Mutation analysis at the cystic fibrosis locus in the British population

Summary The cystic fibrosis (CF) population of South East England seems to reflect a similar distribution in frequency of the ΔF508 mutation and its haplotype association as has been observed in the North American population.     

Abnormal localization of cystic fibrosis transmembrane conductance regulator in primary cultures of cystic fibrosis airway epithelia

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a membrane glycoprotein that forms Cl- channels. Previous work has shown that when some CF-associated mutants of CFTR are expressed in heterologous cells, their glycosylation is incomplete. That observation led to the hypothesis that such mutants are not delivered to the plasma membrane where they can mediate Cl- transport. Testing this hypothesis requires localization of CFTR in nonrecombinant cells and a specific determination of whether CFTR is in the apical membrane of normal and CF epithelia. To test the hypothesis, we used primary cultures of airway epithelia grown on permeable supports because they polarize and express the CF defect in apical Cl- permeability. Moreover, their dysfunction contributes to disease. We developed a semiquantitative assay, using nonpermeabilized epithelia, an antibody directed against an extracellular epitope of CFTR, and large (1 microns) fluorescent beads which bound to secondary antibodies. We observed specific binding to airway epithelia from non-CF subjects, indicating that CFTR is located in the apical membrane. In contrast, there was no specific binding to the apical membrane of CF airway epithelia. These data were supported by qualitative studies using confocal microscopy: the most prominent immunostaining was in the apical region of non-CF cells and in cytoplasmic regions of CF cells. The results indicate that CFTR is either missing from the apical membrane of these CF cells or it is present at a much reduced level. The data support the proposed defective delivery of some CF-associated mutants to the plasma membrane and explain the lack of apical Cl- permeability in most CF airway epithelia.     

K dependence of the Na-K pump is abnormal in erythrocytes from patients with cystic fibrosis and obligate heterozygotes

The affinity of the Na-K pump for K was significantly (P less than .001) lower in erythrocytes from patients with cystic fibrosis (Km 4.6 +/- 0.35 mM; n = 26) or from heterozygotes (Km 3.9 +/- 0.57 mM; n = 12) than in controls (Km 2.2 +/- 0.10 mM; n = 20). The affinity of the Na-K pump for K was lower in normal erythrocytes than in normal fibroblasts which may explain the variability in the severity of involvement of different organs in cystic fibrosis. We have now shown in human skin fibroblasts and erythrocytes, that the K affinity of the Na-K pump is lower in patients with cystic fibrosis than in controls. Since the abnormality is also present in erythrocytes from heterozygotes who are clinically normal, it is likely that this abnormality is closely related to the genetic defect in cystic fibrosis.     

Screening for carriers of cystic fibrosis through primary health care services.

OBJECTIVE--To evaluate the uptake of cystic fibrosis carrier testing offered through primary health care services. DESIGN--Carrier testing for cystic fibrosis was offered to patients of reproductive age through primary health care services. SETTING--Three general practice surgeries and four family planning clinics in South West Hertfordshire District Health Authority. SUBJECTS--Over 1000 patients aged 16-44 attending two general practices and four family planning clinics and a stratified random sample of patients aged 16-44 from one general practice''s age-sex register. RESULTS--When screening was offered opportunistically the uptake was 66% in general practice and 87% in family planning clinics. Ten per cent of those offered a screening appointment by letter took up the invitation. Of the screened population, 76% had previously heard of cystic fibrosis, 35% realised it is inherited, and 18% realised that carriers need not have any family history. If they found themselves in an "at risk" partnership 39% would consider not having children and 26% would consider terminating an affected pregnancy, but in each case most people were unsure how they would react. CONCLUSIONS--Most people offered a cystic fibrosis test opportunistically wish to be tested, and the responses of those tested indicate that knowledge of carrier state would be considered in future reproductive decisions.     

