Cystic fibrosis screening: a fetus with hyperechogenic bowel may be the index case.

BACKGROUND: The potential of hyperechogenic fetal bowel to act as a hallmark for prenatal cystic fibrosis screening in the general population is controversial. METHODS: Our goal was to evaluate the incidence of cystic fibrosis in 209 fetuses with hyperechogenic bowel diagnosed at routine ultrasonography and with no family history of cystic fibrosis. The diagnosis of cystic fibrosis was based on prenatal screening for the eight mutations most frequently observed in France (deltaF508, deltaI507, 1717-1G-->A, G542X, G551D, R553X, W1282X, N1303K) and at postnatal follow up. RESULTS: The overall incidence of cystic fibrosis was 7/209 (3.3%) which is 84 times the estimated risk of CF in the general population (112500). Of these seven cases, six were diagnosed prenatally based on DNA analysis (deltaF508/deltaF508, n=5; deltaF508/G542X, n=1). One case in which only one mutation had been recognised was diagnosed clinically after birth (deltaF508/unidentified mutation). Of the seven cases, none was diagnosed at 16-19 weeks, four at 16-24 weeks, and three after this. The incidence of heterozygous fetuses (15/209, 7%) was not significantly higher than the 5% expected in the general population. The mutations involved in these heterozygous cases were deltaF508 (n=13), G542X (n=1), and G551D (n=1). CONCLUSIONS: Screening for cystic fibrosis should be offered to families in which fetal hyperechogenic bowel is diagnosed at routine ultrasonography. This underlines the need to review genetic counselling in this situation where the fetus is the index case for a genetic disease.     

Demographics of the UK cystic fibrosis population: implications for neonatal screening

The objective was to determine the composition of the Cystic Fibrosis (CF) Population attending specialist UK CF centres in terms of age, gender, age at diagnosis, genotype and ethnicity. With the planned introduction of the national CF screening programme in the UK, cystic fibrosis transmembrane regulator (CFTR) mutations were compared between different ethnic groups enabling a UK-specific frequency of mutations to be defined. Data were analysed from the patient biographies held in the UK CF Database (see www.cystic-fibrosis.org.uk). The currently registered population of 5,274 CF patients is 96.3% Caucasian with a male preponderance that significantly increases with age. The majority of the 196 non-Caucasian CF patients are from the Indian Subcontinent (ISC), of which one in 84 UK CF patients are of Pakistani origin. The commonest CFTR mutation, deltaF508, is found in 74.1% of all CF chromosomes. In the Caucasian CF population, 57.5% are deltaF508 homozygotes but the UK ISC CF population with only 24.7%, has significantly fewer deltaF508 homozygotes patients (95% confidence interval (CI) 0.2-0.4). The distribution of Caucasian patients with deltaF508/deltaF508, deltaF508/Other and Other/Other does not fit the expected distribution with a Hardy-Weinberg model unless those patients without a detected mutation are excluded (P<0.001). The UK CF Database has shown the UK CF population to have distinct characteristics separate from the North American and European CF Registries. The ISC group contains many mutations not recognised by current genetic analysis, and one in four ISC patients have no CFTR mutations identified. The CFTR analysis proposed for the screening programme would detect 96% of patients registered in the database, but is unlikely to achieve the desired >80% detection rates in the ethnic minority groups. Screen-positive, non-Caucasian infants without an identifiable CFTR mutation should be referred for a sweat test and genetic counselling when serum trypsinogen concentrations remain elevated after birth.     

