首次发现国人先天性双侧输精管缺如CFTR基因突变

目的：探讨囊性纤维化跨膜转运调节物（ＣＦＴＲ）基因在国人先天性双侧输精管缺如患者中的突变频率及热点。方法：应用ＰＣＲ－单链构象多态（ＰＣＲ－ＳＳＣＰ）及ＤＮＡ邓技术,对３２例先天性双侧输精管缺如患者ＣＦＴＲ匪帮经２,３,４,５,６ａ,８,１０,１１,１２,１３,１５Ａ,１７ｂ,１９Ａ,２０,２１,２３外显子区域上的突变进行筛查,结果：１例患者在１０号外显子第１６５３至１６５５位三个碱基ＴＴＴ缺失,     

Comprehensive and rapid genotyping of mutations and haplotypes in congenital bilateral absence of the vas deferens and other cystic fibrosis transmembrane conductance regulator-related disorders

Available commercial kits only screen for the most common cystic fibrosis transmembrane conductance regulator (CFTR) mutations causing classic cystic fibrosis and for the Tn variant in IVS8. However, full scanning of CFTR is needed for the diagnosis of patients with cystic fibrosis or CFTR-related disorders (including congenital bilateral absence of the vas deferens) bearing rare mutations. Standard strategies for detecting point mutations rely on extensive scanning of the gene by denaturing gradient gel electrophoresis or denaturing high performance liquid chromatography, which are time-consuming. Moreover, the haplotyping of IVS8-(TG)m and Tn tracts is still challenging despite several recent improvements. We have optimized both the detection of mutations and the haplotyping of IVS8 polyvariants in developing two methods: i) a rapid and robust direct sequence analysis of all exons/flanking introns of the CFTR gene based on single condition touchdown amplification/sequencing in 96-well plates, and ii) a fluorescent assay that allows haplotyping of IVS8-(TG)mTn even without family linkage study. Combined with search for rare large rearrangements, this strategy detected 87.9% of CFTR defects in congenital bilateral absence of the vas deferens patients, a proportion considerably higher than those usually reported. These highly efficient tests, scanning each sample in a few days, greatly improve the genotyping of patients with CFTR-related symptoms and may be particularly important in emergency situations such as fetus with hyperechogenic bowel suggestive of cystic fibrosis.     

Cystic fibrosis transmembrane conductance regulator mutations that disrupt nucleotide binding.

Increasing evidence suggests heterogeneity in the molecular pathogenesis of cystic fibrosis (CF). Mutations such as deletion of phenylalanine at position 508 (delta F508) within the cystic fibrosis transmembrane conductance regulator (CFTR), for example, appear to cause disease by abrogating normal biosynthetic processing, a mechanism which results in retention and degradation of the mutant protein within the endoplasmic reticulum. Other mutations, such as the relatively common glycine-->aspartic acid replacement at CFTR position 551 (G551D) appear to be normally processed, and therefore must cause disease through some other mechanism. Because delta F508 and G551D both occur within a predicted nucleotide binding domain (NBD) of the CFTR, we tested the influence of these mutations on nucleotide binding by the protein. We found that G551D and the corresponding mutation in the CFTR second nucleotide binding domain, G1349D, led to decreased nucleotide binding by CFTR NBDs, while the delta F508 mutation did not alter nucleotide binding. These results implicate defective ATP binding as contributing to the pathogenic mechanism of a relatively common mutation leading to CF, and suggest that structural integrity of a highly conserved region present in over 30 prokaryotic and eukaryotic nucleotide binding domains may be critical for normal nucleotide binding.     

Cystic fibrosis transmembrane conductance regulator (CFTR) activity in nasal epithelial cells from cystic fibrosis patients with severe genotypes

