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The Protein Truncation Test

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Medical Biomethods Handbook

Part of the book series: Springer Protocols Handbooks ((SPH))

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Abstract

Many techniques are available to efficiently pick up (point) mutations, all with their own strong and weak points and limitations. The most powerful qualitative techniques include denaturing gradient gel electrophoresis (DGGE) (see Chapter 8), chemical cleavage of mismatches (CCM) (see Chapter 7), and denaturing high-performance liquid chromatography (DHPLC). Of the quantitative techniques, Southern blotting, quantitative polymerase chain reaction (qPCR) (see Chapter 25) and multiplex ligation-dependent probe amplification (MLPA) are most popular. The protein truncation test (PTT) (1, also known as the in vitro synthesized protein assay (IVSP) (2, is rather exceptional and has several unique features. The most prominent of these are that it uses an RNA template, scans for mutations at the protein level, has a high sensitivity, and hardly gives false positives. In addition, PTT detects truncating mutations only and the large majority of these are disease causing and thus directly clinically relevant. No PTT-detected alterations have been reported that could not be confirmed by sequence analysis; PTT analysis could thus be used directly for diagnostic purposes.

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References

  1. Roest, P. A. M., Roberts, R. G., Sugino, S., Van Ommen, G. J. B., and Den Dunnen, J. T. (1993) Protein truncation test (PTT) for rapid detection of translation-terminating mutations. Hum. Mol. Genet. 2, 1719–1721.

    Article  PubMed  CAS  Google Scholar 

  2. Powell, S. M., Petersen, G. M., Krush, A. A. J., et al. (1993) Molecular Diagnosis of familial adenomatous polyposis. N. Engl. J. Med. 329, 1982–1987.

    Article  PubMed  CAS  Google Scholar 

  3. Mazoyer, S., Dunning, A. M., Serova, O., et al. (1996) A polymorphic stop codon in Brca2. Nature Genet. 14, 253–254.

    Article  PubMed  CAS  Google Scholar 

  4. Koenig, M., Hoffman, E. P., Bertelson, C. J., Monaco, A. P., Feener, C. A., and Kunkel, L. M. (1987) Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell 50, 509–517.

    Article  PubMed  CAS  Google Scholar 

  5. Steimle, V., Otten, L. A., Zufferey, M., and Mach, B. (1993) Complementation cloning of an MHC class II transactivator mutated in hereditary MHC class II deficiency (or bare lymphocyte syndrome). Cell 75, 135–146.

    PubMed  CAS  Google Scholar 

  6. Den Dunnen, J. T., Grootscholten, P. M., Bakker, E., et al. (1989) Topography of the DMD gene: FIGE and cDNA analysis of 194 cases reveals 115 deletions and 13 duplications. Am. J. Hum. Genet. 45, 835–847.

    Google Scholar 

  7. Roberts, R. G., Coffey, A. J., Bobrow, M., and Bentley, D. R. (1992) Determination of the exon structure of the distal portion of the dystrophin gene by vectorette PCR. Genomics 13, 942–950.

    Article  PubMed  CAS  Google Scholar 

  8. Roest, P. A. M., Bakker, E., Fallaux, F. J., Verellen-Dumoulin, C., Murry, C. E., and Den Dunnen, J. T. (1999) New possibilities for prenatal diagnosis of muscular dystrophies: forced myogenesis with an adenoviral MyoD-vector. Lancet 353, 727–728.

    Article  PubMed  CAS  Google Scholar 

  9. Whittock, N. V., Roberts, R. G., Mathew, C. G., and Abbs, S. J. (1997) Dystrophin point mutation screening using a multiplexed protein truncation test. Genet. Test. 1, 115–123.

    Article  PubMed  CAS  Google Scholar 

  10. Van Der Luijt, R. B., Meera Kahn, P., Vasen, H., et al. (1994) Rapid detection of translation-terminating mutations at the adenomatous polyposis coli (APC) gene by direct protein truncation test. Genomics 20, 1–4.

    Article  PubMed  Google Scholar 

  11. Hogervorst, F. B. L., Cornelis, R. S., Bout, M., et al. (1995) Rapid detection of BRCA1 mutations by the protein truncation test. Nature Genet. 10, 208–212.

