ΔF508 Mutation Research Articles

Differentiation of immortalized epithelial cells derived from cystic fibrosis airway submucosal glands

Cystic fibrosis (CF) involves abnormalities in mucus production and secretion of the airway. Studies of the regulation of airway mucin production and secretion has been difficult due to the lack of in vitro models of the airway epithelial cells which express functional differentiation. Because the majority of the mucin in the airway is apparently produced by the submucosal glands, we have focused our attention on the development of cell culture models of human airway submucosal glands. This report describes the propagation of CF airway submucosal gland epithelial cells which continue to express mucin production. The CF bronchus was obtained from a 31-yr-old patient who received a double lung transplant. The glands were dissected out and primary cultures prepared by the explant/outgrowth procedure. The cells were immortalized by infection with Adl2-SV40 hybrid virus. The cultures are maintained in serum-free keratinocyte basal medium supplemented with insulin (5µg/ml), hydrocortisone (0.5µg/ml), epidermal growth factor (10 ng/ml), bovine pituitary extract (25µg/ml), and antibiotics. Cultures were passaged using 0.125% trypsin in Ca(+2) and Mg(+2)-free Hanks', balanced salt solution. Polymerase chain reaction (PCR) analysis demonstrated that the cells were homozygous for the ΔF508 mutation. Morphologic observations showed that the cells were epithelial and were interconnected by sparsely distributed desmosomes. Their cytoplasm contained secretory-type structures including abundant Golgi, rough endoplasmic reticulum, and secretory vesicles. Immunofluorescent studies determined that all cells were positive for cytokeratins, mucin glycoconjugates, and cystic fibrosis transmembrane conductance regulator. The cultures secreted substantial amounts of mucin glycoproteins and expressed the MUC-2 mucin gene. Patch clamp experiments revealed that the cells expressed defective Cl(-) channels which were not activated by Forskolin.

Cystic fibrosis screening in neonates — measurement of immunoreactive trypsin and direct genotype analysis for ΔF508 mutation

This study investigated the clinical usefulness of screening for cystic fibrosis (CF) in 19,992 newborns, over 39 months, in an Austrian population. Immunoreactive serum trypsin (IRT) determination was followed by sweat chloride analysis (sweat test) to establish diagnosis. In a retrospective analysis covering 6 months of the study period, individuals who were considered to be at risk after IRT estimation (n = 22) were analysed for delta F508 mutation, using a new method of DNA extraction from the initial dried blood specimens. A total of 119 infants (0.6%) had values greater than 750 ng trypsin/ml whole blood. In 88 babies sweat tests were performed, leading to the diagnosis of CF in 11 cases. One patient was not initially identified by screening but was later discovered due to his clinical status. Three infants were noted to carry the delta F508 mutation (1 homozygous, 2 heterozygous). Two of these babies already had CF. The second heterozygote was a carrier. A highly efficient three tier screening strategy is presented in which IRT estimation, determination of delta F508 status and sweat chloride testing could lead to a high sensitivity analysis of this population.

Attitudes of high school students toward carrier screening and prenatal diagnosis of cystic fibrosis.

AbstractThe identification of the common mutations of the cystic fibrosis (CF) transmembrane conductance regulator gene has made it feasible to consider population‐based CF carrier screening. However, the demand for such programs will depend largely on the attitudes and perceptions of the general public toward genetic testing. As part of a high school science project, we initiated studies to determine the attitudes of high school students toward carrier screening for CF and other genetic disorders. From a class of 120 biology students, 101 consented to participate in the study. Most of the students were of European ancestry and there were no known family histories of CF. Buccal swabs were obtained for PCR amplification and heteroduplex detection of the most common CF mutation (ΔF508). The students and their corresponding buccal swabs were assigned random numbers and the results were kept confidential unless the student requested his/her test result. A questionnaire was used to assess the students' attitudes toward carrier screening and prenatal diagnosis. This questionnaire was given both before testing and after the class was informed that three individuals were carriers of the ΔF508 mutation. Overall, the data indicate that high school students are receptive to the concepts of carrier screening and prenatal diagnosis. However, their attitudes changed considerably when informed of the possibility that they might actually be a ΔF508 carrier, with marked increases in the level of indecision. At the conclusion of the study, very few students (5/101) requested information about their ΔF508 carrier status.

