutations are rare in other populations, indicating that these mutations could have originated from the region. Homozygosity for the mutations was observed in six of the families, whereas six families were heterozygous for two mutations. One of the families was of Persian origin, and all three children from this family showed homozygous mutations for delta F508.
Lissens et al. (1999) analyzed 20 CBAVD males from Egypt for the presence of 12 common Caucasian CFTR mutations and the intron 8 5T splice variant, IVS-5T (c.1210-12T[5_9]), known to be a major cause of CBAVD in Caucasian patients. In 16 of the males without associated renal abnormalities only one delta F508 carrier was identified, but an exceptionally high frequency of the IVS-5T variant was found (14 of 32 alleles or 44%), confirming that this variant is involved in many cases of CBAVD, even in populations where CF is rare. CFTR mutations or the IVS-5T variant were found neither in the remaining four patients with associated renal abnormalities nor in the spouses of the 20 CBAVD patients. However, one patient was homozygous for a leucine to proline substitution at amino acid position 541 (L541P) of the CFTR. It is as yet not clear whether this change is involved in CBAVD in this male.
Wahab et al. (2004) described a five-month-old Egyptian female with the common homozygous missense mutation N1303K in the exon 21 of the CFTR gene. She was the fourth child of healthy first cousin parents, resident in Qatar. Her birth weight was 3.78 kg. Anti-natal ultrasonography revealed a right multicystic kidney. The pregnancy and delivery were normal. There was no relevant family history. She presented initially with of Pseudo-Bartter's syndrome which has not been reported previously with this mutation. Laboratory tests showed a severe hyponatremia, hypochloremic and hypokalemic metabolic alkalosis. A salty sweat taste was noticed by the father. The diagnosis of CF was made on the basis of an elevated sweat chloride concentration of 90 mmol/l and was confirmed by a second elevated sweat chloride concentration of 105 mmol/l. A pancreatic elastase stool test gave a value of <50 ug El/g.st (Normal 200 to >500 El/gm of stool). She was given pancreatic enzyme, sodium chloride salt replacement and multivitamin supplements. Since then she has been managed as an outpatient and has been clinically well and gaining weight. Since this case is only the second Egyptian patient with CF mutation N1303K to be reported in the literature, Wahab et al. (2004) suggested that CF might not be as rare in the Arab world as once thought and the prevalence of the disease needs to be further defined.
Hussein et al. (2011) studied 30 patients with CBAVD and 30 fertile controls to assess the prevalence of the delta F508 mutation and 5T allele in a sample of Egyptian patients having congenital bilateral absent vas deferens (CBAVD). delta F508 mutation was present in 40% and 5T allele was detected in 47% of the patients. The control group was negative for the delta F508 mutation, while the 5T allele was detected in 10% of these individuals. The total fertilization rate was 75% and pregnancy rate was 60% with no significant difference in fertilization and pregnancy rates between cases positive for delta F508 or the 5T allele and others. Hussein et al. (2011) emphasized the important roles of the delta F508 mutation and 5T allele in the pathogenesis of CBAVD in Egyptians.
Kakish (2001) investigated the CFTR gene mutations in 72 Jordanian children (37 males and 35 females) with cystic fibrosis (CF). Mutations were determined by using a multiplex heteroduplex shift analysis followed by direct sequencing on blood taken from all patients and 42 parents. Twenty different mutations were detected in the CFTR gene and five of those mutations were found for the first time (296+9A-T, T338M, T760M, 3679delA, and G1244D). Since a large number of mutations were identified among Jordanians, Kakish (2001) suggested that it might be the result of ethnic diversity of the Jordanian population reflecting the country's complex history. Surprisingly, low incidence of the delta F508 (c.3882_3885delTATT) mutation was found among the patients (6.3%) that could be explained on the basis of the founding population and the high mortality among patients carrying this severe mutation resulting in under-representation in the studied cases.
