Cystic Fibrosis Gene Research Articles

Spectrum of CFTR Mutations on Reunion Island: Effect of Neonatal Screening

The large heterogeneity in the cystic fibrosis (CF) gene is the main difficulty for genotype characterization. Numerous studies have reported considerable variations in frequencies of CF transmembrane conductance regulator (CFTR) mutations in different populations, such as African, Asian, or European populations. To completely characterize the spectrum of mutations in the CFTR gene in the Réunion Island population, we screened 228 CF chromosomes using denaturing high-pressure liquid chromatography and denaturing gradient gel electrophoresis following by direct sequencing. We identified 27 mutations, accounting for 93% of CF chromosomes. They included three novel mutations (M1T, 3121-3C-->G, and L1324P), which are described in this paper. The detection of such a high proportion of Réunion Island CFTR mutations is important for improving neonatal screening of CF on Réunion Island.

Anti-Inflammatory Medications for Cystic Fibrosis Lung Disease

The lung disease of cystic fibrosis (CF) is characterized by a self-sustaining cycle of airway obstruction, infection, and inflammation. Therapies aimed at decreasing the inflammatory response represent a relatively new strategy for treatment. Attention has focused primarily upon the therapeutic potential of corticosteroids and NSAIDs. Although beneficial, the use of systemic corticosteroids is limited by their unacceptable adverse effects. It is unclear if inhaled corticosteroids are a viable alternative, although their use in CF has dramatically increased in recent years. High-dose ibuprofen has been shown to slow progression of CF lung disease, but its use has not been widely adopted despite a favorable risk-benefit profile. Thus, other anti-inflammatory approaches are under investigation. Since the inflammatory response can be triggered by many stimuli and since the pathways activated by these stimuli produce many mediators, there are a plethora of targets for anti-inflammatory therapeutics. Specific antibodies, receptor antagonists, and counter-regulatory cytokines, such as interleukin (IL)-10 and interferon-gamma, inhibit the pro-inflammatory mediators responsible for the damaging inflammation in the CF airway, including tumor necrosis factor-alpha, IL-1beta and IL-8. Studies of molecules that modulate intracellular signaling cascades that lead to the production of inflammatory mediators, are underway in CF. For patients with established disease, recent and projected advances in therapies that are directed at neutrophil products, such as DNase, antioxidants, and protease inhibitors, hold great promise for limiting the consequences of the inflammatory response. To optimize anti-inflammatory therapy, it is necessary to understand the mechanism of action of these agents in the CF lung to determine which agents will be most beneficial, and to determine which therapies should be initiated at what age and stage of lung disease. Hope remains that correction of the abnormal CF transmembrane conductance regulator protein or gene replacement therapy will be curative. However, correction of the basic defect must also correct the dysregulated inflammatory response in order to be effective. Until those therapies aimed at repairing the basic defect are realized, limiting the effects of the inflammatory process will be important in slowing the decline in lung function and thus prolonging survival in patients with CF.

Global proteomic approach unmasks involvement of keratins 8 and 18 in the delivery of cystic fibrosis transmembrane conductance regulator (CFTR)/ΔF508‐CFTR to the plasma membrane

Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF gene (cftr). Physiologically, CF is characterized by an abnormal chloride secretion in epithelia due to a dysfunction of a mutated cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is a cAMP-dependent chloride channel whose most frequent mutation, deltaF508, leads to an aberrantly folded protein which causes a dysfunction of the channel. However, a growing number of reports suggest that modifier genes and environmental factors are involved in the physiology of CF. To identify proteins whose expression depends on wild-type WT-CFTR or deltaF508-CFTR, we chose a global proteomic approach based on the use of two-dimensional gel electrophoresis (2-DE) and mass spectrometry. The experiments were carried out with HeLa cells stably transfected with WT-CFTR (pTCFWT) or deltaF508-CFTR (pTCFdeltaF508). These experiments unmasked keratin 8 (K8) and 18 (K18) which were differentially expressed in pTCFWT vs. pTCFdeltaF508. An immunoblot of K18 confirmed the 2-DE results. Intracellular localization experiments of WT-CFTR, deltaF508-CFTR, K8, and K18 suggest that the expression of these proteins are linked, and that the concentrations of K8 and K18 and/or their distribution may be involved in the traffic of WT-CFTR/deltaF508-CFTR. A functional assay for CFTR revealed that specifically lowering K18 expression or changing its distribution leads to the delivery of functional deltaF508-CFTR to the plasma membrane. This work suggests a novel function of K18 in CF.

