Detection of over 98% cystic fibrosis mutations in a Celtic population.

We have conducted a large systematic study of 365 cystic fibrosis (CF) chromosomes in a Celtic population from Brittany, France, in which we have been able to identify more than 98% of the cystic fibrosis gene mutations. We detected 19 different CFTR mutations located in 9 exons. Eleven of these mutations have not been described previously and nine of them are presented in this study. The denaturing gradient gel electrophoresis strategy we have used, can be applied to other populations suggesting that population screening for CF on a large scale might be possible.

Distribution of CFTR mutations in cystic fibrosis patients of Tunisian origin: identification of two novel mutations.

Cystic fibrosis (CF), the most common lethal genetic disease in the Caucasian population, is caused by mutations in the CF transmembrane conductance regulator gene (CFTR). More than 500 molecular defects have been reported to date. The distribution of these mutations is both heterogeneous and population related. In Mediterranean populations, 20-30% of CF alleles remain unidentified. We have studied a sample of 39 CF patients of Tunisian origin and have used a GC clamp DGGE assay to scan the CFTR gene. Two novel mutations have been found, but we have been unsuccessful in finding any mutation in 40% of these alleles. These results suggest that, in this Mediterranean population, additional mutations may lie elsewhere in the promoter region or in introns not yet analyzed.

[Cystic fibrosis gene mutations in the West of France: clinical application]. / Les mutations du gène de la mucoviscidose dans l'ouest de la France: application clinique.

The cystic fibrosis transmembrane conductance regulator (CFTR) gene, responsible for the cystic fibrosis phenotype when both alleles are mutated, was cloned and sequenced in 1989. Since then, more than 400 mutations have been reported in the gene, although most of these are rare. We have systematically analysed the entire coding sequence of the CFTR gene in a cohort of patients originating from the West of France (Caen, Brest and Nantes). More than 450 CF children, 914 chromosomes in all, have been exhaustively studied in the three centers. We have been able to characterize more than 90% of the mutations, respectively 93.5%, 99% and 95.8%. Despite the large diversity in the CFTR mutations occurring in CF patients from this area, these results can help to improve genetic counselling, prenatal diagnosis as well as our understanding of the molecular basis of the pathophysiology of cystic fibrosis.

[Gougerot-Sjögren's syndrome. Clinical and biological correlations with the serology of Yersinia infections]. / Syndrome de Gougerot-Sjögren. Corrélations cliniques et biologiques avec la sérologie des infections à Yersinia.

An unusual case of Sjögren's syndrome (SS) induced or revealed by a Yersinia pseudotuberculosis serotype IV infection has prompted the authors to undertake a retrospective study of 32 patients with that syndrome. High titers of antibodies to Y. pseudotuberculosis serotype IV (greater than 1/200 th) were found in 4 patients with primary Sjögren's syndrome and Sharp's syndrome. Seven other patients (5 with primary Sjögren's syndrome, 1 with systemic lupus erythematosus and 1 with Sharp's syndrome) had positive serology with low titres, of doubtful significance. Serology was negative in the 20 remaining patients. Nine out of 25 patients had anti-thyroid antibodies, and their relationship with positive Y. pseudotuberculosis IV serology is discussed. The responsibility of the infection in the induction or disclosure of Sjögren's syndrome may be considered. However, since the syndrome frequently develops before serology becomes positive (8/12 cases with a 1 to 6 years' follow-up), an alternative hypothesis would be that diminished digestive defences in Sjögren's syndrome result in an increased frequency of Yersinia infections.

[Mucoviscidosis: current diagnostic possibilities. Applications in perinatology]. / Mucoviscidose: de nouvelles possibilités diagnostiques. Applications en anténatalogie.

The gene of cystic fibrosis is localised on the long arm of chromosome 7. DNA probes placed close to the gene enable a study of restriction polymorphism to follow the transmission of the gene in index families. It is now possible to counsel those families, who already have an affected child, with an early antenatal diagnosis at ten weeks after the last period. In our personal experience, based on a study of the genotype of 48 families, 70% were informative when they were studied by two probes corresponding to the local pJ3.11 and met. When the latter probes Km19-XV2c were studied concurrently useful information was achieved in 96%. DNA analysis non enables the detection of the chromosome carrying the deleterious gene in practically every family where there is a child suffering from the disease.

[Linkage disequilibrium and DNA markers associated with the gene for cystic fibrosis]. / Déséquilibre de liaison et marqueurs de l'ADN associés au gène de la fibrose kystique.

