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High Throughput Modifications of Single-Strand Conformation Polymorphism Analysis

Mutation Detection in Familial Hypercholesterolemia
  • Steve E. Humphries
  • Vilmundur Gudnason
  • Ros E. Whittall
  • Ian N. M. Day
Part of the Methods in Molecular Medicine™ book series (MIMM, volume 5)

Abstract

In most patients with familial hypercholesterolemia (FH) the disorder is caused by a mutation in the gene coding for the low density lipoprotein receptor (LDL-R) (1). The variety of different defects observed in receptor function at the cellular level reflects mutations in different domains of the gene, and there is an increasing number of pointers to suggest that genetic factors influence clinical severity. The diagnosis of FH on clinical grounds is not 100% accurate, and some hypercholesterolemic individuals may not have a mutation in the LDL-R gene, whereas some individuals who would not be included in the clinical criteria do have such a mutation. The purpose of this chapter is to Illustrate the use of the single-strand conformational polymorphism (SSCP) technique for mutation screening in the LDL-R gene and to discuss several adaptations of published methods that improve throughput, and that we believe are appropriate for a disorder such as FH. In the next few years such techniques will help to tackle molecular diagnosis and family tracing in the large number of FH patients present in Europe and North America.

Keywords

Single Strand Familial Hypercholesterolemia Familial Hypercholesterolemia Polymerase Chain Reaction Fragment SSCP Analysis 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Goldstein, J. L. and Brown, M. S. (1989) Familial hypercholesterolemia, in The Metabolic Basis of Inherited Disease, 6th ed. (Striver, C. R., Beaudet, A. L., Sly, W. W., and Valle, D., eds.), McGraw-Hill, New York, pp. 1215–1250.Google Scholar
  2. 2.
    Cotton, R. G. H. (1993) Current methods of mutation detection. Mutat. Res. 285, 125–144.PubMedGoogle Scholar
  3. 3.
    Condie, A., Eeles, R., Borreson, A.-L., Coles, C., Cooper, C., and Prosser, J. (1993) Detection of point mutations in the P53 gene: comparison of single-stand conformation polymorphism, constant denaturant gel electrophoresis, and hydroxylamine and osmium tetroxide techniques. Hum. Mutat. 2, 58–66.PubMedCrossRefGoogle Scholar
  4. 4.
    Orita, M., Suzuki, Y., Sekiya, T., and Hayashi, K. (1989) Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5, 874–879.PubMedCrossRefGoogle Scholar
  5. 5.
    Britten, R. J. and Davidson, E. H. (1985) Hybridisation strategy, in Nucleic Acid Hybridlsatzon: A Practical Approach (Hames, B. D. and Higgins, S. J., eds.), IRL Press, Oxford, UK, pp. 3–15.Google Scholar
  6. 6.
    Lee, H.-H., Lo, W.-J, and Choo, K.-B. (1992) Mutational analysis by a combined application of the multiple restriction fragment-single strand conformation polymorphism and the direct linear amplification DNA sequencing protocols. Anal. Blochem. 205, 289–293.CrossRefGoogle Scholar
  7. 7.
    Suzuki, Y., Seklya, T., and Hayashi, K. (1991) Allele-specific polymerase cham reaction: a method for amplification and sequence determination of a single component among a mixture of sequence variants. Anal. Biochem. 192, 82–84.PubMedCrossRefGoogle Scholar
  8. 8.
    Lo, Y.-M. D., Patel, P., Mehal, W. Z., Fleming, K. A., Bell, J. I., and Wainscoat, J. S. (1992) Analysis of complex genetic systems by ARMS-SSCP application to HLA genotyping. Nucleic Acids Res. 20, 1005–1009.PubMedCrossRefGoogle Scholar
  9. 9.
    Sarkar, G., Yoon, H.-S., and Sommer, S. S. (1992) Dideoxy fingerprinting (ddF): a rapid and efficient screen for the presence of mutations. Genomics 13, 441–443.PubMedCrossRefGoogle Scholar
