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.     

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.     

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.     

Intra- and extragenic marker haplotypes of CFTR mutations in cystic fibrosis families

Summary In order to facilitate the screening for the less common mutations in the cystic fibrosis (CF) gene viz., the CF transmembrane conductance regulator gene (CFTR), marker haplotypes were determined for German nonCF (N) and CF chromosomes by polymerase chain reaction analysis of four polymorphisms upstream of the CF gene (XV-2c, KM.19, MP6-D9, J44) and six intragenic polymorphisms (GATT, TUB9, M470V, T854T, TUB18, TUB20) that span the CFTR gene from exon 6 through exon 21. Novel informative sequence variants of CFTR were detected in front of exons 10 (1525-61 A or G), 19 (3601-65 C or A), and 21 (4006-200 A or G). The CF locus exhibits strong long-range marker-marker linkage disequilibrium with breakpoints of recombination between XV-2c and KM.19, and between exons 10 and 19 of CFTR. Marker alleles of GATT-TUB9 and TUB18-TUB20 were found to be in absolute linkage disequilibrium. Four major haplotypes encompass more than 90% of German N and CF chromosomes. Fifteen CFTR mutations detected on 421 out of 500 CF chromosomes were each identified on one of these four predominant 7-marker haplotypes. Whereas all analysed F508 chromosomes carried the same KM.19-D9-J44-GATT-TUB9-M470V-T854T haplotype, another frequent mutation in Germany, R553X, was identified on two different major haplotypes. Hence, a priori haplotyping cannot exclude a particular CF mutation, but in combination with population genetic data, enables mutations to be ranked by decreasing probability.     

The occurrence of various non-ΔF508 CFTR gene mutations among Hungarian cystic fibrosis patients

Summary Cystic fibrosis (CF) is an autosomal recessive disease caused by different mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The frequency of the major mutation (F508) in the Hungarian population is 64%. To identify other common mutations in CF families from Hungary, 30 nonF508 CF chromosomes were analyzed for selected mutations in exon 11 (G551D, R553X, G542X), intron 4 (621+1GT), intron 10 (1717–1GA), exon 20 (W1282X), and in exon 21 (N1303K) of the CFTR gene. In 6 of the 30 non-F508 CF chromosomes the following mutations were detected: R553X, G542X, 1717–1GA, W1282X, and N1303K. After analysis of the above eight mutations, 30% of CF chromosomes are as yet undefined and further analysis is planned.     

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.     

Changes in neutral amino acid efflux and membrane potential associated with the expression of CFTR protein

Summary The expression of wild type CFTR facilitates the efflux of neutral amino acids (Rotoli et al., Biochem. Biophys. Res. Commun. 204: 653–658, 1994); as a result, after an extensive depletion of intracellular amino acid pool obtained through an incubation in saline solution, the intracellular leucine levels were lower in murine C127 cells transfected with the wild type CF gene (C127 CFTRw/t) than in cells transfected with either mutant CF (C127 CFTRF508 cells) or mock vector only. No change in amino acid efflux was detected when C127 CFTRw/t and C127 CFTRw/t and C127 CFTRF508 cells were studied under conditions known to activate protein kinase A. Upon an incubation in Cl^– free medium, a permeant analogue of cAMP caused a marked cell depolarization of C127 CFTRw/t cells but not of C127 CFTRF508 cells, thus showing a functional expression of CFTR protein in the former cell line. However, we found that, upon a Cl^– free incubation and in the absence of exogenous cAMP, C127 CFTRw/t cells developed a marked hyperpolarization that was not detected in C127 CFTRF508 cells. It is concluded that the expression of normal CFTR accelerates amino acid efflux and enhances cell hyperpolarization in Cl^– free media; both these effects appear to be independent from PKA stimulation of CFTR.Abbreviations 8-Br-cAMP 8-bromoadenosine 3,5-cyclic monophosphate - C127 CFTRw/t C127i cells expressing CFTR wild type - C127 CFTRF508 C127i cells expressing CFTR bearing F508 mutation - C127 mock C127i cells transfected with the mock vector - CF cystic fibrosis - CFTR cystic fibrosis transmembrane conductance regulator - DMEM Dulbecco's modified Eagle medium - EBSS Earle's balanced salt solution - FBS fetal bovine serum - PKA protein kinase A     

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.     