Use of Nonviral Vectors for Cystic Fibrosis Gene Therapy

Over the last decade, three groups within the United Kingdom (Edinburgh, Oxford, and Imperial College, London) have undertaken key studies in the development of clinical gene therapy for cystic fibrosis. In 2001, catalyzed by the Cystic Fibrosis Trust, these groups came together to form the United Kingdom Cystic Fibrosis Gene Therapy Consortium. The Consortium has removed duplication and competition, developed core facilities playing to the respective strengths of the centers, and introduced the joint strategy described in this article. This is driven by a clinical trial program, with a product pipeline and the necessary development of novel preclinical and human assays. The program is milestone-related, has a structure that lies between the pharmaceutical industry and academia, and has as its endpoint negotiations with industry to undertake a phase III clinical trial of the identified product.

Adeno-associated Virus and Lentivirus Pseudotypes for Lung-directed Gene Therapy

The enthusiasm for cystic fibrosis gene therapy that attended the initial cloning of the gene and in vitro correction of the genetic defect eventually diminished as we learned more about the limitations of vector technologies that were available in the 1980s and 1990s. Substantial progress has been made, however, over the last 5 years in developing second- and third-generation vector constructs that should be more useful in achieving gene transfer to the lung for the treatment of pulmonary diseases such as cystic fibrosis.

Implication du gène CFTR dans la stérilité masculine associée à une absence de canaux déférents

Congenital bilateral absence of vas deferens (CBAVD) is found in most males with cystic fibrosis (CF), but this malformation can be observed without any pulmonary or digestive features. We have analyzed the 27 exons of the CF gene by denaturing high-performance liquid chromatography and the intron-8 polypyrimidine tract length variants by sequencing in a cohort of 291 CBAVD patients. CBAVD patients can be classified in four categories: 72 patients with two CFTR mutations (25%); 65 patients with one CFTR mutation and the 5T allele in trans (22%); 56 patients with only one CFTR mutation or only the 5T allele (19%); 98 patients without CFTR mutations and 5T allele (34%). Seventy different mutations were identified. The most frequent mutations are delF508del (36%), the 5T allele (30%) and R117H (7%). The high proportion of CBAVD who do not have CFTR mutations allows suggesting two hypotheses: other undetected chandes in CFTR may be involved in CBAVD or another gene(s) could be responsible for CBAVD.

High heterogeneity of CFTR mutations and unexpected low incidence of cystic fibrosis in the Mediterranean France

In this report, we present updated spectrum and frequency of mutations of the CFTR gene that are responsible for cystic fibrosis (CF) in Languedoc-Roussillon (L-R), the southwestern part of France. A total of 75 different mutations were identified by DGGE in 215 families, accounting for 97.6% of CF genes and generating 88 different mutational genotypes. The frequency of p.F508del was 60.23% in L-R versus 67.18% in the whole country and only five other mutations (p.G542X, p.N1303K, p.R334W, c.1717-1G>A, c.711+1G>T) had a frequency higher than 1%. The mutations were scattered over 20 exons or their border. This sample representing only 5.7% of French CF patients contributed to 24% of CFTR mutations reported in France. This is one of the highest molecular allelic heterogeneity reported so far in CF. We also present the result of a neonatal screening program based on a two-tiered approach “IRT/20 mutations/IRT” analysis on blood spots, implemented in France with the aim to improve survival and quality of life of patients diagnosed before clinical onset. This 18-month pilot project showed an unexpected low incidence of CF (1/8885) in South of France, with only six CF children detected among 43,489 neonates born in L-R, and 13 among 125,339 neonates born in Provence-Alpes-Côte-d'Azur (PACA).