Frequency and clinical significance of the S1235R mutation in the cystic fibrosis transmembrane conductance regulator gene: results from a collaborative study

More than 900 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been reported to the cystic fibrosis (CF) consortium. A missense mutation, S1235R, was originally reported in a CF patient with a second mutation (G628R) on the same chromosome. The clinical significance of S1235R was not clear. S1235R is not among the commonly reported mutations, and it is not routinely screened for in most laboratories. However, we have detected the S1235R allele at a frequency that is significantly higher than that of many other CF mutations. Among more than 3,000 patients tested for either a possible diagnosis of CF or to determine CF carrier status, we identified 51 patients heterozygous for S1235R. No patients were homozygous for S1235R. Five patients were compound heterozygotes for a second CFTR mutation: two cases (one family) were N1303K/S1235R and three unrelated cases were deltaF508/S1235R. Our data suggest that S1235R, when combined with a second CF mutation, may be pathogenic, although phenotypic manifestations appear to be variable. The possibility that this represents a rare polymorphism cannot be discounted completely. Genetic counseling is difficult when S1235R is identified, even in the presence of a second known mutation, especially in prenatal cases.     

Mutation analysis in 600 French cystic fibrosis patients.

The cystic fibrosis transmembrane conductance regulator (CFTR) gene of 600 unrelated cystic fibrosis (CF) patients living in France (excluding Brittany) was screened for 105 different mutations. This analysis resulted in the identification of 86% of the CF alleles and complete genotyping of 76% of the patients. The most frequent mutations in this population after delta F508 (69% of the CF chromosomes) are G542X (3.3%), N1303K (1.8%), W1282X (1.5%), 1717-1G-->A (1.3%), 2184delA + 2183 A-->G (0.9%), and R553X (0.8%).     

Genetic testing for cystic fibrosis: evaluation of the Elucigene CF20 kit in blood and buccal cells

Routine determination of mutations in cystic fibrosis requires accurate, rapid, reliable and low-cost methods, permitting the simultaneous detection of multiple mutations. The Elucigene CF20 kit developped by Cellmark Diagnostics, uses multiplex ARMS, which allows the screening for 20 CFTR gene mutations (deltaF508, G542X, N1303K, 1717-1G>A, G551D, W1282X, R553X, deltaI507, 1078delT, 2183AA>G, 3849+10kbC>T, R1162X, 621+1G>T, R334W, R347P, 3659delC, R117H, S1251N, E60X, A455E ) in a work day without specific instrumentation. The kit distinguishes between homozygotes and heterozygotes for deltaF508, but not for rare mutations. The kit detects from 68 to 92% of defective alleles in Caucasians. We evaluate the kit in a blind study in two independent laboratories. Thirty blood samples and thirty mouthwash samples from CF patients, carriers and unaffected individuals were analysed by the Elucigene CF20 kit. All the samples were previously analysed by denaturing gradient gel electrophoresis and sequencing. The Elucigene CF20 kit consists of three multiplexes. Each mutiplex contains ARMS specific primers for six to eight mutations and two control reactions. The absence of the upper control fragment indicates that a repeat test is required. We demonstrated a first time amplification rate of 98.3%: of the 60 samples tested, one required a reamplification. Results compared with the reference method demonstrated that in all cases where one or more of the 20 mutations detected by the kit were present in the test set, the kit accurately identified them. Reproducibility was assessed by repeating the analysis of a blood and mouthwash sample five times. Cross reactivity between R117C and R117H, R117P and R117H, R347P and R347H, deltaI507 and deltaF508, G551D and R553X were evaluated. Only a cross reactivity between R347P and R347H was observed. The kit is specially useful for first line study of patients and carrier identification.     

A clinical perspective of cystic fibrosis and new genetic findings: relationship of CFTR mutations to genotype-phenotype manifestations

The present report describes several aspects of the relationship of mutations in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene to phenotype expression of the disease including several clinical vignettes from the authors' experience. The genotype-phenotype relationships in CF are complex, and are affected by many factors, including pollution, smoking, bacterial infection, malnutrition, and certain therapeutic agents. The number of CFTR mutations is growing continuously and rapidly, and more than 1,000 mutations have been discovered so far. From a genetic point of view, the deltaF508 mutation is not only the most frequently encountered but also the most severe genetic lesion for homozygotes. The great clinical variability observed in patients with CF, particularly the severity of lung disease, involvement of the pancreas, and male infertility, are beginning to be better understood through the knowledge, although incomplete, of CFTR mutations and their phenotype expressions. This knowledge has had very significant research and clinical applications in all dimensions of the CF problem. It has not only contributed to the enhancement of better diagnosis and clinical management, but it also has opened new and unanticipated lines of investigation and research.     