Early prenatal direct gene diagnosis of cystic fibrosis in a twin pregnancy and subsequent selective termination

We present a case of prenatal diagnosis of cystic fibrosis (CF) in one twin at 11-12 weeks of gestation. The parents had previously had two children, one of whom is alive and healthy and one who died of CF at the age of 2 1/2 months. The parents were both known to be carriers of the delta F508 mutation. Chorionic villus sampling (CVS) was performed and direct gene analysis showed that one fetus was homozygous for the delta F508 mutation, while the other fetus did not have the mutation at all. Both fetuses had normal karyotypes. Selective termination was subsequently performed. The pregnancy continued without complications except for mild pre-eclampsia at term. The woman had a Caesarean section. The genetic diagnosis was confirmed after birth.

Cystic fibrosis: the delta F508 mutation does not lead to an exceptionally severe phenotype. A cohort study.

In an attempt to ascertain a relationship between genotype and phenotype, we studied the pulmonary and nutritional status of 123 cystic fibrosis patients with known genotype at an age of 8.5-10 years. Patients represent a cohort as they are almost all those born and diagnosed in a given area and period. They were followed at a single centre using uniform diagnostic and treatment protocols. Pulmonary and nutritional status of homozygous delta 508 patients did not differ from that of compound heterozygotes or of patients with other unspecified genotypes. Pulmonary manifestations varied widely in all genotype groups. With the given number of patients, a slightly higher mortality of delta F508 homozygotes could have been coincidental. We conclude that up to the age of 8.5-10 years the severity of pulmonary lesions and nutritional deficiencies is not related to the delta F508 mutation.

Nonsense mutation R1162X of the cystic fibrosis transmembrane conductance regulator gene does not reduce messenger RNA expression in nasal epithelial tissue.

Cystic fibrosis (CF) patients bearing the premature translation termination mutation (nonsense mutation) W1282X present severe pulmonary and pancreatic disease, whereas patients carrying other nonsense mutations such as G542X, R553X, S1255X, R1162X, and W1316X show a severe pancreatic but mild pulmonary illness. CF gene expression was found absent in respiratory tissues with mutations R553X and W1316X, which led to the hypothesis that the absence of the gene product in the lung is more favorable than the presence of an altered one. We asked whether or not all the nonsense mutations characterized by mild pulmonary disease phenotypes do present the absence of CF gene expression. We therefore investigated gene expression at the mRNA level in respiratory cells obtained from nasal polyps from a patient homozygous for the R1162X mutation. Gene expression was studied by amplification with polymerase chain reaction of segments of the CF transmembrane conductance regulator cDNA that was obtained by reverse transcription of RNA. Semiquantitative analysis was performed by Northern analysis. By comparing the data obtained from polyps deriving from non-CF subjects and a CF patient homozygous for dF508 mutation, it is shown that no reduction of CF gene expression is evident in R1162X respiratory tissue. We conclude that CF nonsense mutations have heterogeneous mechanisms of gene expression.

Co-amplification of the cystic fibrosis delta F508 mutation with the HLA DQA1 sequence in single cell PCR: implications for improved assessment of polar bodies and blastomeres in preimplantation diagnosis.