Etiology of azoospermia in 100 consecutive nonvasectomized men

History was taken systematically for 100 azoospermic, nonvasectomized men referred consecutively to a Danish fertility clinic. The men were examined by ultrasound, and their blood samples were analyzed for karyotype, Y microdeletions, and cystic fibrosis transmembrane conductance regulator gene mutations. In 29% of patients, the condition could be explained by genetic abnormalities; in 22%, by diseases or external influence; and in 27%, by former cryptorchidism. The azoospermic condition remained unexplained in only 22%.

Microsphere Bead Arrays and Sequence Validation of 5/7/9T Genotypes for Multiplex Screening of Cystic Fibrosis Polymorphisms

The development of simple and rapid methods for the detection of the common genetic mutations associated with cystic fibrosis (CF) requires access to positive-control samples including the 5/7/9T variants of intron 8. We used PCR and a simple multiplex bead-array assay to identify 5/7/9T control samples from 29 commercially available DNA samples. Unpurified PCR products were directly hybridized to color-coded beads containing allele-specific capture probes for 5/7/9T detection. The performance of the assay was investigated using reverse-complement oligonucleotides, individual PCR products, and multiplex PCR products for 5/7/9T detection within a complex CFTR screening assay. Samples were genotyped by grouping the relative signal intensities from each capture probe. Of 29 commercially available DNA samples analyzed, 2 5T/7T, 2 5T/9T, 9 7T/9T, 11 7T/7T, and 5 9T/9T genotypes were identified. The genotype within each sample group was confirmed by DNA sequencing. The assay was compatible with the analysis of 10 to 1000 ng of genomic DNA isolated from whole blood and allowed for the separate identification of primary CFTR mutations from reflex variants. The correct identification of positive controls demonstrated the utility of a simple bead-array assay and provided accessible samples for assay optimization and for routine quality control in the clinical laboratory.

Advances in cystic fibrosis gene therapy

The first cystic fibrosis gene therapy trials were carried out in 1993, and although proof-of-principle for gene transfer to the lungs was established, efficiency was generally low. The authors review the most recent advances in preclinical airway gene transfer and summarize the results from the latest clinical trials. Recent clinical trials report encouraging results. Repeat administration of adeno-associated virus to the lung was safe. Nonviral nanoparticles used, for the first time, in the nose of cystic fibrosis patients were also safe and led to partial correction of the chloride transport defect in nasal epithelium. Important advances have been made in preclinical research, including the development of new viral and nonviral gene transfer agents and improved plasmid DNA. In addition, physical delivery methods, such a magnetofection and electroporation, are being assessed to improve nonviral gene transfer. Considerable progress has been made in understanding and overcoming the problems associated with gene transfer to airway epithelial cells, the target cells for cystic fibrosis gene therapy. It has also been recognized that novel preclinical and clinical assays are crucial for the success of cystic fibrosis gene therapy, and considerable effort is currently being put into assay development and trial designs.

Genetic Abnormalities among Severely Oligospermic Men Who Are Candidates for Intracytoplasmic Sperm Injection

Recent reports suggest that children born after intracytoplasmic sperm injection performed for male factor infertility are at increased risk of congenital malformations and chromosome aberrations. To explain these observations, we hypothesized that infertile men may be more likely than fertile men to have genetic abnormalities. We studied 750 severely oligozoospermic men (sperm count <5 million/ml) who were candidates for intracytoplasmic sperm injection, and 303 fertile men. We analyzed the peripheral blood karyotype, the Y chromosome long arm for detection of microdeletions in the azoospermia factors, and mutations in the cystic fibrosis gene and the androgen receptor gene. We also analyzed sperm for chromosome aneuploidies among the 421 men who subsequently entered the in vitro fertilization program. A total of 104 genetic abnormalities were diagnosed, corresponding to a frequency of 13.9% (104 of 750). Chromosomal aberrations were present in 5.6% (42 of 750) of infertile men and 0.3% of controls (one of 295), and they were in most cases alterations of the sex chromosomes. Y chromosome long-arm microdeletions were detected in 6.0% (45 of 750) of infertile men and most frequently included the azoospermia factor c, whereas no cases were found in controls (zero of 210). Mutations in the cystic fibrosis gene were diagnosed in 1.2% (nine of 750) of infertile men and 1.0% of controls (three of 303), and mutations in the androgen receptor gene were found in 1.1% (eight of 750) of infertile men and none of the 188 controls. Sperm sex chromosome aneuploidies were increased in men with karyotype anomalies and Y chromosome microdeletions as well as in subjects without constitutional genetic abnormalities. This study shows that the frequency of genetic alterations is increased among men with severe spermatogenic impairment. Genetic tests and genetic counseling should therefore be considered in oligozoospermic men who are candidates for intracytoplasmic sperm injection.

