Clinical evidence that V456A is a Cystic Fibrosis causing mutation in South Asians.

BACKGROUND: Cystic Fibrosis (CF) genotypes in South Asians are variable with a decreased incidence of Delta F508 and an increased incidence of novel mutations. The objective of this study is to provide clinical evidence that V456A, a novel mutation in South Asian Cystic Fibrosis patients, can cause significant lung disease. METHODS: We extracted clinical data from a retrospective chart review of 2 CF patients of South Asian descent. RESULTS: Patient 1, a 10 year and 11 month old Pakistani female at her initial clinic visit, required multiple hospitalizations for bronchiectasis and pulmonary infections. She was pancreatic sufficient but had slow weight gain. Genetic testing revealed that she is homozygous for the CFTR V456A mutation. Patient 2, an Indian female diagnosed with CF on newborn screening, is compound heterozygous for V456A/R709X. She had slow weight gain with BMI ranging from 12.9 to 13.4 kg/m(2) from 3 to 5 years of age and was 14.2 kg/m(2) at 6 years of age. At 6 years of age, pulmonary function tests revealed mild lung disease with FVC of 71%, FEV(1) of 75%, FEF(25-75) of 119%, and FEV(1)/FVC of 86% predicted. Sputum cultures were intermittently positive for Staphylococcus aureus and Haemophilus influenza. CONCLUSIONS: We provide evidence that V456A can cause significant pulmonary disease in South Asian Cystic Fibrosis patients.

Glycosylation and the cystic fibrosis transmembrane conductance regulator.

The cystic fibrosis transmembrane conductance regulator (CFTR) has been known for the past 11 years to be a membrane glycoprotein with chloride channel activity. Only recently has the glycosylation of CFTR been examined in detail, by O'Riordan et al in Glycobiology. Using cells that overexpress wild-type (wt)CFTR, the presence of polylactosamine was noted on the fully glycosylated form of CFTR. In the present commentary the results of that work are discussed in relation to the glycosylation phenotype of cystic fibrosis (CF), and the cellular localization and processing of DeltaF508 CFTR. The significance of the glycosylation will be known when endogenous CFTR from primary human tissue is examined.

Activity of fucosyltransferases and altered glycosylation in cystic fibrosis airway epithelial cells.

Cystic fibrosis (CF) glycoconjugates have a glycosylation phenotype of increased fucosylation and/or decreased sialylation when compared with non-CF. A major increase in fucosyl residues linked alpha 1,3 to antennary GlcNAc was observed when surface membrane glycoproteins of CF airway epithelial cells were compared to those of non-CF airway cells. Importantly, the increase in the fucosyl residues was reversed with transfection of CF cells with wild type CFTR cDNA under conditions which brought about a functional correction of the Cl(-) channel defect in the CF cells. In contrast, examination of fucosyl residues in alpha 1,2 linkage by a specific alpha 1,2 fucosidase showed that cell surface glycoproteins of the non-CF cells had a higher percentage of fucose in alpha 1,2 linkage than the CF cells. Airway epithelial cells in primary culture had a similar reciprocal relationship of alpha 1,2- and alpha 1,3-fucosylation when CF and non-CF surface membrane glycoconjugates were compared. In striking contrast, the enzyme activity and the mRNA of alpha 1,2 fucosyltransferase did not reflect the difference in glycoconjugates observed between the CF and non-CF cells. We hypothesize that mutated CFTR may cause faulty compartmentalization in the Golgi so that the nascent glycoproteins encounter alpha 1,3FucT before either the sialyl- or alpha 1,2 fucosyltransferases. In subsequent compartments, little or no terminal glycosylation can take place since the sialyl- or alpha 1,2 fucosyltransferases are unable to utilize a substrate, which is fucosylated in alpha 1,3 position on antennary GlcNAc. This hypothesis, if proven correct, could account for the CF glycophenotype.

Nuclear translocation of lactosylated poly-L-lysine/cDNA complex in cystic fibrosis airway epithelial cells.

