Cystic fibrosis in Greece: Typing with DNA probes and identification of the common molecular defect

Summary The relative frequency of the ΔF508 mutation in the Greek population is 54.1%; this is similar to that reported in other Southern European populations and contrasts with the considerably higher frequencies encountered in Northern Europe and North America. The low frequency is in agreement with the linkage disequilibrium already reported between cystic fibrosis and haplotype B in this country. In contrast to the common association of pancreatic insufficiency with the homozygous ΔF508 genotype, the present study revealed two homozygous children with no evidence of pancreatic failure.     

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.     

Spatial distribution of the DF508 mutation in cystic fibrosis: a review.

The recent finding of the most common mutation (DF508) in cystic fibrosis in white populations has led to the publication of numerous data regarding its distribution and frequency. A review of the available data shows that there is a gradient in both the incidence of cystic fibrosis and the frequency of the DF508 mutation in Europe, the highest values being found in western Europe. It is postulated that the DF508 mutation was present in a western European population before the arrival of the Indo-Europeans from the Middle East; the mutation then spread into these migrating populations.     

Gradient of distribution in Europe of the major CF mutation and of its associated haplotype

Summary In this collaborative European study, a total of 4871 cystic fibrosis (CF) chromosomes and 3539 normal chromosomes have been characterized for the haplotypes defined by the 2 extragenic polymorphic sequences revealed by XV2c and KM19. The association between one of these haplotypes (B haplotype) and the most frequent CF mutation, ΔF508, suggests for the latter a single origin and a subsequent diffusion according to a South East-North West gradient. The linkage disequilibrium data between CF and the B haplotype in different European populations are compatible with a relatively more recent appearance of the mutation in Northern Europe whereas in Southern Europe a longer history of the same mutation would have allowed time for recombination with other haplotypes. This model is also compatible with a selective advantage of carriers but does not account for (1) the excess of B haplotypes observed among both normal and non-ΔF508 CF chromosomes; (2) the correlation between the B haplotype and the severity of the phenotypic effect caused by CF mutations, as measured by pancreatic insufficiency and meconium ileus.     

XV-2c and KM.19 haplotype analysis in Chilean patients with cystic fibrosis and unknown CFTR gene mutations

Cystic fibrosis (CF) is caused by mutations in the CFTR gene. More than 1600 mutations have been described, with frequencies that differ worldwide according to the ethnic origin of patients. A small group of mutations are recurrent on several populations. It has been shown that they each tend occur on specific chromosome 7 haplotypes, supporting the notion of a single origin for them. Less than 50% of mutations in Chilean patients have been identified to date. To indirectly assess the possible presence of a predominant founder mutation in the remaining unknown alleles, we evaluated 2 polymorphic markers, XV-2c and KM.19, tightly linked to the CFTR locus. The study was done in Chilean CF patients with unknown or deltaF508 (DeltaF508) CFTR mutations and their haplotypes were compared to affected family-based controls. DeltaF508 showed marked linkage disequilibrium with XV-2c/KM.19 haplotype B, with 90% of alleles on that haplotype. There was no difference in haplotype distribution between unknown mutations and normal controls. These results support a European origin for DeltaF508 alleles in Chilean patients, and make unlikely the presence of a predominant founder mutation in the so-far unknown alleles.     

Low frequency of the deltaAF508 mutation of the CFTR gene in a highly admixed population in Bahia, Brazil

Cystic fibrosis (CF) is the most common autosomal recessive disease in the European (Caucasian) population, with an incidence of 1:2000 to 1:8000. The deltaF508 mutation (66%) is predominant among more than 1300 different mutations of the CFTR gene. The population of the state of Bahia, in northeastern Brazil, is highly admixed (mainly African and Portuguese descendants), and so far, no study has been carried out to assess the molecular basis of CF in this population. We determined the deltaF508 mutation frequency in 503 individuals from the general population of Salvador, the capital of the state of Bahia, and in 144 CF patients from several cities in Bahia. In the general population samples we found 4 individuals heterozygous for the deltaF508 mutation (allele frequency of 0.4%). This frequency was lower than that found in the state of Rio de Janeiro, in southeastern Brazil, and similar to that reported for the state of Paraná, in the far south. In the CF patients we found 9 heterozygous individuals and 8 homozygous individuals (allele frequency of 8.68%) for the deltaF508 mutation. This frequency is considerably lower than the average frequency of CF in the world population and in the Brazilian CF population of European ancestry (47%). These data could be explained by the intense admixture among the population in Bahia, and they suggest a heterogeneous molecular basis for CF in this area of Brazil.     

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.     

Frequency of the ΔF508 mutation and XV2c,KM19 haplotypes in cystic fibrosis families from The Netherlands: haplotypes without ΔF508 still in disequilibrium

Summary We have determined the frequency of the major cystic fibrosis (CF) three base pair deletion (ΔF508) mutation in 152 CF chromosomes from patients originating from the northern part of The Netherlands. In these patients, the deletion represents approximately 76% of CF mutations. Meconium ileus is strongly associated with homozygosity for the ΔF508 mutation. The XV2c,KM19 haplotypes on the CF chromosomes without the ΔF508 mutation are in disequilibrium with the population frequency, although showing an increased frequency of the 1 2 haplotype. The surplus of this haplotype is almost entirely made up by the pancreatic insufficient patients.     