R.F.L.P. (Restriction Fragment Length Polymorphism) observed with tightly linked probes to the CF gene allows us to calculate the standardised linkage disequilibrium between CF and these markers. This approach in combination with others strategies permits to situate the gene between D9 and G2. The conditional probabilities observed with these haplotypes modify the classical genetic of CF.

Identification of three novel mutations (457 TAT-->G, D192G, Q685X) in the Slovenian CF patients.

Chromosomes from a cohort of 60 Slovenian families, corresponding to the 121 cystic fibrosis (CF) chromosomes available, were fully scanned for mutations in the coding sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene (The 60 families yielded 121 CF alleles because the mother of one patient was also affected). This corresponds to the 27 exons and intron/exon boundaries that have been studied in chromosomes carrying unidentified alleles. As a result of this survey 84% of the alleles are now clearly identified and we describe in this paper three novel mutations (457 TAT-->G, D192G, and Q685X).

Clinical features of cystic fibrosis patients with rare genotypes in Saguenay Lac-Saint-Jean (Quebec, Canada).

We describe the clinical features of six cystic fibrosis (CF) patients from Saguenay Lac-Saint-Jean who bear rare genotypes. Two patients with a delta F508/I148T genotype had pancreatic insufficiency, as did two patients compound heterozygous for the 621 + 1G-->T mutation who also had a major growth retardation. One CF adult who carried a delta F508/Q890X genotype had meconium ileus and bronchiectasis. The sixth patient (A455E/R117C) had borderline sweat chloride concentrations; the diagnosis of cystic fibrosis had remained doubtful until the molecular analysis showed the presence of two CF mutations. The seventh patient with a delta F508/R1158X genotype experienced several complications and is now 43 years old.

[Direct diagnosis of predominant mutation delta F508 associated with the mucoviscidosis gene]. / Diagnostic direct de la mutation majoritaire delta F508 associée au gène de la mucoviscidose.

The cystic fibrosis locus was mapped on the long arm of the chromosome 7 in 1985. It has recently been cloned and a three base pair deletion has been recognized as the mutation associated with the majority of CF chromosomes (delta F508). CF haplotypes previously defined with tightly associated DNA markers were analysed using PCR (Polymerase Chain Reaction) and allele specific oligonucleotides to determine the presence or absence of this mutation. This mutation was found on 80% of our CF chromosomes and associated predominantly with the B haplotypes. The detection of this mutation is now a major improvement for carrier detection and prenatal diagnosis of the disease.

Asymptomatic carrier of two CFTR mutations: consequences for prenatal diagnosis?

The cystic fibrosis (CF) gene has been observed to have the highest frequency of mutations in the Caucasian population. Prenatal diagnosis can now be performed with a high degree of accuracy since the identification of most of the gene's mutations, as well as the characterization of intragenic markers. However, the observation of a distribution of clinical phenotypes increases the need to identify a mild phenotype and avoid false-negative diagnosis. By screening most of the exons of the CFTR gene, we showed that a supposed obligate carrier of CF was in fact an asymptomatic affected woman.

Prenatal diagnosis of cystic fibrosis in different European populations: application of denaturing gradient gel electrophoresis.

The cystic fibrosis transmembrane regulator gene, one of the most commonly mutated in the European population, was cloned in 1989 and since then has been extensively analysed in patients of various ethnic backgrounds. We have screened the entire coding sequences of the cystic fibrosis transmembrane regulator gene and identified many mutations and polymorphisms. In this paper we propose a general strategy to improve prenatal diagnosis and genetic counselling of cystic fibrosis (CF). As this approach based on denaturing gradient gel electrophoresis is adaptable to different populations, it greatly increases the sensibility and specificity of CF prenatal diagnosis.

Identification of 12 novel mutations in the CFTR gene.

Over 200 mutations, besides the deletion delta F508, have been identified in the CFTR gene and are known to cause CF. In order to characterize the molecular defects of non delta F508 CF chromosomes of various French origin, we have combined the techniques of denaturing gradient gel electrophoresis (DGGE) and direct sequencing to screen for mutations in the whole coding sequence of the CFTR gene corresponding to the 27 exons and their exon-intron boundaries. This approach enabled us to identify 12 novel mutations which are described here. We have systematically tested a large number of other nucleotide changes distributed in the 27 exons, each of them was clearly detected. These data support the notion that the DGGE conditions we have defined for screening coding sequence of the CFTR gene allows the identification of most of, if not all, the CFTR gene mutations.

