Prevalence of CFTR mutations in hypertrypsinaemia detected through neonatal screening for cystic fibrosis.

Nowadays, most of the neonatal screening programs for cystic fibrosis (CF) combine the assay of immunoreactive trypsinogen (IRT) with the analysis of the most common mutations of the CFTR gene. The efficiency of this strategy is now well established, but the identification of heterozygotes among neonates with increased IRT is perceived as a drawback. We proposed to assess the heterozygosity frequency among the children with hypertrypsinaemia detected through the CF screening program implemented in Brittany (France) 10 years ago, to describe the CFTR mutations detected in them and to determine the frequency of the IVS8-5T variant. The molecular analysis relies, in our protocol, on the systematic analysis of three exons of the gene (7-10-11). A total of 160,019 babies were screened for CF in western Brittany between 1992 and 1998. Of the 1964 newborns with increased IRT (1.2%), 60 were CF and 213 were carriers. Heterozygosity frequency was 12.8%), i.e. 3 times greater than in the general population (3.9%; p < 10(-6)), Variability of mutations detected in carriers was greater than in CF children (21 mutations versus 10) and a high proportion of mild mutations or variants (A349V, R297Q, R347H, V317A, G544S, R553G, etc) was observed in carriers. The allelic frequency of the 5T (5.6%) was not significantly increased in this cohort. This study is consistent with previous ones in finding a significantly higher rate of heterozygotes than expected among neonates with hypertrypsinaemia. The strategy of screening used here allows to highlight the variability of mutations detected in heterozygotes and to show that severe mutations, as well as mild mutations, have been observed in neonates with hypertrypsinaemia. If there is no doubt that neonatal hypertrypsinaemia is associated with an elevated frequency of carriers, the underlying mechanisms remain obscure.

Neonatal screening for cystic fibrosis in Brittany, France: assessment of 10 years' experience and impact on prenatal diagnosis.

BACKGROUND: Neonatal screening for cystic fibrosis has been a subject of debate over the past few years. This study assesses 10 years of neonatal screening in Brittany, France, and examines its impact on prenatal screening of subsequent pregnancies in couples with an affected child. METHODS: The study included all the neonates screened for cystic fibrosis in Brittany from Jan 1, 1989, to Dec 31, 1998. The screening consisted of an immunoreactive trypsinogen assay from dried blood spots, plus, from 1993, mutation analysis. Data were collected on incidence of cystic fibrosis, and genotypic and biochemical characteristics. The use of prenatal screening of subsequent pregnancies in affected families was also investigated. FINDINGS: Of the 343,756 neonates screened, 118 children with cystic fibrosis were identified, giving an incidence of one in 2913. All mutated alleles were characterised: 34 different mutations resulting in 36 genotypes were detected. The introduction of DNA analysis into the protocol greatly reduced the recall rate and increased the sensitivity of the test. The mean cost of the screening programme was US$2.32 per screened child. 39 (34%) of the families identified by neonatal screening opted for subsequent prenatal diagnosis at least once. 12 couples would have benefited from this procedure while their first child was still symptom-free. 42 healthy children were born, and 18 pregnancies were terminated (therapeutic abortion rate of 100%). INTERPRETATION: We have shown the feasibility of neonatal screening for cystic fibrosis in Brittany. Through the detection of a large range of mutations, neonatal screening provides the opportunity for more reliable prenatal diagnosis and cascade screening. The neonatal screening programme described here could provide a good model for other countries intending to initiate such a scheme.

Spectrum of CFTR mutations in cystic fibrosis and in congenital absence of the vas deferens in France.

