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.     

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.     

Ethnic heterogeneity and cystic fibrosis transmembrane regulator (CFTR) mutation frequencies in Chicago-area CF families.

The identification of a common mutation, delta F508, in the CFTR gene allowed, for the first time, the detection of cystic fibrosis (CF) carriers in the general population. Further genetic studies revealed > 100 additional disease-causing mutations in this gene, few of which occur on > 1% of CF chromosomes in any ethnic group. Prior to establishing counseling guidelines and carrier risk assessments, we sought to establish the frequencies of the CFTR mutations that are present in CF families living in the Chicago area, a region notable for its ethnic heterogeneity. Our sample included 283 unrelated CF carriers, with the following ethnic composition: 78% non-Ashkenazi Caucasians, 5% Ashkenazi, 9% African-American, 3% Mexican, 0.3% Native American, and 5% mixed ancestry. When a panel of 10 mutations (delta F508, delta I507, G542X, G551D, R553X, S549N, R1162X, W1282X, N1303K, and 1717-1G-->A) was used, detection rates ranged from 75% in non-Ashkenazi Caucasians to 40% in African-Americans. These data suggest that the goal of screening for 90%-95% of CF mutations may be unrealistic in this and other, similar U.S. populations.     

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.     

Molecular characterization of cystic fibrosis: 16 novel mutations identified by analysis of the whole cystic fibrosis conductance transmembrane regulator (CFTR) coding regions and splice site junctions.

The spectrum of cystic fibrosis (CF) mutations was determined in 105 patients by using denaturing gradient gel electrophoresis to screen the entire coding regions and adjacent cystic fibrosis transmembrane conductance regulator (CFTR) gene sequences. The nucleotide substitutions detected included 16 novel mutations, 11 previously described defects, and 11 nucleotide sequence polymorphisms. Among the novel mutations, 6 were of the missense type, 4 were nonsense mutations, 4 were frameshift defects, and 2 affected mRNA splicing. The mutations involved all the CFTR domains, including the R domain. Of the 61 non-delta F508 CF chromosomes studied, mutations were found on 36 (59%), raising the proportion of CF alleles characterized in our patient cohort to 88%. Given the efficacy of the screening method used, the remaining uncharacterized mutations probably lie in DNA sequences outside the regions studied, e.g., upstream-promoter sequences, the large introns, or putative regulatory regions. Our results further document the highly heterogeneous nature of CF mutations and provide the information required for DNA-based genetic testing.     

Identification of common cystic fibrosis mutations in African-Americans with cystic fibrosis increases the detection rate to 75%.

Cystic fibrosis (CF)--an autosomal recessive disorder caused by mutations in CF transmembrane conductance regulator (CFTR) and characterized by abnormal chloride conduction across epithelial membranes, leading to chronic lung and exocrine pancreatic disease--is less common in African-Americans than in Caucasians. No large-scale studies of mutation identification and screening in African-American CF patients have been reported, to date. In this study, the entire coding and flanking intronic sequence of the CFTR gene was analyzed by denaturing gradient-gel electrophoresis and sequencing in an index group of 82 African-American CF chromosomes to identify mutations. One novel mutation, 3120+1G-->A, occurred with a frequency of 12.3% and was also detected in a native African patient. To establish frequencies, an additional group of 66 African-American CF chromosomes were screened for mutations identified in two or more African-American patients. Screening for 16 "common Caucasian" mutations identified 52% of CF alleles in African-Americans, while screening for 8 "common African" mutations accounted for an additional 23%. The combined detection rate of 75% was comparable to the sensitivity of mutation analysis in Caucasian CF patients. These results indicate that African-Americans have their own set of "common" CF mutations that originate from the native African population. Inclusion of these "common" mutations substantially improves CF mutation detection rates in African-Americans.     

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.     

