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

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

Does a primary host defense abnormality involving monocytes-macrophages underlie the pathogenesis of lung disease in cystic fibrosis?

The primary cause of morbidity and mortality in cystic fibrosis (CF) patients is chronic pulmonary disease. Pulmonary disease in CF is characterized in part by: (a) obstruction of the bronchi and bronchioles by inspissated secretions (mucus is hypersecreted and may also be abnormal), (b) recurrent or persistent bacterial infections, and (c) a chronic inflammatory state. We propose herein that much of the pathophysiology of lung disease in CF stems from a genetically inherited metabolic defect in monocyte-macrophages (M-MØ), and we review evidence which indicates that CF M-MØ are innately metabolically abnormal. Once activated by various stimuli, CF M-MØ become metabolically hyperactive and hypersecretory as evidenced by the production of excessive levels of a variety of mediators which could have definite roles in both the initiation of pulmonary obstruction and the accelerated development of a chronic inflammatory response in CF. Evidence is also reviewed which indicates that other CF M-MØ functions crucial to the afferent and efferent phases of the immune response to bacterial infections in the lung may be adversely affected. Mechanisms proposed to explain the abnormal production of mediators by CF M-MØ are discussed, and it is concluded that hyperproduction of mediators by CF M-MØ and their metabolic hyperactivity probably result from a defect in autoregulation. The nature of the metabolic defect in CF M-MØ indicates that CF should be classified as a “new” primary host defense abnormality or alternatively as a “new” primary immune deficiency disease.     

Typing of Pseudomonas aeruginosa strains in Norwegian cystic fibrosis patients

Objectives   Typing of Pseudomonas aeruginosa isolates from Norwegian cystic fibrosis (CF) patients with chronic Pseudomonas lung infection in order to see whether cross-infection might have occurred.
Methods   Isolates from 60 patients were collected during the years 1994–98, and typed by pulsed field gel electrophoresis.
Results   Seventy-one strains were identified. One large cluster of identical strains included 27 patients, and 13 smaller clusters of 2–4 patients were found (26 patients). Seven patients had a strain not shared by other patients (private strains). Harboring the main cluster strain was significantly associated with participation in summer camps and training courses ( P  = 0.004, chi-squared test). There were no associations with regular admissions to hospital (intravenous antibiotic courses) or smaller social gatherings of short duration. Small clusters and private strains were not associated with any of the risk factors. All strains were sensitive to colistin. The minimal inhibitory concentrations were generally lower in Norwegian P. aeruginosa strains compared with isolates from Danish patients.
Conclusions   Our results indicate that cross-infection with P. aeruginosa between cystic fibrosis patients has occurred.     

MicroRNA profiling of cystic fibrosis intestinal disease in mice

Cystic fibrosis (CF) intestinal disease is characterized by alterations in processes such as proliferation and apoptosis which are known to be regulated in part by microRNAs. Herein, we completed microRNA expression profiling of the intestinal tissue from the cystic fibrosis mouse model of cystic fibrosis transmembrane conductance regulator (Cftr) deficient mice (BALBc/J Cftr(tm1UNC)), relative to that of wildtype littermates, to determine whether changes in microRNA expression level are part of this phenotype. We identified 24 microRNAs to be significantly differentially expressed in tissue from CF mice compared to wildtype, with the higher expression in tissue from CF mice. These data were confirmed with real time PCR measurements. A comparison of the list of genes previously reported to have decreased expression in the BALB×C57BL/6J F2 CF intestine to that of genes putatively targeted by the 24 microRNAs, determined from target prediction software, revealed 155 of the 759 genes of the expression profile (20.4%) to overlap with predicted targets, which is significantly more than the 100 genes expected by chance (p=1×10(-8)). Pathway analysis identified these common genes to function in phosphatase and tensin homolog-, protein kinase A-, phosphoinositide-3 kinase/Akt- and peroxisome proliferator-activated receptor alpha/retinoid X receptor alpha signaling pathways, among others, and through real time PCR experiments genes of these pathways were demonstrated to have lower expression in the BALB CF intestine. We conclude that altered microRNA expression is a feature which putatively influences both metabolic abnormalities and the altered tissue homeostasis component of CF intestinal disease.     

First report of cystic fibrosis mutations in Libyan cystic fibrosis patients

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

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

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

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

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

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.     