Blood pressure and the cystic fibrosis gene: evidence for lower pressure rises with age in female carriers.

Individuals homozygous for the autosomal recessive disorder CF are known to have low blood pressure, thought to be caused by greatly increased sweat salt loss. We examined whether carriers of the CF gene also have low blood pressure. Our pilot studies had suggested an effect limited to females, leading to 2 further studies in white females. In the first, blood pressure was measured in 232 known CF mutation carriers and compared with 246 mutation-negative control subjects. The carriers showed a significantly lower rate of increase in systolic blood pressure with age than the controls, especially after age 50 (3.5% per decade compared with 5.4% per decade, P=0.010). In a small substudy, sweat sodium and chloride levels were highest in those CF carriers with the lowest blood pressures. In the second study, CF carrier status was investigated in 563 normotensive females and in 607 women with essential hypertension diagnosed to test whether a lower incidence of carriers in the hypertensives suggested a protective effect. Twenty-five of the normotensives (4.4%) were carriers compared with 21 (3.5%) of the hypertensive group (P=0.45). Older CF carrier females had lower systolic and diastolic pressures than matched control subjects, with a tendency for blood pressure to increase less with age. This could result in significant reduction in stroke and heart disease. The effect on blood pressure is insufficient to prevent hypertension, though it remains conceivable that the severity might be ameliorated in carriers.

Human chromosome 7 circa 2004: a model for structural and functional studies of the human genome.

Human chromosome 7 is arguably amongst the most comprehensively characterized segments of the human genome. By microscopic examination, it belongs to the medium-sized group C submetacentric class, and historical studies involving chromosome-length measurements estimated that it accounts for approximately 5.3% of the human genome (or 160 Mb). Early successes in molecular genetics led to the identification of some of its biomedically important genes, including the T-cell receptor and homeobox families as well as the erythropoietin and cystic fibrosis genes. The Human Genome Project brought chromosome 7-specific and genome-wide initiatives, generating a wealth of genomic resources that have revealed the presence of over 350 disease-associated genes. Two distinct assemblies of the chromosome 7 sequence have been generated-one based largely on mapped large-insert clones and the other based on an integrated whole-genome shotgun sequencing strategy. These two sequences are mainly identical (<1% difference), and both estimate the unit length of chromosome 7 to be just over 158 Mb, remarkably similar to the originally predicted size. Systematic annotation efforts have anchored to the sequence, amongst many features, over 900 known genes and some 1000 other gene structures, as well as over 650 chromosomal breakpoints identified in patients with characterized phenotypic differences. Chromosome 7 has also been shown to contain the highest content of intra-autosomal segmental duplications in the human genome. The orthologous regions of roughly 22 Mb of chromosome 7 are currently being sequenced in multiple other vertebrate species. Examining these comparative sequence data, in conjunction with the other accumulating genomic information about these regions and the rest of the chromosome, should provide a model for the next generation of structural and functional analyses of the human genome.

Cystic fibrosis gene mutation reduces epithelial cell acidification and injury in acid-perfused mouse duodenum

