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.     

Contribution of molecular biology to the prevention of cystic fibrosis. Experience in Lyon

Enzymatic prenatal diagnosis of cystic fibrosis was performed in 113 amniotic fluids and DNA polymorphism was studied in 104 families, including 28 cases with prenatal material analysis. According to the results, the enzymatic diagnosis should be cautiously interpreted when the risk is less than 1/4. In these situations DNA analysis in the parents is very helpful to assess the reliability of enzymatic diagnosis.     

Determination of haplotypes of DNA markers linked to the mucoviscidosis locus: detection of heterozygotes

Linked DNA probes have been used in three families presenting an affected child with cystic fibrosis. The strategy used for the determination of haplotypes associated with parental normal and mutated genes is presented as well as its application to the detection of cystic fibrosis carriers among healthy children.     

Linkage of DNA markers to cystic fibrosis in 26 families.

Two DNA markers, the met oncogene and the anonymous probe, pJ3.11, previously reported to be tightly linked to cystic fibrosis (CF), were used for linkage analysis in 26 families with two or more individuals affected with CF. A new high frequency polymorphism was identified using BanI and the pmetD probe. The results of linkage analysis were as follows: between met and CF, lod score of 18.2 at theta of .009; between pJ3.11 and CF, lod score of 12.1 at theta of 0; and between met and pJ3.11, lod score of 16.7 at theta of 0. These data indicate that most or all of CF is due to an abnormality at a single locus and that the DNA markers are useful for prenatal diagnosis and heterozygote detection within affected families.     

Homogeneity of cystic fibrosis in Italy.

In 12 unrelated Italian cystic fibrosis (CF) families the frequencies of four DNA polymorphisms closely linked to the CF gene on chromosome 7 were quite similar to those reported for other population samples. Among the 23 affected children from the 12 families, only one recombinant occurred between the CF gene and the met locus, thus confirming the hypothesis of genetic homogeneity of CF previously suggested by the analysis of consanguineous marriages among 624 couples of CF parents. Chi-square test of association indicates a possible linkage disequilibrium between the CF gene and the DNA polymorphism that is most informative in our sample (pmetH TaqI).     

The frequency of the CF ΔF508 deletion in CF chromosomes of different ethnic origin

Summary The identification of the molecular defect in a significant proportion of cystic fibrosis families (in our series up to 60%) allows direct DNA diagnosis.     

Approaches to localizing disease genes as applied to cystic fibrosis.

Using chromosome jumping and walking and restriction fragment length polymorphism (RFLP) analysis, we have defined the region which must contain the cystic fibrosis gene. DNA segments spanning approximately 250 kb in the direction of the gene were isolated and used to identify several new polymorphisms informative in cystic fibrosis families. These RFLPs include a highly polymorphic, CA/GT repeat, and a 10 bp insertion uncovered using the polymerase chain reaction. By analyzing a family with a recombination near the gene, we can exclude this region as containing the mutation. Data on the extent of linkage disequilibrium of these markers provides additional information on where the gene is located.     

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.     

Analysis of the complete coding region of the CFTR gene in ten Algerian cystic fibrosis families

The spectrum of cystic fibrosis (CF) mutations in the North African population remains poorly known. In order to offer an effective diagnostic service and to determine accurate risk estimates, we decided to identify the CF mutations in 10 Algerian CF families. We carried out a chemical-clamp denaturing gradient gel electrophoresis analysis of the CFTR gene and automated direct DNA sequencing. We identified 5 mutations and we characterized 60% of the CF chromosomes. Taking advantage of the homogeneity of the sample, we report clinical features of homozygous CF patients.     

Allele polymorphism of the DNA loci MET, D7S8, D7S23, linked to the cystic fibrosis gene in some populations of the USSR, in high risk families and in cystic fibrosis patients

RELP analysis of DNA loci MET, D7S8 and D7S23 was carried out in Leningrad population and partially in populations of Moscow, Azerbaijan, Ukraine, Buryatia as well as in individuals from high risk families and in cystic fibrosis (CF) patients by means of blot hybridization and polymerase chain reaction. Allelic polymorphism of all loci studied in these three groups was found to be quite similar to that in the North-Western Europe and in whites of the North America. Linkage disequilibrium of the alleles studied with the CF gene was especially pronounced for alleles of the D7S23 locus and gradually decreases from KM-19 through CS-7 to XV-2c DNA probes. The data witness genetic homogeneity of the CF mutation in European populations of the USSR and its similarity to this mutation in Western Europe. The significance of these data for potential diagnosis of CF and for heterozygous carrier detection is discussed.     