We have developed a heminested PCR (polymerase chain reaction) method, performed on single cells, for the analysis of the most common cystic fibrosis (CF) mutation (delta F508). As a quality control, the polymorphic exon 2 of the HLA DQA1 locus was co-amplified from the same cell. With a non-radioactive reverse dot-blot assay, the genotype of these two loci could be determined. Experiments on 98 single fibroblasts, heterozygous for the CFTR and the DQA1 locus, showed that amplification of either locus could be obtained in 97 per cent of the cases, but only 90 per cent showed heterozygosity for CF, 75 per cent showed heterozygosity for DQA1, and 74 per cent showed heterozygosity for both CF and DQA1. Contaminations detected only after DQA1 typing occurred in 3 per cent of our samples. Error rate calculations based on our experimental PCR data indicate that single blastomere diagnosis would lead to unacceptable errors, i.e., an affected fetus, in less than 1 per cent of the cases. The risk of undetected crossing-over or the dubious results that crossing-over could generate, would make isolated polar body diagnosis at the present time very difficult. The combined approach of PCR on polar bodies followed by confirmation of the diagnosis on blastomeres, however, should give a solid base for preimplantation diagnosis of monogenic disorders.

Passive smoking and lung function in cystic fibrosis.

The relationship between passive exposure to cigarette smoking and objective measures of health was examined in 340 patients with cystic fibrosis attending a large hospital-based clinic. Patients who came from households with smokers did not differ from those living in smoke-free households in terms of nutritional status, clinical scores, spirometry, or colonization with Pseudomonas. The number of cigarettes smoked in the household was not significantly related to nutritional status, clinical score, spirometry, or hospitalization. Similar results were found when children 6 to 11 yr of age were analyzed separately, except that height percentile was negatively related to the number of cigarettes smoked in the household. The effects of household exposure to cigarette smoke were further evaluated by analyzing changes in nutritional status, clinical score, and spirometry over a 15-yr period among patients whose families never, always, stopped, or started smoking during this time. Height percentile increased slightly during this interval among those whose households never smoked, whereas no change occurred among patients whose households always smoked, and a decline was seen among patients whose households quit. These differences were statistically significant. Patients whose households never smoked had consistently higher pulmonary function measurements than did patients whose families always smoked, although the differences were not statistically significant. The rates of decline were similar in these two groups. Patients whose households stopped smoking had significantly lower pulmonary functions at the end of the study than did subjects whose households never smoked.(ABSTRACT TRUNCATED AT 250 WORDS)

Linkage Disequilibrium between Cystic Fibrosis Mutations and Polymorphic 4-bp Repeat within CFTR Gene

The PCR technique was used in a study of the linkage of cystic fibrosis mutations and a polymorphic (GATT)n repeat in intron 6 of the CFTR gene. Absolute linkage disequilibrium was found between the common delta F-508 mutation and the (GATT)6 allele. This allele was also in linkage disequilibrium with other unidentified mutations in the CFTR gene resulting in the pancreatic insufficient form of disease. The frequency of (GATT)n alleles in the pancreatic sufficient form of CF did not differ significantly from the data obtained in the total population. The significance of the (GATT)n polymorphic repeat for the diagnosis of CF is discussed.

Polymerase chain reaction analysis of the cystic fibrosis ΔF508 mutation in human blastomeres following oocyte injection of a single sperm from a carrier

The efficiency of the polymerase chain reaction (PCR) in detecting the cystic fibrosis (CF) delta F508 mutation (which is the most common mutation of CF) was assessed in single human blastomeres. Twenty-one human immature oocytes (germinal-vesicle-stage oocytes) that had been donated for research were matured in vitro and a single spermatozoon from a carrier of the CF delta F508 mutation was injected into the ooplasm. Fourteen embryos were obtained after intracytoplasmic sperm injection (ICSI). PCR analysis was carried out on 70 single blastomeres isolated from these 14 embryos. The results showed that the efficiency of DNA amplification by PCR in single nucleate blastomeres was 94 per cent (59/63). There were no false-positive results since none of the blank samples or the blastomeres without a nucleus showed an amplified signal. We found that nine embryos were homozygous for the unaffected genotype and that four embryos were heterozygous since they contained both the unaffected and the delta F508 genotype. In a four-cell embryo, we observed the homozygous unaffected genotype in one blastomere and a heterozygous delta F508/unaffected genotype in the other three blastomeres.

Results of retroactive testing of human semen donors for cystic fibrosis and human immunodeficiency virus by polymerase chain reaction.

