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The identification of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has led to the identification of more than 225 presumed disease-causing mutations at the locus. The diagnosis of cystic fibrosis or the carrier state by direct DNA analysis is hindered by this large number. A practical assay must be able to detect enough mutations to achieve clinically significant sensitivity. The use of allele-specific oligonucleotide probes is the most promising of the available methods. However, to date this has generally involved tedious probe-by-probe hybridizations, due to variations in the oligonucleotides' denaturation temperatures caused by differences in their G-C base-pair content. We have developed a rapid, cost-effective assay that simultaneously detects 12 CFTR mutations after multiplex polymerase-chain-reaction amplification of genomic DNA. The test may be readily extended to detect additional mutations at minimal increase in the cost per test or the turnaround time. We improve specificity and avoid the need for individual hybridizations by the use of tetramethylammonium chloride to virtually eliminate the effects of G-C differences. Coupled with non-invasive sample-collection methods, this is an immediately practical assay for cystic fibrosis. More generally, it will serve as a model for the development of diagnostic tests in other genetic disorders involving complex mutation analysis.
Hum Mol Genet 1993 Feb
PMID:Efficient 12-mutation testing in the CFTR gene: a general model for complex mutation analysis. 768 36

Traditionally, DNA used for PCR-based diagnostic analysis has originated from white cells fractionated from whole blood. Although this method yields substantial quantities of DNA, there are some drawbacks to the procedure, including the inconvenience of drawing blood, risk of exposure to blood-borne pathogens, liquid sample handling, and the somewhat involved extraction procedure. Alternatively, DNA for genetic diagnosis has been derived from finger stick blood samples, hair roots, cheek scrapings, and urine samples. Oral saline rinses have also been used extensively as a means of collecting buccal epithelial cells as a DNA source. However, this method still requires liquid sample handling. Herein, we present our results involving the rapid extraction of DNA from buccal cells collected on cytology brushes and swabs for use in PCR reactions, specifically the multiplex amplification of 5 exons within the CFTR gene. The quality of DNA isolated from buccal cells, collected in this manner, has been sufficient to reproducibly support multiplex amplification. Cheek cell samples and the DNA prepared from them as described here are highly stable. The success rate of PCR amplification on DNA prepared from buccal cells is 99%. In a blind study comparing the analysis of 12 mutations responsible for cystic fibrosis in multiplex products amplified with DNA from both blood and buccal cell samples from 464 individuals, there was 100% correlation of results for blood and cheek cell DNA, validating the use of DNA extracted from cheek cells collected on cytology brushes for use in genetic testing.
Hum Mol Genet 1993 Feb
PMID:Multiplex PCR amplification from the CFTR gene using DNA prepared from buccal brushes/swabs. 768 37

In order to examine the onset of the cystic fibrosis (CF) disease process, the expression of the cystic fibrosis gene (CFTR) has been examined in mid-trimester human foetal tissues by in situ hybridization. CFTR mRNA was detected in the epithelia of pancreatic ducts, small intestine, colon, genital ducts, lung and trachea. The majority of these sites of CFTR expression in the foetus are similar to those seen in adult tissues. However, epithelia of the lung, that contain very little CFTR mRNA in the adult, express high levels of CFTR mRNA in the foetus. Since the lung is the major site of pathology and morbidity in CF these findings have implications for treatment.
Hum Mol Genet 1993 Mar
PMID:Expression of the cystic fibrosis gene in human foetal tissues. 768 39

An improved understanding of the expression of the cystic fibrosis gene (CFTR) will assist our approach to preventing the organ damage caused by cystic fibrosis (CF). We have studied the expression of CFTR in human fetal tissues at different gestational ages using in situ hybridization to detect CFTR mRNA. CFTR was principally expressed in less differentiated cells of endodermal origin. The highest levels were seen in specific areas of the developing pancreas, liver, gall bladder and intestine, with lower but significant levels in lung and trachea. Expression was also seen in reproductive tissues, such as epididymis and third trimester uterus and fallopian tubes, and in addition, sweat and salivary glands. No detection of CFTR mRNA was found in many other relevant tissues. The detection of CFTR transcript in these organs is consistent with the clinical manifestations of CF and the function of CFTR as a chloride channel early in development. The localization and levels of expression described have implications regarding the pathogenesis of organ damage and the potential gains that can be achieved by early therapy in the disease.
Hum Mol Genet 1993 Mar
PMID:Cell-specific localization of CFTR mRNA shows developmentally regulated expression in human fetal tissues. 768 40