Poly-l-lysine, with 40% of its amino groups substituted with lactose, is an effective vector to transfer the CFTR gene into CF airway epithelial cells and correct the chloride channel dysfunction. The intracellular fate of the lactosylated poly-l-lysine/cDNA complex was studied using confocal microscopy. In the presence of chloroquine the complex remained intact during internalization, intracellular transport, and, most importantly, transport into the nucleus. When cells were transfected in the presence of agents that enhance transfection efficiency such as E5CA peptide, a fusogenic peptide, or glycerol a similar fate of the lactosylated poly-l-lysine/cDNA complex was seen. However, when these agents were omitted from the transfection medium, the complex remained in the perinuclear region. Uncomplexed lactosylated poly-l-lysine reached the nucleus efficiently. In contrast mannosylated poly-l-lysine or unsubstituted poly-l-lysine complexed to plasmid did not. Therefore the nuclear accumulation of the complex may be attributed to the substitution of poly-l-lysine with lactose. It is hypothesized that the lactose residues provide for nuclear localization by means of targeting a potential lectin-like protein with galactose/lactose specificity. This mechanism may be responsible for the nuclear internalization of the complex.

Terminal glycosylation in cystic fibrosis (CF): a review emphasizing the airway epithelial cell.

Altered terminal glycosylation, with increased fucosylation and decreased sialylation is a hallmark of the cystic fibrosis (CF) glycosylation phenotype. Oligosaccharides purified from the surface membrane glycoconjugates of CF airway epithelial cells have the Lewis x, selectin ligand in terminal positions. This review is focused on the investigations of the glycoconjugates of the CF airway epithelial cell surface. Two of the major bacterial pathogens in CF, Pseudomonas aeruginosa and Haemophilus influenzae, have binding proteins which recognize fucose in alpha-1,3 linkage and asialoglycoconjugates. Therefore, consideration has been given to the possibility that the altered terminal glycosylation of airway epithelial glycoproteins in CF contributes to both the chronic infection and the robust, but ineffective, inflammatory response in the CF lung. Since the glycosylation phenotype of CF airway epithelial cells have been modulated by the expression of wtCFTR, the hypotheses which have been proposed to relate altered function of CFTR to the regulation of the glycosyltransferases are discussed. Understanding the effects of mutant CFTR on glycosylation may provide further insight into the regulation of glycoconjugate processing as well as new approaches to the therapy of CF.

Gene therapy of cystic fibrosis (CF) airways: a review emphasizing targeting with lactose.

Cystic fibrosis is a disease for which a number of Phase I clinical trials of gene therapy have been initiated. Several factors account for the high level of interest in a gene therapy approach to this disease. CF is the most common lethal inherited disease in Caucasian populations. The lung, the organ that is predominantly responsible for the morbidity and mortality in CF patients, is accessible by a non-invasive method, the inhalation of aerosols. The vectors employed in the Phase I trials have included recombinant adenoviruses, adeno-associated viruses and cationic lipids. While there have been some positive results, the success of the vectors until now has been limited by either immunogenicity or low efficiency. A more fundamental obstacle has been the absence of appropriate receptors on the apical surface of airway epithelial cells. Molecular conjugates with carbohydrate substitution to provide targeting offer several potential advantages. Lactosylated polylysine in which 40% of the lysines have been substituted with lactose has been shown to provide a high efficiency of transfection in primary cultures of CF airway epithelial cells. Other important features include a relatively low immunogenicity and cytotoxicity. Most importantly, the lactosylated polylysine was demonstrated to give nuclear localization in CF airway epithelial cells. Until now, most non-viral vectors did not have the capability to provide nuclear localization. These unique qualities provided by the lactosylation of non-viral vectors, such as polylysine may help to advance the development of molecular conjugates sufficiently to warrant their use in future clinical trials for the gene therapy of inherited diseases of the lung.