Factors associated with mucoid transition of Pseudomonas aeruginosa in cystic fibrosis patients

Although the mucoid form of Pseudomonas aeruginosa (Pa) is largely responsible for the progression of lung disease in cystic fibrosis (CF), the relationship between factors relating daily-care regimes to mucoidy acquisition are as yet poorly investigated. Fifty-two CF patients registered at the CF centre of Dijon, France, were retrospectively evaluated from the date of Pa colonization either to the first -positive sputum culture for mucoid Pa (n = 26) or to the last culture in which the Pa remained non-mucoid (n = 26). All clinical, pathological and therapeutic events were recorded. The association between the parameters collected and mucoid transition of Pa was assessed in a Cox model with time-dependant covariables. The mean follow-up was 4.7 ± 4.3 years. Three independent parameters were associated with the higher risk of mucoid transition of Pa: persistence of Pa in sputum (OR 7.89; p <0.01), use of inhaled bronchodilators (OR 3.40; p = 0.04), and the use of inhaled colimycin (OR 4.04; p = 0.02). Isolation of Staphylococcus aureus, Haemophilus influenzae or Streptococcus pneumoniae in sputum was associated with a lower risk (OR 0.24; p < 0.01). Mucoid transition of Pa was associated with variables that reflected the severity of both lung disease and Pa colonization. Although they do not lead to prophylactic measures, these results corroborate the need to avoid Pa persistence.     

Vitamin E status and its determinants in patients with cystic fibrosis

Purpose

The risk of vitamin E deficiency is of primary concern in cystic fibrosis patients. However, early diagnosis and routine vitamin E supplementation can lead to its normal or even high levels. In the present study, we assessed vitamin E status in a large group of cystic fibrosis patients. Moreover, we also aimed to establish determinants of its body resources in cystic fibrosis patients.

Chloride transport in cultured nasal epithelium of cystic fibrosis patients

In this study, nasal polyp epithelial cells from control and cystic fubrosis (CF) patients were cultured using a method which allows multiple passages. The cells were tested in Ussing chamber experiments to study transcellular ion transport. Cultured CF nasal polyp cells did not exhibit spontaneous transcellular chloride transport in the presence of amiloride, in contrast to normal cells. Forskolin increased the short circuit current (I _sc) in control but not CF cells. Forskolin and isoproterenol increased the cAMP levels in control and CF cells. Histamine, bradykinin and isoproterenol transiently increased the intracellular calcium level and caused a parallel increase of the transcellular chloride current in both normal and CF cells. The transient effects of isoproterenol were not sensitive to the beta blocker atenol and could not be mimicked by forskolin. We conclude that in cultured nasal polyp cells a difference in chloride transport activity between CF and control cells is retained following multiple passages. Our results suggest that the active state of chloride channels in nasal polyp cells does not require activation of a second messenger pathway. This apparently spontaneous activity appears to be reduced in CF cells. The calcium- but not the cAMP-dependent activation of transepithelial chloride secretion is at least partially preserved in cultured CF airway epithelium.     

Multiplex PCR reveals that viruses are more frequent than bacteria in children with cystic fibrosis

BackgroundCystic fibrosis is a degenerative disease characterized by progressive epithelial secretory gland dysfunction associated with repeated respiratory infections. Bacterial infections are very frequent in children with cystic fibrosis, but because rapidMethodsfor screening for the wide variety of potentially involved viruses were unavailable until recently, the frequency of viral presence is unknown. Multiplex PCR enables screening for many viruses involved in respiratory infections.ObjectivesThis study aimed to evaluate the frequency of viruses and bacteria in respiratory specimens from children with cystic fibrosis and to clarify the incidence and characteristics (seasonality and age of patients) of different viruses detected in children with cystic fibrosis.Study designIn this 2-year prospective study, we obtained paired nasopharyngeal-swab and sputum specimens from children with cystic fibrosis during clinical respiratory examinations separated by at least 14 days. We analyzed viruses in nasopharyngeal-swab specimens with multiplex PCR and bacteria in sputum with standard methods.ResultsWe analyzed 368 paired specimens from 33 children. We detected viruses in 154 (41.8%) and bacteria in 132 (35.9%). Bacteria were commoner in spring and summer; viruses were commoner in autumn and winter. In every season, Staphylococcus aureus was the commonest bacteria and rhinovirus was the commonest virus. Nearly all infections with Haemophilus influenzae occurred in autumn and winter.Viruses were more prevalent in children <5 years old, and bacteria were more prevalent in children ≥12 years old.ConclusionsMultiplex PCR screening for respiratory viruses is feasible in children with cystic fibrosis; the clinical implications of screening warrant further study.     