The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes a cAMP-activated chloride (Cl-) channel, and expression of the full length gene in vitro is sufficient to correct the Cl- conductance defect that is characteristic of cystic fibrosis (CF) epithelial cells. Alternatively spliced forms of CFTR mRNA have been identified in several tissues from normal individuals. One of the alternative transcripts, often present at high levels, results in the in-frame deletion of exon 9. Translation of this transcript would result in a CFTR protein missing the amino terminal portion of the first nucleotide binding fold (NBF). To evaluate the possible function of this form of CFTR, a cDNA representing this transcript (CFTR delta 9) was transduced into CFPAC cells, which are derived from a CF patient. CFTR delta 9 RNA was expressed in the transduced cell lines, but only immature, incompletely glycosylated protein was detectable by Western blot analysis. No increase in cAMP-activated anion permeability was detectable by 125I efflux assay or by means of the halide sensitive dye 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ). In a second assay system, in vitro synthesized mRNA representing CFTR delta D9 was injected into Xenopus oocytes, but expression of this alternatively spliced form of CFTR was not associated with the appearance of Cl- conductance. These results suggest that the protein produced by the CFTR delta 9 transcript is not properly processed and is not capable of generating Cl- conductance in response to cAMP. Whether this alternative transcript has some other function or represents 'noise' in the mRNA splicing mechanism remains unresolved.
Hum Mol Genet 1993 Mar
PMID:Expression of an abundant alternatively spliced form of the cystic fibrosis transmembrane conductance regulator (CFTR) gene is not associated with a cAMP-activated chloride conductance. 768 41

Single sperm typing provides an accurate method to order tightly linked loci by single cell DNA high resolution segregation analysis. We have used similar methods to type individual sperm from a known delta F508 cystic fibrosis heterozygote to determine the frequency of the mutation within his germ cell population, and to test possible explanations for the reported sex ratio distortion of the cystic fibrosis (CF) mutation to male offspring. Using a nested polymerase chain reaction we have been able to amplify a single locus sequence, cystic fibrosis transmembrane conductance regulator (CFTR), from a single target sperm haploid genome to detectable amounts without the use of radioactivity. The same sperm from a single male delta F508 carrier were simultaneously typed for the presence of the sex chromosomes to verify the ratio of X- to Y-bearing sperm and to determine the association, if any, between sex and delta F508 in male gametes. We have demonstrated that there is a significant difference in the proportions of 'normal' and 'delta F508' sperm (X2 = 7.36, p < 0.01), although when the same sperm are sexed the difference between delta F508/X and delta F508/Y sperm is not significant (X2 = 1.71, p = 0.192). Single sperm typing can address questions about segregation distortion in man, and it is unlikely that sex ratio distortion for CF carriers is due to events which occur pre-fertilisation. As these data are from one individual only, they should be confirmed for other male carriers, including those with different CF mutations.
Hum Mol Genet 1993 Apr
PMID:Segregation of delta F508 and normal CFTR alleles in human sperm. 768 44

Strains of Pseudomonas aeruginosa causing pulmonary infections in cystic fibrosis patients have an unusual mucoid phenotype because of production of the capsule-like exopolysaccharide, alginate. Transcriptional activation of algD, the first gene of a large alginate biosynthetic gene cluster, is associated with conversion to the alginate-producing (Alg+) phenotype. In this study, we examined the regulation of alginate genes immediately downstream of algD. Mutants of the Alg+ strain FRD1 were constructed by gene replacement with defined Tn501 (8.2kb) insertions in the alginate biosynthetic gene cluster, resulting in an Alg- phenotype. The Alg+ phenotype of these mutants was restored by integration of narrow-host-range plasmids containing DNA fragments from P. aeruginosa that reconstructed a continuous alginate gene cluster. A broad-host-range plasmid containing the entire alginate gene cluster except for the terminal gene, algA, was unable to complement an alG::Tn501 mutant unless algA was transcribed from a second plasmid. This indicated that any Tn501 insertion in the cluster was polar on downstream alginate genes. Northern blot hybridization experiments also showed that a transposon insertion downstream of algD adversely affected algG and algA transcription. These results provided evidence that the alginate biosynthetic gene cluster has an operonic structure and is cotranscribed from the algD promoter.
Mol Microbiol 1993 May
PMID:Genetic analysis of the alginate biosynthetic gene cluster of Pseudomonas aeruginosa shows evidence of an operonic structure. 768 97