Retroactive testing for cystic fibrosis carrier status was performed on 149 semen donors and 100 donor applicants; 162 human immunodeficiency virus (HIV-1) sero-negative non-active semen donors and 45 HIV-1 sero-negative actively donating semen donors were also retroactively tested for HIV-1 by polymerase chain reaction (PCR). The 249 individuals tested for cystic fibrosis were examined for seven mutations including delta F508, G542X, S549I, S549N, G551D, R553X, and W1282X. Of 149 retroactively tested donors, five (3.4%) were determined to be heterozygous carriers of the delta F508 mutation for cystic fibrosis. One of the 100 donor applicants was also heterozygous for delta F508. The 207 HIV-1 sero-negative donors had an average of 15.4 (range 2-45) HIV antibody tests during an average of 15.6 (range 1-68) months of donations. No donors had positive seroconversion. These donors also tested negative for HIV by PCR. Cystic fibrosis testing of donor applicants prevents known carriers from being used for artificial insemination by donor therapy and reduces substantially the risk of an offspring being born with the disease. With stringent donor selection and exclusion of high-HIV-risk applicants, the occurrence of HIV infection and subsequent seroconversion in donor populations should be very infrequent.

DNA analysis of cystic fibrosis in Brazil by direct PCR amplification from Guthrie cards.

A 3 bp deletion of codon 508 (phenylalanine) of the cystic fibrosis (CF) gene constitutes the mutation of most CF chromosomes. The frequency of this mutation (referred to as delta F508), varies considerably between populations, ranging from 26% of the CF mutations in Turkey to 88% in Denmark. To determine the frequency of the delta F508 mutation in Brazilian Caucasoid CF patients, we used direct polymerase chain reaction (PCR) amplification of DNA obtained from dried blood spots on Guthrie cards, followed by ethidium bromide staining of gels. Although the overall frequency of the delta F508 mutation was 47% of 380 CF chromosomes from Brazilian Caucasoids born in five different states, significant interstate differences were observed, ranging from a delta F508 frequency of 27% to 53%. While our method could be used to screen patients and their relatives for carrier testing and prenatal diagnosis, the efficacy of screening only for the delta F508 mutation would be low, and would vary from state to state. Screening for a panel of local mutations will be needed to increase the mutation detection rate and optimize genetic counseling.

Identification of revertants for the cystic fibrosis ΔF508 mutation using STE6-CFTR chimeras in yeast

Mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis; the most common mutation is deletion of phenylalanine at position 508 (ΔF508). We constructed STE6-CFTR chimeras with portions of the first nucleotide-binding domain (NBD1) of the yeast STE6 a-factor transporter replaced by portions of CFTR NBD1. The chimeras were functional in yeast, but mating efficiency decreased when ΔF508 was introduced into NBD1. We isolated two ΔF508 revertant mutations (R553M and R553Q) that restored mating; both were located within the CFTR NBD1 sequence. Introduction of these revertant mutations into human CFTR partially corrected the processing and CI − channel gating defects caused by the ΔF508 mutation. These results suggest that the NBD1s of CFTR and STE6 share a similar structure and function and that, in CFTR, the regions containing F508 and R553 interact. They also indicate that the abnormal conformation produced by ΔF508 can be partially corrected by additional alterations in the protein.

Mutation analysis and haplotype correlation for 139 cystic fibrosis patients from the Nebraska Regional Cystic Fibrosis Center.

Cystic fibrosis (CF) is the most common autosomal recessive disorder in Caucasian populations with an approximate frequency of one in 2,500 live births and a carrier frequency of one in 25. We studied 400 individuals seen at The Nebraska Regional Cystic Fibrosis Center that included 139 CF patients, 206 parents, and 55 unaffected siblings to determine the frequency of the delta F508, R117H, G542X, S549R/N, G551D, R553X, R560T, and W1282X mutations. In addition, we determined haplotypes on each of these individual's chromosomes using four markers that included XV-2c, KM-19, pMP6d.9, and G2. Results from this study showed that the delta F508 mutation was present in 70% of CF chromosomes. Of the 139 CF patients 74 (53%) were homozygous for the delta F508 deletion, 47 (34%) were heterozygous for the delta F508 deletion and an unknown mutation, and 18 (13%) carried two unknown mutations. Four additional mutations were also found in our population and included G542X (6%), G551D (5%), R553X (4%), and R560T (1%). One patient was documented to be a compound heterozygote for G542X/G551D. A polymorphism, F508C, that has previously been reported in several families was also present in our study. The most common haplotype associated with the delta F508 deletion in our CF patients was the E haplotype (CF Consortium B) while other mutations were associated with a variety of haplotypes.