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-delta F508 mutations was associated with a single haplotype: 1078deIT with 16-31-13, G55ID 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 delta F508 chromosomes are the same as those found in Ireland, Spain, and Italy. This suggests that some haplotypes, associated or not with the delta F508 mutation, were present in an ancestral population from which all four populations descended.

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.

Complete screening of mutations in the coding sequence of the CFTR gene in a sample of CF patients from Russia: identification of three novel alleles.

To date, a large number of mutations causing the disease, cystic fibrosis, have been reported worldwide. Having analysed the coding sequence of a sample of cystic fibrosis (CF) patients from Russia, we have identified three novel CF mutations. Two of them, 175 del C in exon 1 and 624 del T in exon 5, are frameshift mutations, predicted to result in premature termination of the CFTR transcript. The third mutation is missense and occurs in exon 12 (D572N). The profile of mutations in this sample of Russian CF patients is particular, with two mutations in exon 13 (2143 del T and 2184 ins A), accounting for 12% of the non-delta F508 alleles.

Identification of six novel CFTR mutations in a sample of Italian cystic fibrosis patients.

The spectrum of cystic fibrosis (CF) mutations has been determined in many populations of different ethnic and geographic origins. However, in the south of Europe, the commonest mutation, delta F508, accounts for only about 50% of CF chromosomes, while identification of most of the other mutant alleles has not been achieved. In an ongoing effort to identify these alleles, we have scanned the entire coding sequences of the CF gene using a GC clamp denaturing gradient gel electrophoresis assay in a sample of 57 chromosomes from patients of italian origin. We have identified six novel mutations (C276X, H139R, R117L, S42F, A1006E and 3121-2A---> T). Each has only been found once in this sample of CF patients.

Retrospective study of the cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in Guthrie cards from a large cohort of neonatal screening for cystic fibrosis.

The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes a cAMP-activated chloride channel, and in individuals with both alleles of the gene mutated, symptoms of CF disease are manifest. With more than 300 mutations so far described in the gene the profile of mutant alleles in a population is specific to its ethnic origin. For an analysis with an unbiased recruitment of the CF alleles in neonates of similar origin (Normandy, France), we have retrospectively analyzed the Guthrie cards of affected newborns, diagnosed by the immunoreactive trypsinogen (IRT) assay. Analysis of the 27 exons of the CFTR gene using a GC clamp denaturing gradient gel electrophoresis (DGGE) assay has enabled us to identify over 96% of the mutated alleles. Two of these were novel mutations. We would like to propose this strategy as an efficient method of retrospective molecular genetic diagnosis that can be performed wherever Guthrie cards can be obtained. Knowledge of rare alleles could be a prerequisite for CF therapy in the future.

Absence of mutations in the interspecies conserved regions of the CFTR promoter region in cystic fibrosis (CF) and CF related patients.

This study was aimed at testing if a 5.2 kb untranslated region on both sides of the first CFTR exon, shown to contain regulatory elements, could carry mutations responsible for cystic fibrosis (CF) or CF related phenotypes. Selection of the DNA segments studied within this region was based upon the identification of conserved sequences throughout evolution (phylogenetic footprints, PFs). Comparison of the CFTR sequences in eight species representing four orders of mammals (man, gibbon, rhesus monkey, squirrel, monkey, rabbit, cow, rat, and mouse) identified four clusters of PFs within the 3.9 kb of DNA sequence upstream from the initiation codon, as well as two nearby PFs at +1 kb within intron 1. Six DNA segments containing PFs were scanned for mutations by denaturing gradient gel electrophoresis (DGGE) in patients with CF (n = 29), congenital bilateral absence of the vas deferens (n = 143), or disseminated bronchiectasis (n = 33), for whom only one or no mutations had been identified despite extensive DGGE analysis of the 27 CFTR exons and exon/intron boundaries. Only one polymorphism (-966 T-->G) was identified with a frequency of 2.2% and no other sequence variations were found. This study reinforces the idea that the promoter region in the CFTR is not frequently mutated.

Complete identification of cystic fibrosis transmembrane conductance regulator mutations in the CF population of Saguenay Lac-Saint-Jean (Quebec, Canada).

Over the past few years, we have conducted a systematic study of 230 cystic fibrosis (CF) chromosomes in the Saguenay Lac-Saint-Jean (SLSJ) population which has a high CF incidence (1/936 live births). We identified 11 mutations accounting for 100% of the CF chromosomes found in patients born in SLSJ. Our results indicate that denaturing gradient gel electrophoresis (DGGE) is a powerful method of identifying CF mutations. They have also considerable implications for genetic counselling and molecular characterization of doubtful patients. They make carrier screening technically feasible in this population.