Three point mutations in the CFTR gene in French cystic fibrosis patients: Identification by denaturing gradient gel electrophoresis

Summary The cystic fibrosis (CF) gene was recently identified as a gene spanning 250 kilobases (kbp) and coding for a 1480 amino acid protein, cystic fibrosis transmembrane conductance regulator (CFTR). Approximately 70% of CF mutations involve a three-base-pair deletion in CFTR exon 10, resulting in the loss of a phenylalanine at position 508 in the gene product (ΔF508). In order to screen for other molecular defects, we have used a strategy based on denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified gene segments. This method, which permits rapid detection of any sequence change in a given DNA stretch, was used successfully to analyse 61 non-ΔF508 CF chromosomes from French CF patients. A study of CFTR exons 10, 11, 14a, 15 and 20 detected three mutations located in exons 14a, 15 and 20, along with several nucleotide sequence polymorphisms. These nucleotide changes were identified by direct sequencing of PCR fragments displaying altered electrophoretic behaviour, together with some of the polymorphisms and mutations previously characterized by others. The strategy presented here constitutes a valuable tool for the development of carrier testing for individuals or couples with a family history of cystic fibrosis, and will contribute to deciphering the functionally important regions of the CFTR gene.     

Analysis of four diverse population groups indicates that a subset of cystic fibrosis mutations occur in common among Caucasians.

To determine the nature and frequency of non-delta F508 cystic fibrosis (CF) mutations among diverse populations, we have sequenced exons 9-12 and 19-23 of the CF transmembrane conductance regulator (CFTR) gene from 128 CF chromosomes (39 U.S. Caucasian, 27 African-American, 42 Northern Irish, and 20 Israeli chromosomes). These regions were chosen because they encode the two putative ATP-binding folds of CFTR, domains which appear to have functional significance. In addition, CFTR exons 1 and 2 were analyzed in the American patients. Mutations were found on 49 of the 128 CF chromosomes. Nineteen different mutations were observed; six were novel, while the remaining 13 had been reported previously by our group or by other investigators. Six of nine different mutations found in African-American patients were unique to that population. However, the vast majority of the mutations found in U.S. Caucasians (eight of nine), Northern Irish (four of five), and Israelis (three of three) also occurred in other Caucasian groups. The preponderance of previously reported mutations in these three groups suggested that a subset of the non-delta F508 mutations occur in common among Caucasians. A survey of mutation frequencies in other Caucasian groups confirmed this observation. Unfortunately, this subset accounts for less than half of non-delta F508 CF mutations in most groups. These data suggest that screening for delta F508 and this select group of mutations will efficiently and economically maximize the number of CF mutations identified in Caucasian groups. However, it will be difficult to detect more than 90% of mutant CFTR alleles except in ethnically and geographically discrete populations where CF is the result of founder effect.     

Identification of the M1101K mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and complete detection of cystic fibrosis mutations in the Hutterite population.

The Hutterite population is a genetic isolate with an increased incidence of cystic fibrosis (CF). Previously we identified three CF haplotypes defined by polymorphisms flanking the CF transmembrane conductance regulator (CFTR) gene. delta F508 was present on one of the haplotypes in only 35% of CF chromosomes. We hypothesized that the other two CF haplotypes, one of which was the most common and the other of which is rare, each harbored different non-delta F508 mutations. Single-strand conformation polymorphism analysis detected a missense mutation, M1101K, in both chromosomes of a Hutterite patient carrying the two non-delta F508 haplotypes. M1101K appears to have originated on an uncommon CFTR allele and to be infrequent outside the Hutterite population. The presence of M1101K on two haplotypes is likely the result of a CFTR intragenic recombination which occurred since the founding, 10-12 generations ago, of the Hutterite population. The crossover was located between exons 14a and 17b, an interval of approximately 15 kbp. delta F508 and M1101K accounted for all of the CF mutations in patients from 16 CF families representing the three subdivisions of the Hutterite population.     

Development, multiplexing, and application of ARMS tests for common mutations in the CFTR gene.