Can a Place of Origin of the Main Cystic Fibrosis Mutations Be Identified?

The genetic background of the mutations that most often cause cystic fibrosis (CF) is different from that of non-CF chromosomes in populations of European origin. It is not known whether these haplotype backgrounds could be found at high frequencies in populations in which CF is, at present, not common; such populations would be candidates for the place of origin of CF mutations. An analysis of haplotypes of CF transmembrane conductance regulator, together with their variation in specific CF chromosomes, in a worldwide survey of normal chromosomes shows (1) a very low frequency or absence of the most common CF haplotypes in all populations analyzed and (2) a strong genetic variability and divergence, among various populations, of the chromosomes that carry disease-causing mutations. The depth of the gene genealogy associated with disease-causing mutations may be greater than that of the evolutionary process that gave rise to present-day human populations. The concept of "population of origin" lacks either spatial or temporal meaning for mutations that are likely to have been present in Europeans before the ethnogenesis of present populations; subsequent population processes may have erased the traces of their geographic origin.     

Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in allergic bronchopulmonary aspergillosis.

The etiology of allergic bronchopulmonary aspergillosis (ABPA) is not well understood. A clinical phenotype resembling the pulmonary disease seen in cystic fibrosis (CF) patients can occur in some individuals with ABPA. Reports of familial occurrence of ABPA and increased incidence in CF patients suggest a possible genetic basis for the disease. To test this possibility, the entire coding region of the cystic fibrosis transmembrane regulator (CFTR) gene was analyzed in 11 individuals who met strict criteria for the diagnosis of ABPA and had normal sweat electrolytes (< or = 40 mmol/liter). One patient carried two CF mutations (deltaF508/R347H), and five were found to carry one CF mutation (four deltaF508; one R117H). The frequency of the deltaF508 mutation in patients with ABPA was significantly higher than in 53 Caucasian patients with chronic bronchitis (P < .0003) and the general population (P < .003). These results suggest that CFTR plays an etiologic role in a subset of ABPA patients.     

Cystic fibrosis in the population of Reunion Island

The frequencies of the delta F 508 mutation and haplotypes linked to the cystic fibrosis (CF) gene and detected with DNA probes XV-2C and KM-19 have been studied in the population of Reunion Island, a French province located in the Indian Ocean. The deletion was present in 41.3% of CF chromosomes, whereas this proportion is about 70% in the French population. The delta F 508 mutation was associated with the haplotype B defined by the DNA markers XV-2C (allele 1) and KM-19 (allele 2) in 76.4% of CF chromosomes, while this proportion is over 90% in the French population. Founder effect, genetic drift and admixture can explain these differences.     

Global proteomic approach unmasks involvement of keratins 8 and 18 in the delivery of cystic fibrosis transmembrane conductance regulator (CFTR)/deltaF508-CFTR to the plasma membrane

Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF gene (cftr). Physiologically, CF is characterized by an abnormal chloride secretion in epithelia due to a dysfunction of a mutated cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is a cAMP-dependent chloride channel whose most frequent mutation, deltaF508, leads to an aberrantly folded protein which causes a dysfunction of the channel. However, a growing number of reports suggest that modifier genes and environmental factors are involved in the physiology of CF. To identify proteins whose expression depends on wild-type WT-CFTR or deltaF508-CFTR, we chose a global proteomic approach based on the use of two-dimensional gel electrophoresis (2-DE) and mass spectrometry. The experiments were carried out with HeLa cells stably transfected with WT-CFTR (pTCFWT) or deltaF508-CFTR (pTCFdeltaF508). These experiments unmasked keratin 8 (K8) and 18 (K18) which were differentially expressed in pTCFWT vs. pTCFdeltaF508. An immunoblot of K18 confirmed the 2-DE results. Intracellular localization experiments of WT-CFTR, deltaF508-CFTR, K8, and K18 suggest that the expression of these proteins are linked, and that the concentrations of K8 and K18 and/or their distribution may be involved in the traffic of WT-CFTR/deltaF508-CFTR. A functional assay for CFTR revealed that specifically lowering K18 expression or changing its distribution leads to the delivery of functional deltaF508-CFTR to the plasma membrane. This work suggests a novel function of K18 in CF.     

Estimating the age of alleles by use of intraallelic variability.

A method is presented for estimating the age of an allele by use of its frequency and the extent of variation among different copies. The method uses the joint distribution of the number of copies in a population sample and the coalescence times of the intraallelic gene genealogy conditioned on the number of copies. The linear birth-death process is used to approximate the dynamics of a rare allele in a finite population. A maximum-likelihood estimate of the age of the allele is obtained by Monte Carlo integration over the coalescence times. The method is applied to two alleles at the cystic fibrosis (CFTR) locus, deltaF508 and G542X, for which intraallelic variability at three intronic microsatellite loci has been examined. Our results indicate that G542X is somewhat older than deltaF508. Although absolute estimates depend on the mutation rates at the microsatellite loci, our results support the hypothesis that deltaF508 arose < 500 generations (approximately 10,000 years) ago.     