Effects of the ΔF508 mutation on the structure,function, and folding of the first nucleotide-binding domain of CFTR

The fatal autosomal recessive disease cystic fibrosis (CF) is caused by mutations in the gene which encodes the cystic fibrosis transmembrane conductance regulator (CFTR). Many of these disease-causing mutations, including the deletion of F508 (F508) which accounts for approximately 70% of the disease alleles, occur in one of the two consensus nucleotide binding sequences. Peptide studies have directly demonstrated that the N-terminal nucleotide binding sequences bind adenine nucleotides. Structurally, circular dichroism spectropolarimetry indicates that this region of CFTR assumes a -stranded structure in solution. The F508 mutation causes a diminution in the amount of -stranded structure and a concomitant increase in the amount of random coil structure present, indicating that either the mutant peptide has a different native structure or that the conformational equilibrium is shifted toward a more disordered form. Furthermore, the mutant peptide is more sensitive to denaturation, indicating that F508 is a stability, or protein-folding mutant. Here we review these results and discuss their implications for interpreting the behavior of F508in situ and for the rational design of new CF drugs.     

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.     

Extended haplotype analysis of cystic fibrosis mutations and its implications for the selective advantage hypothesis

The major cystic fibrosis (CF) mutation, F508, is associated with one haplotype (B) determined by the two polymorphic markers, XV2C and KM19. This haplotype is rare (15%) among non-F chromosomes. Its frequency among non-F508 CF chromosomes is 50% with variation between populations. One hypothesis for the high frequency of CF haplotype B chromosomes suggests that there was a selective advantage for CF mutations on this specific background as a result of epistatic selection at other closely linked loci. Since the XV2C and KM19 markers are located 200kb 5 to the CF gene and span only 60 kb, an extended haplotype analysis was needed to test this hypothesis. Haplotypes were determined for 183 CF and 120 non-CF Israeli chromosomes at the XV2C and KM19 loci and at three intragenic polymorphic sites (GATT in intron 6A, TUB18 in intron 19, and 24M in exon 24). Among the studied chromosomes the frequency of non-F508 CF chromosomes associated with haplotype B was 70% (88% among Ashkenazi CF chromosomes). Nine mutations (F508, W1282X, G542X, N1303K, 3849+10 kb CT, Q359K/T360K, S549I, S549R, and 1717-1GA) were identified among the studied chromosomes. These mutations accounted for 96% of CF chromosomes of Ashkenazi origin. Haplotype B was associated with seven of these (F508, W1282X, G542X, N1303K, Q359K/ T360K, S549R, and 1717-1GA). The extended haplotype analysis revealed that in five of the seven mutations associated with the haplotype B, 97% of the chromosomes shared the same intragenic haplotype, 212. The variation found in 3% of the chromosomes was only in the GATT repeat. Two mutations, W1282X and 1717-1GA, were associated with a completely different intragenic haplotype, 121. The results of this study indicate that grouping of CF chromosome by haplotype analysis spanning a small extragenic region might not be sufficient. In addition, the results of the extended haplotype analysis indicate that all the studied CF chromosomes that carry the same mutation derived from the same origin. Furthermore, the results indicate that the majority of the CF mutations are associated with the same extended haplotype, supporting the selective advantage hypothesis.     

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.     

