ABCMdb

PMID: 7693946

Will K, Reiss J, Dean M, Schlosser M, Slomski R, Schmidtke J, Stuhrmann M
CFTR transcripts are undetectable in lymphocytes and respiratory epithelial cells of a CF patient homozygous for the nonsense mutation R553X.
J Med Genet. 1993 Oct;30(10):833-7., [PubMed]

Sentences

No. Mutations Sentence Comment

0 ABCC7 p.Arg553* ABCC7 p.Arg553* ABCC7 p.Arg553* ABCC7 p.Arg553* ABCC7 p.Arg553* J Med Genet 1993; 30: 833-837 CFTR transcripts are undetectable in lymphocytes and respiratory epithelial cells of a CF patient homozygous for the nonsense mutation R553X Katrin Will, Jochen Reiss, Michael Dean, Manfred Schlosser, Ryzard Slomski, Jorg Schmidtke, Manfred Stuhrmann Abstract In order to analyse the influence of the nonsense mutation R553X on CFTR gene expression, transcripts from epithelial cells and lymphocytes were examined from nine subjects (one CF patient homozygous for R553X, one CF patient compound heterozygous for R553X/ AF508, four CF carriers heterozygous for R553X, one CF carrier with the genotype AF508/N, and two uncharacterised normal adults). Login to comment 3 ABCC7 p.Arg553* In repeated experiments it was not possible to detect any CFTR mRNA in cells derived from the R553X homozygous patient. Login to comment 4 ABCC7 p.Arg553* ABCC7 p.Arg553* Furthermore, in subjects heterozygous for R553X we could not detect by hybridisation with a specific oligonucleotide probe and direct sequencing any CFTR mRNA derived from the R553X allele. Login to comment 11 ABCC7 p.Trp1282* ABCC7 p.Arg553* ABCC7 p.Gly542* The most frequent mutation, AF508,6 located in the first nucleotide binding fold, is usually associated with severe CF,7 but in exceptional cases AF508 homozygotes are only mildly affected.8 Three nonsense mutations, G542X,9 R553X,'0 and W1282X," represent the second, fourth, and fifth most frequent CF mutations worldwide (Cystic Fibrosis Genetics Analysis Consortium, unpublished data). Login to comment 13 ABCC7 p.Arg553* ABCC7 p.Trp1316* ABCC7 p.Gly542* ABCC7 p.Gly542* ABCC7 p.Ser1255* Two CF patients, homozygous for the G542X mutation, have been described as mildly affected."213 Cutting et al14 found two patients heterozygous for W1316X/R553X and S1255X/G542X, respectively, to exhibit severe pancreatic but only mild pulmonary disease. Login to comment 14 ABCC7 p.Trp1282* ABCC7 p.Arg553* In contrast, Shoshani et al"5 showed that the most common CF mutation in the Ashkenazi Jewish population, W1282X, which is located in exon 20, is clearly associated with a severe phenotype. We previously classified the only known homozygous R553X patient as being moderately severely affected. Login to comment 15 ABCC7 p.Arg553* ABCC7 p.Arg553* '6 We report here on CFTR mRNA analysis in this patient, who is homozygous for R553X (a CGA to TGA substitution at position 1789 in exon 11 of the CFTR gene), and in R553X heterozygotes, in order to investigate the influence of nonsense mutations on gene expression. Login to comment 16 ABCC7 p.Arg553* Methods FAMILY A Case 1 was a 15 year old male CF patient of German origin, who was homozygous for the stop mutation R553X, with severely reduced pancreatic function, but only mildly affected lung function. Login to comment 19 ABCC7 p.Arg553* '6 Case 2 was a male CF carrier (R553X) of German origin. He was a symptomless adult and the father of case 1 and a second cousin of case 3. Login to comment 20 ABCC7 p.Arg553* Case 3 was a female CF carrier (R553X) of German origin. She was a symptomless adult and the mother of case 1 and a second coetsin of case 2. Login to comment 27 ABCC7 p.Arg553* 834 Will, Reiss, Dean, Schlosser, Slomski, Schmidtke, Stuhrmann FAMILY B Case 4 was a 6 year old male CF patient of German origin and a compound heterozygote for AF508 and R553X, with insufficient pancreatic function and recurrent pneumonia. Login to comment 31 ABCC7 p.Arg553* Case 6 was a female CF carrier (R553X) of German origin. She was a symptomless adult and the mother of case 4. Login to comment 32 ABCC7 p.Arg553* CONTROLS Case 7 was a male CF carrier (R553X) of American origin and was a symptomless adult. Case 8 was a female of German origin and was a symptomless adult. Case 9 was a male of Polish origin and was a symptomless adult. Login to comment 53 ABCC7 p.Arg553* '9 Filters were prehybridised for one hour at 60'C (ASO for wild type allele) and 62'C (R553X mutation specific ASO) in a hybridisation solution containing 5 x SSPE, 5 x Denhardt's solution, and 0-5% Triton