Cystic fibrosis genotype and bacterial infection: a possible connection

Patients with cystic fibrosis (CF) have repeated bacterial infection of the airways, which can lead to chronic infection. There is evidence that disease severity is determined by the genetic mutations present. This study aims to establish if CF genotype is related to the frequency and types of airway bacterial infection. Adult patients attending the regional CF unit are followed for two years and assigned to one of three groups depending on whether they are chronically infected with Burkholderia cepacia complex (BCC) organisms, Pseudomonas aeruginosa, or neither of these organisms. Genotype analysis is performed on all patients to determine which of the cystic fibrosis transmembrane regulator (CFTR) gene mutations are present. The numbers and types of organism with the CFTR mutations isolated from sputum are identified. Data are available on 59 patients: 15 colonised with BCC, 24 colonised with P. aeruginosa, and 20 not colonised with either organism. Twenty patients were homozygous for deltaF508, 25 were heterozygous, and the deltaF508 mutation was not present in the remaining 14 patients. Patients homozygous or heterozygous for the deltaF508 mutation had an increased likelihood of colonisation with BCC or P. aeruginosa, an increased number of positive sputum cultures and a higher frequency of multiple infecting organisms. Cystic fibrosis mutational analysis identified seven patients who had the R117H mutation. These patients were less likely to be colonised with BCC or P. aeruginosa. In conclusion, patients homozygous or heterozygous for the deltaF508 deletion are more likely to suffer airway colonisation with BCC or P. aeruginosa, with increased numbers of positive sputum cultures and infecting organisms. Those with the R117H mutation are less likely to be colonised by Gram-negative organisms.     

A cystic fibrosis allele encoding missense mutations in both nucleotide binding folds of the cystic fibrosis transmembrane conductance regulator.

German cystic fibrosis (CF) chromosomes were screened for molecular lesions in exon 20 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by chemical cleavage of mismatch. An 3884G-to-A transition was detected in two patients which leads to an exchange of a serine by an asparagine in the Walker motif A of the second nucleotide binding fold. The affected serine residue is evolutionarily strongly conserved among the pro- and eukaryotic members of the protein superfamily of traffic ATPases. The two S1251N alleles were linked to the benign missense mutation F508C which is located in another conserved region of CFTR, the center region of the first nucleotide binding fold. Both patients with the complex allele F508C-S1251N are carrying delta F508 on the other CF chromosome and are suffering from severe pulmonary and gastrointestinal CF disease. Although F508C has been classified as a neutral sequence variation because of its discovery in healthy delta F508 gene carriers, it may nevertheless influence CFTR dysfunction caused by the S1251N mutation.     

First report of cystic fibrosis mutations in Libyan cystic fibrosis patients

Background: There are few data on the molecular basis of Cystic Fibrosis (CF) in North Africa, probably due to under-diagnosis.

Aim: This is the first study of cystic fibrosis transmembrane conductance regulator (CFTR) mutations in the Libyan population.

Subjects and methods: This study analysed the complete coding region and flanking intronic sequences of the CFTR gene in 10 unrelated Libyan CF patients.

Results: This study identified four mutations (F508del, c.1670delC, N1303K and E1104X), with a high frequency of the latter.

Conclusion: Identification of CF mutations facilitates molecular investigation of cystic fibrosis in the Libyan population and helps to provide effective genetic counselling among CF families.     