    Article  PubMed  CAS  Google Scholar 

  12. Den Dunnen, J. T. (1996) Protein truncation test, in Current Protocols In Human Genetics (Dracopoli, N. C., Haines, J. L., Korf, B. R., et al., eds.), Wiley, New York.

    Google Scholar 

  13. Traverso, G., Diehl, F., Hurst, R., et al. (2003) Multicolor in vitro translation. Nature Biotechnol. 21, 1093–1097.

    Article  CAS  Google Scholar 

  14. Ars, E., Serra, E., De La Luna, S., Estivill, X., and Lazaro, C. (2000) Cold shock induces the insertion of a cryptic exon in the neurofibromatosis type 1 (NF1) mRNA. Nucleic Acids Res. 28, 1307–1312.

    Article  PubMed  CAS  Google Scholar 

  15. Culbertson, M. R. (1999) RNA surveillance. Unforeseen consequences for gene expression, inherited genetic disorders and cancer. Trends. Genet. 15, 74–80.

    Article  PubMed  CAS  Google Scholar 

  16. Lamande, S. R., Bateman, J. F., Hutchison, W., et al. (1998) Reduced collagen VI causes Bethlem myopathy: a heterozygous COL6A1 nonsense mutation results in mRNA decay and functional haploinsufficiency. Hum. Mol. Genet. 7, 981–989.

    Article  PubMed  CAS  Google Scholar 

  17. Chelly, J., Concordet, J., Kaplan, J. C., and Kahn, A. (1989) Illegitimate Transcription Of Any Gene In Any Cell Type. Proc. Natl. Acad. Sci. USA 86, 2617–2621.

    Article  PubMed  CAS  Google Scholar 

  18. Hope, I. A. and Struhl, K. (1985) GCN4 protein synthesized in vitro, binds HIS3 regulatory sequences: implications for general control of amino acid biosynthetic genes in yeast. Cell 43, 177–188.

    Article  PubMed  CAS  Google Scholar 

  19. Rowan, A. J. and Bodmer, W. F. (1997) Introduction of a myc reporter tag to improve the quality of mutation detection using the protein truncation test. Hum. Mutat. 9, 172–176.

    Article  PubMed  CAS  Google Scholar 

  20. Kahmann, S., Herter, P., Kuhnen, C., et al. (2002) A non-radioactive protein truncation test for the sensitive detection of all stop and frameshift mutations. Hum. Mutat. 19, 165–172.

    Article  PubMed  CAS  Google Scholar 

  21. Gite, S., Lim, M., Carlson, R., Olejnik, J., Zehnbauer, B., and Rothschild, K. (2003) A high-throughput nonisotopic protein truncation test. Nature Biotechnol. 21, 194–197.

    Article  CAS  Google Scholar 

  22. Garvin, A. M., Parker, K. C., and Haff, L. (2000) Maldi-tof based mutation detection using tagged in vitro synthesized peptides. Nature Biotechnol. 18, 95–97.

    Article  CAS  Google Scholar 

  23. Martinez-Gimeno, M., Gamundi, M. J., Hernan, I., et al. (2003) Mutations in the pre-mRNA splicing-factor genes PRPF3, PRPF8, and PRPF31 in Spanish families with autosomal dominant retinitis pigmentosa. Invest. Ophthalmol. Vis. Sci. 44, 2171–2177.

    Article  PubMed  Google Scholar 

  24. Chang, C. C. and Gould, S. J. (1998) Phenotype-genotype relationships in complementation group 3 of the peroxisome-biogenesis disorders. Am. J. Hum. Genet. 63, 1294–1306.

    Article  PubMed  CAS  Google Scholar 

  25. Laken, S. J., Petersen, G. M., Gruber, S. B., et al. (1997) Familial colorectal cancer in ashkenazim due to a hypermutable tract in APC. Nature Genet. 17, 79–83.

    Article  PubMed  CAS  Google Scholar 

  26. Roberts, R. G., Bentley, D. R., and Bobrow, M. (1993) Infidelity in the structure of ectopic transcripts: a novel exon in lymphocyte dystrophin transcripts. Hum. Mutat. 2, 293–299.

    Article  PubMed  CAS  Google Scholar 

  27. Yates, J., Aksmanovic, V., Mcmahon, R., Bione, S., and Toniolo, D. (1996) Mutation analysis in emery-dreifuss muscular dystrophy. Eur. J. Hum. Genet. 4, 62–63.