In a study of genetic disorders in Kuwait, Samilchuk (2005) analyzed the CFTR gene in patients with CF and/or congenital bilateral aplasia of the vas deferens (CBAVD). Commercial kits could identify only three mutations in CF patients; delta F508, G542N, and W1282X, which together accounted for 55% of all CF alleles among Arabs. Specific tests designed for other commonly reported mutations led to the detection of three other mutations; 1548delG, I1234V, and 3120+1G>A (c.2988+1G>A). These mutations accounted for 24% of the Arab CF alleles, and Samilchuk (2005) strongly recommended their testing in Arab populations. Mutation analysis of CFTR exons revealed two additional mutations, C128R and I560T, the latter being rare and the former, novel. Among patients with CBAVD, an unusually high 33% of the Arab patients were found to carry the delta F508 mutation.
In order to identify the distribution of CFTR mutations in the Lebanese population Desgeorges et al. (1997) studied 20 unrelated Lebanese Arab families, with at least one CF-affected child. DNA was collected from a total of 89 individuals from these families, including patients, their parents, and healthy siblings. Analysis of the DNA using denaturing gradient gel electrophoresis and direct sequencing following PCR, allowed identification of 10 mutations that accounted for 87.5% of the CFTR alleles. The most common of these mutations were: delta F508 (38%), W1282X (16%), and N1303K (9%). Two novel putative mutations were also identified; E672del, a single amino acid deletion in the regulatory domain caused by deletion of nucleotides 2145 to 2148 (GAA), and 4096-28G-A, occurring at the putative branch point involved in the splicing of intron 21. The E672del mutation was identified in a 4-year old boy, who presented with pancreatic insufficiency without pulmonary disease, whereas the girl with the 4096-28G-A mutation presented with major malnutrition and severe pulmonary distress, and deceased at 4-months of age. In both these patients, the second defective allele could not be characterized. About 48.3% of the mutant genes were identified on haplotype backgrounds that were found to be absent on wild-type genes. Interestingly, about 66% of the delta F508 chromosomes from the Maronite community were associated with the 7T allele at locus IVS8(T)n; an association representing Central and Northern Europe. In all other communities the mutation was linked to the 9T allele, which is more Mediterranean in its occurrence. Desgeorges et al. (1997) hypothesized that this was the original haplotype on which the delta F508 mutation occurred, and a recombination later with the Central European haplotype was responsible for the 7T-delta F508 association.
Romey et al. (1999) undertook a study aiming at comparing ethnic, clinical, and genetic data from patients with the p.S549R mutation in the CFTR gene. Of the cystic fibrosis patients selected for the study, a total of 16 (10 males and 6 females) were from UAE and Oman. DNA analysis identified all the 16 patients to be homozygous for the p.S549R mutation. Although none of the children had presented with meconium ileus at birth, all of them presented with insufficiency of the pancreas, severe lung disease, high rate of pulmonary infection, and rapid pulmonary decline. Three of the patients died of pulmonary failure. This phenotypic severity was found to be in contrast to that seen by the authors in patients of other ethnicities (French and Spanish) who carried the combined complex allele [-102T>A+ p.S549R (c.1647T>G)]. Romey et al. (1999) surmised that the [-102T>A] modification may alter the severity of the p.S549R mutation.
Frossard et al. (2000) performed mutation analysis on 15 Omani families (12 true Omani Bedouin families, and three Omani nationals of Baluchi origin) with a total of 16 CF-affected children. The CFTR genes of these patients were screened for p.S549R (c.1647T>G) and delta F508 (c.3882_3885delTATT) mutations. Eleven patients (69%), all of Bedouin descent, were found to be homozygous for the p.S549R (c.1647T>G) mutation. On the other hand, two patients (12%) of Baluch descent were found to be homozygous for delta F508. Thus, these two mutations accounted for 81% of the CF patients and 87% of the families with CF. CFTR mutations in the remaining three patients could not be detected.