Immunoreactive trypsin and a comparison of two ΔF508 mutation analyses in newborn screening for cystic fibrosis: An anonymous pilot study in Denmark

A total of 1081 newborn screening cards were anonymously selected for cystic fibrosis (CF) screening by quantitation of immunoreactive trypsin (IRT, Delfia) and by ΔF508 mutational analyses using polymerase chain reaction followed by both gel retardation test and timeresolved fluorescence hybridization assay (Delfia). The IRT values showed a log normal distribution and was significantly higher in girls than boys and in 28 carriers compared with 1052 normals. In 12 newborns corresponding to 1.02% an IRT concentration greater than 70 μg/l was found. One of these was a ΔF508 homozygote with an IRT concentration of 380 μg/l. A comparison between the two types of ΔF508 mutational analyses gave identical results (1052 normals, 28 heterozygotes, and one homozygote), i.e., a carrier frequency of 1:38.6. However, the Delfia ΔF508 assay was easier to perform and the various controls makes the assay more reliable. In future newborn CF screening programs we therefore recommend Delfia IRT followed by Delfia ΔF508 analyses for IRT values greater than 70 μg/l.

Genetic determinants of airways' colonisation with Pseudomonas aeruginosa in cystic fibrosis

Exocrine pancreatic insufficiency and lung infection with Pseudomonas aeruginosa are major features of cystic fibrosis (CF). This monogenic disease is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. 267 children and adolescents with CF who were regularly seen at the same centre were assessed for an association of the CFTR mutation genotype with exocrine pancreatic function and the age of onset of chronic colonisation with P aeruginosa.The major mutation ΔF508 accounted for 74% of CF alleles; 33 further CFTR mutations had been detected on the CF chromosomes of the study population by June, 1992. With the exception of ΔF508/R347P compound heterozygotes, patients of the same mutation genotype were either pancreas insufficient (PI) or pancreas sufficient (PS). The age-specific colonisation rates with P aeruginosawere significantly lower in PS than in PI patients. The missense and splice site mutations that are "mild" CF alleles with respect to exocrine pancreatic function were also "low risk" alleles for the acquisition of P aeruginosa. On the other hand, the proportion of P aeruginosa-positive patients increased most rapidly in the PI ΔF508 compound heterozygotes who were carrying a termination mutation in the nucleotide binding fold-encoding exons. Pancreatic status and the risk of chronic airways' colonisation with P aeruginosa are predisposed by the CFTR mutation genotype and can be differentiated by the type and location of the mutations in the CFTR gene.

Wheezing in Infants with Cystic Fibrosis: Clinical Course, Pulmonary Function, and Survival Analysis

Wheezing is a common finding in infants with cystic fibrosis (CF). This study was undertaken to determine the prevalence of wheezing in infants with CF and to compare the clinical outcome of those who wheezed in infancy with that of those who did not. The study cohort included 229 CF patients born between 1965 and 1979 with CF diagnosed before 2 years of age. Fifty-seven (25%) had physician-documented wheezing during the first 2 years of life. Wheezing had resolved by the age of 2 years in 50% of the patients and by the age of 4 years in 75%. Although wheezing seemed to be linked to a family history of allergy and asthma, the frequency of the delta F508 mutation was similar to that of the non-wheezers. There was no significant difference in survival at the age of 13 years between the two groups. At the age of 7 years, patients who had wheezed had significantly lower forced expiratory flow rate at mid-expiratory phase (85 +/- 34% predicted) compared with those with no wheezing history (101 +/- 34% predicted). At the age of 13 years, forced expiratory volume in 1 second values was lower in the wheezing group (69 +/- 24% predicted vs 78 +/- 21% predicted), as was forced expiratory flow rate at mid-expiratory phase (56 +/- 33% predicted vs 69 +/- 30% predicted). In conclusion, although wheezing in infants with CF seems to have diminished with age, pulmonary function abnormalities were more evident at 7 and 13 years of age in the group that wheezed than in the group that did not.