The amplification refractory mutation system (ARMS) is a simple, rapid and reliable method for the detection of any mutation involving single base changes or small deletions. We have applied ARMS methodology to the detection of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Single ARMS tests have been developed for 11 CFTR mutations found in the northwest of England. ARMS reactions for the most common mutations have been multiplexed to give a test which will detect the presence of the delta F508, G551D, G542X, and 621 + 1G----T mutations in a DNA sample. The multiplex test has been validated by the analysis of over 500 previously genotyped samples and has been found to be completely accurate. The rapid detection of the most common mutations has enabled early molecular confirmation of suspected cystic fibrosis in neonates, rapid typing of cystic fibrosis patients and their relatives, and testing of sperm and egg donors.     

Association of a nonsense mutation (W1282X), the most common mutation in the Ashkenazi Jewish cystic fibrosis patients in Israel, with presentation of severe disease

Only about 30% of the cystic fibrosis chromosomes in the Israeli cystic fibrosis patient populations carry the major CF mutation (delta F508). Since different Jewish ethnic groups tended to live as closed isolates until recent times, high frequencies of specific mutations are expected among the remainder cystic fibrosis chromosomes of these ethnic groups. Genetic factors appear to influence the severity of the disease. It is therefore expected that different mutations will be associated with either severe or mild phenotype. Direct genomic sequencing of exons included in the two nucleotide-binding folds of the putative CFTR protein was performed on 119 Israeli cystic fibrosis patients from 97 families. One sequence alteration which is expected to create a termination at residue 1282 (W1282X) was found in 63 chromosomes. Of 95 chromosomes, 57 (60%) are of Ashkenazi origin. Together with the delta F508 (23% in this group), G542X, N1303K, and 1717-1G----A mutations, the identification of 92% of cystic fibrosis chromosomes of Ashkenazi origin becomes possible. Patients homozygous for the W1282X mutation (n = 16) and patients heterozygous for the delta F508 and W1282X mutations (n = 22) had similarly severe disease, reflected by pancreatic insufficiency, high incidence of meconium ileus (37% and 27%, respectively), early age at diagnosis, poor nutritional status, and variable pulmonary function. In conclusion, the W1282X mutation is the most common cystic fibrosis mutation in the Ashkenazi Jewish patient population in Israel. This nonsense mutation is associated with presentation of severe disease.     

Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis.

The gene associated with cystic fibrosis (CF) encodes a membrane-associated, N-linked glycoprotein called CFTR. Mutations were introduced into CFTR at residues known to be altered in CF chromosomes and in residues believed to play a role in its function. Examination of the various mutant proteins in COS-7 cells indicated that mature, fully glycosylated CFTR was absent from cells containing delta F508, delta 1507, K464M, F508R, and S5491 cDNA plasmids. Instead, an incompletely glycosylated version of the protein was detected. We propose that the mutant versions of CFTR are recognized as abnormal and remain incompletely processed in the endoplasmic reticulum where they are subsequently degraded. Since mutations with this phenotype represent at least 70% of known CF chromosomes, we argue that the molecular basis of most cystic fibrosis is the absence of mature CFTR at the correct cellular location.     

Screening for non-delta F508 mutations in five exons of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in Italy.

Analysis of exons 10, 11, 14a, 15, and 20 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by denaturing-gradient-gel electrophoresis (DGGE) allowed the identification of mutations causing cystic fibrosis (CF) in 25 of 109 non-delta F508 chromosomes, as well as identification of a number of polymorphisms and sequence variations. Direct sequencing of the PCR fragments which showed an altered electrophoretic behavior not attributable to known mutations has led to the characterization of four new mutations, two in exon 11, and one each in exons 15 and 20. Screening for the different mutations thus far identified in our patients by the DGGE analysis and other independent methods should allow detection of about 70% of the molecular defects causing CF in Italy. Mutations located in exons 11 and 20 account for at least 30% of the non-delta F508 mutations present in Italian CF patients.     

Immunocytochemical analysis reveals differences between the subcellular localization of normal and delta Phe508 recombinant cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis (CF) is caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). The most common mutation responsible for CF is the deletion of amino acid residue Phe508, with an average allelic frequency of 70%. We have isolated an anti-CFTR monoclonal antibody which specifically recognizes recombinant normal and delta Phe508-CFTR produced by a vaccinia virus expression system. Immunocytochemical analysis of L cells expressing either normal or delta Phe508-CFTR showed a marked difference in subcellular distribution. Normal CFTR had a distinct localization in the perinuclear area and was also associated with the plasma membrane. delta Phe508-CFTR essentially lacked the membrane-associated distribution and was present throughout the cytoplasm. This heterologous expression system thus provides a model system for studying the subcellular localization of different mutant forms of CFTR.     