Prospective evaluation of growth, nutritional status, and body composition in children with cystic fibrosis.

BACKGROUND: Several cross-sectional studies have shown improvement in the growth of children with cystic fibrosis (CF) because of increased awareness of and more comprehensive care of their special nutritional needs. However, longitudinal data on the nutritional status of these children are rare. OBJECTIVE: The objective was to compare changes in growth, body composition, and nutritional status between children with and without CF. DESIGN: This was a prospective 3-y cohort study of 25 children aged 5-10 y with CF, mild pulmonary disease, and pancreatic insufficiency and of 26 healthy control children. Three methods were used to assess body composition: measurements of skinfold thickness, total body water by deuterium oxide, and total-body electrical conductivity. Growth and body-composition changes over time were analyzed by a longitudinal mixed-effects model. RESULTS: Over the 3 y of the study, the statural growth of the boys with CF was slower than that of the control subjects (P = 0.004). The same divergence over time between the boys with and without CF was observed for fat-free mass assessed by skinfold-thickness measurements and total body water (P = 0.008 and 0.02, respectively) and for fat mass assessed by skinfold-thickness measurements and total-body electrical conductivity (P = 0.009 and 0.001, respectively). The differences in the pattern of changes in growth and body composition were less striking for girls. CONCLUSIONS: Despite comprehensive care, the growth of boys with CF was impaired on the basis of height, fat-free mass, and fat mass, when observed longitudinally. Caution should be used when interpreting cross-sectional measurements because they often do not detect suboptimal growth.

A prospective study of body composition changes in children with cystic fibrosis.

Recent cross-sectional studies of children with cystic fibrosis (CF) have shown an improvement in their growth with improved nutritional status, but there are only a few longitudinal studies in this population. A four-year prospective study was conducted in 25 children with CF and 26 controls, ages 5 to 10 years at baseline, to describe changes in body composition using three independent methods of assessment: skinfold thickness, total body water by deuterium dilution, and total body electrical conductivity (TOBEC). The statural growth of the boys with CF was slower than that of the control boys. Using different methods, the fat-free mass and fat-mass increases were shown to be slower in boys with CF than in controls. These differences were less significant in girls. Despite comprehensive care, the growth of boys with CF may still not be optimal, as observed longitudinally.

Terminal glycosylation of cystic fibrosis airway epithelial cells.

Cystic fibrosis (CF) has a characteristic glycosylation phenotype usually expressed as a decreased ratio of sialic acid to fucose. The glycosylation phenotype was found in CF/T1 airway epithelial cells (deltaF508/deltaF508). When these cells were transfected and were expressing high amounts of wtCFTR, as detected by Western blot analysis and in situ hybridization, the cell membrane glycoconjugates had an increased sialic acid content and decreased fucosyl residues in alpha1,3/4 linkage to antennary N-acetyl glucosamine (Fuc(alpha)1,3/4GlcNAc). After the expression of wtCFTR decreased, the amount of sialic acid and Fuc(alpha)1,3/4GlcNAc returned to levels shown by the parent CF cells. Sialic acid was measured by chemical analysis and Fuc(alpha)1,3/4GlcNAc was detected with a specific alpha1,3/4 fucosidase. CF and non-CF airway cells in primary culture also had a similar reciprocal relationship between fucosylation and sialylation. It is possible that the glycosylation phenotype is involved in the pathogenesis of CF lung disease by facilitating bacterial colonization and leukocyte recruitment.

Terminal glycosylation and disease: influence on cancer and cystic fibrosis.

Terminal glycosylation has been a recurring theme of the laboratory. In cystic fibrosis (CF), decreased sialic acid and increased fucosyl residues in alpha1,3 position to antennary N-acetyl glucosamine is the CF glycosylation phenotype. The glycosylation phenotype is reversed by transfection of CF airway cells with wtCFTR. In neuronal cells, polymers of alpha2,8sialyl residues are prominent in oligodendrocytes and human neuroblastoma. These findings are discussed in relationship to early studies in our laboratories and those of other investigators. The potential extension of these concepts to future clinical therapeutics is presented.