More than 250 mutations have been detected in the cystic fibrosis (CF) transmembrane regulator (CFTR) gene, most of which are single point mutations or small deletions or insertions of a few nucleotides. Here we report the first large deletion identified in the CFTR gene, which involves 50 kb in two stretches of DNA: one of 10 kb from exon 4 to exon 7, and another of 40 kb, spanning exons 11 to 18. The deletion has been detected via uniparental inheritance of CFTR microsatellite alleles (IVS17BTA and IVS17BCA) in 3 independent CF families. Clinical status of the 3 CF patients, of which two have the delta F508 mutation as the other CF allele, suggests that this mutation is responsible for a severe clinical phenotype, indistinguishable from homozygous delta F508 patients. The deletion detected here suggests that other large, but less complex molecular defects could also exist in the CFTR gene.
Hum Mol Genet 1993 Jun
PMID:Uniparental inheritance of microsatellite alleles of the cystic fibrosis gene (CFTR): identification of a 50 kilobase deletion. 768 7

We have analyzed the CFTR mRNA populations in a cystic fibrosis patient heterozygous for the 621 + 1G-->T and 711 + 1G-->T mutations. Total RNA isolated from the nasal epithelial cells and Epstein-Barr virus-transformed lymphoblasts derived from this patient was reversely transcribed and a region extending from exon 3 to exon 7 of the gene was amplified by the polymerase chain reaction and analyzed. Three abnormal products were identified, suggesting the presence of three aberrant transcripts, and their profiles were identical in both cell types. Two of the products were found to be missing either exon 4 or exon 5 as anticipated from the transcripts from the 621 + 1G-->T or 711 + 1G-->T alleles, respectively. The third product was apparently derived from an alternatively spliced mRNA species in the absence of the nominal splice site (in 621 + 1G-->T) through the use of a cryptic splice donor sequence (TT528/GTGAGG) within exon 4. Although reading frames appeared to be preserved in all three putative transcripts, significant portions of the presumed first and second transmembrane spans as well as the immediately following cytoplasmic domain would be deleted from the mutant CFTR polypeptides, if made. These observations are consistent with a loss of CFTR function in this cystic fibrosis patient.
Hum Mol Genet 1993 Jun
PMID:Analysis of CFTR transcripts in nasal epithelial cells and lymphoblasts of a cystic fibrosis patient with 621 + 1G-->T and 711 + 1G-->T mutations. 768 8

Three different putative splicing mutations in the CFTR gene have been studied by analysing mRNA extracted from nasal epithelial cells harvested from patients with cystic fibrosis. Six patients were analysed, all of whom had classical symptoms of cystic fibrosis (CF). Two patients carried the 621 + 1G-->T mutation, 3 patients carried the 1717 - 1G-->A mutation and 1 patient carried the 1898 + 1G-->A mutation. All patients carried the delta F508 mutation on the other chromosome. Ten non-CF control subjects were also studied. The 621 + 1G-->T mutation resulted in activation of an alternative splice site within exon 4 in one patient and activation of this site or skipping of exon 4 in the other patient. The 1717 - 1G-->A mutation resulted in skipping of exon 11 in all 3 patients studied and the 1898 + 1G-->T mutation resulted in skipping of exon 12. These experiments demonstrate that these mutations do result in aberrant splicing of CFTR mRNA as predicted from the changes in genomic sequence.
Hum Mol Genet 1993 Jun
PMID:Abnormal mRNA splicing resulting from three different mutations in the CFTR gene. 768 9


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