By direct sequencing of exon 21, Shoshani et al. (1994) revealed a 4-bp deletion, TATT, at position 4010 of the coding sequence (c.3882_3885delTATT). This frameshift mutation is expected to create a termination codon (TAG) 34 amino acids downstream of the mutation. This alteration is likely to be a disease-causing mutation since it is predicted to create a truncated polypeptide that lacks the second ATP binding domain. The patient is of Arab origin and inherited this deletion from her father. The CFTR chromosome carries the D121 haplotype. Her other CFTR chromosome has the p.Asn1303Lys mutation.
Wahab (2003) described a late diagnosis of cystic fibrosis in a 36 year old Qatari female, in whom the main presenting symptoms were those of chronic lung disease. She was diagnosis with CF at the age of 35 years. There was a positive history of consanguinity with her parents being first-degree cousins. She had been married twice and had four healthy children. She has a younger sister with a similar history. Genetic analysis showed that the patient has a homozygous pathogenic mutation I1234V in exon 9 in both alleles of the CFTR gene. This led Wahab (2003) to suggest that this mutation has a variable age presentation from early infancy to childhood with variable expression of clinical severity and long survival. Wahab (2003) recommended that CF should be considered in patients with bronchiectasis and chronic lung disease regardless of age.
For the first time in Qatar, Abdul Wahab et al. (2005) reported the isolation of Stenotrophomonas Maltophilla from the respiratory secretion of a 17 year old Qatari female with homozygous cystic fibrosis mutation I123V. The patient later died.
Abdel Rahman et al. (2006) assessed exocrine pancreatic function among cystic fibrosis patients with CFTR I1234V mutation by measuring fecal pancreatic elastase-1 (FE1) activity through a cross-sectional study. Forty CF patients with homozygous CFTR I1234V mutation (22 males and 18 females) belonging to a large Arab kindred family were included in the study. Fifteen of the 40 patients (38%) were on pancreatic enzyme replacement during the study. The control group comprised 25 age- and sex-matched healthy subjects (14 males and 11 females) without any chronic diseases. The measurement of the FE1 concentration was performed by using enzyme-linked immunosorbent assay (ELISA) for each CF patient over a period of 12 months. CF patients were divided into two groups: the first group comprised the CF patients who underwent FE1 testing in Qatar using polyclonal ELISA technique, whereas the second group comprised the CF patients who underwent FE1 testing in Germany by the monoclonal ELISA technique. Control subjects were tested with the polyclonal ELISA technique in Qatar. By comparing the results of the two groups, no significant difference was found between the two methods for both CF groups. This finding is in agreement with a previous study, which concluded that both monoclonal and polyclonal ELISA kits are equally suitable for evaluating exocrine pancreatic function both in children and adults. Moreover, the concentration of FE1 in healthy controls was not significantly different from that of CF patients with or without pancreatic enzyme replacement, indicating that CFTR I1234V mutation is a mild genotype mutation associated with pancreatic sufficiency. Thus, Abdel Rahman et al. (2006) recommended that pancreatic replacement therapy not be supplemented in case of cystic fibrosis with CFTR I1234V.
[See also: Egypt > Wahab et al., 2004; Syria > Wahab et al., 2002].
El-Harith et al. (1997) undertook mutation analysis of 15 Saudi Arabian children affected with CF from 12 families. They identified six mutations in the CTFR gene, detectable by PCR with subsequent restriction enzyme digestion, that would allow the detection of 70% of Saudi CFTR mutations. The most frequent of these were: 3120+1G>A (c.2988+1G>A), c.3909C>G (p.N1303K), and 1548delG. Other less frequent mutations included: 2043delG (c.1911delG), c.3700A>G (p.I1234V), and 406-2A>G. Two mutations, 1548delG and 406-2A>G, were identified for the first time. Most of the patients were found to present with severe forms of the disease.