CFTR Haplotype Analysis Reveals Genetic Heterogeneity in the Etiology of Congenital Bilateral Aplasia of the Vas Deferens

Congenital bilateral aplasia of the vas deferens (CBAVD) was suggested to be a mild form of cystic fibrosis (CF). Mutation analysis of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in males with CBAVD revealed that in some males CBAVD is caused by two defective CFTR alleles. The genetic basis of CBAVD in the other males and its association with CF remained unclear. We undertook this study to test the hypothesis of commonality of CBAVD and CF by haplotype analysis, in the CFTR locus, of males suffering from CBAVD and of their families. According to the hypothesis of commonality of CBAVD and CF, two brothers with CBAVD are expected to carry the same two CFTR alleles, while their fertile brothers are expected to carry at least one different allele. Eleven families were studied, of which two families, with unidentified CFTR mutations, did not support this hypothesis. In these families two brothers with CBAVD inherited different CFTR alleles. Their fertile brothers inherited the same CFTR alleles as their brothers with CBAVD. These results provide evidence for genetic heterogeneity in CBAVD. Though in some families CBAVD is associated with two CFTR mutations, we suggest that in others it is caused by other mechanisms, such as mutations at other loci or homozygosity or heterozygosity for partially penetrant CFTR mutations.     

Usefulness of linked DNA probes for prenatal diagnosis of cystic fibrosis: report of a case in a 1:4 risk pregnancy

Prenatal diagnosis of cystic fibrosis was performed with linked DNA probes in a couple with a 1:4 risk. The limits and the future of molecular prenatal diagnosis are discussed.     

A 3′ splice site consensus sequence mutation in the cystic fibrosis gene

Summary In the cystic fibrosis (CF) gene, recently cloned, a three base pair deletion (ΔF508) has been identified in a majority of CF patients. This deletion has been found in 80% of CF chromosomes in families from north west Brittany. In order to identify new mutations we have selected 43 chromosomes negative for the three base pair deletion from these families and directly sequenced exon 11 after DNA amplification by the polymerase chain reaction. We have detected a base change (G→A) at the 3′ end of the consensus sequence of intron ten (namely 1717-1). This mutation destroys a splice site in the cystic fibrosis gene which probably produces a mutant allele. This single nucleotide mutation has been reported on two other CF chromosomes.     

Analysis of DNA polymorphism haplotypes linked to the cystic fibrosis locus in North American black and Caucasian families supports the existence of multiple mutations of the cystic fibrosis gene.

Strong linkage disequilibrium (LD) was found between DNA marker XV2c and the cystic fibrosis (CF) locus (delta = 0.46) and between DNA marker KM19 and CF (delta = 0.67) in 157 CF and 138 normal chromosomes from U.S. Caucasians. DNA haplotypes with nine polymorphic sites were created in 54 Caucasian families. There is a strong LD between the haplotypes and the presence of the mutant CF genes. This implies that the DNA polymorphisms examined are close to the CF gene and that one mutation of the CF gene predominates in the Caucasian population. Haplotype analysis can also be used to refine estimates of CF carrier risk in Caucasians. Data for XV2c and MET markers in 16 American black patients and their families revealed a different haplotype distribution and LD pattern with the CF locus. These data suggest that racial admixture alone does not explain the occurrence of CF in American blacks and that multiple alleles of the CF gene may exist in this population.     

Carrier detection and prenatal diagnosis of cystic fibrosis using an intragenic TA-repeat polymorphism

Summary We have analysed the segregation of a TA-repeat polymorphism in intron 17b of the cystic fibrosis transmembrane conductance regulator gene responsible for cystic fibrosis (CF) in 23 French CF families non-informative for the F508 mutation (i.e. with at least one parent not carrying F508) or closely linked DNA markers. At least 13 different alleles ranging from 7 to 45 repeats were observed and the detected heterozygosity was 89%. Of the 23 families studied, 19 were fully informative for prenatal diagnosis or carrier detection, 3 were partially informative and one was not informative. In 6 families, prenatal diagnosis for CF or carrier detection in siblings of CF cases were performed using this polymorphism.     