Frequency of the ΔF508 mutation and flanking marker haplotypes at the CF locus from 167 Czech families

Summary This study analyses distribution patterns of the ΔF508 mutation of the cystic fibrosis transmembrane conductance regulator gene (CFTR) gene and the cystic fibrosis (CF)-linked marker loci MET, D7S23, D7S399, and D7S8 in a sample of 167 (116 complete) CF families from Bohemia and Moravia (Czechoslovakia). DNA typing was performed by polymerase chain reaction amplification, restriction analysis, and agarose or polyacrylamide gel electrophoresis. The frequency of the ΔF508 mutation in this sample is 67% and the frequency of the B haplotype is 77.6% on CF chromosomes. Linkage disequilibrium was found between ΔF508 and all markers tested.     

A novel donor splice site in intron 11 of the CFTR gene, created by mutation 1811+1.6kbA-->G, produces a new exon: high frequency in Spanish cystic fibrosis chromosomes and association with severe phenotype.

mRNA analysis of the cystic fibrosis transmembrane regulator (CFTR) gene in tissues of cystic fibrosis (CF) patients has allowed us to detect a cryptic exon. The new exon involves 49 base pairs between exons 11 and 12 and is due to a point mutation (1811+1.6kbA-->G) that creates a new donor splice site in intron 11. Semiquantitative mRNA analysis showed that 1811+1.6kbA-->G-mRNA was 5-10-fold less abundant than delta F508 mRNA. Mutation 1811+1.6kbA-->G was found in 21 Spanish and 1 German CF chromosomes, making it the fourth-most-frequent mutation (2%) in the Spanish population. Individuals with genotype delta F508/1811+1.6kbA-->G have only 1%-3% of normal CFTR mRNA. This loss of 97% of normal CFTR mRNA must be responsible for the pancreatic insufficiency and for the severe CF phenotype in these patients.     

Frequency of the F508 deletion in the CFTR gene in Turkish cystic fibrosis patients

Summary The F508 deletion in the cystic fibrosis transmembrane conductance regulator (CFTR) gene was found in 8 out of 30 Turkish cystic fibrosis (CF) chromosomes (27%). Five Turkish ΔF508 CF chromosomes were associated with the risk haplotype B in KM19 (2 allele)/XV2c (1 allele). In the Turkish population, cystic fibrosis is predominantly caused by mutations other than the F508 deletion.     

Analysis of the spectra of mutations and polymorphic loci of cystic fibrosis transmembrane conductance regulator in the population of Bashkortostan

The spectra of mutations and polymorphic loci of the gene of cystic fibrosis transmembrane conductance regulator (CFTR) was studied in 60 cystic fibrosis (CF) families from Bashkortostan. Mutations delF508, 394delTT, CFTRdele2,3(21 kb), R334W, and S1196X (33.3, 3.3, 1.7, 0.8, and 0.8%, respectively) were identified. The frequencies of tandem tetranucleotide repeat (TTR) alleles were determined for locus IVS6a-GATT of intron 6 of the CFTR gene and two extragenic loci flanking the CFTR gene, D7S23 and MET (probes CS.7 and MetH) in mutant and normal chromosomes. Allelic and haplotypic associations of these loci with the mutations found were estimated. An absolute linkage between the 6TTR allele of locus IVS6a-GATT and the delF508 mutation was ascertained. A considerable linkage disequilibrium between the delF508 mutation and the C2 allele of locus D7S23 and between this mutation and the A1 allele of locus MET was found. Most of the other mutant chromosomes carried marker alleles 7TTR, C1, and A2. It was demonstrated that 67% of CF chromosomes carrying delF508 had haplotype 6-2-1 for loci IVS6a-GATT/D7S23/MET, respectively. The frequency distribution of haplotypes in CF chromosomes without delF508 had a high variance and did not differ significantly from the distribution in normal chromosomes (chi 2 = 9.415; p > 0.05).     

A molecular genetic analysis of the mutations in the exons of the CFTR gene in cystic fibrosis patients in Ukraine

The results of DNA analysis of major mutations in CFTR gene in 260 families from Ukraine with high risk of cystic fibrosis are presented. The perspectives of molecular diagnosis as a main tool for cystic fibrosis prevention are discussed.     