Terminal glycosylation in cystic fibrosis.

Cystic fibrosis (CF) is a common genetic disease for which the gene was identified within the last decade. Pulmonary disease predominates in this ultimately fatal disease and current therapy only slows the progression. CF transmembrane regulator (CFTR), the gene product, is an integral membrane glycoprotein that normally functions as a chloride channel in epithelial cells. The most common mutation, deltaF508, results in mislocalization and altered glycosylation of CFTR. Altered fucosylation and sialylation are hallmarks of both membrane and secreted glycoproteins in CF and the focus here is on these investigations. Oligosaccharides from CF membrane glycoproteins have the Lewis x, selectin ligand in terminal positions. In addition, two major bacterial pathogens in CF, Pseudomonas aeruginosa and Haemophilus influenzae, have binding proteins, which recognize fucose in alpha1,3 linkage and asialoglycoconjugates. We speculate that the altered terminal glycosylation of airway epithelial glycoproteins in CF contributes to the chronic infection and robust inflammatory response in the CF lung. Understanding the effects of mutant CFTR on glycosylation may provide further insight into the regulation of glycoconjugate processing as well as therapy for CF.

Enhanced efficiency of lactosylated poly-L-lysine-mediated gene transfer into cystic fibrosis airway epithelial cells.

Lactosylated poly-L-lysine is a nonviral vector that transfers genes into airway epithelial cells, including those from individuals with cystic fibrosis (CF). Substitution of 40% of the epsilon-amino groups of poly-L-lysine with lactosyl residues not only provided a ligand for receptor-mediated endocytosis, but also reduced the toxicity when compared with nonsubstituted poly-L-lysine. Lactosylated poly-L-lysine/pCMVLuc complex is not toxic to cells in amounts that gave the maximum gene expression. The level of gene expression was regulated by using different combinations of chloroquine, glycerol, and E5CA peptide. Using cultured CF cells, chloroquine, combined with E5CA peptide, increased the transfer of the pCMVLuc/ lactosylated poly-L-lysine complex 10,000-fold compared with transfer without additives. In many systems, a high efficiency is of paramount importance and the enhancing agents can be used to modulate the expression of the gene. For example, transfer of pCMVLacZ/lactosylated poly-L-lysine complexes with chloroquine added to the transfection medium gave only 20% transfection efficiency of the reporter gene. However, when chloroquine was combined with glycerol, the efficiency was increased to 90%, thus approaching that reported with viral vectors. This highly efficient vector may be of great value for the future development of gene transfer systems.

High-efficiency transfer of cystic fibrosis transmembrane conductance regulator cDNA into cystic fibrosis airway cells in culture using lactosylated polylysine as a vector.

To find more efficient vectors for the transfer of CFTR cDNA, lactosylated polylysine was explored for transfer into airway epithelial cells in primary culture. The efficacy and high efficiency of transfection were shown by several criteria: expression of both mRNA and protein for CFTR and the functional correction of the Cl- channel activity. Using specific combinations of agents to enhance the transfection, an efficiency of 90% was obtained as detected by in situ hybridization with digoxigenin-labeled probes generated against exon 14 of CFTR. The highest efficiency was observed by adding E5CA peptide (10 microg) and 5% glycerol to the transfection mixture. The degree of transfection could be controlled by the enhancing agents, thus modulating the efficiency of transfection. The highest level of transfection efficiency is equivalent to that reported for viral vectors. None of the agents or their combinations in the concentrations used were cytotoxic to the primary cells. Antibody pAb3145 was used to detect the expression of the CFTR protein in the cells. When an N-terminal GFP-CFTR fusion gene was used to transfect the CF cells a functional correction of the CFTR Cl- channel was detected by patch-clamp electrophysiology. The high efficiency of CFTR gene transfer with lactosylated polylysine leads to the conclusion that lactosylated polylysine is a promising vector to transfer the CFTR gene into human airway cells in culture.