Tumour recurrence following chemotherapy remains a major obstacle to the cure of many cancers. This is exemplified by small-cell lung cancer (SCLC). Host-tumour interactions are central to tumour survival and proliferation. We hypothesized that a factor(s) within the local environment of SCLC cells could provide a survival signal or block a death signal, thereby accounting for the protection of SCLC cells from chemotherapy-induced apoptosis. Here we review recent work undertaken in our laboratory addressing this issue. We have shown that, in vivo, SCLC cells are surrounded by an extensive stroma of extracellular matrix (ECM) at both primary and metastatic sites which contains, among other proteins, fibronectin, laminin and collagen IV. Furthermore, adhesion of SCLC cells to fibronectin, laminin and collagen IV through beta1 integrins enhances tumorigenicity and confers resistance to apoptosis induced by standard chemotherapeutic agents, including etoposide, cis-platinum and adriamycin. Adhesion to ECM proteins stimulated protein tyrosine kinase (PTK) activity in both untreated and etoposide-treated cells. This effect could be completely blocked by a selective PTK inhibitor or by a function-blocking beta1 integrin antibody. PTK activation was found to block chemotherapy-induced activation of the death protease caspase-3 and, hence, apoptosis. Adhesion to ECM or treatment with a PTK inhibitor did not affect etoposide inhibition of topoisomerase II. Thus adhesion to ECM through beta1 integrins protects SCLC cells from chemotherapy-induced caspase-3 activation and apoptosis by activating PTK signalling downstream of DNA damage. Survival of tumour cells attached to ECM within this microenvironment could explain the local recurrence of SCLC and other tumours that is often seen clinically after chemotherapy.     

Scanning the cystic fibrosis transmembrane conductance regulator gene using high-resolution DNA melting analysis

BACKGROUND: Complete gene analysis of the cystic fibrosis transmembrane conductance regulator gene (CFTR) by scanning and/or sequencing is seldom performed because of the cost, time, and labor involved. High-resolution DNA melting analysis is a rapid, closed-tube alternative for gene scanning and genotyping. METHODS: The 27 exons of CFTR were amplified in 37 PCR products under identical conditions. Common variants in 96 blood donors were identified in each exon by high-resolution melting on a LightScanner(R). We then performed a subsequent blinded study on 30 samples enriched for disease-causing variants, including all 23 variants recommended by the American College of Medical Genetics and 8 additional, well-characterized variants. RESULTS: We identified 22 different sequence variants in 96 blood donors, including 4 novel variants and the disease-causing p.F508del. In the blinded study, all 40 disease-causing heterozygotes (29 unique) were detected, including 1 new probable disease-causing variant (c.3500-2A>T). The number of false-positive amplicons was decreased 96% by considering the 6 most common heterozygotes. The melting patterns of most heterozygotes were unique (37 of 40 pairs within the same amplicon), the exceptions being p.F508del vs p.I507del, p.G551D vs p.R553X, and p.W1282X vs c.4002A>G. The homozygotes p.G542X, c.2789 + 5G>A, and c.3849 + 10kbC>T were directly identified, but homozygous p.F508del was not. Specific genotyping of these exceptions, as well as genotyping of the 5T allele of intron 8, was achieved by unlabeled-probe and small-amplicon melting assays. CONCLUSIONS: High-resolution DNA melting methods provide a rapid and accurate alternative for complete CFTR analysis. False positives can be decreased by considering the melting profiles of common variants.     

Simultaneous screening for 11 mutations in the cystic fibrosis transmembrane conductance regulator gene by multiplex amplification and reverse dot-blot.

An assay is described in which 11 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene can be screened simultaneously. Six different exons of the CFTR gene are amplified in a single multiplex amplification. Biotinylated dUTP is incorporated into the different fragments during the amplification process. A sample of this mixture is then hybridized to 21 different poly-dT tailed oligonucleotide probes which are bound to a nylon membrane. In order to screen the different mutations in a single step hybridization, the length of the different oligonucleotides and the amount used in the assay were optimized. The detection is performed by binding avidin-alkaline phosphatase to the biotin, followed by a chemiluminescent reaction. By means of this fast and sensitive assay, about 85% of all the cystic fibrosis mutations in the Belgian population can be detected.     