Deletion ΔF508 and haplotype analysis of CFTR gene region in Slovak CF patients

Summary Analysis of a sample of 50 unrelated cystic fibrosis (CF) patients and 46 nuclear families from Slovakia (Czechoslovakia) by the polymerase chain reaction and Southern hybridization revealed that the proportion of the F508 mutation was 58% in this population, and that the frequency of the B (i.e., KM19/XV2c [1–2]) haplotype was increased in both F508 and nonF508 CF chromosomes (98% and 46%, respectively). These results support the view that the trans-European gradient of the F508 frequency is of a geographical rather than of an ethnic origin, and that in Slavonic populations, there exists an as yet unidentified but frequent CF mutation other than F508, associated with the B haplotype.     

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.     

Distinct spectrum of CFTR gene mutations in congenital absence of vas deferens

Congenital absence of the vas deferens (CAVD) is a frequent cause for obstructive azoospermia and accounts for 1%–2% of male infertility. A high incidence of mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has recently been reported in males with CAVD. We have investigated a cohort of 106 German patients with congenital bilateral or unilateral absence of the vas deferens for mutations in the coding region, flanking intron regions and promotor sequences of the CFTR gene. Of the CAVD patients, 75% carried CFTR mutations or disease-associated CFTR variants, such as the “5T” allele, on both chromosomes. The distribution of mutation genotypes clearly differed from that observed in cystic fibrosis. None of the CAVD patients was homozygous for ΔF508 and none was compound heterozygous for ΔF508 and a nonsense or frameshift mutation. Instead, homozygosity was found for a few mild missense or splicing mutations, and the majority of CAVD mutations were missense substitutions. Twenty-one German CAVD patients were compound heterozygous for ΔF508 and R117H, which was the most frequent CAVD genotype in our study group. Haplotype analysis indicated a common origin for R117H in our population, whereas another frequent CAVD mutation, viz. the “5T allele” was a recurrent mutation on different intragenic haplotypes and multiple ethnic backgrounds. We identified a total of 46 different mutations and variants, of which 15 mutations have not previously been reported. Thirteen novel missense mutations and one unique amino-acid insertion may be confined to the CAVD phenotype. A few splice or missense variants, such as F508C or 1716 G→A, are proposed here as possible candidate CAVD mutations with an apparently reduced penetrance. Clinical examination of patients with CFTR mutations on both chromosomes revealed elevated sweat chloride concentrations and discrete symptoms of respiratory disease in a subset of patients. Thus, our collaborative study shows that CAVD without renal malformation is a primary genital form of cystic fibrosis in the vast majority of German patients and links the particular expression of clinical symptoms in CAVD with a distinct subset of CFTR mutation genotypes. Received: 15 April 1997 / Accepted: 29 April 1997     

Congenital bilateral absence of the vas deferens (CBAVD) and cystic fibrosis transmembrane regulator (CFTR): correlation between genotype and phenotype

To assess better the link between congenital bilateral absence of the vas deferens (CBAVD) and cystic fibrosis (CF), we compared sweat chloride values, analysis of the CFTR intron 8 poly(T) tract length and analysis of 10 exons in a population of 38 patients with CBAVD. The data indicate that this population can be divided into three groups of patients. In the first group of 15 patients with abnormal sweat chloride (> 60 mmol/l), the frequency of CF mutations is high. In the second group of 18 patients with equivocal sweat chloride (between 40 and 60 mmol/l), the frequency of the 5T variant is high; 6 patients have a F508 mutation and a 5T variant and 1 patient is homozygous for the 5T variant; a 5T variant has been detected in 3 other patients, and a F508 mutation in another patient. A third group of 5 patients is probably not related to CF: these patients have other congenital abnormalities of the urogenital tract, low chloride values (< 40 mmol/l) and apparently no abnormality of the CF gene.     

Frequency of ΔF508 and haplotype association in Austrian cystic fibrosis families

Summary The frequency of the major mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene was analyzed for 113 Austrian cystic fibrosis (CF) patients. An overall frequency of 55% for F508 was found with values of 72% and 13% for patients with pancreatic insufficiency (CF-PI) and those with pancreatic sufficiency (CF-PS), respectively. Furthermore, the distribution of the alleles of the closely linked DNA markers XV2c/KM19/MP6d-9 in our families is described.     

