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.     

DNA analysis and gene diagnosis of cystic fibrosis]

Cystic fibrosis (CF) is the most common autosomal recessive disorder in the Caucasian population, affecting approximately 1 in 2,000 newborns but the actual estimate varies with the geographic location. The incidence of CF in non-Caucasian populations is low. Intensive efforts using genetic linkage information ultimately led to the cloning of the CF gene prior to the identification of the gene product or its function. The gene encodes what is believed to be a transmembrane protein, which has been named the cystic fibrosis transmembrane conductance regulator (CFTR). The CFTR contains two nucleotide-binding folds (NBF) which show homology to numerous transport proteins with the greatest homology to the P-glycoproteins that are encoded by the multiple drug-resistance loci. A three- base deletion resulting in the loss of phenylalanine residue (delta F508) in the tenth exon of the CFTR gene is the mutation occurring on the majority of CF chromosomes. The overall frequency of delta F508 in the present mutant CF gene pool is about 70%, but the study populations are not equally represented: there is marked variation in the population of delta F508 among different geographic populations. Recently, numerous additional, less common mutations have been found. Some mutations occur on 2-5% of the CF chromosomes. Many of these are rare 'private' mutations, occurring in individual families of all racial and ethnic backgrounds. By contrast over 80% of Western European CF mutations have been identified. The highly heterogeneous nature of the remaining CF mutations provides important insights into the structure and function of the protein, but further improvements are needed in DNA-based genetic screening for CF carrier status.     

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.     

The molecular basis of cystic fibrosis in South Africa

The spectrum of CFTR mutations in three South African populations is presented. To date. a total of 192 white patients (384 chromosomes) with confirmed CF have been tested. deltaF508 accounts for 76% of the CF chromosomes in this group, with 3272-26A-->G, 394delTT and G542X occurring at the following frequencies: 4, 3.6 and 1.3%, respectively. A further 11 mutations account for 6% of CF chromosomes. A total of 91% of the CF-causing mutations can now be detected in the South African white population. Haplotype analysis suggests a founder effect in South Africans of European origin for the two common CFTR mutations, 3272-26A-->G and 394delTT. The diagnosis of CF has been confirmed in 14 coloured and 12 black CF patients. In the coloured population, both the deltaF508 and 3120 + 1G-->A mutations occur at appreciable frequencies of 43 and 29%, respectively. In the black population, the most common CF-causing mutation, the 3120 + 1G-->A mutation, occurs at an estimated frequency of 46%. Four other mutations have been detected, resulting in the identification of a total of 62.5% of mutations in this population.     

Partial CFTR genotyping and characterisation of cystic fibrosis patients with myocardial fibrosis and necrosis

Myocardial necrosis and fibrosis is a rare complication of cystic fibrosis (CF) causing sudden and unexpected death in infancy due to cardiac arrest. Characteristic morphological lesions are recognisable postmortem. The 18 CF patients with this complication had varied clinical features including mild pulmonary involvement, early onset severe pancreatic insufficiency, and profound electrocardiogram (ECG) changes. In this group of patients, 5 were deltaF508 homozygotes, 1 was deltaF508/ N1303K and 1 was a deltaF508/M compound heterozygote. A pair of affected siblings (deltaF508 homozygotes) were fully concordant for myocardial involvement and for the general course of the disease. The co-existence of a genetic predisposition to myocardial lesions resulting most probably from severe cystic fibrosis transmembrane (CFTR) genotypes (such as deltaF508/deltaF508, deltaF508/N1303K) and deficiency of certain trophic factors necessary for metabolism of the myocardium, are postulated to cause myocardial complications in CF leading to circulatory failure and early death.     

Calculating posterior cystic fibrosis risk with echogenic bowel and one characterized cystic fibrosis mutation: avoiding pitfalls in the risk calculations

We describe a general approach to derive fetal risk following two separate test results that each raise the likelihood of the same fetal abnormality without clearly determining whether the abnormality exists. Echogenic bowel observed on fetal ultrasonography may have multiple causes, including an a priori risk of approximately 1% of cystic fibrosis (CF). On numerous occasions our laboratory tests have detected only normal cystic fibrosis transmembrane regulator (CFTR) alleles in fetuses with echogenic bowel. This result indicates that another cause most likely explains the abnormal ultrasound finding. One of our tested fetuses was heterozygous for the deltaF508 CFTR mutation and had a normal karyotype. Over 770 CFTR mutations have been described, and a significant proportion of parental mutant alleles could not be detected by our 25-mutation test. Further mutation analysis demonstrated that the fetus' mother carried the deltaF508 mutation but the father (of different ethnic background than the mother) did not carry a detectable mutation. Thus, this test result substantially increased the risk of the fetus having CF, while still not giving a definitive answer to whether the fetus was affected. A rigorous mathematical analysis determined that the 1% risk of CF following ultrasound study was increased to slightly under 12% following DNA analysis. The case is described, and the mathematical formulas are explained and illustrated with examples, along with a review of conditional probability (Appendix 2).     