Characterization of a recurrent novel large duplication in the cystic fibrosis transmembrane conductance regulator gene

Recently, DNA rearrangements in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been described with increasing frequency. These large DNA rearrangements are not detected using conventional methods of DNA sequencing, single-strand conformational polymorphism, or denaturing high-performance liquid chromatography. We and others have described methods to detect such rearrangements in the CFTR gene. With one exception, all rearrangements reported thus far are single or multiple exon deletions, whereas only one report has described a large duplication. We describe here the detection and characterization of a novel large duplication in the CFTR gene. This duplication, referred to as gIVS6a + 415_IVS10 + 2987Dup26817bp, was detected in a classic CF female patient whose other mutation was DeltaF508. The duplication was inherited paternally. The duplication encompassed exons 6b to 10 and occurred on the IVS8-11TG/IVS8-7T/G1540 haplotype. This large duplication is predicted to result in the production of a truncated CFTR protein lacking the terminal part of NBD1 domain and beyond and thus can be considered a null allele. The combination of the DeltaF508 and gIVS6a + 415_IVS10 + 2987Dup26817bp mutation probably causes the severe CF phenotype in this patient. We designed a simple polymerase chain reaction test to detect the duplication, and we further detected the same duplication from another independent laboratory. The duplication breakpoint is identical in all three patients, suggesting a likely founder mutation.     

Localization of the cystic fibrosis transmembrane conductance regulator in pancreas.

Cystic fibrosis (CF) is characterized by an abnormality in cAMP-regulated chloride transport that results from a primary defect in the protein product of the CF gene, the CF transmembrane conductance regulator (CFTR). In this report, antibodies against CFTR peptides were used to localize the CFTR protein in human pancreas. An affinity purified antibody (alpha-1468) raised against a synthetic CFTR peptide identified a 155-170-kD protein on immunoblot. Cytochemical studies with alpha-1468 localized CFTR to small branching, tubular structures. The same structures were recognized by two other antibodies raised against different regions of the CFTR molecule. To identify the cells being stained, double-label immunofluorescence studies were performed using alpha-1468 and a monoclonal antibody which stains pancreatic centroacinar and intralobular duct cells. Both antibodies localized to the same population of cells, with alpha-1468 being confined to the apical domain of these cells. No conclusive staining of acinar cells was evident. These findings suggest that proximal duct epithelial cells play a key role in the early events leading to pancreatic insufficiency in CF, and imply that apical chloride transport by these cells is essential for normal pancreatic secretory function.     

Recovery of airway cystic fibrosis transmembrane conductance regulator function in mice with cystic fibrosis after single-dose lentivirus-mediated gene transfer

The potential for gene therapy to be an effective treatment for cystic fibrosis (CF) airway disease has been limited by inefficient gene transfer vector particle delivery and lack of persistent gene expression. We have developed an airway conditioning process that, when combined with a human immunodeficiency virus (HIV)-derived lentivirus (LV) vector, resulted in persistent in vivo expression of transgenes in airway epithelium. Pretreatment of mouse nasal epithelium with the detergent lysophosphatidylcholine (LPC) prior to instillation of a single dose of an LV vector carrying the LacZ marker gene produced significant LacZ gene expression in nasal airway epithelium for at least 92 days. Transduction of the cystic fibrosis transmembrane conductance regulator (CFTR) gene using the same LV vector system resulted in partial recovery of electrophysiologic function in the nasal airway epithelium of CF mice (cftr(tm1Unc) knockout) for at least 110 days. This first demonstration of LV-mediated in vivo recovery of CFTR function in CF airway epithelium illustrates the potential of combining a preconditioning of the airway surface with a simple and brief LV vector exposure to produce therapeutic gene expression in airway.     

Localization of cystic fibrosis transmembrane conductance regulator in chloride secretory epithelia.

Cystic fibrosis is caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). To further our understanding of CFTR's function and regulation, we used confocal immunofluorescence microscopy to localize CFTR in cells stained with monoclonal antibodies against different regions of the protein: the R (regulatory) domain (M13-1), the COOH terminus (M1-4), and a predicted extracellular domain (M6-4). All three antibodies immunoprecipitated a 155-170-kD polypeptide from cells expressing CFTR. Each antibody stained HeLa and 3T3 cells expressing recombinant CFTR, but not cells lacking endogenous CFTR: HeLa, NIH-3T3, and endothelial cells. For localization studies, we used epithelial cell lines that express endogenous CFTR and have a cAMP-activated apical Cl- permeability: T84, CaCo2, and HT29 clone 19A. Our results demonstrate that CFTR is an apical membrane protein in these epithelial cells because (a) staining for CFTR resembled staining for several apical membrane markers, but differed from staining for basolateral membrane proteins; (b) thin sections of cell monolayers show staining at the apical membrane; and (c) M6-4, an extracellular domain antibody, stained the apical surface of nonpermeabilized cells. Our results do not exclude the possibility that CFTR is also located beneath the apical membrane. Increasing intracellular cAMP levels did not change the apical membrane staining pattern for CFTR. Moreover, insertion of channels by vesicle fusion with the apical membrane was not required for cAMP-mediated increases in apical membrane Cl- conductance. These results indicate that CFTR is located in the apical plasma membrane of Cl(-)-secreting epithelia, a result consistent with the conclusion that Cl TR is an apical membrane chloride channel.     

