Abstract
Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR). Analysis of DNA from a pancreatic sufficient patient by means of denaturing gradient gel electrophoresis (DGGE) and subsequent DNA sequencing led to the identification of a novel potential splice mutation and a novel missense mutation in the CFTR gene. One C to T substitution (297-3C→T) was found at the splice acceptor site of intron 2 and a T to C substitution at 1213 was found in exon 7. To determine the effect of the potential splicing mutation on the patient's CFTR transcripts and by taking advantage of the “illegitimate” transcription phenomenon, RNA from EBV-lymphoblastoid cells was reverse transcribed and amplified by the polymerase chain reaction (PCR). Direct sequencing of the PCR product revealed that the transcript from the chromosome with the 297-3C→T mutation exhibited the skipping of exon 3.
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Abadie V, Jaruzelska J, Lyonnet S, Millasseau P, Berthelon M, Rey F, Munnich A, Rey L (1993) Illegitimate transcription of the phenylalanine hydroxylase gene in lymphocytes for identification of mutations in phenylketonuria. Hum Mol Genet 2:31–34
Boat TF, Welsh MJ, Beaudet AL (1989) Cystic fibrosis. In: Scriver CR, Beaudet AL, Sly WS, Valle D (eds) The metabolic basis of inherited disease, 6th edn. McGraw-Hill, New York, pp 2649–2680
Chalkey G, Harris A (1991) Lymphocyte mRNA as a resource for detection of mutations and polymorphisms in the CF gene. J Med Genet 28:777–780
Chelly J, Concordet JP, Kaplan JC, Kahn A (1989) Illegitimate transcription: transcription of any gene in any cell. Proc Natl Acad Sci USA 86:2617–2621
Costes B, Girodon E, Chassignol M, Thuong NT, Dupret D, Goossens M (1993) Psoralen-modified oligonucleotide primers improve detection of mutations by denaturing gradient gel electrophoresis and provide an alternative to GC-clamping. Hum Mol Genet 2:393–397
Cystic Fibrosis Genetic Analysis Consortium (1990) Worldwide survey of the ΔF508 mutation — report from the Cystic Fibrosis Genetic Analysis Consortium. Am J Hum Genet 47:354–359
Fernandez E, Bienvenu T, Desclaux-Arramond F, Beldjord K, Kaplan JC, Beldjord C (1993) Use of chemical clamps in denaturing gradient gel electrophoresis: application in the detection of the most frequent Mediterranean β-thalassemic mutations. PCR Methods Appl 3:122–124
Fonknechten N, Chelly J, Lepercq J, Kahn A, Kaplan JC, Kitzis A, Chomel JC (1992a) CFTR illegitimate transcription in lymphoid cells: quantification and applications to the investigation of pathological transcripts. Hum Genet 88:508–512
Fonknechten N, Chomel JC, Kitzis A, Kahn A, Kaplan JC (1992b) Skipping of exon 5 as a consequence of the 711+1G→T mutation in the CFTR gene. Hum Mol Genet 1:281–282
Gotoda T, Senda M, Murase T, Yamada N, Takaku F, Furuichi Y (1989) Gene polymorphism identified byPvuII in familial lipoprotein lipase deficiency. Biochem Biophys Res Commun 164:1391–1396
Jeanpierre M (1987) A rapid method for the purification of DNA from blood. Nucleic Acids Res 15:9611
Kerem B, Rommens JM, Buchanan JA, Markiewicz D, Cox TK, Chakravarti A, Buchwald M, Tsui LC (1989) Identification of the cystic fibrosis gene: genetic analysis. Science 245:1073–1080
Riordan JR, Rommens JM, Kerem BS, Alen N, Rozmahel R, Grzelczak Z, Zielenski J, Lok S, Plavsic N, Chou JL, Drumm M, Iannuzi MC, Collins FS, Tsui LC (1989) Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245:1066–1080
Rommens JM, Iannuzzi MC, Kerem BS, Drumm ML, Melmer G, Dean M, Rozmahel R, Cole J, Kennedy D, Hidaka N, Zsiga M, Buchwald M, Riordan J, Tsui LC, Collins FS (1989) Identification of the cystic fibrosis gene: chromosome walking and jumping. Science 245:1059–1065
Sarkar G, Sommer SS (1989) Access to a messager RNA sequence or its protein product is not limited by tissue or species specificity. Science 244:331–334
Tsui LC (1992) The spectrum of cystic fibrosis mutations. Trends Genet 8:392–398
Wong C, Antonarakis SE, Goff SC, Orkin SH, Forget BG, Nathan DG, Giardina PJV, Kazazian HH Jr (1989) β-Thalassemia due to two novel nucleotide substitutions in consensus acceptor splice sequences of the β-globin gene. Blood 73:914–918
Zielenski J, Bozon D, Kerem B, Markiewicz D, Rommens JM, Tsui LC (1991) Identification of mutations in exons 1 through 8 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Genomics 10:229–235
Zielenski J, Bozon D, Markiewicz D, Aubin G, Simard F, Rommens JM, Tsui LC (1993) Analysis of CFTR transcripts in nasal epithelial cells and lymphoblasts of a cystic fibrosis patient with 621+1G→T and 711+1G→T. Hum Mol Genet 2:683–687
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Bienvenu, T., Hubert, D., Fonknechten, N. et al. Unexpected inactivation of acceptor consensus splice sequence by a −3 C to T transition in intron 2 of the CFTR gene. Hum Genet 94, 65–68 (1994). https://doi.org/10.1007/BF02272843
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DOI: https://doi.org/10.1007/BF02272843