Cystic fibrosis with three mutations in the cystic fibrosis transmembrane conductance regulator gene

Summary Three mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene were discovered in a pancreas-insufficient patient with cystic fibrosis (CF) who displayed an uncommon combination of almost normal chloride concentration in sweat tests and typical symptoms of gastrointestinal and pulmonary disease. The R553Q mutation was found on the maternal F508-CFTR gene. Codon 553 is located within a consensus motif of the ATP-binding cassette transport proteins at a less conserved position. Other members of this protein superfamily contain a glutamine instead of arginine at the homologous position, suggesting a modulating rather than disease-causing role of the R553Q mutation in CFTR. The amplification refractory mutation system did not detect the R553Q mutation in a further 65 normal, 113 F508, and 91 non-F508 CF chromosomes. The index case carried the R553X nonsense mutation on the paternal chromosome. The R553X mutation was present on a further 9 out of 86 German nonF508 CF chromosomes linked with the XV2c-KM19Mp6d9-J44-GATT haplotypes 2-2-2-1-1 and 1-1-2-1-2. The location of R553X on separate haplotypes including both alleles of the intragenic GATT repeat suggests an ancient and/or multiple origins of the R553X mutations. The association of the genotype of the CFTR mutation and the clinical phenotype was assessed for the patients carrying the related genotypes F508/F508 (n = 80), F508/R553X (n = 9) and F508-R553Q/R553X (n = 1). In compound heterozygotes, the median chloride concentration in pilocarpine iontophoresis sweat tests was significantly lower than in the F508 homozygotes (P < 0.01). The patient groups were significantly different with respect to the distributions of the centiles for height (P < 0.001) and weight (P < 0.01) as the most sensitive predictors of the course and prognosis in CF. Growth retardation was more pronounced in the compound heterozygotes.     

Frequency of the F508 deletion in cystic fibrosis patients from the European part of the USSR

Summary The frequency of the F508 deletion (F508) has been analyzed in 189 cystic fibrosis (CF) patients from the European part of the USSR, viz. 127 nothern Slavonians (Leningrad region), 30 southern Slavonians (the Ukraine), 10 central Slavonians (Moscow region), 14 Moldavians (Kishenev region) and 8 Lithuanians (Vilnius region). The distribution of CF⁺ chromosomes with and without F508 varied significantly in the different ethnic groups studied and correlated with the clinical manifestation of CF. The overall frequency of F508 in Slavonian patients is equal to 62.5%, approximately 90% of them being heterozygous or homozygous for this mutation. The frequency of the deletion among 99 Slavonian patients with severe disease manifestation (pancreatic insufficiency, PI) is equal to 67.5%, only 12 patients having pancreatic sufficiency (PS, 17.5%). The highest value of F508 (77.4%) is registered in PI/CF patients of the southern Slavonian group; it is much less frequent (about 57%) in relevant groups of Slavonians from the northern and central parts of the country. Unusually low frequencies (24% and 26%) of F508 are detected in a few samples of Lithuanian and Moldavian CF patients, respectively. All F508⁺CF-chromosomes of Slavonian origin are associated with haplotypes 2.2.2. defined by the restriction fragment length polymorphism sites KM19/PstI, CS.7/Hin6I and MP6d-9/MspI, although a high proportion (about 25%) of unknown mutations is associated with the same haplotype. Haplotype B (allele 1XV2c/TaqI; allele 2 KM19/PstI) accounts for 91% of F508⁺CF chromosomes. Our data are consistent with the hypothesis of a single origin and subsequent diffusion of this major CF mutation; however, its interpopulational dissemination in Eastern Europe does not follow the suggested south-east to north-west gradient in Western Europe. The significance of these data for prenatal diagnosis and carrier screening of CF mutations is briefly discussed.     