Dysfunction of the cystic fibrosis transmembrane regulator (CFTR) is associated with diminished duodenal HCO3- secretion, despite a reported lack of clinical duodenal ulceration in affected subjects. We hypothesized that duodenal epithelial cells expressing a mutant CFTR have enhanced resistance to acid-induced injury. To test this hypothesis, we measured duodenal epithelial cell intracellular pH (pHi), injury, and acid back-diffusion in response to a luminal acid challenge in transgenic mice. A murine colony was established for the CFTR DeltaF508 (DeltaF) mutation. Epithelial cell pH i was measured by microscopy with a trapped, fluorescent pH-sensitive dye in living C57BL/6 and DeltaF/DeltaF, +/DeltaF, and +/+ mice. In vivo confocal microscopy confirmed the localization of the dye in the cytoplasm of the epithelial cells. Epithelial injury was measured fluorometrically using propidium iodide. Duodenal epithelial bicarbonate secretion and proton permeability were measured by back-titration. Bicarbonate secretion and acid back-diffusion were measured in a perfused duodenal loop. Basal and post-acid exposure bicarbonate secretion were reduced in DeltaF/DeltaF mice, although acid back-diffusion was similar to controls. Epithelial pHi of CFTR DeltaF/DeltaF mice during luminal acid exposure was significantly higher than pHi in +/DeltaF, +/+, or C57BL/6 mice. Acid-related epithelial injury was markedly less in DeltaF/DeltaF mice in comparison with the other groups. Increased cellular buffering power of the epithelial cells of DeltaF/DeltaF mice likely protects against acidification and injury during acid exposure. We speculate that this protective mechanism partially underlies the perceived relative lack of peptic ulceration in patients affected by cystic fibrosis.

Cystic fibrosis-associated liver disease (CFALD): what is the risk?

As life expectancy for patients with cystic fibrosis (CF) is extended through improved nutritional and pulmonary care, hepatobiliary lesions are recognized more frequently and are being characterized more precisely. Currently, liver disease is the most common nonpulmonary cause of death in patients with CF. Given the increasing prevalence with age, the question arises whether liver disease is an inevitable consequence of CF. The answer is no—this was recognized as early as 1956 by DiSant'Agnese and Blanc who reported that “liver disease has a variable occurrence despite the propensity for most patients to develop inspissated secretions; this suggests additional factors contribute.” So, why does only a subset of patients with CF develop liver disease? A partial explanation relates to the variable and inconsistent definition of “liver disease” in various centers and in the sensitivity of detection. CFALD is not easy to detect; clinical identification of the developing fibrotic liver lesion is difficult due to a lack of symptoms until portal hypertension is obvious. There are also likely to be factors intrinsic to the patient that determine whether liver disease will develop. A recent study found elevated endogenous biliary concentrations of ursodeoxycholic acid (UDCA) in patients with CF but without liver disease compared with those with CFALD. This suggests a possible protective role of UDCA against liver injury (Smith et al. Hepatology 2004;39:1673). What about specific risk factors for CFALD? No mutation in the CF gene is more frequent in patients with CFALD; however, liver disease is confined to patients with severe mutations (those associated with pancreatic insufficiency) (Colombo et al. JPediatr 1994;124:393, and Hepatology 2002;36:1374). These studies also cited male sex, a history of meconium ileus (MI), and certain HLA types as risk factors. In this issue of The Journal, Corbett et al report a case-controlled study of all children with CF in the Republic of Ireland in whom a diagnosis of CFALD was established over an 18-month period. They sought clinical factors associated with the onset of liver disease. Delayed diagnosis seemingly predisposed the development of CFALD, and children with liver disease had early evidence of impaired growth and nutrition, altered body compositions, and worsened pulmonary status. MI was not a risk factor in their patients. These new data add support to the recommendation for enhanced CF screening programs and may add some insight into the risk factors for development of CFALD.

Information on commonly asked questions about genetic evaluation and counseling for infertile couples

Information on commonly asked questions about genetic evaluation and counseling for infertile couples

Functional characterization of a recombinant adeno-associated virus 5-pseudotyped cystic fibrosis transmembrane conductance regulator vector.

Despite extensive experience with recombinant adeno-associated virus (rAAV) 2 vectors in the lung, gene expression has been low in the context of cystic fibrosis (CF) gene therapy, where the large size of the cystic fibrosis transmembrane conductance regulator (CFTR) coding sequence has prompted the use of compact endogenous promoter elements. We evaluated the possibility that gene expression from recombinant adeno-associated virus (rAAV) could be improved by using alternate AAV capsid serotypes that target different cell-surface receptors (i.e., rAAV5) and/or using stronger promoters. The relative activities of the cytomegalovirus (CMV) Rous sarcoma virus (RSV) promoter, the CMV enhancer/beta-actin (CB) promoter combination, and the CMV enhancer/RSV promoter hybrid were assessed in vitro in a CF bronchial cell line. The CB promoter was the most efficient. AAV capsid serotypes, rAAV2 and rAAV5, were also compared, and rAAV5 was found to be significantly more efficient. Based on these studies a rAAV5-CB-promoter-driven CFTR minigene vector was then used to correct the CF chloride transport defect in vitro, as well as the hyperinflammatory lung phenotype in Pseudomonas-agarose bead challenged CF mouse lungs in vivo. These studies provide functional characterization of a new version of rAAV-CFTR vectors.