Prenatal diagnosis of cystic fibrosis using linked DNA markers and microvillar intestinal enzyme analysis

Summary Prenatal dignosis was performed for 47 pregnancies with 1 in 4 risk of cystic fibrosis, including 7 cases analyzed with linked DNA markers, 16 cases analyzed by microvillar intestinal enzyme testing, and 24 cases where both methods of testing were attempted. DNA was obtained by chorionic villus sampling in 10 cases and by amniocentesis in 21 cases, and diagnosis was based on analysis with the tightly linked DNA markers D7S8 and met. DNA analysis using these probes was fully informative in 74.4% of 90 couples with 1 in 4 risk. In 18 cases where both DNA results and microvillar intestinal enzyme data were diagnostic, there was agreement regarding the predicted status of the fetus. No adequate diagnosis was achieved in two cases where both diagnostic tests were attempted. Ourcome is known for 24 pregnancies including 10 where DNA analysis was diagnostic, and no errors in diagnosis were detected. Prenatal diagnosis of cystic fibrosis using DNA markers is highly informative and accurate, but microvillar intestinal enzyme analysis remains a valuable part of a complete diagnostic program.     

An Algerian child homozygous for the M470V polymorphism and for a deletion of two nucleotides in exon 10 of the CFTR gene, shows severe cystic fibrosis symptoms.

When screening for the presence of major cystic fibrosis mutations in Algerian cystic fibrosis families by heteroduplex formation, aberrant heteroduplexes were observed for exon 10 in one family. Here we describe the clinical and molecular findings in a severely affected child of this family, homozygous for the 1609delCA and for the M470V polymorphism.     

Studies of cystic fibrosis in Hutterite families by using linked DNA probes.

Several multigenerational S-leut Hutterite families with cystic fibrosis (CF) were ascertained. Linkage studies with DNA marker loci MET and pJ3.11 (D7S8) were performed to determine whether (1) the CF gene in this inbred population is linked to DNA markers on chromosome 7, as it is in outbred populations of European origins, and (2) ancestral origin(s) of the CF gene could be determined. Our results indicate that the CF gene in Hutterite families segregates with chromosome 7 markers, identified by probes metH and pJ3.11. Thus, the CF mutation in Hutterites is likely to be either at the same locus as or at one closely linked to that reported in outbred populations. Heterozygous carriers could be distinguished from normal homozygous sibs of affected individuals. In the families studied, three different chromosome 7 haplotypes carried the CF mutation, raising the possibility that the CF gene may have been introduced into this population by as many as three different ancestors.     

Screening for cystic fibrosis: feasibility of molecular genetic analysis of dried blood specimens

Direct genotypic analysis for the common Caucasian cystic fibrosis mutation (delta F508) was performed using dried blood specimens in a filter paper matrix (neonatal screening blotter). DNA was obtained from dried and liquid blood samples, amplified, and analyzed by polyacrylamide gel electrophoresis. Additionally, intact 4-mm-diameter punched discs from blotters containing dried blood specimen were used in the amplification reactions and analyzed by electrophoresis. The results agreed completely between these three sample types, demonstrating the feasibility of molecular genetic confirmation of the delta F508 mutation from the neonatal screening blotter among those with positive CF screening results. Such a program could reduce follow-up testing by at least 50% in a CF newborn screening program and would identify immediately those families who would benefit from carrier detection for the delta F508 allele.     

A linkage study of cystic fibrosis in extended multigenerational pedigrees.

The linkage of polymorphic DNA markers on chromosome 7 to cystic fibrosis (CF) was examined in two pedigrees and a number of smaller nuclear families. The pedigrees are multigenerational and together consist of more than 300 members including 30 affected individuals, while the nuclear families each have two generations and either two or three children with CF. Tight linkage was observed between the CF locus and the met oncogene locus theta = 0, zeta = 15.45), pJ3.11 (theta = 0, zeta = 10.07), and 7C22 (theta = 0, zeta = 6.64) in both the pedigrees and nuclear families with no evidence for recombination between CF and any of the DNA markers. Weaker linkage between the CF locus and the locus for the serum enzyme activity marker paraoxonase (PON) was detected, theta = 0.18, zeta = 0.76. The two pedigrees were sufficiently informative to detect significant linkage between CF and each of the three DNA markers previously shown to be tightly linked to the CF locus. These results establish a locus for CF in these pedigrees in the region of chromosome 7 nearest the three DNA markers met, pJ3.11, and 7C22 and are consistent with locus homogeneity for the defect causing CF in these populations and others that have been examined to date.