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.     

Molecular analysis of the cystic fibrosis gene reveals a high frequency of the intron 8 splice variant 5T in Egyptian males with congenital bilateral absence of the vas deferens

It has previously been shown that defects in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are largely responsible for the condition of congenital bilateral absence of the vas deferens (CBAVD), without associated renal abnormalities, in Caucasian populations. To assess the involvement of the CFTR in CBAVD in a population with presumed low cystic fibrosis (CF) frequency, we have analysed 20 CBAVD males from Egypt for the presence of 12 common Caucasian CFTR mutations and the intron 8 5T splice variant, IVS-5T, known to be a major cause of CBAVD in Caucasian patients. In 16 of the males without associated renal abnormalities only one deltaF508 carrier was identified, but an exceptionally high frequency of the IVS-5T variant was found (14 of 32 alleles or 43.7%), confirming that this variant is involved in many cases of CBAVD, even in populations where CF is rare. CFTR mutations or the IVS-5T variant were found neither in the remaining four patients with associated renal abnormalities nor in the spouses of the 20 CBAVD patients. However, one patient was homozygous for a leucine to proline substitution at amino acid position 541 (L541P) of the CFTR. It is as yet not clear whether this change is involved in CBAVD in this male.     

CFTR gene mutations in sarcoidosis

Sarcoidosis is a complex disease of multiorgan granulomatous inflammation. Genetic susceptibility is involved in the pathogenesis of the disorder. Two successive studies from Italy have shown a high frequency of mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in patients suffering from sarcoidosis. We have genotyped a panel of 63 families with two or more affected siblings for the CFTR gene mutation R75Q, which was found to be present in three of 26 cases of the Italian study. Although R75Q was present in seven families, it was neither associated with the sarcoidosis phenotype in the German population (P=0.5), nor was it linked to sarcoidosis (P=0.54). In addition, a screening for 34 functional CFTR mutations was performed in a subset of 54 patients from 25 families. These patients were known to be concordant for at least one parental copy of the CFTR gene. With the exception of the mayor CF mutation deltaF508, which was present in three patients and absent in one patient from two families, we did not find any other CF mutation in these 54 patients. Our results do not support the hypothesis that CFTR mutations have a major influence on the pathogenesis of sarcoidosis.     

Screening for CF mutations in adult cystic fibrosis patients with a directed and optimized SSCP strategy

Twenty adolescent and adult cystic fibrosis (CF) patients have been studied for the presence of mutations in the CFTR gene. Mutations other than deltaFSOS have been detected by comparison to the single-stranded conformation polymorphism (SSCP) pattern of known mutations in eight exons, in which 80% of the more common mutations are present. Each mutation was confirmed by direct sequencing. For each of the analyzed exons, optimal SSCP conditions have been determined that allow all available known mutations in that exon to be distinguished from each other. This approach allowed mutations to be defined in 75% of the non deltaF508 alleles and 92% of all CF alleles in this cohort. © 1994 Wiley-Liss, Inc.     

Cystic fibrosis transmembrane conductance regulator gene mutations: do they play a role in the aetiology of allergic bronchopulmonary aspergillosis?

BACKGROUND: Previous work suggests that cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations may be implicated in the aetiology of allergic bronchopulmonary aspergilosis (ABPA). OBJECTIVE: To compare the frequency of CF gene mutations in asthmatics with ABPA of varying severity with asthmatics who were skin prick test (SPT)-positive to Aspergillus fumigatus (Af) without evidence of ABPA and asthmatics SPT-negative to Af. METHODS: Thirty-one Caucasian patients with ABPA were identified, together with asthmatics SPT positive to Af without evidence of ABPA (n = 23) and SPT negative to Af (n = 28). Genomic DNA was tested for 16 CF mutations accounting for approximately 85% of CF alleles in Caucasian New Zealanders. RESULTS: Four (12.9%) ABPA patients were found to be carriers of a CF mutation (DeltaF508 n = 3, R117H n = 1), one (4.3%) asthmatic SPT positive to Af without ABPA (DeltaF508), and one (3.6%) asthmatic SPT negative to Af (R117H). All patients with a CF mutation had normal sweat chloride (< 40 mM). There was no significant difference between the frequency of CF mutations in the ABPA patients and asthmatics without ABPA. However, the frequency of CF mutations in the ABPA patients was significantly different (P = 0.0125) to the expected carrier rate in the general population. CONCLUSION: These results lend further support to a possible link between CF mutations and ABPA.     