Molecular basis of cystic fibrosis in Lithuania: incomplete CFTR mutation detection by PCR-based screening protocols

Mutational analysis of the cystic fibrosis transmembrane regulator (CFTR) gene was performed in 98 unrelated CF chromosomes from 49 Lithuanian CF patients through a combined approach in which the p.F508del mutation was first screened by allele-specific PCR while CFTR mutations in nonp.F508del chromosomes have been screened for by denaturing gradient gel electrophoresis analysis. A CFTR mutation was characterized in 62.2% of CF chromosomes, two of which (2.0%) have been previously shown to carry a large gene deletion CFTRdele2,3(21 kb). The most frequent Lithuanian CF mutation is p.F508del (52.0%). Seven CFTR mutations, p.N1303K (2.0%), p.R75Q (1.0%), p.G314R (1.0%), p.R553X (4.2%), p.W1282X (1.0%), and g.3944delGT (1.0%), accounted for 10.1% of Lithuanian CF chromosomes. It was not possible to characterize 35.8% of the CF Lithuanian chromosomes. Analysis of intron 8 (TG)mTn and M470V polymorphic loci did not permit the characterization of the CFTR dysfunction underlying the CF phenotype in the patients for which no CFTR mutation was identified. Thus, screening of the eight CFTR mutations identified in this study and of the large deletion CFTRdele2,3(21 kb) allows the implementation of an early molecular or confirmatory CF diagnosis for 65% of Lithuanian CF chromosomes.     

CFTR: folding, misfolding and correcting the ΔF508 conformational defect

Cystic fibrosis (CF), the most common lethal genetic disease in the Caucasian population, is caused by loss-of-function mutations of the CF transmembrane conductance regulator (CFTR), a cyclic AMP-regulated plasma membrane chloride channel. The most common mutation, deletion of phenylalanine 508 (ΔF508), impairs CFTR folding and, consequently, its biosynthetic and endocytic processing as well as chloride channel function. Pharmacological treatments may target the ΔF508 CFTR structural defect directly by binding to the mutant protein and/or indirectly by altering cellular protein homeostasis (proteostasis) to promote ΔF508 CFTR plasma membrane targeting and stability. This review discusses recent basic research aimed at elucidating the structural and trafficking defects of ΔF508 CFTR, a prerequisite for the rational design of CF therapy to correct the loss-of-function phenotype.     

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.     

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.     

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.     

Cystic fibrosis in Lebanon: distribution of CFTR mutations among Arab communities

Cystic fibrosis (CF) is thought to be rare among the Arab populations from the Middle East and little data have been reported so far. We have studied a sample of 20 families living in Lebanon for several generations and who have at least one child with CF. These families are mainly from the Maronite, Greek Catholic, Greek Orthodox, Shiite or Sunnite groups. We found a 50% rate of consanguineous marriage, independent of the community of origin. The distribution of CF genotypes was determined through the screening of all exons of the CFTR (cystic fibrosis transmembrane conductance regulator) gene by the technique of denaturing gradient gel electrophoresis combined with asymmetric amplification DNA sequencing. A total of ten different mutations accounting for 87.5% of 32 unrelated CF alleles was identified, including two novel putative mutations (E672del and IVS21-28G→A). Three mutations, ΔF508 (37.5%), W1282X (15.6%), and N1303K (9.4%) accounted for 62.5% of CF alleles. Interestingly, in the Maronite group, 66.7% of the ΔF508 chromosomes were found to be associated with allele 7 of the IVS8(T)tract, contrasting with the absolute linkage disequilibrium between European ΔF508 chromosomes and allele 9. During this study, two previously undescribed polymorphisms (IVS14a + 17del5 and 2691T/C) were also identified. Received: 2 January 1997 / Accepted: 16 March 1997     

XV-2c/KM19 haplotypes analysis of cystic fibrosis patients from western Mexico

The analysis of polymorphic markers within or closely linked to the cystic fibrosis transmembrane regulator (CFTR) gene is useful as a molecular tool for carrier detection of known and unknown mutations. To establish the association between mutations in the CFTR gene in western Mexican cystic fibrosis (CF) patients, the distribution of XV2c/KM19 haplotypes was analyzed by PCR and restriction enzyme digestion in 384 chromosomes from 74 CF patients, their unaffected parents, and normal subjects. The haplotype analysis revealed that haplotype B was present in 71.9% of CF chromosomes compared to 0% of non-CF chromosomes. The F508del and G542X mutations were strongly associated with haplotype B (96.7% and 100% of chromosomes, respectively). The haplotype distribution of the CF chromosomes carrying other CFTR mutations had a more heterogeneous background. Our results show that haplotype B is associated with CFTR mutations. Therefore, haplotype analysis is a suitable alternate strategy for screening CF patients with a heterogeneous clinical picture from populations with a high molecular heterogeneity where carrier detection programs are not available. In addition, it may be a helpful diagnostic tool for genetic counseling and carrier detection in the relatives of CF patients and in couples who are planning to have children.     