To examine whether the 3120+1G>A (c.2988+1G>A) mutation has a common origin in the diverse populations in which it has been observed or whether its widespread distribution is the result of recurrent mutational events, Dork et al. (1998) analyzed DNA samples obtained from 17 unrelated CF patients in 4 different populations and from 8 unrelated African CF carriers. They found identical extended CFTR haplotypes for the 3120+1G>A (c.2988+1G>A) alleles in Arab, African, and African American patients, strongly suggesting that the mutation had a common origin. This finding was not surprising in the case of Africans and African Americans; it was not as easy to explain the presence of the 3120+1G>A (c.2988+1G>A) mutation in African and Saudi Arab patients. Recent ethnic admixture accounts for a few percent of Africans in Saudi Arabia; however, this was considered an unlikely explanation of the finding, since none of the Saudi families with the mutation had any anthropomorphologic signs of an African descent. In the past, a continuous gene flow between Arab and African populations probably persisted for many centuries, in association with trading and with the spread of the Islamic religion. Thus far, the Greeks are the only Caucasian population in which the 3120+1G>A (c.2988+1G>A) mutation has been identified. A recurrent mutational event seems to be unlikely, because the Greek haplotype differs from the others in only minor respects. Historical contacts, e.g., under Alexander the Great or during the ancient Minoan civilization, may provide an explanation for the common ancestry of the disease mutation in these ethnically diverse populations. Dork et al. (1998) concluded that 3120+1G>A (c.2988+1G>A) is an ancient mutation that may be more common than previously thought in populations of the tropical and subtropical belt, where CF probably is an underdiagnosed disorder.
In a study performed by Eskandarani (2002) to identify the CFTR gene mutations in Bahraini CF patients, one of the families studied was of Syrian origin. Mutation analysis of two affected siblings from this family identified homozygous mutation for 2043delG [See also Bahrain >Eskandarani, 2002].
Wahab et al. (2002) described a Syrian child with typical features of severe cystic fibrosis (CF) phenotype and a positive sweat test. DNA analysis confirmed homozygosity for the delta F508 (c.3882_3885delTATT) mutation on chromosome 7.
Messaoud et al. (2005) performed mutation analysis on 390 Tunisian CF patients belonging to 383 families to identify the CFTR mutations present in the Tunisian population. A total of 17 mutations were identified in this population, the most frequent of which were: delta F508 (51%), followed by G542X, W1282X, and N1303K. Four novel mutations were also identified; these included: T665S, 2766 del8, F1166C, and L1043R.
Ghorbel et al. (2011) investigated the possible roles of the delta F508 mutations and the IVS8-polyT polymorphism in CFTR gene in Tunisian infertile men without CBAVD. The study included 148 infertile patients and 126 fertile individuals. The polymorphic IVS8-polyT tract in CFTR gene was analyzed in only 129 infertile patients and 54 individuals of the control group. Y chromosome microdeletions were also screened in all infertile patients. delta F508 was not found either in infertile patients or in controls. The 5T allele of IVS8-polyT tract was found in both infertile men (4%) and fertile individuals (8%). A 5T/5T genotype was observed only in two azoospermic patients without Y microdeletions. The most frequent genotype of IVS8-polyT tract in infertile men and controls was 7T/7T (70% and 59% respectively). There was no association between the IVS8-polyT polymorphism and reduced semen quality. Ghorbel et al. (2011) concluded that neither the delta F508 mutation nor the 5T allele is involved in pathogenesis of male infertility in Tunisian infertile patients without CBAVD.