Fluorescence-based, multiplex allele-specific PCR (MASPCR) detection of the ΔF508 deletion in the cystic fibrosis transmembrane conductance regulator (CFTR) gene

Cystic fibrosis (CF) is a common genetic disorder in Caucasians, and in some populations 70% of cases are associated with a 3 base pair (bp) deletion (ΔF508) in the CFTR gene. We have implemented a fluorescence-based, multiplex allele-specific polymerase chain reaction (MASPCR) assay for deletion of the ΔF508 mutation. Different allele-specific fluorescently-tagged primers are used in the PCR reaction to distinguish between normal and ΔF508 alleles. Fluorescent PCR products are then visualized in a single lane on an agarose gel following electrophoresis combined with real-time multicolour fluorescence detection. The approach simplifies diagnosis of the most common mutation in the CFTR gene, and holds promise for a multiplex allele-specific, fluorescence-tagged gene amplification strategy for detection of additional CF mutations which may result in more cost-effective testing without increasing the risk of missed or erroneous diagnoses.

A new mutation (1078delT) in exon 7 of the CFTR gene in a southern French adult with cystic fibrosis

Disparities in the spectrum of mutations within the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene are commonly observed in populations from different ethnical and/or geographical origins. The occurrence of CF in Brittany (western France) is one of the highest in populations from Caucasian origin (< 1/2000 in specific areas). The W846X2, 1078delT and G551D mutations, as well as the I1027T polymorphism in cis with the ΔF508 mutation (currently referred to as p.F508del) are particularly frequent in this area. We investigated the age of the respective variants in the region of interest.Several polymorphic markers surrounding the CFTR gene were genotyped. Allele frequencies as well as mutation rates and other parameters were used to calculate the respective age of the most recent common ancestors in the region of interest by a previously employed, simple likelihood-based method.Following haplotype reconstruction and simulation, the ages were estimated to be approximately 600, 1000, 1200 and 600 years, respectively (with a 95% confidence interval).These datings thus provide historical insights in the context of understanding population migrations. They also underline the usefulness of this method for estimating the age of rare mutations with a limited number of carriers.

Efficiency and accuracy of polymerase-chain-reaction assay for cystic fibrosis allele ΔF508 in single cell

Diagnosis of genetic disorders in the embryo before implantation, though possible by removal of one or two blastomeres at the eight-cell stage, is still experimental because the procedures of gene analysis of DNA from a single cell are not yet reliable enough for clinical application. We have evaluated the efficiency and accuracy of polymerase-chain-reaction (PCR) amplification of a single-copy gene, wild-type or cystic fibrosis ΔF508 allele, on single sperm cells from a donor known to be a heterozygous carrier of the ΔF508 mutation. DNA from single spermatozoa was decontaminated by restriction-enzyme treatment, then the region around the ΔF508 site was amplified by nested PCR. The distribution of the wild-type and mutant alleles (59 [55%] and 48 [45%, respectively]) in the 107 single spermatozoa did not differ from that expected (50% each). 1 sample did not provide an amplified signal. To check that the two alleles would be amplified with equal efficiency when they were both present within a cell, we did PCR for 51 two-sperm samples. Again the distribution did not deviate from that expected (17 [33%] both wild-type; 21 [41 %] one wild-type, one ΔF508; 13 [26%] both ΔF508 vs 25%; 50%; 25% expected). None of the 74 blanks in these experiments was contaminated. We conclude that our ΔF508 single-cell assay is efficient and accurate and can be used for analysis of blastomere DNA to diagnose cystic fibrosis before embryo implantation.