Lung involvement,the ΔF508 mutation and DNA haplotype analysis in cystic fibrosis

Summary Molecular studies of cystic fibrosis (CF) have allowed the genetic analysis of patients by means of DNA markers and the direct analysis of the CF gene. Some limited observations are available on the correlation between phenotype and genotype. Here, we report a study on the correlation of DNA haplotypes identified by KM-19 and XV-2c, the presence of the F508 mutation and lung involvement in 82 unrelated CF patients. Pulmonary involvement was defined by Chrispin's chest X-ray score, pulmonary function, sputum microbiology, serum immunoglobulin (SIg) levels and Shwachman's clinical score. Patients homozygous for haplotype B showed worse X-ray and clinical scores, more frequent sputum colonization byPseudomonas aeruginosa andStaphylococcus aureus, lower spirometric values and raised concentrations of SIg G, A and M, compared with patients with other haplotypes. When lung involvement parameters were examined in patients homozygous, heterozygous or null for the F508 mutation, no difference was found among the three groups. Our data indicate a significant occurrence of severe pulmonary involvement in patients homozygous for the B haplotype; this is not influenced by the F508 mutation. We suggest that simple DNA haplotypes may provide data of both diagnostic and prognostic value, without the need for extensive and expensive molecular analyses.     

Novel cystic fibrosis mutation associated with mild disease in Cypriot patients

Cyprus is an island in the eastern Mediter-ranean basin inhabited by people of Caucasian extraction, mostly Greek-Cypriots. The most common inherited disease among Caucasians is cystic fibrosis (CF). Although no careful scientific study had ever been done the impression was that CF was extremely rare among the Greek-Cypriots, with an incidence estimated at around 130,000. About 2 years ago, we introduced molecular diagnostic methodology in an effort to assist clinicians in safer diagnosis of patients presenting with atypical CF symptomatology, and also for testing the hypothesis that mutations that cause milder phenotypes might be responsible for misdiagnosis or for missing entirely some cases of CF. Initial screening for F508 revealed that it is indeed rare in the general population. Further screening of suspected CF patients revealed a novel mutation that converted leucine at position 346 to proline (L346P) in two unrelated families. The second CF mutation was F508 and 1677delTA in the two families respectively, both reportedly associated with severe phenotypes. Yet our patients did not present with typical CF pictures possibly because of the dominant nature of this novel mild mutation in exon 7. Symptoms included failure to thrive, chest infections and electrolyte disturbances. These findings raise the possibility that Cyprus might have been spared very severe CF phenotypes but not cystic fibrosis transmembrane conductance regulator (CFTR) mutations.     

Identification of a cystic fibros is mutation: deletion of isoleucine506

Summary The recent isolation of the cystic fibrosis (CF) gene has resulted in the identification of a common mutation (AF₅₀₈) that is found on about 70% of CF chromosomes and that comprises a deletion of 3 bp and results in the omission of Phe₅₀₈ from within a putative ATP-binding domain of the predicted gene product. We describe a CF mutation that involves the deletion of 3 bp encoding Ile₅₀₈ or Ile₅₀₇. This is a rare mutation found in less than 1% of CF chromosomes and could be mistaken for AF₅₀₈ using the current methods for the molecular diagnosis of CF.     

Molecular analysis of cystic fibrosis in the Hungarian population

Summary Hungarian cystic fibrosis (CF) families (n = 33) including 114 family members have been analysed for the presence of the F508 mutation within the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and have been haplotyped with probes for restriction fragment length polymorphisms (RFLPs) known to be linked to the CFTR gene. The F508 deletion was present in 64% of CF chromosomes. As in many other populations, linkage disequilibrium was found between the CF locus and the haplotype B (XV-2c: allele 1, KM1-9: allele 2), which accounts for 95% of F508 CF chromosomes in our families.     

Analysis of 160 CF chromosomes: detection of a novel mutation in exon 20

The cystic fibrosis (CF) gene has been cloned and a major mutation identified (F508). This 3-bp deletion has been found in approximately 70% of CF chromosomes. We have used the strategy of denaturing gradient gel electrophoresis followed by direct sequencing of the polymerase chain reaction products, in order to detect other mutations in exons 10, 11 and 20 of the CF transmembrane conductance regulator gene. A new mutation, F1286-S, was found in exon 20. It involves a nucleotide change of TC at nucleotide 3989 and changes a phenylalanine into serine at position 1286 of the protein.     