The first study to identify the genetic mutations responsible for cystic fibrosis among the UAE national population was undertaken by Frossard et al. (1994). Eight families with one child suffering from CF and a group of 30 random unrelated UAE nationals were used for the study. Mutation analysis was performed on the patients, following PCR amplification of 17 CFTR exons, by denaturing gradient gel electrophoresis (DGGE) and direct genomic sequencing. Surprisingly, the delta F508 mutation was not detected in any of the patients. Instead multiplex amplification of exons 11, 14b, and 17b enabled the identification of a CF causing mutation in exon 11, designated as p.S549R, in 75% of the mutated chromosomes. This mutation is expected to disrupt the function of the first nucleotide binding region and, therefore, expresses in a severe form of CF. Confirmation of this high frequency for this mutation in CF patients in larger scale studies was expected to be helpful for screening for CF mutations in the UAE population. Two other polymorphisms, though not causing CF, detected in the population included M470V and a silent E528E. The M470V polymorphism was present in 50% of the patients and 40% of the studied UAE national population. The E528E polymorphism was noted in 10% of the national population. The identification of these polymorphisms was expected to be of importance in following the co-segregation of the CF alleles within family members affected with the disease and enabling easy prenatal detection. Few years later, Frossard et al. (1998) and Dawson and Frossard (1999) studied 17 unrelated UAE national families, with a total of 20 children affected with cystic fibrosis. Patients from all 10 families of Bedouin descent were found to be homozygous for the p.S549R (c.1647T>G) mutation on exon 11, whereas their parents were found to be heterozygous. On the other hand, all patients from families of Baluch origin were homozygous for the delta F508 mutation, suggesting that this mutation was introduced to the UAE as well as Europe from Baluchistan some 40,000-50,000 years ago (Dawson and Frossard, 2000a and 2000b). Both these mutations identified have been shown to affect the processing of the CFTR protein, leading to its degradation, and therefore, resulting in very severe forms of the disease. It was also noted that all patients were homozygous for the respective mutation; a fact attributable to the consanguinity prevalent in the population. A study of the variables associated with the presentation of the disease (Frossard et al., 1999a) revealed that in individuals homozygous for the R549, the symptoms were very severe, with early age of presentation, very high sweat chloride levels, universal pancreatic insufficiency, and early colonization with P. aeruginosa. Furthermore, Frossard et al. (1999b) studied the clinical severity of the disease in UAE national CF affected patients, homozygous for the p.S549R mutation. Results indicated that the mutation was severe in its form, with low age of diagnosis (1.0 year). Detailed radiological analyses in 12 children with CF who were homozygous for p.S549R (c.1647T>G) revealed a diversity of pulmonary changes that included marked hyperinflation in early infancy in conjunction with inflammation of the interstitium. After 2 years of age, signs of central airway involvement occurred in association with early signs of pulmonary hypertension.
In order to determine the prevalence of these mutated alleles in the national population, Dawson and Frossard (1999) and Frossard et al. (1999c) screened a sample of 400 unrelated UAE nationals (200 males, 200 females) for these mutations. Six carriers were detected by this screening, and the carrier rate was calculated at 1:100 for p.S549R, and 1:200 for delta F508. The number of UAE nationals affected with cystic fibrosis in the national population was estimated at 1:15,876.
In 2001, Dawson and Frossard (2001a) compared the clinical severity associated with the two cystic fibrosis (CF) mutations p.S549R (c.1647T>G) and deltaF508. Clinical and biochemical variables of CF were compared in two age- and sex-matched groups of CF children in the United Arab Emirates. The clinical severity of mutations p.S549R (c.1647T>G) and deltaF508 showed comparable patterns, with very low Shwachman scores and high sweat chloride levels. Dawson and Frossard (2001a) concluded that patients homozygous for the CF mutations deltaF508 and p.S549R have a severe clinical presentation and illness and are indistinguishable on clinical grounds. Dawson and Frossard (2001b) further suggested that the founding chromosomes for the p.S549R may have originated in Bedouins of eastern Arabia. More recently, Saleheen and Frossard (2006) reported an Emirati CF patient homozygous for the 3120+1G>A (c.2988+1G>A) mutation.
[See also: Oman > Romey et al., 1999].