A six-month study of growth and energy expenditure in children with cystic fibrosis taking a pulmonary inhalation medication (rhDNase).

OBJECTIVE: To characterize the effects of recombinant human deoxyribonuclease (rhDNase) on growth velocity, body composition, resting energy expenditure (REE) and food intake in children with cystic fibrosis (CF). METHODS: A prospective, six-month pilot study was conducted in twenty-one subjects with CF (twelve male, nine female, ages 11.5+/-3.1 years) measured at baseline, two and six months post-baseline. Repeated measures ANOVA was used to examine the change in variables across time. RESULTS: The majority (75%) of subjects had minimal lung disease at baseline (FEV1: 80%-119% predicted). As expected for growing children, weight and height gains (1.6 kg and 2.5 cm) were observed between baseline and six months (p=0.0001). No change was observed in weight z-scores from six months prior to initiation of rhDNase therapy to six months post, though a significant decline (p=0.049) in Ht z-score was observed over this twelve-month period. Triceps skinfolds and mid-arm muscle circumference increased from baseline to six months (p<0.01); respective z-scores remained stable. Energy intake remained constant during the period it was studied from baseline to two months of therapy: 120%+/-27% RDA. REE, though slightly elevated compared to healthy children (baseline 106%+/-8% predicted), remained stable throughout the study and at a level which may be expected for children with minimal lung disease. A trend (p=0.057) towards a decrease in the number of subjects requiring hospitalization for pulmonary exacerbations during the trial period was observed. CONCLUSIONS: In summary, these pilot data from younger children with milder CF-related lung disease do not confirm anecdotal reports of improved rate of weight gain, caloric intake or decreases in the elevated REE. Future research might focus on documentation of the possible nutritional effects of rhDNase in clinical trials of children with more severe lung disease.

Acute pulmonary exacerbation is not associated with increased energy expenditure in children with cystic fibrosis.

OBJECTIVE: The objective of this study was to document alterations in resting energy expenditure (REE), energy intake, and body composition that occur during admission for management of a pulmonary exacerbation in children with cystic fibrosis. STUDY DESIGN: A prospective study of 14 children with cystic fibrosis (8 male, 12.5 +/- 3.3 years) was conducted throughout a 14-day hospitalization and again 3 weeks after discharge. Repeated measures analysis of variance was used to examine the change in variables across time. RESULTS: Significant increases were documented from days 1 to 14 in weight (37.1 +/- 13.1 kg to 38.4 +/- 13.5 kg) and pulmonary function (forced expiratory volume in 1 second: 67.6% +/- 19.8% to 78.5% +/- 24.5%). Energy intake was not different between hospitalization and follow-up and averaged 175% of the Recommended Dietary Allowance. REE on day 1 was elevated (122% +/- 11% predicted); however, when expressed with fat free mass as a covariate, no significant changes were noted in REE throughout the eight measurements. CONCLUSIONS: These data suggest that an acute pulmonary exacerbation is not associated with an increase above a subject's baseline REE in children with cystic fibrosis with mild to moderate lung disease. Management of an acute pulmonary exacerbation should be associated with sustained improvement in pulmonary function and nutritional status; in contrast, weight loss or failure to improve pulmonary function should be seen as atypical.

Prospective evaluation of resting energy expenditure, nutritional status, pulmonary function, and genotype in children with cystic fibrosis.