A new insertion/deletion of the cystic fibrosis transmembrane conductance regulator gene accounts for 3.4% of cystic fibrosis mutations in Sardinia: implications for population screening

Previous studies performed on Sardinian patients affected by cystic fibrosis (CF) have led to the identification of molecular defects in 87 of 88 patients. Two mutations, the F508del and T338I, were quite prevalent and accounted for 50% and 20% of the molecular defects, respectively. T338I has been detected rarely in other populations, most likely because of the genetic isolation of Sardinians. In the present study, we have performed a molecular analysis of the CF gene in eight Sardinian patients in whom only a single mutation has been defined. Using DNA analyses (Southern blot, single nucleotide polymorphisms, microsatellite analyses, and Extra-Long polymerase chain reaction) selected to detect gross gene rearrangement and by using mRNA studies, we detected a novel mutation c.54-5811_164 + 2186del8108ins182 in six of the eight patients investigated. This mutation consists of a gross deletion of 8108 bp spanning exon 2 with an insertion of 182 bp at the deletion junction, between nucleotide 54-5811 of intron 1 (IVS1 nt16864) and nucleotide 164 + 2186 of intron 2 (IVS2 nt 2186). By including the novel mutation in our mutation panel we are now able to reach a 95% detection rate, thereby improving the process of carrier detection and genetic counseling in Sardinia.     

Complete gene scanning by temperature gradient capillary electrophoresis using the cystic fibrosis transmembrane conductance regulator gene as a model

Many inherited diseases involve large genes with many different mutations. Identifying a wide spectrum of mutations requires an efficient gene-scanning method. By differentiating thermodynamic stability and mobility of heteroduplexes from heterozygous samples, temperature gradient capillary electrophoresis (TGCE) was used to scan the entire coding region of the cystic fibrosis transmembrane conductance regulator gene. An initial panel (29 different mutations) showed 100% agreement between TGCE scanning and previously genotyped results for heterozygous samples. Different peak patterns were observed for single base substitutions and base insertions/deletions. Subsequently, 12 deidentified clinical samples genotyped as wild type for 32 mutations were scanned for the entire 27 exons. Results were 100% concordance with the bidirectional sequence analysis. Ten samples had nucleotide variations including a reported base insertion in intron 14b (2789 + 2insA) resulting in a possible mRNA splicing defect, and an unreported missense mutation in exon 20 (3991 G/A) with unknown clinical significance. This methodology does not require labeled primers or probes for detection and separation through a temperature gradient eliminates laborious temperature optimization required for other technologies. TGCE automation and high-throughput capability can be implemented in a clinical environment for mutation scanning with high sensitivity, thus reducing sequencing cost and effort.     

Macromolecular complexes of cystic fibrosis transmembrane conductance regulator and its interacting partners

The cystic fibrosis transmembrane conductance regulator (CFTR) is the product of the gene mutated in patients with cystic fibrosis (CF). CFTR is a cAMP-regulated chloride channel localized primarily at the apical or luminal surfaces of epithelial cells lining the airway, gut, exocrine glands, etc., where it is responsible for transepithelial salt and water transport. CFTR chloride channel belongs to the superfamily of the ATP-binding cassette (ABC) transporters, which bind ATP and use the energy to drive the transport of a wide variety of substrates across extra- and intracellular membranes. A growing number of proteins have been reported to interact directly or indirectly with CFTR chloride channel, suggesting that CFTR might regulate the activities of other ion channels, receptors, or transporters, in addition to its role as a chloride conductor. The molecular assembly of CFTR with these interacting proteins is of great interest and importance because several human diseases are attributed to altered regulation of CFTR, among which cystic fibrosis is the most serious one. Most interactions primarily occur between the opposing terminal tails (N- or C-) of CFTR and its binding partners, either directly or mediated through various PDZ domain-containing proteins. These dynamic interactions impact the channel function as well as the localization and processing of CFTR protein within cells. This review focuses on the recent developments in defining the assembly of CFTR-containing complexes in the plasma membrane and its interacting proteins.     