Cystic fibrosis mutations in Costa Rica

Using polymerase chain reaction amplification of DNA in dried blood spots and a nonisotopic reverse dot blot hybridization method, we performed molecular genetic analysis for 6 and for 16 of the most common mutations of the cystic fibrosis transmembrane conductance regulator gene (CFTR) in 24 unrelated Costa Rican individuals with cystic fibrosis (CF). While many countries and ethnic groups have been surveyed for CF mutations since the cloning of CFTR, Costa Rica has not heretofore been studied. Moreover, Costa Rica represents an especially intriguing population because of its mixed European-African-Amerindian origins and the existence of a detailed historical record of the founding Spanish families. Thus, such a study may reveal not only the population frequencies of various mutant alleles in this country, but also something about their geographic migrations and ethnic founder effects. The most common CF mutation in Caucasians, deltaF508, was found in only 11 (23%) of the CF chromosomes studied, while the G542X mutation, relatively rare in the general population but more common in southern Europe, was observed in 12 (25%). None of the other mutations tested was found in any of the subjects. We failed to detect the second mutant allele in 17 subjects and could not detect either allele in 4 subjects. The high prevalence of the G542X mutation in our cohort, which exceeds that of both the general Caucasian population and the American Hispanic population, reflects the strong genetic influence of the original Spanish founding families of Costa Rica. These results highlight important differences in Costa Rican CF genotypes as compared both to other North American and European populations and to American Hispanics, raising important implications about isolated founder effects and strategies for population screening in that country.     

Preliminary results on the frequency of the ΔF508 mutation in cystic fibrosis patients from the USSR

Summary The frequency of the ΔF508 mutation in a sample of 29 cystic fibrosis (CF) families from the USSR is equal to 44.8%, in agreement with previously reported haplotype data. If confirmed on a larger sample of CF patients from the USSR, this result might be indicative of the presence of a larger heterogeneity of CF mutations in the Soviet population than in other European populations.     

Linkage disequilibrium between the four most common cystic fibrosis mutations and microsatellite haplotypes in the Celtic population of Brittany

Microsatellite haplotypes were determined for 117 chromosomes carrying the four most frequent mutations in the cystic fibrosis (CF) gene identified in the Breton population of Celtic origin, as well as for 83 normal chromosomes (noncarriers of a CF mutation). Each of the three non-ΔF508 mutations was associated with a single haplotype: 1078delT with 16-31-13, G551D with 16-7-17, and W846X with 16-32-13. Although these results suggest identity-by-descent for each mutation, recurrent mutations, although unlikely, could not be completely ruled out. The four most frequent haplotypes on normal chromosomes and the three most frequent haplotypes on ΔF508 chromosomes are the same as those found in Ireland, Spain, and Italy. This suggests that some haplotypes, associated or not with the ΔF508 mutation, were present in an ancestral population from which all four populations descended. Received: 27 November 1995 / Revised: 1 February 1996     

The deletion F508 is the major gene mutation in a representative Belgian cystic fibrosis population

Summary The cystic fibrosis (CF) gene deletion F508 was studied in a Belgian population of 74 families and their 83 CF children. The haplotypes for CF and normal chromosomes had previously been determined with several linked DNA probes. In our CF population, the gene deletion F508 was found in 76% of the mutant alleles. Of the deletion F508, 97% segregated with the highest risk haplotype for the CF carrier status. Some 61% of our families were found to be homozygous for this major CF mutation. Each of our three pancreatic sufficiency patients (two of whom were siblings) was heterozygote for the F508 deletion.     

Recombinant synthesis, purification, and nucleotide binding characteristics of the first nucleotide binding domain of the cystic fibrosis gene product.

The majority of mutations which lead to clinical cystic fibrosis are located within the two predicted nucleotide binding domains of the cystic fibrosis gene product. We have used a prokaryotic expression system to synthesize and purify the first nucleotide binding domain (NBD-1, amino acids 426-588) with and without the most common mutation associated with the disease (the deletion of phenylalanine at position 508, delta F508). Both wild type and delta F508 NBD-1 bind ATP-agarose in a quantitatively comparable manner; this binding was inhibited by excess Na2ATP, trinitrophenol-ATP, or 8-azido-ATP. Irreversible NBD-1 labeling by an ATP analog was demonstrated using [32P]8-azido-ATP. This covalent labeling was inhibited by preincubation with Na2ATP, with half-maximal inhibition for Na2ATP occurring at approximately 5 mM for both the wild type and delta F508 nucleotide binding domain. These experiments are among the first to confirm the expectation that the cystic fibrosis transmembrane conductance regulator NBD-1 binds nucleotide. Since, under the conditions used in our study, NBD-1 without phenylalanine 508 displays very similar nucleotide binding characteristics to the wild type protein, our results support previous structural models which predict that the delta F508 mutation should not cause an alteration in ATP binding.     

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.