不同肺腺癌细胞系N－ras、p53基因突变核酸测序分析

应用聚合酶链反应（ｐｏｌｙｍｅｒａｓｅｃｈａｉｎｒｅａｃｔｉｏｎＰＣＲ）和ｄｓＤＮＡｃｙｃｌｅｓｅｑｕｅｎｃｉｎｇｓｙｓｔｅｍ技术对体外培养的人肺腺癌细胞系ＬＴＥｐ－ａ＿２和ｈＬＡ中Ｎ－ｒａｓ癌基因及ｐ５３抑癌基因外显子５、７进行核酸序列测定分析。结果表明Ｎ－ｒａｓ突变热点第１２、１３、６１密码子未见异常。ｐ５３抑癌基因第１５４密码子均发现ＧＧＣ→ＧＴＣ突变（Ｇｌｙ→Ｖａｌ变异）。经光盘检索（美国Ｓｉｌｖｅｒｐｌａｔｔｅｒ公司提供的ＣＯ－ＲＯＭＭＥＤＩＬＩＮＥ）分析了１９８８～１９９３年间的专题文献，尚未见肺癌ｐ５３基因第１５４密码子突变的报道。     

Direct sputum analysis of Pseudomonas aeruginosa macrorestriction fragment genotypes in patients with cystic fibrosis

The distribution of bacterial populations in the airways of 13 patients with cystic fibrosis who were colonized for 6 – 23 years with Pseudomonas aeruginosa was investigated by genotyping of bacterial chromosomes directly isolated from 21 sputa. After removal of host material from sputum by hypotonic cell lysis and repetitive washing and centrifugation steps, agarose-embedded bacterial cells were lysed, residual eukaryotic DNA separated by field inversion gel electrophoresis, and the purified bacterial chromosomes subjected to macrorestriction fragment pattern and Southern analyses. Bacterial populations consisted of a single P. aeruginosa clone in 17 sputa, of which more than one clonal variant was apparent in two SpeI fragment fingerprints. Two clones of P. aeruginosa and another species co-existed in four samples. Genomically homogeneous populations of P. aeruginosa are characteristic for chronically colonized lungs in most cases of cystic fibrosis. Received: 25 April 1997     

Expression of cystic fibrosis transmembrane conductance regulator in the human distal lung

The determination of the expression of cystic fibrosis transmembrane conductance regulator (CFTR) in the lung is essential for a full understanding of the normal lung physiology and the pathogenesis of the lung disease in cystic fibrosis (CF). However, studies on the expression of CFTR in the distal adult human lung have yielded conflicting results despite functional evidence of expression of CFTR in bronchiolar and alveolar epithelial cells. We used 2 high-affinity monoclonal anti-CFTR antibodies, MAb24-1 and MAb13-1, to determine the expression of CFTR in samples of bronchiolar and alveolar tissues obtained from the same non-CF individuals. CFTR immunostaining was detected in the epithelium of bronchiolar and alveolar tissues. The staining pattern was similar with both antibodies. In bronchioles, CFTR labeling was present mostly in ciliated cells; in alveoli, CFTR labeling was detected in both type I and type II cells. We conclude that CFTR is expressed in human bronchiolar and alveolar epithelial cells. The potential importance of CFTR expression in alveoli should be further investigated, particularly with respect to the CF lung disease and the physiology of the alveolar region.     

中国人WD基因第14和18号外显子的错义突变

目的了解中国人肝豆状核变性（Ｗｉｌｓｏｎ＇ｓｄｉｓｅａｓｅ,ＷＤ）基因第１４和１８号外显子的突变情况,与国外报道的这两个外显子的高突变频率进行比较。方法应用聚合酶链反应－单链构像多态（ＰＣＲ－ＳＳＣＰ）结合ＤＮＡ测序技术,对４４例无亲缘关系的ＷＤ患者及６０例正常对照进行突变检测。结果一例患者在１４号外显子发生了Ａｒｇ１０４１Ｐｒｏ（３１２１Ｇ→Ｃ）纯合错义突变,另一例患者在１８号外显子发生了Ａｓｎ１２７０Ｓｅｒ（３８０９Ａ→Ｇ）杂合错义突变。结论Ａｒｇ１０４１Ｐｒｏ为未报道过的新型错义突变,Ａｓｎ１２７０Ｓｅｒ突变与国外报道一致。但均未呈热区分布。     

Single-stranded conformation polymorphism analysis of the CFTR gene in slovenian cystic fibrosis patients: Detection of mutations and sequence variations

Cystic fibrosis (CF) mutations have been identified in Slovenian CF patients using single-stranded conformation polymorphism (SSCP) analysis. The entire coding region and all of the splice junction sites were screened in 24 patients. By varying the electrophoretic conditions and composition of the gel, 16 different nucleotide changes have been observed in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Three newly described mutations and four previously reported mutations were found. In addition two new polymorphisms have been identified. Of 35 non-ΔF508 chromosomes examined, mutations were detected on 25.7%, raising the proportion of Slovenian CF alleles characterized to 67.5%. Because of the high sensitivity of the SSCP technique most of the remaining uncharacterized CF mutations probably lie in large introns, promoter sequences, or putative regulatory regions not yet analyzed. © 1993 Wiley-Liss, Inc.     