SERCA pump inhibitors do not correct biosynthetic arrest of deltaF508 CFTR in cystic fibrosis

Deletion of phenylalanine 508 (deltaF508) accounts for nearly 70% of all mutations that occur in the cystic fibrosis transmembrane conductance regulator (CFTR). The deltaF508 mutation is a class II processing mutation that results in very little or no mature CFTR protein reaching the apical membrane and thus no cAMP-mediated Cl- conductance. Therapeutic strategies have been developed to enhance processing of the defective deltaF508 CFTR molecule so that a functional cAMP-regulated Cl- channel targets to the apical membrane. Sarcoplasmic/endoplasmic reticulum calcium (SERCA) inhibitors, curcumin and thapsigargin, have been reported to effectively correct the CF ion transport defects observed in the deltaF508 CF mice. We investigated the effect of these compounds in human airway epithelial cells to determine if they could induce deltaF508 CFTR maturation, and Cl- secretion. We also used Baby Hamster Kidney cells, heterologously expressing deltaF508 CFTR, to determine if SERCA inhibitors could interfere with the interaction between calnexin and CFTR and thereby correct the deltaF508 CFTR misfolding defect. Finally, at the whole animal level, we tested the ability of curcumin and thapsigargin to (1) induce Cl- secretion and reduce hyperabsorption of Na+ in the nasal epithelia of the deltaF508 mouse in vivo, and (2) induce Cl- secretion in intestine (jejunum and distal colon) and the gallbladder of the deltaF508 CF mouse. We conclude that curcumin and thapsigargin failed to induce maturation of deltaF508 CFTR, or induce Cl- secretion, as measured by biochemical and electrophysiologic techniques in a variety of model systems ranging from cultured cells to in vivo studies.     

Novel and characteristic CFTR mutations in Saudi Arab children with severe cystic fibrosis.

More than 600 different CFTR (cystic fibrosis transmembrane conductance regulator) gene mutations have been identified so far that are considered to cause the fatal genetic disorder cystic fibrosis (CF). We have investigated 15 Arab children from 12 families, who were diagnosed as having CF, for mutations in the coding region and in the flanking intron sequences of the CFTR gene. Six different CFTR mutations were identified including two novel mutations, 1548delG in exon 10 and 406-2A-->G in intron 3. Prominent mutations were the splice mutation 3120 + 1G-->A (intron 16) followed by N1303K (exon 21) and 1548delG (exon 10). Most CF children were homozygotes who presented with a severe form of the disease including failure to thrive, recurrent chest infections, particularly with Pseudomonas aeruginosa, and frequent hospital admissions. Identification of the CFTR mutations facilitates molecular investigation of the disease and better understanding of its pathophysiology in Arab children, among whom CF is probably an underdiagnosed disease.     

Three common CFTR mutations should be included in a neonatal screening programme for cystic fibrosis in Sweden

Children with cystic fibrosis (CF) diagnosed by neonatal screening have a better nutritional development and other advantages compared with those in a nonscreened group. The two-tier immunoreactive trypsinogen (IRT)/DNA screening protocol has been found superior to the single-tier IRT approach, improving the positive predictive value and thus reducing the false-positive rate. However, variations of the DNA test are required for different populations. In this study we examined CFTR (cystic fibrosis transmembrane conductance regulator) mutations in 331 CF patients attending the centres in Stockholm, Lund and Uppsala, comprising about 75% of the CF population in Sweden. The frequency of deltaF508 among CF alleles was 68.3%. There were two other mutations, 394delTT and 3659delC, found to be fairly frequent, amounting to 8.5 and 7.9%, respectively. Other mutations were comparatively rare. A simple and effective method of analysing the three mutations from Guthrie cards has been developed. Assuming Hardy-Weinberg equilibrium, 90% of our CF patients will be expected to carry at least one deltaF508 allele and 97.6% to carry at least one deltaF508, 394delTT or 3659delC copy. Including the latter two in a screening programme would thus substantially reduce the risk of a false-negative outcome.     

Genotype-phenotype relationship in 12 patients carrying cystic fibrosis mutation R334W.

