A rapid,efficient, and sensitive assay for simultaneous detection of multiple cystic fibrosis mutations

We describe the use of DGGE multiplex systems for rapid analysis of 15 exons of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, in which about half of the known CF molecular defects are clustered. We have previously determined the spectrum of mutation affecting the CFTR gene in the French population using a strategy based on denaturing gradient gel electrophoresis (DGGE) of amplified gene segments. Analysis of CF patients' DNA with five DGGE multiplex systems permitted us to characterize nearly 35% of non-ΔF508 CF alleles and increased the CF allele detection rate to almost 82% in this population. This simple and rapid multiplex analysis strategy, which allows a significant proportion of the most frequent CF mutations in Caucasians to be detected, will be helpful in the implementation of genetic screening programs. © 1993 Wiley-Liss, Inc.     

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.     

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.     

Sequence analysis of the cystic fibrosis gene in patients with disseminated bronchiectatic lung disease

Summary The diagnosis of classical cystic fibrosis (CF) is easily made by clinical assessment alone, but may be missed or delayed in cases with an atypical clinical course. In a recent major study the age at diagnosis varied between 2 months and 47 years. For diagnostic purposes we have investigated the cystic fibrosis transmembrane regulator (CFTR) gene in 10 adult patients (age 18 to 45 years) with chronic obstructive pulmonary disease since childhood or adolescence and bronchiectases disseminated through both lungs. Only one subject (a 29-year-old male) had exocrine pancreatic insufficiency (PI); all others were pancreatic-sufficient (PS). The first nucleotide (ATP)-binding fold of the CFTR was analyzed by direct sequencing of polymerase chain reaction (PCR)-amplified genomic DNA in these cases. Two patients with different phenotypes (one PI, one PS) were found to be homozygous for the common F508 mutation of the CFTR gene, which proved the diagnosis of cystic fibrosis in their cases and allowed genetic counselling. The PS patient had normal sweat tests and had not previously been recognized as having CF. Four other patients were heterozygous for F508, with no other mutation in exons 10 or 11 of the gene, and four patients had normal sequences of these exons. Because only about 70% of all CF chromosomes carry F508, the unexpectedly high frequency (4/8=50%) of heterozygosity for F508 among the non-F508/F508 patients with bronchiectases suggests that some of these might also have unrecognized CF with rare genotypes and mutations in any of the 22 exons not sequenced. About 90 different mutations have already been detected in coding regions of the CFTR gene and a very broad and so far not fully recognized spectrum of clinical phenotypes may be caused by mutations of this gene. Screening for the most frequent CFTR gene mutations, which is practicable using recent technology, may provide significant new diagnostic information in patients with CF-like pulmonary phenotypes, especially if they have normal or borderline sweat tests and no pancreatic insufficiency.Abbreviations PCR polymerase chain reaction - CF cystic fibrosis - CFTR cystic fibrosis transmembrane conductance regulator - COPD chronic obstructive pulmonary disease - PI pancreatic insufficiency - PS pancreatic sufficiency     

Prevalence of cystic fibrosis mutations in the East German population.

A representative multicenter cystic fibrosis (CF) mutation analysis on about half of all known cystic fibrosis patients of the 5 East German L?nder is reported. Analyses for 17 mutations, among them Delta F508, R553X, G542X, S549R,N,I, G551D, S1255X, R347P,H, and Y122X, were performed. As expected, the delta F508 mutation in exon 10 of the CFTR gene is the major gene alteration causing CF in our patients. However, in comparison to studies from Western Germany, a significantly lower percentage of just over 60% is found in our patients, resembling data obtained from slavonic populations. The severe phenotype of cystic fibrosis is most frequently associated with homozygosity for the delta F508 mutation. No particular allele association could be found with the intermediate and mild phenotypes of this disease. The next most frequent of the investigated mutations is R553X (13.3% of non-delta F chromosomes) followed by R347P (9.2%) and G542X (4.4%).     