Two new mutations detected by single-strand conformation polymorphism analysis in cystic fibrosis from Russia

Single-strand conformation polymorphism (SSCP) analysis followed by direct sequencing of exons containing ATP-binding domains of the cystic fibrosis transmembrane conductance regulator (CFTR) gene was performed on 80 Russian DNA samples. Two new alterations — S1196X (exon 19) and W1282R (exon 20) — and two novel polymorphisms — 1525-61 (intron 9) and 1716+12 T-C (intron 10) — were identified. Mutation S1196X changes a TCA codon to TGA and destroys an EcoRI site. Alteration W1282R results from a T-to-C change at position 3976. It was found in one Russian patient and creates an AciI site; however, it is unclear whether this is a disease-causing mutation or a polymorphism. Polymorphism 1525-61 results from an A-to-G change. Alteration 1716+12 T-C was found in a Moldovian patient and creates a new MaeII site. It is not known whether this alteration affects the splicing of the mRNA. The previously described A4002G polymorphism was encountered in approximately 9% of Russian CF chromosomes. In addition, we have found the previously described 3732delA mutation in 7 CF chromosomes, making it the second (after F508) most frequent mutation in the Russian population.     

Clinical characteristics of 16 cystic fibrosis patients with the missense mutation R334W,a pancreatic insufficiency mutation with variable age of onset and interfamilial clinical differences

We present the genotype/phenotype correlation analysis for 16 cystic fibrosis (CF) patients who carry mutation R334W. Current age and age of diagnosis was significantly higher in the R334W/any-mutation group (P < 0.05 and P < 0.01), compared with the 508/508 group. A slightly, but not significantly, worse lung function was found in the R334W/any-mutation group, when compared with the 508/508 patients. The proportion of patients with lung colonization with bacterial pathogens was slightly, but not significantly, higher in the R334W/any-mutation group (71.4%), compared with the 508/508 or R334W/508 groups (55.5%). None of the R334W patients had meconium ileus but 60% were pancreatic insufficient (PI), a significantly lower proportion (P 0.001) than 508/508 patients. Two R334W/N1303K compound heterozygous sisters were PI but discrepant for lung function. Two groups of three sibs with genotype R334W/508 showed interfamilial discordant clinical data for lung and pancreatic function. The data provided here for mutation R334W demonstrate that this mutation is responsible for a less severe form of CF than 508. Interfamilial differences for PI and lung function suggest that other factors, viz. genetic, environmental and medical, contribute to the wide spectrum of clinical differences observed in CF patients with the same CF transmembrane conductance regulator genotypes.     

Cystic fibrosis in Spain: high frequency of mutation G542X in the Mediterranean coastal area

We have determined the frequency of deletion F508 and mutation G542X, a nonsense mutation in exon 11 of the cystic fibrosis (CF) gene, in a sample of 400 Spanish CF families. Mutation G542X represents 8% of the total number of CF mutations in Spain, making it the second most common mutation after the F508 deletion, which accounts for 48% of CF chromosomes. G542X has a higher frequency in the Mediterranean coastal area (14%) and in the Canary Islands (25%). About 70% of G542X chromosomes are from Andalucia, Múrcia, Valencia, Catalunya and the Canary Islands. The F508 deletion has its highest frequency in the Basque Country (83%). Mutation G542X is associated with the same rare haplotype that is found in association with the F508 mutation. The haplotype homogeneity found for G542X, even when intragenic microsatellites (IVS8CA, IVS17BTA and IVS17BCA) are considered, allows us to postulate that this mutation arose from a single mutational event. The geographic distribution of mutations F508 and G542X suggests that F508 was present in the Iberian Peninsula before the Indo-European invasions, and that G542X was introduced into Spain, via the Mediterranean Sea, probably by the Phoenicians, between 2500 and 3000 years ago.     