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).     

A step forward on the path towards understanding osteoporosis

Li W, Sun L, Corey M, Zou F, Lee S, Cojocaru AL, Taylor C, Blackman SM, Stephenson A, Sandford AJ, Dorfman R, Drumm ML, Cutting GR, Knowles MR, Durie P, Wright FA, Strug LJ. Understanding the population structure of North American patients with cystic fibrosis. It is generally presumed that the cystic fibrosis (CF) population is relatively homogeneous, and predominantly of European origin. The complex ethnic make‐up observed in the CF patients collected by the North American CF Modifier Gene Consortium has brought this assumption into question, and suggested the potential for population substructure in the three CF study samples collected from North America. It is well appreciated that population substructure can result in spurious genetic associations. To understand the ethnic composition of the North American CF population, and to assess the need for population structure adjustment in genetic association studies with North American CF patients, genome‐wide single‐nucleotide polymorphisms on 3076 unrelated North American CF patients were used to perform population structure analyses. We compared self‐reported ethnicity to genotype‐inferred ancestry, and also examined whether geographic distribution and cystic fibrosis transmembrane regulator (CFTR) mutation type could explain the population structure observed. Although largely Caucasian, our analyses identified a considerable number of CF patients with admixed African‐Caucasian, Mexican‐Caucasian and Indian‐Caucasian ancestries. Population substructure was present and comparable across the three studies of the consortium. Neither geographic distribution nor CFTR mutation type explained the population structure. Given the ethnic diversity of the North American CF population, it is essential to carefully detect, estimate and adjust for population substructure to guard against potential spurious findings in CF genetic association studies. Other Mendelian diseases that are presumed to predominantly affect single ethnic groups may also benefit from careful analysis of population structure.     

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.     

Cystic fibrosis mutations delta F508 and G542X in Jewish patients.

We have screened our CF patients for mutations in exons 10 and 11 of the CFTR gene. Two mutations, delta F508 and G542X, have been found in 66 Jewish CF patients. The average frequency of the delta F508 mutation in the Jewish population is 33.8%. The G542X mutation accounts for 13% of the Ashkenazi CF mutations and has been found in three out of seven chromosomes of Jewish patients from Turkey (probably descended from Ashkenazi immigrants). The G542X mutation was not found in any of the other non-Ashkenazi patients. All the G542X bearing chromosomes have the same haplotype. Based on these observations it is concluded that the G542X mutation was introduced into the Jewish people after the split into Ashkenazi and non-Ashkenazi.     

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.     

CFTR (TG)m(T)n polymorphism in patients with CBAVD in a population expressing low incidence of cystic fibrosis

As it is well established that an association exists between congenital bilateral absence of the vas deferens (CBAVD) and cystic fibrosis gene mutations, we investigated CFTR(TG)m(T)n polymorphism within a Taiwanese population that exhibits a very low incidence of CF. Sixty-three patients with CBAVD and 86 age-matched normal control subjects were evaluated. Temporal temperature gradient gel electrophoresis was used for CFTR mutational analysis. No major CFTR mutation was found in the patient series. A single prominent CFTR mutation, IVS8-5T, was present; however, (50.8% of 63 cases and 33.3% of 126 alleles), and exhibited a high prevalence of 12 or 13 TG repeats (93.8% of 32 cases and 95.2% of 42 alleles with IVS8-5T). Although these results are similar to those of Japanese CBAVD patients, they are higher than the common frequency (about 21%) found among Caucasian CBAVD patients. The very high percentage (42.9%) of patients with no CFTR mutations is also an ethnic characteristic. We concluded that CBAVD patients from Taiwan, who express a very low incidence of CF, were less affected by CFTR mutations, with the exception of IVS8-5T linked to either 12 or 13 TG repeats, which does exhibit a high prevalence among CBAVD patients tested.     

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.