Mutation analysis of the cystic fibrosis transmembrane regulator gene in native American populations of the southwest

We report DNA and clinical analyses of cystic fibrosis (CF) in two previously unstudied, genetically isolated populations: Pueblo and Navajo Native Americans. Direct mutation analysis of six mutations of the CFTR gene--namely, delta F508, G542X, G551D, R553X, N1303K, and W1282X--was performed on PCR-amplified genomic DNA extracted from blood samples. Haplotype analyses with marker/enzyme pairs XV2c/TaqI and KM19/PstI were performed as well. Of the 12 affected individuals studied, no delta F508 mutation was detected; only one G542X mutation was found. None of the other mutations was detected. All affected individuals have either an AA, AC, or CC haplotype, except for the one carrying the G542X mutation, who has the haplotype AB. Clinically, six of the affected individuals examined exhibit growth deficiency, and five (all from the Zuni Pueblo) have a severe CF phenotype. Four of the six Zunis with CF are also microcephalic, a finding not previously noted in CF patients. Our DNA data have serious implications for risk assessment of CF carrier status for these people.     

Membrane function in cystic fibrosis. I. Putrescine transport in normal and cystic fibrosis fibroblasts

Putrescine transport was examined in normal and cystic fibrosis fibroblasts. No differences were observed in accumulation pattern, kinetics of uptake, or efflux between CF and normal cells. In both growing and growth-arrested CF and normal fibroblasts, exogenously supplied putrescine remained unchanged for at least 60 min. Some differences were observed in the response of CF and normal cells to environmental (media) changes.This research was supported by a grant from the Cystic Fibrosis Foundation and by a grant from the National Institutes of Health, Training Grant (GM01316 11 GNC).     

Hypertrophic osteoarthropathy in cystic fibrosis.

Seven adult patients with cystic fibrosis who had radiological evidence of hypertrophic osteoarthropathy were reviewed. In five of the patients symptoms were particularly pronounced at times of acute infective exacerbations; appropriate treatment of the infective episodes resulted in reduction or resolution of the bone pain and joint effusions. Despite this symptomatic relief periosteal changes persisted radiologically and their chronic nature was indicated by changes in the midshafts of long bones. Four of the seven patients had transient gynaecomastia or mastalgia related to infective exacerbations. It is hypothesised that a neuroendocrine mechanism--namely, release of vasoactive intestinal polypeptide--might account for the osteoarthropathy.     

Membrane function in cystic fibrosis. II. Methionine transport in normal and cystic fibrosis fibroblasts

Initial rate kinetics of methionine transport, time course of accumulation of methionine, and efflux of accumulated methionine were studied in three normal and four CF human diploid fibroblast strains. The range of apparent Km's was 12.7-32.1 micrometer for the CF strains and 18.3-39.2 micrometer for the normal strains. The range of apparent Vmax's was 6.69-9.22 nmole mg-1 min-1 for the CF strains and 5.59-7.87 nmole mg-1 min-1 for the normal strains. The patterns of accumulation and efflux are quite similar in all the strains studied except for WI-38, which showed somewhat higher efflux and lower accumulation than for others. There was no significant difference in the kinetic parameters of methionine transport between CF and normal skin fibroblasts, and methionine transport will not serve as a marker for cystic fibrosis in cultured fibroblasts.     

Insulin iontophoresis in cystic fibrosis.

Insulin and its vehicle without insulin were administered separately by iontophoresis to patients with cystic fibrosis (CF), obligate heterozygotes, and healthy controls. The resultant sweat chloride concentration after treatment with both preparations was compared in each individual. No difference after the two treatments was found in the control sample. A decrease in sweat chloride concentration after insulin iontophoresis in comparison with the vehicle was observed in both the CF (P smaller than 0.005) and heterozygote (P smaller than 0.01) samples. These observations suggest an involvement of insulin in CF and a possible role of insulin in sweat gland function.     

Probing the basic defect in cystic fibrosis.

The concurrent developments in electrophysiology studies and the identification of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has provided a unique opportunity to probe the basic cellular defect underlying cystic fibrosis. Various properties of the CFTR protein have been deduced from its primary sequence, the variety of mutations in patients and genotype-phenotype correlations, as well as the results of more recent DNA transfection studies. The most exciting observation is the fact that CFTR acts like a cAMP-regulated Cl- channel.