A cystic fibrosis allele encoding missense mutations in both nucleotide binding folds of the cystic fibrosis transmembrane conductance regulator.

German cystic fibrosis (CF) chromosomes were screened for molecular lesions in exon 20 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by chemical cleavage of mismatch. An 3884G-to-A transition was detected in two patients which leads to an exchange of a serine by an asparagine in the Walker motif A of the second nucleotide binding fold. The affected serine residue is evolutionarily strongly conserved among the pro- and eukaryotic members of the protein superfamily of traffic ATPases. The two S1251N alleles were linked to the benign missense mutation F508C which is located in another conserved region of CFTR, the center region of the first nucleotide binding fold. Both patients with the complex allele F508C-S1251N are carrying delta F508 on the other CF chromosome and are suffering from severe pulmonary and gastrointestinal CF disease. Although F508C has been classified as a neutral sequence variation because of its discovery in healthy delta F508 gene carriers, it may nevertheless influence CFTR dysfunction caused by the S1251N mutation.     

Screening of 62 mutations in a cohort of cystic fibrosis patients from north eastern Italy: Their incidence and clinical features of defined genotypes

The frequency of 62 different CFTR mutations in 225 chromosomes from a CF birth cohort, which includes all the affected subjects born in northeast Italy during a 10-year period of time, was investigated. New mutations were also searched by the analysis of 15 different exons. The total proportion of CF chromosomes with detectable mutations is 73.78%. Therefore although a considerable improvement in CF mutation detection in our population has been achieved, the search for other common and uncommon mutations should be continued. Moreover a carrier screening program should be postponed until reaching a cumulative proportion of known CF alleles of at least 90%. The correlations between the genotypes which have been identified and the main clinical features added some new information to the classification of CF mutations as pancreatically severe or mild ones. © 1993 Wiley-Liss, Inc.     

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.     

Screening for cystic fibrosis mutations in southern France: identification of a frameshift mutation and two missense variations.

In the search for mutations in the cystic fibrosis gene in patients from the Mediterranean area, we have analysed exons 4, 9, 10, 19, and 21 by the single-strand conformation polymorphism (SSCP) technique in 50 patients with at least one non-delta F508 chromosome. Ten samples demonstrated a shifted band, four in exon 19 and six in exon 21. Sequencing of the PCR fragments has led to the identification of three new sequence alterations, two in exon 19 (3737 delA and I1234V), and one in exon 21 (N1303H). We also analysed the frequency of two known intronic polymorphisms in front of exon 19 (C to A change at nucleotide 3601-65) and exon 21 (G to A change at position 4006-200).     

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.     

Association between cystic fibrosis transmembrane conductance regulator gene mutations and susceptibility for childhood asthma in Korea

Purpose

Classic cystic fibrosis is now known part of cystic fibrosis transmembrane conductance regulator (CFTR)-related disorders. These include a wide spectrum, from multi-system disorders, such as cystic fibrosis, to mono-symptomatic conditions, such as chronic pancreatitis or congenital bilateral absence of the vas deferens. However, respiratory disease is considered typical for the multi system disorder, cystic fibrosis, and is the major cause of morbidity and mortality. The purpose of this study was to evaluate the potential effects of CFTR gene mutations in Korean children with asthma.

Materials and Methods

We selected 14 mutations identified in Korea and each of the 48 children with and without asthma were genotyped for the case-control study.

Results

No significant differences were found in genotype and allele frequencies of the 9 polymorphisms observed between the non-asthma and asthma groups. In a haplotype determination based on a Bayesian algorithm, 8 haplotypes were assembled in the 98 individuals tested. However, we also did not find any significant differences in haplotype frequencies between the non-asthma and asthma groups.

Conclusion

We have concluded that this study did not show any evidence in support of providing that CFTR genetic variations significantly contribute to the susceptibility of asthma in Korean children.     

