Advertisement

Springer Nature is making Coronavirus research free. View research | View latest news | Sign up for updates

Methods for analysis of multiple cystic fibrosis mutations

  • 123 Accesses

  • 17 Citations

Summary

A large number of mutations causing cystic fibrosis (CF) have been reported. In an attempt to improve methods for genetic diagnosis and for heterozygote screening, we evaluated methods for efficient analysis of the ΔF508, G542X, G551D, R553X, and N1303K mutations. We found that multiple mutations can be analyzed simultaneously using hybridization with allelespecific oligonucleotides. Alternatively all of these mutations can be detected by amplification of DNA followed by restriction enzyme digestion and analysis on polyacrylamide gels. A previously reported method for use of modified primers for DNA amplification to allow detection of virtually any single-base change by restriction enzyme analysis proved particularly useful. The common ΔF508 mutation and three mutations in exon 11 were analyzed using a multiplex amplification reaction followed by double digestion with restriction enzymes and electrophoresis in a single lane on a polyacrylamide gel. In a sample of 439 CF chromosomes from North American Caucasians, the frequencies of various mutations were as follows: ΔF508=75.8%, G542X=2.7%, G551D=3.2%, R553X=1.4%, and N1303K=1.4% for a total of 84.5% detection of CF chromosomes by analysis for these five mutations.

This is a preview of subscription content, log in to check access.

References

  1. Beaudet AL (1990) Invited editorial: Carrier screening or cystic fibrosis. Am J Hum Genet 47:603–605

  2. 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. McGraw-Hill, New York, pp 2649–2680

  3. Cutting GR, Kasch LM, Rosenstein BJ, Zielenski J, Tsui L-C, Antonarakis SE, Kazazian HH (1990) A cluster of cystic fibrosis mutations in the first nucleotide-binding fold of the cystic fibrosis conductance regulator protein. Nature 346:366–369

  4. Davies K (1990) Complementary endeavours. Nature 348:110–111

  5. Gibbs RA, Nguyen P-N, Edwards A, Civitello AB, Caskey CT (1990) Multiplex DNA deletion detection and exon sequencing of the hypoxanthine phosphoribosyltransferase gene in LeschNyhan families. Genomics 7:235–244

  6. Haliassos A, Chomel JC, Tesson L, Baudis M, Kruh J, Kaplan JC, Kitzis A (1989) Modification of enzymatically amplified DNA for the detection of point mutations. Nucleic Acids Res 17:3606

  7. Kerem B, Rommens JM, Buchanan JA, Markiewicz D, Cox TK, Chakravarti A, Buchwald M, Tsui L-C (1989) Identification of the cystic fibrosis gene: genetic analysis. Science 245:1073–1080 (Erratum Science 245: 1437)

  8. Kerem B, Zielenski J, Markiewicz D, Bozon D, Gazit E, Yahav J, Kennedy D, Riordan JR, Collins FS, Rommens JM, Tsui L-C (1990) Identification of mutations in regions corresponding to the two putative nucleotide (ATP)-binding folds of the cystic fibrosis gene. Proc Natl Acad Sci USA 87:8447–8451

  9. Lemna WK, Feldman GL, Kerem B, Fernbach SD, Zevkovich EP, O'Brien WE, Riordan JR, Collins FS, Tsui L-C, Beaudet AL (1990) Mutation analysis for heterozygote detection and prenatal diagnosis of cystic fibrosis. N Engl J Med 322:291–296

  10. Mathew CG, Roberts RG, Harris A, Bentley DR, Borrow M (1989) Rapid screening for ΔF508 deletion in cystic fibrosis. Lancet II: 1345–1346

  11. Nozari G, Rahbar S, Wallace RB (1986) Discrimination among the transcripts of the allelic human beta-globin genes β A, βS and βC using oligodeoxynucleotide hybridization probes. Gene 43:23–28

  12. Osborne L, Knight R, Santis G, Hodson M (1991) A mutation in the second nucleotide binding fold of the cystic fibrosis gene. Am J Hum Genet 48:608–612

  13. Riordan JR, Rommens JM, Kerem B, Alon N, Rozmahel R, Grzelczak Z, Zielenski J, Lok S, Plavsic N, Chou J-L, Drumm ML, Iannuzzi MC, Collins FS, Tsui L-C (1989) Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245:1066–1073

  14. Romeo G, Devoto M (1990) Population analysis of the major mutation in cystic fibrosis. Hum Genet 85:391–392

  15. Rommens JM, Iannuzzi MC, Kerem B, Drumm ML, Melmer G, Dean M, Rozmahel R, Cole JL, Kennedy D, Hidaka N, Zsiga M, Buchwald M, Riordan JR, Tsui L-C, Collins FS (1989) Identification of the cystic fibrosis gene: chromosome walking and jumping. Science 245:1059–1065

  16. Rommens JM, Kerem B, Greer W, Chang P, Tsui L-C, Ray P (1990) Rapid nonradioactive detection of the major cystic fibrosis mutation. Am J Hum Genet 46:395–396

  17. Saiki RK, Walsh PS, Levenson CH, Erlich HA (1989) Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes. Proc Natl Acad Sci USA 86:6230–6234

  18. Wilfond BS, Fost N (1990) The cystic fibrosis gene: medical and social implications for heterozygote detection. JAMA 263:2777–2783

  19. Wood WI, Gitschier J, Lasky LA, Lawn RM (1985) Base composition-independent hybridization in tetramethylammonium chloride: a method for oligonucleotide screening of highly complex gene libraries. Proc Natl Acad Sci USA 82:1585–1588

  20. Workshop on Population Screening for the Cystic Fibrosis Gene (1990) Statement from the National Institutes of Health Workhop on Population Screeni for the Cystic Fibrosis Gene. N Engl J Med 323:70–71

  21. Zielenski J, Rozmahel R, Bozon D, Kerem B, Grzelczak Z, Riordan JR, Rommens J, Tsui L-C (1991) Genomic DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Genomics 10:214–228

Download references

Author information

Correspondence to Arthur L. Beaudet.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Ng, I.S.L., Pace, R., Richard, M.V. et al. Methods for analysis of multiple cystic fibrosis mutations. Hum Genet 87, 613–617 (1991). https://doi.org/10.1007/BF00209023

Download citation

Keywords

  • Cystic Fibrosis
  • Restriction Enzyme
  • Enzyme Analysis
  • Modify Primer
  • Genetic Diagnosis