Geographic distribution and origin of CFTR mutations in Germany

The geographic distribution and origin of CFTR mutations in Germany was evaluated in 658 three-generation families with cystic fibrosis (CF). Fifty different mutations were detected on 1305 parental CF chromosomes from 22 European countries and overseas. The major mutation ΔF508 was identified on 71.5% of all CF chromosomes, followed by R553X (1.8%), N1303K (1.3%), G542X (1.1%), G551D (0.8%) and R347P (0.8%). According to the grandparents’ birthplace, 74% of CF chromosomes had their origin in Germany; the ΔF508 percentage was 77%, 75%, 70% and 62% in northern, southern, western and eastern Germany, respectively. Ten or more mutant alleles in the investigated CF gene pool originated from Austria, the Czech Republic, Poland, Russia, Turkey and the Ukraine. This widespread geographic origin of CFTR mutations in today’s Germany reflects the many demographic changes and migrations in Central Europe during the 20th century. Received: 10 October 1995 / Revised: 9 January 1995     

Is congenital bilateral absence of vas deferens a primary form of cystic fibrosis? Analyses of the CFTR gene in 67 patients.

Congenital bilateral absence of the vas deferens (CBAVD) is an important cause of sterility in men. Although the genetic basis of this condition is still unclear, it has been shown recently that some of these patients carry mutations in their cystic fibrosis transmembrane conductance regulator (CFTR) genes. To extend this observation, we have analyzed the entire coding sequence of the CFTR gene in a cohort of 67 men with CBAVD, who are otherwise healthy. We have identified four novel missense mutations (A800G, G149R, R258G, and E193K). We have shown that 42% of subjects were carriers of one CFTR allele and that 24% are compound heterozygous for CFTR alleles. Thus, we have been unable to identify 76% of these patients as carrying two CFTR mutations. Furthermore, we have described the segregation of CFTR haplotypes in the family of one CBAVD male; in this family are two male siblings, with identical CFTR loci but displaying different phenotypes, one of them being fertile and the other sterile. The data presented in this family, indicating a discordance between the CBAVD phenotype and a marked carrier (delta F508) chromosome, support the involvement of another gene(s), in the etiology of CBAVD.     

Benign missense variations in the cystic fibrosis gene.

The common mutation causing cystic fibrosis is a deletion of phenylalanine 508 (delta F508), which occurs in a putative nucleotide-binding fold of the gene product. We report two additional mutations, substitution of cysteine for phenylalanine 508 (F508C) and substitution of valine for isoleucine 506 (I506V). Three compound heterozygous persons, two delta F508/F508C and one delta F508/I506V, had normal clinical and epithelial physiological studies indicating that the F508C and I506V mutations are benign. This opportunity to study the in vivo function of these mutations suggests that amino acid substitutions are more benign than changes in the length of this portion of the putative nucleotide-binding fold. These mutations must be taken into account when performing molecular diagnosis and carrier detection for cystic fibrosis.     

A mouse model for the cystic fibrosis delta F508 mutation.

Most cystic fibrosis (CF) patients produce a mutant form (delta F508) of the cystic fibrosis transmembrane conductance regulator (CFTR), which is not properly processed in normal cells but is active as a chloride channel in several experimental systems. We used a double homologous recombination ('Hit and Run') procedure to generate a mouse model for the delta F508 mutation. Targeted embryonic stem (ES) cells (Hit clones) were found; of these either 80 or 20% of the clones had lost the delta F508 mutation, depending on the distance between the linearization site in the targeting construct and the delta F508 mutation. Correctly targeted clones underwent a second selection step resulting in ES cell clones (Run clones) heterozygous for the delta F508 mutation with an efficiency of 2-7%. Chimeric mice were generated and offspring homozygous for the delta F508 mutation showed electrophysiological abnormalities in nasal epithelium, gallbladder and in the intestine, and histological abnormalities in the intestine, typical of CF. Our data suggest that the delta F508 mice have residual delta F508 CFTR activity which would explain the mild pathology of the delta F508 mice. The delta F508 mouse may provide a useful model for the study of the processing defect of delta F508 CFTR and for the development of novel therapeutic approaches based on circumvention of the processing block.     