Analysis of linkage disequilibrium between different cystic fibrosis mutations and three intragenic microsatellites in the Italian population

Three intragenic microsatellites of the CFTR gene, a TA and a CA repeats, namely IVSl7bTA and IVSl7bCA, located in intron 17b and a CA repcat (IVS8CA) located in intron 8 of the CFTR gene, were analyzed in a large sample of Italian cystic fibrosis (CF) and normal chromosomes. Linkage disequilibrium was evaluated between each marker and different CF mutations on a total of 377 CF and 358 normal chromosomes. Our results are consistent with the hypothesis that all AF508 chromosomes derive from a single mutational event. The same hypothesis is valid for mutations G542X, N1303K, 1717-1IG→, which might have been originated more recently than δF508. © 1995 Wiley- Liss, Inc.     

Intracellular retention of lysosomal enzymes in cystic fibrosis

Multiple leakage of lysosomal enzymes from cystic fibrosis fibroblasts into the extracellular space has been suggested to occur but was not observed in these experiments. Both extracellular and intracellular levels of enzyme activity were within the normal range. The release of lysosomal enzymes from individual cell lines was seen to be affected both by intrinsic properties of each cell line and by many external factors.     

肠出血性大肠埃希氏菌的多重PCR检测方法

目的开发肠出血性大肠埃希氏菌（ｅｎｔｅｒｏ－ｈｅｍｏｒｒｈａｇｉｃＥ．ｃｏｌｉ,ＥＨＥＣ）的多重ＰＣＲ检测方法。方法以溶血素（ｈｅｍｏｌｙｓｉｎ,ｈｌｙ）基因和志贺样毒素（Ｓｈｉｇａｌｉｋｅｔｏｘｉｎ,ＳＬＴ）基因为靶基因,进行了研究。结果发现ＳＬＴ１－ｈｌｙ组合能够检测出产生ＳＬＴ１毒素的ＥＨＥＣ菌株,ＳＬＴ２－ｈｌｙ组合能够检测出产生ＳＬＴ２的ＥＨＥＣ菌株,ＳＬＴｓ－ｈｌｙ组合能够检测出所有产生ＳＬＴ１和ＳＬＴ２的ＥＨＥＣ菌株。结论实验证明,把这３种多重ＰＣＲ方法结合使用,能够快速地检测和鉴定ＥＨＥＣ菌株,能够比较准确地了解所检测菌株产生志贺样毒素的情况,为ＥＨＥＣ的检测工作提供了一种更加方便易行的技术     

Detection of more than 94% cystic fibrosis mutations in a sample of belgian population and identification of four novel mutations

We have analysed 194 Belgian CF chromosomes using a variety of techniques: ΔF508 was detected by polyacrylamide gel electrophoresis; dot blotting of PCR products was used to identify the mutations G542X, 1717-1 G → A, and N1303K; molecular defects in exons 2, 3, 4, 5, 6b, 7, 11, 12, 13, 14a, 14b, 17b, 19, 20, and 21 were screened for by DGGE. We identified 17 mutations, which accounted for 94.3% of the Belgian CF chromosomes. Four novel mutations and a novel polymorphism were characterized. The detection of such a high proportion of Belgian CF mutations is important in understanding the functional role of the molecule and in improving prenatal and genetic diagnosis of CF. © 1993 Wiley-Liss, Inc.     

Psychological intervention for adults with cystic fibrosis.

This study assessed the effects of a stress management and life-style modification program on the physical and psychological well-being of young adults with cystic fibrosis. Four fibrocystic patients recently transferred to an adult rehabilitation centre completed this 6-week program. Instruction in stress management consisted of both behavioural (e.g. relaxation training) and cognitive techniques. Discussion on life-style included the topics of drug and alcohol abuse, leisure, and nutrition. Individual assessments were conducted before and after a 6-week non-intervention control period and after a 6-week training program. Assessments included a battery of psychometric inventories as well as electromyographic testing of trapezius muscle tension. At the end of the program, participants reported that they experienced fewer physical and psychological symptoms of stress. Electromyographic measures provided some suggestion of a trend toward greater stability at posttest.     