  10. 10.
    Keen, J., Lester, D., Inglebeam, C., Curtis, A., and Bhattacharya, S. (1991) Rapid detec tion of single base mismatches as heteroduplexes on Hydrolink gels. Trends Genet. 7, 5.PubMedCrossRefGoogle Scholar
  11. 11.
    Sarkar, G., Yoon, H-S., and Sommer, S. S. (1992) Screening for mutations by RNA single-strand conformation polymorphism (rSSCP): comparison with DNA-SSCP. Nucleic Acids Res. 20, 871–878.PubMedCrossRefGoogle Scholar
  12. 12.
    Patterson, D. and Slack, J. (1972) Lipid abnormalities in male and female survivors of myocardial infarction and their first degree relatives. Lancet i, 1393–1399.Google Scholar
  13. 13.
    Betteridge, D. J., Dodson, P. M., Durrington, P. N., Hughes, E. A., Laker, M. F., Nicholls, D. P., et al. (1993) Management of hyperlipidaemia: guidelines of the British Hyperlipidaemia Association. Postgrad. Med. J. 69, 359–369.PubMedCrossRefGoogle Scholar
  14. 14.
    Kane, J. P., Malloy, M. J., Ports, T. A., Phillips, N. R., Diehl, J. C., and Havel, R. J. (1990) Regression of coronary atherosclerosis during treatment of familial hypercholesterolemia with combined drug regimens. JAMA 264, 3007–3012.PubMedCrossRefGoogle Scholar
  15. 15.
    Tybjaerg-Hansen, A. and Humphries, S. (1992) Familial defectrve apolipoprotein B-100: a single mutation that causes hypercholesterolaemia and premature coro nary artery disease. Atherosclerosis 96, 91–107.PubMedCrossRefGoogle Scholar
  16. 16.
    Starzl, T. E., Bilheimer, D. W., Bahnson, H. T., Hardesty, R L., Griffith, B. T., Iwatsuki, S., et al. (1984) Heart-liver transplantation in a patient with familial hypercholesterolaemia. Lancet 23, 1382,1383.CrossRefGoogle Scholar
  17. 17.
    Grossman, M., Raper, S. E., Kozarsky, K., Stein, E. A., Engelhardt, J. F., Muller, D., et al. (1994) Successful ex vzvo gene therapy directed to liver in a patient with familial hypercholesterolaemia. Nat. Genet. 6, 335–341PubMedCrossRefGoogle Scholar
  18. 18.
    Leonard, J. V., Whitelaw, A. G. L., Wolff, 0. H., Lloyd, J. K, and Slack, J. (1977) Diagnosing FH in children by measuring serum cholesterol. Br. Med. J, i, 1566–1568.CrossRefGoogle Scholar
  19. 19.
    Schmitz, G., Bruning, T., Kovacs, E., and Barlage, S. (1993) Fluorescence flow cytometry of human leukocytes in the detection of LDL receptor defects in the differential diagnosis of hypercholesterolemia. Arterioscl. Thromb. 13, 1053–1065.PubMedGoogle Scholar
  20. 20.
    Kessling, A. M., Seed, M., Taylor, R., Wynn, V., and Humphries, S. E (1990) Rising cholesterol levels m children with famrhal hypercholesterolaemia. Biomed Pharmacother. 44, 373–379.PubMedCrossRefGoogle Scholar
  21. 21.
    Hobbs, H. H., Brown, M S., and Goldstein, J. L. (1992) Molecular genetics of the LDL receptor gene in familial hypercholesterolaemia. Hum. Mutat. 1, 445–466.PubMedCrossRefGoogle Scholar
  22. 22.
    King-Underwood, L., Gudnason, V., Humphries, S., Seed, M., Patel, D., Knight, B, et al. (1991) Identification of the 664 proline to leucme mutation in the low density lipoprotein receptor in four unrelated patients with famihal hypercholesterolaemia in the UK. Clzn. Genet. 40, 17–28.CrossRefGoogle Scholar
  23. 23.
    Sun, X.-M., Webb, J. C., Gudnason, V., Humphries, S., Seed, M., Thompson, G. R., Knight, B. L., and Soutar, A. K. (1992) Characterization of deletions in the LDL receptor gene in patients with FH in the UK. Arterzoscl Thromb 12, 762–770.Google Scholar
  24. 24.
    Gudnason, V., King-Underwood, L., Seed, M., Sun, X-M., Soutar, A. K, and Humphries, S. E. (1993) Identification of recurrent and novel mutations in exon 4 of LDL receptor gene in patients with familial hypercholesterolaemia in the United Kingdom. Arterzoscl Thromb. 13, 6–63.Google Scholar