The major cystic fibrosis mutation in a British population

Summary We have studied 72 families with at least one child with cystic fibrosis (CF); they were referred because they had requested pre-natal diagnosis in a future pregnancy. The F508 mutation was found in 108/140 CF chromosomes (77%). In 41/72 families (57%), both parents carried a deleted chromosome and the child was doubly deleted. In only 4 families, 2 of them being consanguineous, did neither parent carry a deleted chromosome. Meconium ileus was associated with children who were F508/F508, F508/non-deleted and non-deleted/ non-deleted.This paper should have been published in Human Genetics, Vol.85, No.4, 1990, together with the other European data on Population analysis of the major mutation in cystic fibrosis. Its publication was delayed for technical reasons     

Extensive analysis of 40 infertile patients with congenital absence of the vas deferens: in 50% of cases only one CFTR allele could be detected

Mutations in the cystic fibrosis (CF) conductance transmembrane regulator (CFTR) gene have been detected in patients with CF and in males with infertility attributable to congenital bilateral absence of the vas deferens (CBAVD). Thirty individuals with CBAVD and 10 with congenital unilateral absence of the vas deferens (CUAVD) were analyzed by single-strand conformation analysis and denaturing gradient gel electrophoresis for mutations in most of the CFTR gene. All 40 individuals were pancreatic sufficient, but twenty patients had recurrent or sporadic respiratory infections, asthma/asthmatic bronchitis, and/or rhino-sinusitis. Agenesia or displasia of one or both seminal vesicles was detected in 30 men and other urogenital malformations were present in six subjects. Among the 40 samples, we identified 13 different CFTR mutations, two of which were previously unknown. One new mutation in exon 4 was the deletion of glutamic acid at codon 115 (E115). A second new mutation was found in exon 17b, viz., an AC substitution at position 3311, changing lysine to threonine at codon 1060 (K1060T). CFTR mutations were detected in 22 out of 30 (73.3%) CBAVD patients and in one out of 10 (10%) CUAVD individuals, showing a significantly lower incidence of CFTR mutations in CBAVD/CUAVD patients (P 0.0001), compared with that found in the CF patient population. Only three CBAVD patients were found with more than one CFTR mutation (F508/L206W, F508/R74W+D1270N, Rl 17H/712-1GT), highlighting L206W, R74W/ D1270N, and R117H as benign CF mutations. Sweat electrolyte values were increased in 76.6% of CBAVD patients, but three individuals without CFTR mutations had normal sweat electrolyte levels (10% of the total CBAVD patients), suggesting that factors other than CFTR mutations are involved in CBAVD. The failure to identify a second mutation in exons and their flanking regions of the CFTR gene suggests that these mutations could be located in introns or in the promoter region of CFTR. Such mutations could result in CFTR levels below the minimum 6%–10% necessary for normal protein function.     

Cystic Fibrosis: A Brief Look at Some Highlights of a Decade of Research Focused on Elucidating and Correcting the Molecular Basis of the Disease

The disease Cystic Fibrosis (CF) is caused by mutations in the protein called CFTR, cystic fibrosis transmembrane conductance regulator, an ABC-transporter–like protein found in the plasma membrane of animal cells. CFTR is believed to function primarily as a Cl^– channel, but evidence is mounting that this protein has other roles as well. Structurally, CFTR consists of a single polypeptide chain (1480 amino acids) that folds into 5 distinct domains. These include 2 transmembrane domains that are involved in channel formation; 2 nucleotide-binding domains (NBF1 and NBF2), the first of which clearly binds and hydrolyzes ATP; and 1 regulatory domain (R) that is phosphorylated in a cAMP-dependent process. Currently, the 3D structure of neither CFTR nor its domains has been elucidated, although both nucleotide domains have been modeled in 3D, and solution structures in 3D have been obtained for peptide segments of NBF1. The most common mutation causing CF is the deletion () of a single phenylalanine (F) in position 508 within a putative helix located in NBF1. CF patients bearing this F508 mutation frequently experience chronic lung infections, particularly by Pseudomonas aeruginosa, and have a life span that rarely exceeds the age of 30. Since the CFTR gene was cloned and sequenced in 1989, there has been over a decade of research focused on understanding the molecular basis of CF caused by the F508 mutation, with the ultimate objective of using the knowledge gained to carry out additional research designed to correct the underlying defect. In general, this pioneering or ground roots research has succeeded according to plan. This brief review summarizes some of the highlights with a focus on those studies conducted in the authors' laboratory. For us, this research has been both exciting and rewarding mainly because the results obtained, despite very limited funding, have provided considerable insight, not only into the chemical, molecular, and pathogenic basis of CF, but have made it possible for us and others to now develop novel, chemically rational, and cost effective strategies to identify agents that correct the structural defect in the F508 CFTR protein causing most cases of CF.     