多重PCR方法检测多发性骨髓瘤患者重排基因的研究

目的　探讨分子生物学方法在多发性骨髓瘤诊断中的意义。方法　采用多重 Ｐ Ｃ Ｒ 联合 Ｓｏｕｔｈｅｒｎ 杂交方法检测２９ 例多发性骨髓瘤患者骨髓及外周血标本 Ｉｇ Ｈ Ｃ Ｄ ＲⅢ及 Ｔ Ｃ Ｒ ＶγⅠ Ｊγ重排基因。结果　８９ ．７ ％ （２６／２９） 和６５ ．５ ％ （１９／２９） 的患者骨髓标本存在 Ｉｇ Ｈ 及 Ｔ Ｃ Ｒ ＶγⅠ Ｊγ重排基因,而在外周血标本,仅５５ ．２ ％ （ １６／２９） 和３７ ．９ ％ （１１／２９） 存在 Ｉｇ Ｈ 及 Ｔ Ｃ Ｒ 重排基因。联合检测 Ｉｇ Ｈ Ｃ Ｄ ＲⅢ和 Ｔ Ｃ Ｒ ＶγⅠ Ｊγ重排基因,９３ ．１ ％ （２７／２９） 骨髓标本及５８ ．６ ％ （１７／２９） 外周血标本存在 Ｉｇ Ｈ 或／ 和 Ｔ Ｃ Ｒ 重排基因,外周血标本Ⅱ期和Ⅲ期患者重排基因阳性率（７１ ．４ ％ ） ,显著高于Ⅰ期患者（２５ ％ ）（ Ｐ＜ ０ ．０５） 。结论　 Ｐ Ｃ Ｒ 技术检测多发性骨髓瘤患者重排基因对诊断是有帮助的, 尤其是骨髓标本。多重 Ｐ Ｃ Ｒ 方法可在一个 Ｐ Ｃ Ｒ 循环中同时扩增两个重排基因,更简便、经济,更适宜临床实际应用。     

多重PCR检测方法和液态芯片技术在呼吸道病毒检测中的应用研究

目的 评估澳大利亚维多利亚感染性疾病参比实验室（The Victorian Infectious Diseases Reference Laboratory,VIDRL）的呼吸道病毒多重PCR检测方法和Luminex公司多指标同步分析液态芯片技术-呼吸道病毒检测试剂盒（xTAG RVP）,应用于多种常见呼吸道病毒检测的优势和缺点,为更好地、有针对性地选择合适技术进行临床实验诊断或公共卫生应急检测提供依据.方法 使用VIDRL的呼吸道病毒多重PCR检测方法和Luminex公司xTAG RVP试剂盒对198份临床流感样症状患者的咽拭子标本进行常见呼吸道病毒的核酸检测.结果 198份临床标本经VIDRL多重PCR方法和xTAG RVP方法检测,阳性率分别为38.89％和45.45％,符合率为72.22％.单一病毒的符合率为87.88％～100％.结论 xTAG RVP方法与VIDRL多重PCR方法相比,两者在常见呼吸道病毒检测中的特异性和敏感性相近,xTAG RVP方法具有明显的快速和高通量的优势.     

A robotics-assisted procedure for large scale cystic fibrosis mutation analysis

We describe a convenient, efficient, semiautomated protocol for assaying large numbers of DNA samples for over 20 mutations causing cystic fibrosis. The protocol uses the following: (1) a programmable robotic workstation to perform rapid pipetting and dot-blotting operations, (2) an allele-specific oligonucleotide hybridization in a single water bath without correcting for G + C content of oligonucleotides, and (3) a combinatorial system that allows direct determination of the genotype for more frequent mutations. We have used this system routinely for 16 months for carrier detection and for diagnosis of cystic fibrosis. The method can be readily applied to any combination of allele-specific oligonucleotide assays whether for multiple alleles at one locus or for a few alleles at multiple loci. © 1994 Wiley-Liss, Inc.     

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.