We present a phenotype-genotype correlation analysis in 12 patients with cystic fibrosis (CF) carrying the mutation R334W in the CFTR gene. The clinical data obtained for this group were compared with the clinical data of deltaF508/deltaF508 patients. Current age and age at diagnosis were significantly higher in the R334W mutation group (p=0.028 and p=0.0001). We found a lower rate of Pseudomonas aeruginosa colonisation in patients carrying the R334W mutation, although the difference was not found to be statistically significant. However, we found a statistically significant higher age of onset of Pseudomonas aeruginosa colonisation (p=0.0036) in the group of patients with the R334W mutation. Thirty three percent of R334W patients were pancreatic insufficient, significantly lower than the deltaF508/deltaF508 patients (p=0.004). We also found that the weight expressed as a percentage of ideal weight for height was significantly higher in patients with the R334W mutation (p=0.0028).     

Cystic fibrosis mutations among African Americans in the southeastern United States.

Since the cloning of the cystic fibrosis (CF) gene and the identification of delta F508, the most common CF mutation, screening the general population for CF has been vigorously debated. Adding to the controversy is the question of whether screening should be offered to African Americans, whose incidence of CF (1/17,000) is much lower than that of whites (1/2500). We tested for five common mutations (delta F508, G551D, G542X, R553X, and N1303K) in order to determine the frequency of common mutations in African Americans with CF from the southeastern United States. delta F508 was found on 50% of CF chromosomes; 46% of CF mutations were undetermined mutations. Our data indicate that at the current detection rate, the sensitivity of CF screening in African Americans would be appreciably lower than that of whites, and thus their inclusion in screening programs probably would not be warranted.     

Analysis of 40 known cystic fibrosis mutations in South African patients

A total of 140 South African (SA) Caucasoid cystic fibrosis (CF) families were analysed for the common CF mutation, ΔF508. The 52 non-ΔF508 CF chromosomes in a subset of 127 of these families were also tested for 39 other known CF mutations. The most frequent mutation, apart from ΔF508 (which occurs at a frequency of 79%), was G542X (1.3%). Four other mutations, R553X, S549N, 621 + 1G→T and N1303K, were each found in single families. The other 35 mutations remained unidentified in this sample of CF families. Since 83% of SA Caucasoid CF mutations have been identified, diagnosis by mutation analysis will be possible in only 69% of CF cases. When a diagnosis has been confirmed by a positive sweat test, a combination of linked marker analysis and mutation detection will be necessary if prenatal diagnosis and carrier detection are to be offered in the remaining families.     

Pancreatic insufficiency and pulmonary disease in German and Slavic cystic fibrosis patients with the R347P mutation

Cystic fibrosis (CF) is caused by mutations in the gene for the cystic fibrosis transmembrane conductance regulator (CFTR) that codes for a cAMP-regulated chloride channel. The R347P is a missensemutation located within the first membrane spanning domain (MSD1,) of the CFTR protein. This mutation occurs with an overall worldwide frequency of about 0.2%. The patients, originally described with this mutation were compound heterozygotes with the ΔF508 mutation and had a very mild course of CF, suggesting that R347P, similar to other missense mutations affecting the MSDl domain, causes a mild phenotype. We report here a group of 19 CF patients with the R347P mutation of German, Bulgarian, Czech, and Slovak origin, including two homozygotes. Most patients presented with early disease onset, pancreas insufficiency (PI), and early pulmonary involvement, suggesting that this mutation can lead to a severe course of CF. Most R347P alleles in the group studied share a common polymorphic haplotype. In addition, these analyses gave evidence for recurrence of the mutation in two CF patients of German and Czech origin. © 1995 Wiley-Liss, Inc.     