    Google Scholar 

  28. De Koning Gans, P. A. M., Ginjaar, H. B., Bakker, E., Yates, J. R. W., and Den Dunnen, J. T. (1999) A protein truncation test for emery-dreifuss muscular dystrophy (EMD): detection of N-terminal truncating mutations. Neuromusc. Disord. 9, 247–250.

    Article  PubMed  Google Scholar 

  29. Shattuck-Eidens, D., Mcclure, M., Simard, J., et al. (1995) A collaborative survey of 80 mutations in the brca1 breast and ovarian cancer susceptibility gene. J. Am. Med. Assoc. 273, 535–541.

    Article  CAS  Google Scholar 

  30. Van Ommen, G. J. B., Bakker, E., and Den Dunnen, J. T. (1999) The human genome project and the future of diagnostics, treatment, and prevention. Lancet 354, Si5–Si10.

    PubMed  Google Scholar 

  31. Den Dunnen, J. T. and Van Ommen, G. J. B. (1999) The protein truncation test: a review. Hum. Mutat. 14, 95–102.

    Article  Google Scholar 

  32. Lancaster, J. M., Wooster, R., Mangion, J., et al. (1996) BRCA2 mutations in primary breast and ovarian cancers. Nature Genet. 13, 238–240.

    Article  PubMed  CAS  Google Scholar 

  33. Farrington, S. M., Lin-Goerke, J., Ling, J., et al. (1998) Systematic analysis of hMSH2 and hMLH1 in young colon cancer patients and controls. Am. J. Hum. Genet. 63, 749–759.

    Article  PubMed  CAS  Google Scholar 

  34. Kohonen-Corish, M., Ross, V. L., Doe, W. F., et al. (1996) RNA-based mutation screening in hereditary nonpolyposis colorectal cancer. Am. J. Hum. Genet. 59, 818–824.

    PubMed  CAS  Google Scholar 

  35. Heim, R. A., Silverman, L. M., Farber, R. A., Kam-Morgan, L. N. W., and Luce, M. C. (1995) Screening for truncated NF1 proteins. Nature Genet. 8, 218–219.

    Article  Google Scholar 

  36. Parry, D. M., Maccollin, M. M., Kaiser Kupfer, M. I., et al. (1996) Germ-line mutations in the neurofibromatosis 2 gene: correlations with disease severity and retinal abnormalities. Am. J. Hum. Genet. 59, 529–539.

    PubMed  CAS  Google Scholar 

  37. Roelfsema, J. H., Spruit, L., Saris, J. J., et al. (1997) Mutation detection in the repeated part of the PKD1 gene. Am. J. Hum. Genet. 61, 1044–1052.

    Article  PubMed  CAS  Google Scholar 

  38. Romey, M. C., Tuffery, S., Desgeorges, M., Bienvenu, T., Demaille, J., and Claustres, M. (1996) Transcript analysis of CFTR frameshift mutations in lymphocytes using the revesre transriptionpolymerase chain reaction technique and the protein truncation test. Hum. Genet. 98, 328–332.

    Article  PubMed  CAS  Google Scholar 

  39. Petrij, F., Giles, R. H., Dauwerse, J. G., et al. (1995) Rubinstein-Taybi syndrome caused by mutations in the transcriptional co-activator CBP. Nature 376, 348–351.

    Article  PubMed  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Center for Human and Clinical Genetics, Leiden University Medical Center, Leiden, Nederland

    Johan T. den Dunnen

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  1. Johan T. den Dunnen
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Editors and Affiliations

  1. University of Hertfordshire, Hatfield, UK

    John M. Walker  & Ralph Rapley  & 

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© 2005 Humana Press Inc., Totowa, NJ

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den Dunnen, J.T. (2005). The Protein Truncation Test. In: Walker, J.M., Rapley, R. (eds) Medical Biomethods Handbook. Springer Protocols Handbooks. Humana Press. https://doi.org/10.1385/1-59259-870-6:195

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  • DOI: https://doi.org/10.1385/1-59259-870-6:195

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-288-9

  • Online ISBN: 978-1-59259-870-0

  • eBook Packages: Springer Protocols

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