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     

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.     

Reduced number of CFTR molecules in erythrocyte plasma membrane of cystic fibrosis patients

Cystic fibrosis (CF), the most common genetic disease among Caucasians, is caused by mutations in the gene encoding CFTR (cystic fibrosis transmembrane conductance regulator). The most frequent mutation, ΔF508, results in protein misfolding and, as a consequence, prevents CFTR from reaching its final location at the cell surface. CFTR is expressed in various cell types including red blood cells. The functional role of CFTR in erythrocytes is still unclear. Since the number of CFTR copies in a single erythrocyte of healthy donors and CF patients with a homozygous ΔF508 mutation is unknown, we counted CFTR, localized in erythrocyte plasma membrane, at the single molecule level. A novel experimental approach combining atomic force microscopy with quantum-dot-labeled anti-CFTR antibodies, used as topographic surface markers, was employed to detect individual CFTR molecules. Analysis of erythrocyte plasma membranes taken from healthy donors and CF patients with a homozygous ΔF508 mutation reveals mean (SEM) values of 698 (12.8) (n=542) and 172 (3.8) (n=538) CFTR molecules per red blood cell, respectively. We conclude that erythrocytes reflect the CFTR status of the organism and that quantification of CFTR in a blood sample could be useful in the diagnosis of CFTR related diseases.     

Genetic determination of exocrine pancreatic function in cystic fibrosis.

We showed elsewhere that the pancreatic function status of cystic fibrosis (CF) patients could be correlated to mutations in the CF transmembrane conductance regulator (CFTR) gene. Although the majority of CF mutations--including the most common, delta F508--strongly correlated with pancreatic insufficiency (PI), approximately 10% of the mutant alleles may confer pancreatic sufficiency (PS). To extend this observation, genomic DNA of 538 CF patients with well-documented pancreatic function status were analyzed for a series of known mutations in their CFTR genes. Only 20 of the 25 mutations tested were found in this population. They accounted for 84% of the CF chromosomes, with delta F508 being the most frequent (71%), and the other mutations accounted for less than 5% each. A total of 30 different, complete genotypes could be determined in 394 (73%) of the patients. The data showed that each genotype was associated only with PI or only with PS, but not with both. This result is thus consistent with the hypothesis that PI and PS in CF are predisposed by the genotype at the CFTR locus; the PS phenotype occurs in patients who have one or two mild CFTR mutations, such as R117H, R334W, R347P, A455E, and P574H, whereas the PI phenotype occurs in patients with two severe alleles, such as delta F508, delta I507, Q493X, G542X, R553X, W1282X, 621 + 1G----T, 1717-1G----A, 556delA, 3659delC, I148T, G480C, V520F, G551D, and R560T.     

Cystic fibrosis mutations in the Hutterite Brethren.

The presence or absence of the major cystic fibrosis (CF) mutation, delta F508, in the general patient population was determined by Kerem et al. using allele-specific oligonucleotides for the mutant and normal sequences in the polymerase chain reaction (PCR). delta F508 was identified by Riordan et al., and it is a 3-bp deletion of the phenylalanine codon at position 508. The Hutterite Brethren are an inbred North American population who have three different DNA marker haplotypes of CF chromosomes. Genomic DNA from both a CF child and one parent from each of 10 Hutterite families was analyzed for the presence or absence of the deletion mutation. delta F508 is associated with one of the three CF haplotypes in the Hutterite population, and this is the most common haplotype in a subset of the linkage family data of Kerem et al. The other two Hutterite CF haplotypes are generally rate in Caucasian populations. Since these two CF haplotypes do not carry the deletion mutation, they must carry a different CF mutation(s). The results of the PCR analysis for the deletion mutation lend additional support to our previous conclusion that there were at least three original carriers of CF mutations among the founders of the Hutterite population and that all copies of the same CF haplotype were identical by descent. One Hutterite CF patient has both of the haplotypes which do not carry delta F508. Analysis of this individual's DNA should allow identification of two additional CF mutations in this population.     

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.