The spectrum of CFTR mutations in south-west German cystic fibrosis patients

The cystic fibrosis transmembrane conductance regulator (CFTR) gene of 110 cystic fibrosis (CF) patients from the south-west of Germany was screened for 12 different mutations. This analysis resulted in an identification of 79% of all CF mutations and a complete genotype in 66% of the families. The most common mutation found was F508 (67%). Another 5 mutations accounted for a further 12.5% (4% G542X; 3% R553X; 3% N1303K; 2% 1717-1 GA; 0.5% G551D) whereas 6 mutations (R117H, A455E, I507, S549I, S549N, and R1162X) were not found. Fifty-four (49%) patients were AF508 homozygotes and 18 (16.5%) were compound heterozygotes for F508 and one of the rarer mutations. These frequencies differ slightly from those found in the north of Germany and considerably from those reported from the south of Europe, which seems to be consistent with a north to south decline of the relative abundance of F508. Two patients, age 6 and 25 years, were compound heterozygotes for G542X and N1303K. The clinical features of the 6 year old were characterised by severe gastrointestinal and as yet only mild pulmonary complications whereas the 25 year old manifested severe pulmonary and gastrointestinal symptoms indicating that the N1303K mutation of the C-terminal CFTR nucleotide binding fold significantly impairs protein function in both the pancreas and the lungs.     

Identification of mutations in exons 1 through 8 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.

Five different mutations have been identified in the gene causing cystic fibrosis (CF) through sequencing regions encompassing exons 1-8, including the 5' untranslated leader. Two of these apparent mutations are missense mutations, one in exon 3 (Gly to Glu at position 85; G85E) and another in exon 5 (Gly to Arg at 178; G178R), both causing significant changes in the corresponding amino acids in the encoded protein--cystic fibrosis transmembrane conductance regulator (CFTR). Two others affect the highly conserved RNA splice junction flanking the 3' end of exons 4 and 5 (621 + 1G----T, 711 + 1G----T), resulting in a probable splicing defect. The last mutation is a single-basepair deletion in exon 4, causing a frameshift. These five mutations account for the 9 of 31 non-delta F508 CF chromosomes in our Canadian CF family collection and they are not found in any of the normal chromosomes. Three of the mutations, 621 + 1G----T, 711 + 1G----T, and G85E, are found in the French-Canadian population, with 621 + 1G----T being the most abundant (5/7). There are two other sequence variations in the CFTR gene; one of them (129G----C) is located 4 nucleotides upstream of the proposed translation initiation codon and, although present only on CF chromosomes, it is not clear whether it is a disease-causing mutation; the other (R75Q) is most likely a sequence variation within the coding region.     

Methods for analysis of multiple cystic fibrosis mutations

Summary A large number of mutations causing cystic fibrosis (CF) have been reported. In an attempt to improve methods for genetic diagnosis and for heterozygote screening, we evaluated methods for efficient analysis of the F508, G542X, G551D, R553X, and N1303K mutations. We found that multiple mutations can be analyzed simultaneously using hybridization with allelespecific oligonucleotides. Alternatively all of these mutations can be detected by amplification of DNA followed by restriction enzyme digestion and analysis on polyacrylamide gels. A previously reported method for use of modified primers for DNA amplification to allow detection of virtually any single-base change by restriction enzyme analysis proved particularly useful. The common F508 mutation and three mutations in exon 11 were analyzed using a multiplex amplification reaction followed by double digestion with restriction enzymes and electrophoresis in a single lane on a polyacrylamide gel. In a sample of 439 CF chromosomes from North American Caucasians, the frequencies of various mutations were as follows: F508=75.8%, G542X=2.7%, G551D=3.2%, R553X=1.4%, and N1303K=1.4% for a total of 84.5% detection of CF chromosomes by analysis for these five mutations.