急性心肌梗死后囊性纤维化跨膜转运调节体氯通道基因表达的变化

目的：研究猪急性心肌梗死（AMI）后囊性纤维化跨膜转运调节体（CFTR）氯通道基因表达的变化，探讨急性心肌梗死后早期室性心律失常发生的分子机制。 方法：通过结扎猪左前降支远端1 /3-1 /2处2h然后再灌注建立AMI模型，同时设立相应的假手术(SH)组。术后24h取左心室梗死区、边缘区和正常区内层（Endo）、中层（Mid）和外层（Epi）心肌（SH组取对应位置心肌），应用逆转录－聚合酶链反应(RT－PCR)半定量分析CFTR氯通道基因表达的改变。 结果： 与SH组相比，AMI组CFTR mRNA表达在梗死区三层心肌中均明显下降（P<0.05），在边缘区三层心肌中均明显上升（P<0.05），在正常区三层心肌中均没有显著性改变（P>0.05）。AMI组CFTR mRNA表达在梗死区和边缘区的三层心肌之间以及在梗死区、边缘区和正常区同一层心肌之间有显著性差异（P<0.05）。 结论：AMI后CFTR氯通道基因表达在梗死区、边缘区和正常区心肌之间以及局部心肌三层间呈不均一性改变，可能是AMI后室性心律失常发生的分子生物学基础之一。     

Direct visualization of cystic fibrosis transmembrane regulator mutations in the clinical laboratory setting

BACKGROUND: The recommendation for population- based cystic fibrosis (CF) carrier screening by the American College of Medical Genetics for the 25 most prevalent mutations and 6 polymorphisms in the CF transmembrane regulatory gene has greatly increased clinical laboratory test volumes. We describe the development and technical validation of a DNA chip in a 96-well format to allow for high-throughput genotype analysis. METHODS: The CF Portrait chip contains an 8 x 8 array of capture probes and controls to detect all requisite alleles. Single-tube multiplex PCR with 15 biotin-labeled primer pairs was used to amplify sequences containing all single-nucleotide polymorphisms to be interrogated. Detection of a thin-film signal created by hybridization of multiplex PCR-amplified DNA to complementary capture probes was performed with an automated image analysis instrument, NucleoSight. Allele classification, data formatting, and uploading to a laboratory information system were fully automated. RESULTS: The described platform correctly classified all mutations and polymorphisms and can screen approximately 1300 patient samples in a 10-h shift. Final validation was performed by two separate 1000-sample comparisons with Roche CF Gold line probe strips and the Applera CF OLA, Ver 3.0. The CF Portrait Biochip made no errors during this validation, whereas the Applera assay made seven miscalls of the IVS-8 5T/7T/9T polymorphism CONCLUSIONS: The CF Portrait platform is an automated, high-throughput, DNA chip-based assay capable of accurately classifying all CF mutations in the recommended screening panel, including the IVS-8 5T/7T/9T polymorphism.     

A phase I study of adenovirus-mediated transfer of the human cystic fibrosis transmembrane conductance regulator gene to a lung segment of individuals with cystic fibrosis

A third-generation adenoviral vector containing recombinant human cystic fibrosis transmembrane conductance regulator (CFTR) gene was delivered by bronchoscope in escalating doses to the conducting airway of 11 volunteers with cystic fibrosis. Assessments of dose-limiting toxicity (DLT), efficiency of gene transfer, and cell-mediated and humoral immune responses to vector administration were performed. DLT, manifest by flulike symptoms and transient radiographic infiltrates, was seen at 2.1 x 10(11) total viral particles. A highly specific assay for gene transfer was developed using in situ hybridization with an oligoprobe against unique vector sequence. Detectable gene transfer was observed in harvested bronchial epithelial cells (<1%) 4 days after vector instillation, which diminished to undetectable levels by day 43. Adenovirus-specific cell-mediated T cells were induced in most subjects, although only mild increases in systemic humoral immune response were observed. These results demonstrate that gene transfer to epithelium of the lower respiratory tract can be achieved in humans with adenoviral vectors but that efficiency is low and of short duration in the native CF airway.