X-100. Login to comment 59 ABCC7 p.Arg553* ABCC7 p.Arg553* As positive controls, PCR products including exon 11, amplified with primers 1 li-5 and 1 li-32" from genomic DNA of a R553X carrier, a patient homozygous for R553X, and a normal adult were blotted and hybridised. Login to comment 63 ABCC7 p.Arg553* With the exception of the R553X homozygous patient (lane 5), cDNA amplification was possible from all nasal epithelial cells and lymphocytes that were attempted. Login to comment 74 ABCC7 p.Arg553* (A) 0-8% agarose gel of 'nested' PCR products amplifiedfrom the R553X homozygous patient (lane 5), the patient's mother (lane 3), the patient's father (lane 4), and two healthy controls (lanes 1 and 2). Login to comment 82 ABCC7 p.Arg553* Exon 12 skipping, accounting for over 90% of the detectable mRNA, has been previously reported.2223 In repeated experiments, it was not possible to detect any CFTR cDNA from the R553X homozygous patient on ethidium bromide stained agarose gel. Login to comment 85 ABCC7 p.Arg553* In order to increase the level of detection sensitivity, we performed allele specific oligonucleotide (ASO) hybridisations for the R553X and normal alleles. Login to comment 86 ABCC7 p.Arg553* ABCC7 p.Arg553* As shown in fig 3, only the CFTR exon 11 controls, amplified from genomic DNA of homozygotes and heterozygotes for R553X, hybridised to the R553X mutant oligonucleotide. Login to comment 87 ABCC7 p.Arg553* CFTR exon 11 DNA of a person heterozygous for R553X hybridised to both the wild type and the mutant oligonucleotides. Login to comment 89 ABCC7 p.Arg553* The PCR reaction from the homozygous patient hybridised to neither the normal nor the R553X ASO. Login to comment 91 ABCC7 p.Arg553* These results show that the levels of CFTR transcripts carrying the R553X mutation are severely reduced or even absent in both tissues. Login to comment 92 ABCC7 p.Arg553* ABCC7 p.Arg553* Discussion We show here that patients, either homozygous or heterozygous for the nonsense mutation R553X, have no detectable CFTR mRNA derived from the R553X allele in nasal epithelial cells and lymphocytes. Login to comment 99 ABCC7 p.Arg553* Figure 3 Allele specific oligonucleotide hybridisation (ASO) for the R553X and normal alleles. Login to comment 100 ABCC7 p.Arg553* ABCC7 p.Arg553* Each membrane was dotted with amplified cDNAs from all subjects (subjects 1 to 9 from left to right) and PCR products including exon 11, amplified with primers 1 li-5 and 1 li-320 from genomic DNA of the R553X homozygous patient, a R553X carrier, and a normal adult. Login to comment 101 ABCC7 p.Arg553* ABCC7 p.Arg553* Hybridisation was carried out with an ASO for the wild type allele (normal) and a R553X mutation specific ASO (R553X). Login to comment 102 ABCC7 p.Arg553* Solid symbols: R553X, hatched symbols: AF508, open symbols: normal. Login to comment 105 ABCC7 p.Arg553* If stop codon mutations, such as R553X, within the first half of the CFTR gene, generally lead to severely reduced or even absent intracytoplasmic mRNA levels, it is surprising that the 583 bp splice variant carrying a stop codon within the same region appears to be rather stable2' (this study). Login to comment 110 ABCC7 p.Trp1282* Transcription studies of patients homozygous for the stop mutation W1282X within exon 20 of the CFTR gene (that is, its 3' end), which is associated with a severe phenotype,'5 as well as mRNA analysis of other patients homozygous for nonsense mutations will help to come to a better understanding of the relationship between the location of nonsense mutations, and their influence on the mRNA level and the clinical phenotype. We thank C Stewart and B Gerrard, PRI/ DynCorp, Frederick, Maryland, for technical assistance. Login to comment 147 ABCC7 p.Trp1282* Association of a nonsense mutation (W1282X), the most common mutation in the Ashkenazi Jewish cystic fibrosis patients in Israel, with presentation of severe disease. Login to comment 150 ABCC7 p.Arg553* A cystic fibrosis patient homozygous for the nonsense mutation R553X. Login to comment 169 ABCC7 p.Arg553* ABCC7 p.Trp1316* Severe deficiency of cystic fibrosis transmembrane conductance regulator messenger RNA carrying nonsense mutations R553X and W1316X in respiratory epithelial cells of patients with cystic fibrosis. Login to comment 183 ABCC7 p.Arg553* Mol Cell Biol 1989;9:2868-80. doi: 10.1136/jmg.30.10.833 1993 30: 833-837J Med Genet K Will, J Reiss, M Dean, et al. nonsense mutation R553X. Login to comment