Mutation analysis of ten exons of the CFTR gene in Greek cystic fibrosis patients: characterization of 74.5% of CF alleles including one novel mutation

To initiate the complete characterization of mutations in the CFTR gene in Greek cystic fibrosis (CF) patients, we screened 184 patients for six relatively common mutations (AF 508, G542X, G551D, 621+1 GT, N1303K, W1282X) using allele-specific hybridization and, in addition, analyzed exons 4, 5, 7, 8, 10, 11, 17b, 19, 20 and 21 using the method of denaturing gradient gel electrophoresis (DGGE). Six mutations accounted for 65.9% of the CF alleles in Greek patients, of which the F 508 mutation had a frequency of 52.7%. A further 15 previously described mutations accounted for another 8.3% CF alleles and one previously undescribed mutation (3272-4AG) was found in one chromosome. The W1282X mutation was not detected at all. Thus, so far, we have identified 21 mutations in the CFTR gene in Greek CF patients, accounting for 74.5% of the CF alleles.     

Incidence and expression of the N1303K mutation of the cystic fibrosis (CFTR) gene

Summary The N1303K mutation was identified in the second nucleotide binding fold of the cystic fibrosis (CF) gene last year. We have gathered data from laboratories throughout Europe and the United States of America in order to estimate its frequency and to attempt to characterise the clinical manifestations of this mutation. N1303K, identified on 216 of nearly 15000 CF chromosomes tested, accounts for 1.5% of all CF chromosomes. The frequency of the N1303K allele varies significantly between countries and ethnic groups, being more common in Southern than in Northern Europe. This variation is independent of the AF508 allele. It was not found on UK Asian, American Black or Australian chromosomes. N1303K is associated with four different linked marker haplotypes for the polymorphic markers XV-2c, KM.19 and pMP6d-9. Ten patients are homozygous for this mutation, whereas 106 of the remainder carry one of 12 known CF mutations in the other CF allele. We classify N1303K as a severe mutation with respect to the pancreas, but can find no correlation between this mutation, in either the homozygous or heterozygous state, and the severity of lung disease.     

Cystic Fibrosis: A Disease of Altered Protein Folding

Cystic fibrosis (CF) is caused by mutations in the gene that encodes the cystic fibrosis transmembrane conductance regulator, CFTR. Previously we demonstrated that the common F508 mutation in the first nucleotide binding domain (NBD1) alters the ability of the domain to fold into a functional three-dimensional structure, providing a molecular explanation for the observation that the mutant CFTR is retained in the endoplasmic reticulum and does not traffic to the apical membrane of affected epithelial cells. Notably, when conditions are altered to promote folding of the mutant protein, it can assume a functional conformation. Correcting the folding defect may have therapeutic benefit for the treatment of cystic fibrosis. Here we summarize these results and discuss the implications in vitro folding studies have for understanding the pathobiology of CF.     