Analysis of cystic fibrosis gener product (CFTR) function in patients with pancreas divisum and recurrent acute pancreatitis.

The mechanism by which pancreas divisum may lead to recurrent episodes of acute pancreatitis in a subset of individuals is unknown. Abnormalities of the cystic fibrosis gene product (CFTR) have been implicated in the genesis of idiopathic chronic pancreatitis. The aim of this study was to determine if CFTR function is abnormal in patients with pancreas divisum and recurrent acute pancreatitis (PD/RAP). A total of 69 healthy control subjects, 12 patients with PD/RAP, 16 obligate heterozygotes with a single CFTR mutation, and 95 patients with cystic fibrosis were enrolled. CFTR function was analyzed by nasal transepithelial potential difference testing in vivo. The outcomes of the PD/RAP patients following endoscopic and surgical treatments were concomitantly analyzed. Direct measurement of CFTR function in nasal epithelium in response to isoproterenol demonstrated that the values for PD/RAP were intermediate between those observed for healthy controls and cystic fibrosis patients. The median value was 13 mV for PD/RAP subjects, which was statistically different from healthy controls (22 mV, p= 0.001) and cystic fibrosis pancreatic sufficient (-1 mV, p < 0.0001) and pancreatic insufficient (-3 mV, p < 0.0001) patients. These results suggest a link between CFTR dysfunction and recurrent acute pancreatitis in patients with pancreas divisum and may explain why a subset of patients with pancreas divisum develops recurrent acute pancreatitis.

Causes of azoospermia and their management.

Azoospermia may occur because of reproductive tract obstruction (obstructive azoospermia) or inadequate production of spermatozoa, such that spermatozoa do not appear in the ejaculate (non-obstructive azoospermia). Azoospermia is diagnosed based on the absence of spermatozoa after centrifugation of complete semen specimens using microscopic analysis. History and physical examination and hormonal analysis (FSH, testosterone) are undertaken to define the cause of azoospermia. Together, these factors provide a >90% prediction of the type of azoospermia (obstructive v. non-obstructive). Full definition of the type of azoospermia is provided based on diagnostic testicular biopsy. Obstructive azoospermia may be congenital (congenital absence of the vas deferens, idiopathic epididymal obstruction) or acquired (from infections, vasectomy, or other iatrogenic injuries to the male reproductive tract). Couples in whom the man has congenital reproductive tract obstruction should have cystic fibrosis (CF) gene mutation analysis for the female partner because of the high risk of the male being a CF carrier. Patients with acquired obstruction of the male reproductive tract may be treated using microsurgical reconstruction or transurethral resection of the ejaculatory ducts, depending on the level of obstruction. Alternatively, sperm retrieval with assisted reproduction may be used to effect pregnancies, with success rates of 25-65% reported by different centres. Non-obstructive azoospermia may be treated by defining the cause of low sperm production and initiating treatment. Genetic evaluation with Y-chromosome microdeletion analysis and karyotype testing provides prognostic information in these men. For men who have had any factors potentially affecting sperm production treated and remain azoospermic, sperm retrieval from the testis may be effective in 30-70% of cases. Once sperm are found, pregnancy rates of 20-50% may be obtained at different centres with in vitro fertilisation and intracytoplasmic sperm injection.

Gene delivery systems—gene therapy vectors for cystic fibrosis

Gene delivery systems (GDS) play a central role in the development of gene therapy strategies for Cystic Fibrosis (CF). Further, these systems are important tools in studies with cultured cells and in animal models. In this review, we describe the properties of several viral and synthetic gene delivery systems, and evaluate their possible application in gene therapy of CF. While many gene delivery systems give satisfactory results in cultured or animal studies, none of these systems has been shown to fulfil all the requirements of safety and efficacy for use in CF patients. The intact airway epithelium, the most important target in CF gene therapy, proves to be well protected against invading vector systems.