Respiratory epithelial gene expression in patients with mild and severe cystic fibrosis lung disease

Despite having identical cystic fibrosis transmembrane conductance regulator genotypes, individuals with DeltaF508 homozygous cystic fibrosis (CF) demonstrate significant variability in severity of pulmonary disease. This investigation used high-density oligonucleotide microarray analysis of nasal respiratory epithelium to investigate the molecular basis of phenotypic differences in CF by (1) identifying differences in gene expression between DeltaF508 homozygotes in the most severe 20th percentile of lung disease by forced expiratory volume in 1 s and those in the most mild 20th percentile of lung disease and (2) identifying differences in gene expression between DeltaF508 homozygotes and age-matched non-CF control subjects. Microarray results from 23 participants (12 CF, 11 non-CF) met the strict quality control guidelines and were used for final data analysis. A total of 652 of the 11,867 genes identified as present in 75% of the samples were significantly differentially expressed in one of the three disease phenotypes: 30 in non-CF, 53 in mild CF, and 569 in severe CF. An analysis of genes differentially expressed by severity of CF lung disease demonstrated significant upregulation in severe CF of genes involved in protein ubiquination (P < 0.04), mitochondrial oxidoreductase activity (P < 0.01), and lipid metabolism (P < 0.03). Analysis of genes with decreased expression in patients with CF compared with control subjects demonstrated significant downregulation of genes involved in airway defense (P < 0.047) and protein metabolism (P < 0.048). This study suggests that differences in CF lung phenotype are associated with differences in expression of genes involving airway defense, protein ubiquination, and mitochondrial oxidoreductase activity and identifies specific new candidate modifiers of the CF phenotype.     

Analysis of linkage disequilibrium between different cystic fibrosis mutations and three intragenic microsatellites in the Italian population

Three intragenic microsatellites of the CFTR gene, a TA and a CA repeats, namely IVSl7bTA and IVSl7bCA, located in intron 17b and a CA repcat (IVS8CA) located in intron 8 of the CFTR gene, were analyzed in a large sample of Italian cystic fibrosis (CF) and normal chromosomes. Linkage disequilibrium was evaluated between each marker and different CF mutations on a total of 377 CF and 358 normal chromosomes. Our results are consistent with the hypothesis that all AF508 chromosomes derive from a single mutational event. The same hypothesis is valid for mutations G542X, N1303K, 1717-1IG→, which might have been originated more recently than δF508. © 1995 Wiley- Liss, Inc.     

A Single-tube multiplex system for the simultaneous detection of 10 common cystic fibrosis mutations

We describe a nonisotopic single-tube polymerase chain reaction (PCR) multiplex system that detects 10 of the more common cystic fibrosis (CF) mutations (ΔF508, ΔI507, V520F, G551D, G542X, R553X, R117H, 621 + lG→T, N1303K, A455E). The use of this method detects approximately 90% of the CF alleles in the British population. Five exons of the CF gene are amplified simultaneously by PCR, followed by an overnight triple enzyme restriction digest, and then resolved by high-resolution gel acrylamide electrophoresis. © 1995 Wiley-Liss, Inc.     

Informativity of intragenic microsatellites for carrier detection and prenatal diagnosis of cystic fibrosis in the Italian population

Molecular diagnosis of cystic fibrosis (CF) in the Italian population, based on the detection of the deltaF508 mutation (51.2% of CF chromosomes), provides full informativity for prenatal diagnosis (PDN) in about 28% of families at risk. Identification of the predominant non-deltaF508 mutations allows the characterization of about 70% of CF chromosomes, making approximately 48% of couples fully informative. In families where at least one chromosome remains uncharacterized, allele segregation is still determined using RFLPs closely linked to the CF gene. The recent identification of three polymorphic clusters of dinucleotide repeats (IVS8/ GT, IVS17b/TA and IVS17b/CA) led us to evaluate whether their analysis might improve feasibility studies for prenatal diagnosis or hetero-zygote identification. One hundred nuclear families with a CF child, reflecting the general Italian deltaF508 mutation distribution, were geno-typed for the three microsatellites. In this study microsatellite analysis using IVS8/GT and IVS17b/TA allowed the identification of both parental CF chromosomes in 94% of couples; inclusion in the study of the less polymorphic repeat locus, IVS17b/CA, slightly improved this percentage (97%). Hence, a strategy involving primarily the detection of the delta F508 mutation and secondarily microsatellite analysis makes possible PDN of CF in virtually all Italian CF families.