Functional genomic responses to cystic fibrosis transmembrane conductance regulator (CFTR) and CFTR(delta508) in the lung

Cystic fibrosis (CF), a common lethal pulmonary disorder in Caucasians, is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) that disturbs fluid homeostasis and host defense in target organs. The effects of CFTR and delta508-CFTR were assessed in transgenic mice that 1) lack CFTR expression (Cftr-/-); 2) express the human delta508 CFTR (CFTR(delta508)); 3) overexpress the normal human CFTR (CFTR(tg)) in respiratory epithelial cells. Genes were selected from Affymetrix Murine Gene-Chips analysis and subjected to functional classification, k-means clustering, promoter cis-elements/modules searching, literature mining, and pathway exploring. Genomic responses to Cftr-/- were not corrected by expression of CFTR(delta508). Genes regulating host defense, inflammation, fluid and electrolyte transport were similarly altered in Cftr-/- and CFTR(delta508) mice. CFTR(delta508) induced a primary disturbance in expression of genes regulating redox and antioxidant systems. Genomic responses to CFTR(tg) were modest and were not associated with lung pathology. CFTR(tg) and CFTR(delta508) induced genes encoding heat shock proteins and other chaperones but did not activate the endoplasmic reticulum-associated degradation pathway. RNAs encoding proteins that directly interact with CFTR were identified in each of the CFTR mouse models, supporting the hypothesis that CFTR functions within a multiprotein complex whose members interact at the level of protein-protein interactions and gene expression. Promoters of genes influenced by CFTR shared common regulatory elements, suggesting that their co-expression may be mediated by shared regulatory mechanisms. Genes and pathways involved in the response to CFTR may be of interest as modifiers of CF.     

Pitfalls in the immunohistochemical localization of the cystic fibrosis transmembrane conductance regulator in paraffin embedded sweat glands

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause the hereditary disease cystic fibrosis. The most frequent mutant F508 has been shown in vitro to be retained in the endoplasmic reticulum. Ex vivo studies using immunohistochemical labelling in cryofixed skin biopsies have confirmed the mislocalization of F508 CFTR in sweat glands. The purpose of this study was to test CFTR antibodies in paraffin-embedded skin biopsies to take advantage of the superior tissue preservation as compared to cryofixation. A panel of 7 CFTR antibodies was applied to skin sections of healthy controls and of cystic fibrosis patients homozygous for the F508 mutation. Sweat gland labelling consistent with CFTR localization and different between control and cystic fibrosis tissue was obtained with 2 antibodies. Conventional staining controls confirmed the labelling specificity. The antibodies were subsequently tested in a series of 237 sections of 16 biopsy specimens. However, the sweat gland labelling pattern proved not to be dependent on CFTR genotype. This finding was the sole indicator of non-specificity of the staining which was revealed only by the size of our random sample. Our results emphasize that CFTR immunolabelling following formalin fixation has to be interpreted with the utmost caution.     

Increased frequency of CFTR gene mutations identified in Indian infertile men with non-CBAVD obstructive azoospermia and spermatogenic failure

High incidence of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene is associated with congenital bilateral absence of the vas deferens (CBAVD) and is considered as the genital form of cystic fibrosis (CF). The CFTR gene may also be involved in the etiology of male infertility in cases other than CBAVD. The present study was conducted to identify the spectrum and frequency of CFTR gene mutations in infertile Indian males with non-CBAVD obstructive azoospermia (n = 60) and spermatogenic failure (n = 150). Conspicuously higher frequency of heterozygote F508del mutation was detected in infertile males with non-CBAVD obstructive azoospermia (11.6%) and spermatogenic failure (7.3%). Homozygous IVS(8)-5T allele frequency was also significantly higher in both groups in comparison to those in normal healthy individuals. Two mutations in exon 25 viz., R1358I and K1351R were identified as novel mutations in patients with non-CBAVD obstructive azoospermia. Mutation R1358I was predicted as probably damaging CFTR mutation. This is the first report from the Indian population, emphasizing increased frequency of CFTR gene mutations in male infertility other than CBAVD. Thus, it is suggested that screening of CFTR gene mutations may be required in infertile Indian males with other forms of infertility apart from CBAVD and willing for assisted reproduction technology.