  25. 25.
    Webb, J C., Sun, X.-M., Patel, D. D., McCarthy, S. N., Knight, B. L., and Soutar, A. K. (1992) Characterisation of two new point mutations in the low density lipoprotein (LDL) receptor genes of an English patient with homozygous familial hypercholesterolemia. J Lzpzd Res. 33, 689–698.Google Scholar
  26. 26.
    Gudnason, V., Mak, Y.-T., Betteridge, J., McCarthy, S. N., and Humphries, S (1993) Use of the single strand conformatronal polymorphism method to detect recurrent and novel mutations in the low density lipoprotein receptor gene in patients with familial hypercholesterolaemia: detection of a novel mutation Asp200 + Gly. Clin. Invest. 71, 331–33CrossRefGoogle Scholar
  27. 27.
    Gudnason, V., Day, I, and Humphries, S. E. (1994) Effect on plasma lipid levels of mutations in exon 4 of the low-density hpoprotem receptor gene in patients with familial hypercholesterolaemia. Arterioscl Thromb. 14, 1717–1721.PubMedGoogle Scholar
  28. 28.
    Sun, X.-M., Patel, D. D., Bhatnagar, D., Knight, B. L., and Soutar, A.K. (1995) Characterization of a splice-site mutation in the gene for the LDL receptor assocrated with an unpredictably severe clinical phenotype in English patients with heterozygous FH. Arterioscl Thromb. 15, 219–22Google Scholar
  29. 29.
    Graham, C. A., Ward, A. I, Nevin, N. C., Trinick, T., Young, I., O’Kane, M., and Nicholls, D. P. (1995) Automated sequencing has identified 70% of mutations in 30 patients with familial hypercholesterolaemia in Northern Ireland. Atherosclerosis 112, 261–267.CrossRefGoogle Scholar
  30. 30.
    Esser, V., Limbird, L. E., Brown, M. S., Goldstein, J. L, and Russell, D. W. (1988) Mutational analysis of the ligand binding domain of the low density lipoprotein receptor. J. Biol. Chem. 263, 13,282–13,29PubMedGoogle Scholar
  31. 31.
    Hobbs, H. H., Leltersdorf, E., Leffert, C. C., Cryer, D. R., Brown, M. S., and Goldstein, J. L. (1989) Evtdence for a dominant gene that suppresses hypercholesterolemia in a family with defective low density lipoprotein receptors. J. Clm. Invest. 84, 656–66CrossRefGoogle Scholar
  32. 32.
    Chamberlain, J. S., Gibbs, R. A., Ranier, J. E., Nguyen, P. N., and Caskey, C. T. (1988) Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nuclezc Acids Res. 16, 11,141–11,156.CrossRefGoogle Scholar
  33. 33.
    Whittall, R., Gudnason, V., Weavind, G., Day, L. B., Humphries, S., and Day, I. N. M. (1995) Utilities for high throughput use of the single strand conformational polymorphism method: screening of 791 patients with familial hypercholesterolaemia for mutations in exon 3 of the low density lipoprotein receptor gene. J. Med. Genet., 32, 509–515.PubMedCrossRefGoogle Scholar
  34. 34.
    Day, I. N. M., Whittall, R., Gundason, V., and Humphries, S. E. (1995) Dried template DNA, and dried PCR oligonucleottdes and mailing in 96-well plates: LDL receptor gene mutation screening. BioTechniques 18, 981–984.PubMedGoogle Scholar
  35. 35.
    Day, I. N. M. and Humphries, S. E. (1994) Electrophoresis for genotyping: microtitre array diagonal gel electrophorests (MADGE) on hortzontal polyacrylamide (H-PAGE) gels, Hydrolink or agarose. Anal. Biochem. 222, 389–395.PubMedCrossRefGoogle Scholar
  36. 36.
    Sheffield, V. C., Beck, J. S., Kwitek, A. E., Sandstrom, D. W., and Stone, E. M. (1993) The sensitivity of single-strand conformation polymorphism analysis for the detection of single base substitutions. Genomics 16, 325–332.PubMedCrossRefGoogle Scholar
  37. 37.
    Wood, W I., Gitschier, J., Lasky, L. A., and Lawn, R. M. (1985) Base composition independent hybridization in tetramethylammonium chloride: a method for oligonucleotide screening of highly complex gene libraries. Proc Natl Acad Sci. USA 82, 1585–1588PubMedCrossRefGoogle Scholar