We have collated the results of cystic fibrosis (CF) mutation analysis conducted in 19 laboratories in France. We have analyzed 7, 420 CF alleles, demonstrating a total of 310 different mutations including 24 not reported previously, accounting for 93.56% of CF genes. The most common were F508del (67.18%; range 61-80), G542X (2.86%; range 1-6.7%), N1303K (2.10%; range 0.75-4.6%), and 1717-1G>A (1.31%; range 0-2.8%). Only 11 mutations had relative frequencies >0. 4%, 140 mutations were found on a small number of CF alleles (from 29 to two), and 154 were unique. These data show a clear geographical and/or ethnic variation in the distribution of the most common CF mutations. This spectrum of CF mutations, the largest ever reported in one country, has generated 481 different genotypes. We also investigated a cohort of 800 French men with congenital bilateral absence of the vas deferens (CBAVD) and identified a total of 137 different CFTR mutations. Screening for the most common CF defects in addition to assessment for IVS8-5T allowed us to detect two mutations in 47.63% and one in 24.63% of CBAVD patients. In a subset of 327 CBAVD men who were more extensively investigated through the scanning of coding/flanking sequences, 516 of 654 (78. 90%) alleles were identified, with 15.90% and 70.95% of patients carrying one or two mutations, respectively, and only 13.15% without any detectable CFTR abnormality. The distribution of genotypes, classified according to the expected effect of their mutations on CFTR protein, clearly differed between both populations. CF patients had two severe mutations (87.77%) or one severe and one mild/variable mutation (11.33%), whereas CBAVD men had either a severe and a mild/variable (87.89%) or two mild/variable (11.57%) mutations.

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.

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.

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.

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.

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.

Neonatal screening for cystic fibrosis: result of a pilot study using both immunoreactive trypsinogen and cystic fibrosis gene mutation analyses.

We have evaluated a two-tier neonatal cystic fibrosis (CF) screening of immunoreactive trypsinogen (IRT) followed by CFTR gene mutation analysis using a systematic scanning of exons 7, 10, and 11, and, if necessary, by direct DNA sequencing. Over an 18-month period we screened 32,300 neonates born in the western part of Britanny. The first tier, involving IRT screening at 3 days of age, utilizes a low elevation of the trypsinogen level (600 ng/ml), which is highly sensitive. The second tier, which corresponds to the exhaustive screening for mutations in three exons of the gene, is highly specific for this population (Britanny). The false positive rate is very low, and no false negatives have been reported to date. This strategy has allowed the identification of five novel alleles (V322A, V317A, 1806 del A, R553G, G544S).

Mutations in the cystic fibrosis gene in patients with congenital absence of the vas deferens.

BACKGROUND: Congenital bilateral absence of the vas deferens (CBAVD) is a form of male infertility in which mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been identified. The molecular basis of CBAVD is not completely understood. Although patients with cystic fibrosis have mutations in both copies of the CFTR gene, most patients with CBAVD have mutations in only one copy of the gene. METHODS: To investigate CBAVD at the molecular level, we have characterized the mutations in the CFTR gene in 102 patients with this condition. None had clinical manifestations of cystic fibrosis. We also analyzed a DNA variant (the 5T allele) in a noncoding region of CFTR that causes reduced levels of the normal CFTR protein. Parents of patients with cystic fibrosis, patients with types of infertility other than CBAVD, and normal subjects were studied as controls. RESULTS: Nineteen of the 102 patients with CBAVD had mutations in both copies of the CFTR gene, and none of them had the 5T allele. Fifty-four patients had a mutation in one copy of CFTR, and 34 of them (63 percent) had the 5T allele in the other CFTR gene. In 29 patients no CFTR mutations were found, but 7 of them (24 percent) had the 5T allele. In contrast, the frequency of this allele in the general population was about 5 percent. CONCLUSIONS: Most patients with CBAVD have mutations in the CFTR gene. The combination of the 5T allele in one copy of the CFTR gene with a cystic fibrosis mutation in the other copy is the most common cause of CBAVD: The 5T allele mutation has a wide range of clinical presentations, occurring in patients with CBAVD or moderate forms of cystic fibrosis and in fertile men.

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.

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.

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.

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).

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.

Association of 1078 del T cystic fibrosis mutation with severe disease.

Apart from the high frequency of the delta F508 mutation (81.81%) in Breton cystic fibrosis chromosomes, one mutation, 1078 del T, is also observed frequently (4.96%) in this group, in comparison with the rest of the French where it occurs with a frequency of 0.57%. These two mutations account for more than 86.5% of the total CF mutations identified on Breton chromosomes. We have conducted an unblinded retrospective analysis of 25 patients with the 1078 del T mutation and compared their phenotypes with those of a group of 70 delta F508 homozygous patients. Both groups of patients had the same ethnic origin and were regularly attending the same CF centre in Brittany, which makes this sample highly homogeneous despite the small size. The 1078 del T mutation appeared to be associated with severe presentation of the disease with, however, a trend to reduced mortality and less Pseudomonas aeruginosa colonisation.

[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.