Prospective study on nontuberculous mycobacteria in patients with and without cystic fibrosis

Recent reports indicated an increase of nontuberculous mycobacteria (NTM) in cystic fibrosis (CF) patients. However, it is still a matter of discussion whether criteria for diagnosis of NTM pulmonary infection established by the American Thoracic Society (ATS) are applicable for CF patients. We determined incidence and prevalence of NTM in CF patients and non-CF patients without HIV infection, and validity of ATS criteria for CF patients. Over a period of 2 years, 1,251 respiratory samples were investigated for mycobacteria from 91 CF and 162 non-CF patients. For all patients with NTM recovery, we reviewed clinical charts and determined outcome for up to 2 years. Incidence and prevalence for repeated recovery of NTM were higher in CF patients, but not significantly. CF patients with repeated recovery of NTM met clinical and bacteriological ATS criteria, but radiographic criteria were not met. Treated CF patients showed favorable clinical outcomes, as opposed to untreated patients. We propose a modified definition for diagnosis and hence treatment of NTM pulmonary infection in CF patients.     

Fluorescence-based oligonucleotide ligation assay for analysis of cystic fibrosis transmembrane conductance regulator gene mutations

Isolation of the gene for cystic fibrosis (CF), the cystic fibrosis transmembrane conductance regulator (CFTR), provided a basis for analyzing its molecular pathology and resulted in the identification of < 400 mutations associated with disease. Except for the ΔF508 mutation, no other single mutation accounts for > 5% of CF chromosomes in most populations, and most mutation frequencies are < 1%. A strategy based on multiplex PCR followed by multiplex allele-specific oligonucleotide probe ligation was used to detect 30 mutations, distributed throughout ten exons and seven introns of the CFTR gene, that together account for > 96% of CF mutant chromosomes worldwide. Mutations were detected by competitive oligonucleotide probe ligation to detect normal and/or mutant genotypes in one reaction. Three probes (one common and two allelic probes) were needed for analysis of each mutation. Probes hybridized to target DNA were joined by a thermostable ligase if there were no mismatches at their junctions; temperature cycling resulted in a linear increase in product. Common probes were labeled with fluorochromes, and allelic probes each had different lengths. Ligation products were analyzed electrophoretically on a fluorescent DNA sequencer. The results show that combined PCR and probe ligation amplification rapidly and reliably screen for CF homozygotes and carriers. © Wiley-Liss, Inc.     

Simultaneous detection of the two prevalent mutations in the cystic fibrosis gene in Reunion Island

A rapid method for the diagnosis of the most frequent cystic fibrosis mutations in the Reunion Island is described based on a coamplification polymerase chain reaction (PCR) followed by a single digestion using MseI. We have used this strategy to detect the two most frequent mutations in this area: ΔF508 (in exon 10) and Y122X (in exon 4). These two mutations account for 70% of the CF chromosomes. This diagnosis method, which is rapid, easy, direct, and inexpensive, allows adult and neonatal carrier screening in this population. © 1993 Wiley-Liss, Inc.     

Prevalence and dynamics of Lactobacillus sp. in the lower respiratory tract of patients with cystic fibrosis

No prevalence or dynamics analysis of Lactobacilli in the lung of cystic fibrosis (CF) patients has yet been conducted. In order to use them as probiotics in the treatment of Pseudomonas aeruginosa infection, we describe their lung epidemiology.Over a period of 8 months, we analyzed 279 sputum samples from 124 CF patients classified according to their P. aeruginosa Leeds status of colonization. A total of 137 strains belonging to 11 species were isolated. The prevalence of carriage was 61%. No difference in species diversity or frequency was observed according to Leeds criteria. The next step will be to focus on the strain level.     

Epidemiology of chronic Pseudomonas aeruginosa infections in cystic fibrosis.

Chronic lung infection with Pseudomonas aeruginosa is primarily responsible for pulmonary deterioration of cystic fibrosis patients. The purpose of this study was to type the P. aeruginosa isolates collected sequentially from cystic fibrosis patients, chronically colonized with P. aeruginosa, by random amplified polymorphic DNA fingerprinting-PCR (RAPD-PCR). Sequential P. aeruginosa isolates (n: 130) that had been collected from 20 CF patients over at least 9 years were investigated. The isolates were analyzed by RAPD-PCR using two arbitrary primers. Antimicrobial susceptibility testing of all isolates was performed by the disc diffusion method. RAPD-PCR typing demonstrated that strains dissimilar in colony morphotype and of different antibiotic susceptibility patterns could be of the same genotype. Some CF patients were colonized with a rather constant P. aeruginosa flora, with strains of different phenotypes but of one genotype. However, some patients may be colonized with more than one genotype. The results also demonstrated that there might be a risk of cross-colonization between CF patients followed-up at the same center.