Growth failure and malnutrition are common clinical features in cystic fibrosis (CF), but the relationships among resting energy expenditure (REE), pulmonary function, and nutritional status, are poorly understood. To better understand these relationships, REE, growth, nutritional status, and pulmonary function were measured prospectively in 25 prepubertal children with CF and 26 prepubertal control subjects of similar age and gender over a 3-y period. All subjects with CF had pancreatic insufficiency and mild pulmonary disease. REE was elevated for the CF children compared with control subjects throughout the study. This increased REE was not associated with declining pulmonary function. Longitudinal analyses revealed different patterns of change over time in boys and girls, such that REE significantly increased in the girls with CF and pulmonary function decreased in the boys. Boys with CF experienced a decline in weight Z score and percent ideal body weight, whereas the girls with CF experienced a decline in height Z score. Pulmonary function was not associated with REE, but nutritional status (percent ideal body weight) and genotype (delta F508 homozygotes versus others) were predictive of changes in pulmonary function over time. Fat free mass and height were found to be the best predictors of REE, and after accounting for these important body size and composition variables, differences in REE between boys and girls and CF and control groups increased over time. These findings identify the importance of investigating gender differences in the course of disease and considering REE as an early indicator of disease severity independent of pulmonary function.

Gluconoylated and glycosylated polylysines as vectors for gene transfer into cystic fibrosis airway epithelial cells.

To provide an alternative to viral vectors for the transfer of genes into airway epithelial cells in cystic fibrosis (CF), a novel set of substituted polylysines were employed. Polylysine was partially neutralized by blocking a number of positively charged residues with gluconoyl groups. In addition, polylysine was substituted with sugar residues on a specified number of amino groups. Using the gluconoylated polylysine as vector, the pCMVLuc plasmid gave high expression of the reporter gene luciferase in immortalized CF/T43 cells. The luciferase activity was 75-fold greater in the presence of 100 microM chloroquine. Luciferase gene expression persisted at high levels for up to at least 120 hr following transfection. Glycosylated polylysines/pCMVLuc complexes were compared to the gluconoylated polylysine/pCMVLuc complex and beta-Gal-, alpha-Glc-, and Lac-substituted polylysines gave 320%, 300%, and 290%, respectively, higher expression of the reporter gene luciferase. Luciferase expression ranged from 35 to 2 ng of luciferase per milligram of cell protein in the order: beta-Gal = alpha-Glc = Lac > alpha-Gal = Rha = Man > beta-GalNAc > alpha-GalNAc = alpha-Fuc, suggesting that the transfection efficiency is sugar dependent. Most importantly, in primary cultures of both CF and non-CF airway epithelial cells grown from tracheal tissue explants, lactosylated polylysine gave uniformly high expression of luciferase. The glycosylated polylysines provide an attractive nonviral approach for the transfer of genes into airway epithelial cells.

Turnover of the cystic fibrosis transmembrane conductance regulator (CFTR): slow degradation of wild-type and delta F508 CFTR in surface membrane preparations of immortalized airway epithelial cells.

The protein product of the cystic fibrosis (CF) gene, termed the cystic fibrosis transmembrane conductance regulator (CFTR), is known to function as an apical chloride channel at the surface of airway epithelial cells. It has been proposed that CFTR has additional intracellular functions and that there is altered processing of mutant forms. In examining these functions we found a stable form of CFTR with slow turnover in surface membrane preparations from CF and non-CF immortalized airway epithelial cell lines. The methods used to study the turnover of CFTR were pulse/chase experiments utilizing saturation labeling of [35S] Met with chase periods of 5-24 h in the presence of 8 mM Met and cell fractionation techniques. Preparations of morphologically identifiable surface membranes were compared to total cell membrane preparations containing intracellular membranes. Surface membrane CFTR had lower turnover defined by pulse/chase ratios than that of the total cell membrane preparations. Moreover, mutant CFTR was stable in the surface membrane fraction with little degradation even after a 24 h chase, whereas wild-type CFTR had a higher pulse/chase ratio at 24 h. In the presence of 50 microM castanospermine, which is an inhibitor of processing alpha-glucosidases, a more rapid turnover of mutant CFTR was found in the total cell membrane preparation, whereas wild-type CFTR had a lower response. The results are compatible with a pool of CFTR in or near the surface membranes which has an altered turnover in CF and a glycosylation-dependent alteration in the processing of mutant CFTR.