  38. 38.
    Cariello, N. F. and Skopek, T. R. (1993) Mutational analysis using denaturing gradient gel electrophoresis and PCR. Mutut Res. 288, 103–112.Google Scholar
  39. 39.
    Leren, T. P., Solberg, K., Rodningen, O. K., Rosby, O., Tonstad, S., Ose, L., et al. (1993) Screening for point mutations in exon 10 of the low density lipoprotein receptor gene by analysis of single-strand conformation polymorphisms: detection of a nonsense mutation-FH469 ➔ Stop. Hum. Genet. 92, 6–10.PubMedCrossRefGoogle Scholar
  40. 40.
    Sekiya, T. (1993). Detection of mutant sequences by single-strand conformation polymorphism analysis. Mutat. Res. 288, 79–83.PubMedGoogle Scholar
  41. 41.
    Cai, Q.-Q. and Touitou, I. (1993) Excess PCR primers may dramatically affect SSCP efficiency. Nucleic Acids Res. 21, 3909,3910.PubMedCrossRefGoogle Scholar
  42. 42.
    Hongyo, T., Buzard, G. S., Calvert, R. J., and Weghorst, C. M. (1993) “Cold SSCP”: a simple, rapid and non-radioactive method for optimized single-strandconformation polymorphism analyses. Nucleic Acids Res 21, 3637–3642.PubMedCrossRefGoogle Scholar
  43. 43.
    Yap, E. P. H. and McGee, J. O. (1993) Nonisotoptc discontinuous phase single strand conformation polymorphism (DP-SSCP): genetic profiling of D-loop of human mitochondrial (mt) DNA. Genomes 21, 4155.Google Scholar
  44. 44.
    Spmardi, L., Mazars, R., and Theillet, C. (1991) Protocols for an improved detection of point mutations by SSCP. Nucleic Acids Res 19, 4009.CrossRefGoogle Scholar
  45. 45.
    Savov, A., Angehcheva, D., Jordanova, A., Eigel, A., and Kalaydjieva, L. (1992) High percentage acrylamide gels improve resolution in SSCP analysis. Nucleic Aads Res. 20, 6741,6742.CrossRefGoogle Scholar
  46. 46.
    Baxter, S. M., Greizerstein, M B., Kushlan, D. M., and Ashley, G. W (1993) Conformational properties of DNA hairpins with TTT and TTTT loops. Biochemistry 32, 8702–871PubMedCrossRefGoogle Scholar
  47. 47.
    Mayer, P., Slater, G. W., and Drouin, G. (1993) Exact behavir of single-stranded DNA electrophoretic mobilities in polyacrylamtde gels. Appl. Theoret. Electrophor 3, 145–155.Google Scholar
  48. 48.
    Ainsworth, P J., Surh, L. C., and Coulter-Mackte, M. B. (1991) Diagnostic single strand conformatronal polymorphism, (SSCP): a simplified non-radioisotopic method as applied to a Tay-Sachs Bl variant. Nucleic Acids Res. 19, 405,406.PubMedCrossRefGoogle Scholar
  49. 49.
    Mohabeer, A J., Hiti, A. L., and Martin, W. J. (1991) Non-radioacttve single strand conformation polymorphtsm (SSCP) using the Pharmacia “PhastSystem.” Nucleic Acids Res. 19, 3154.PubMedCrossRefGoogle Scholar
  50. 50.
    Bassam, B. J., Caetano-Anollës, G., and Gresshoff, P. M. (1991) Fast and sensrtive silver staining of DNA in polyacrylamrde gels. Anal Biochem. 196, 80–83.PubMedCrossRefGoogle Scholar
  51. 51.
    Beck, S. and Pohl, F. M. (1984) DNA sequencing with direct blotting electrophoresis. EMBO. J. 3, 2905–2909.PubMedGoogle Scholar
  52. 52.
    Ellison, J, Dean, M., and Goldman, D. (1993) Efficacy of fluorescence-based PCR-SSCP for detection of point mutatons. BioTechmques 15 684–691Google Scholar
  53. 53.
    Yang, M. M. and Youvan, D. C. (1989) A prospectus for multispectral-multiplex DNA sequencing. Biotechnology 7, 576–581.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc, Totowa, NJ 1996

Authors and Affiliations

  • Steve E. Humphries
    • 1
  • Vilmundur Gudnason
    • 1
  • Ros E. Whittall
    • 1
  • Ian N. M. Day
    • 1
  1. 1.Division of Cardiovascular Genetics, Department of MedicineUniversity College London Medical SchoolLondonUK

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