Iron limitation and its effect on membrane proteins and siderophore transport inNeurospora crassa

Summary Cells of the fungusNeurospora crassa were grown under iron-deficient and iron-sufficient conditions and their plasma membrane proteins were compared. Three strains were studied:N. crassa 74A (wild type), a siderophore-free mutantN. crassa (arg-5 ota aga) as well as a slime variant ofN. crassa which lacks a cell wall. Plasma membranes were purified, solubilized and analyzed by one-dimensional SDS/polyacrylamide gel electrophoresis yielding approximately 50 distinct protein bands with molecular masses in the range 14–160 kDa. Iron-sufficient and iron-deficient growth resulted in nearly identical plasma membrane protein profiles in all strains. Although minor alterations in the proportion of certain proteins could be detected, significant overproduction of certain membrane proteins during iron limitation could not be observed. Transport of⁵⁵ Fe-labeled siderophores seems to be correlated to the degree of iron limitation. For example, transport rates were enhanced five-fold after 16 h of growth in iron-deficient medium compared to growth in iron-sufficient medium. Extraction and HPLC measurement of siderophores from conidiospores yielded approximately 10^–15 mol/spore, indicating that germination tubes and young cells used for transport measurements are not iron-deficient. It is suggested that the putative transport systems for siderophores in fungal plasma membranes are constitutively expressed and enhanced uptake of siderophores during iron limitation is rather the result of cellular transport regulation mechanisms.     

A termination mutation (2143delT) in the CFTR gene of German cystic fibrosis patients

German patients with cystic fibrosis (CF) were screened for molecular lesions in exon 13 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by single strand conformation polymorphism (SSCP) and chemical cleavage of mismatch analyses. Direct sequencing of four samples that displayed the same SSCP pattern and that were susceptible to cleavage of heteroduplexes by osmium tetroxide revealed, in all cases, a deletion of a single T residue at nucleotide position 2143 within codon 671 of the CFTR gene. As a result, leucine codon 671 is changed into a termination codon. In total, the 2143delT mutation was confirmed in 6 out of 271 German non-F508 CF chromosomes by artificial restriction fragment length polymorphism analysis, indicating that this frameshift mutation accounts for about 2% of German non-508 mutations. The 6 pancreas insufficient patients who are compound heterozygous for 2143-delT suffer from the typical features of pulmonary and gastrointestinal CF disease. The 2143delT mutation completes the panel of the more frequent CFTR mutations that reside on the F508 haplotype and that contribute to its overpresentation among German non-F508 alleles that are associated with severe forms of disease.     

Exon 9 of the CFTR gene: splice site haplotypes and cystic fibrosis mutations

The alternatively spliced exon 9 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene codes for the initial part of the amino-terminal nucleotide-binding fold of CFTR. A unique feature of the acceptor splice site preceding this exon is a variable length polymorphism within the polypyrimidine tract influencing the extent of exon 9 skipping in CFTR mRNA. We investigated this repeat for its relationship to CFTR mutations and intragenic markers on 200 chromosomes from German patients with cystic fibrosis (CF). Four frequent length variations were strongly associated with the four predominant haplotypes previously defined by intragenic marker dimorphisms. One of these alleles displayed absolute linkage disequilibrium to the major CF mutation F508. Other frequent CFTR mutations were linked to one particular splice site haplotype indicating that differential exon 9 skipping contributes little to the clinical heterogeneity among CF patients with an identical mutation. We also identified a novel missense mutation (V456F) and a novel nonsense mutation (Q414X) within the coding region of exon 9. The missense mutation V456F adjacent to Walker motif A was present in a pancreas-sufficient CF patient. In contrast, the pancreas-insufficient Q414X/F508 compound heterozygote suffered from a severe form of the disease, indicating that alternative splicing of exon 9 does not overcome the deleterious effect of a stop codon within this exon.     

Carrier detection and prenatal diagnosis of cystic fibrosis using an intragenic TA-repeat polymorphism

Summary We have analysed the segregation of a TA-repeat polymorphism in intron 17b of the cystic fibrosis transmembrane conductance regulator gene responsible for cystic fibrosis (CF) in 23 French CF families non-informative for the F508 mutation (i.e. with at least one parent not carrying F508) or closely linked DNA markers. At least 13 different alleles ranging from 7 to 45 repeats were observed and the detected heterozygosity was 89%. Of the 23 families studied, 19 were fully informative for prenatal diagnosis or carrier detection, 3 were partially informative and one was not informative. In 6 families, prenatal diagnosis for CF or carrier detection in siblings of CF cases were performed using this polymorphism.