Skip to main content
PMC home page
Journal of Medical Genetics logoLink to Journal of Medical Genetics
. 1999 Oct;36(10):747–753. doi: 10.1136/jmg.36.10.747

A highly accurate, low cost test for BRCA1 mutations

N J van Orsouw 1, R Dhanda 1, Y Elhaji 1, S Narod 1, F Li 1, C Eng 1, J Vijg 1
PMCID: PMC1734249  PMID: 10528853

Abstract

The hereditary breast and ovarian cancer syndrome is associated with a high frequency of BRCA1 mutations. However, the widespread use of BRCA1 testing has been limited to date by three principal concerns: the fear of loss of health and life insurance, the uncertain clinical value of a positive test result, and the current lack of an inexpensive and sensitive screening test for BRCA1 mutations. We have developed an inexpensive system for gene mutational scanning, based on a combination of extensive multiplex PCR amplification and two dimensional electrophoresis. The efficiency of this system, as a screening test for BRCA1 mutations, was evaluated in a panel of 60 samples from high risk women, 14 of which contained a previously identified mutation in BRCA1. All 14 mutations were identified, as well as an additional five that had previously escaped detection. In addition to the 19 mutations, a total of 15 different polymorphic variants were scored, most of which were recurring. All were confirmed by nucleotide sequencing. The cost of screening per sample was calculated to be approximately US$70 for the manual technique used in this study, and may be reduced to approximately US$10 with the introduction of commercially available PCR robotics and fluorescent imaging. Implementation of this method of mutation screening in the research and clinical setting should permit rapid accrual of quantitative data on genotype-phenotype associations for the evaluation of diagnostic testing.


Keywords: genetic testing; two dimensional gene scanning (TDGS)

Full Text

The Full Text of this article is available as a PDF (156.4 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Dhanda R. K., Smith W. M., Scott C. B., Eng C., Vijg J. A simple system for automated two-dimensional electrophoresis: applications to genetic testing. Genet Test. 1998;2(1):67–70. doi: 10.1089/gte.1998.2.67. [DOI] [PubMed] [Google Scholar]
  2. Dhanda R. K., van Orsouw N. J., Sigalas I., Eng C., Vijg J. Critical factors in the performance and cost of two-dimensional gene scanning: RB1 as a model. Biotechniques. 1998 Oct;25(4):664-8, 670, 672-5. doi: 10.2144/98254dt06. [DOI] [PubMed] [Google Scholar]
  3. FitzGerald M. G., Bean J. M., Hegde S. R., Unsal H., MacDonald D. J., Harkin D. P., Finkelstein D. M., Isselbacher K. J., Haber D. A. Heterozygous ATM mutations do not contribute to early onset of breast cancer. Nat Genet. 1997 Mar;15(3):307–310. doi: 10.1038/ng0397-307. [DOI] [PubMed] [Google Scholar]
  4. Ford D., Easton D. F., Bishop D. T., Narod S. A., Goldgar D. E. Risks of cancer in BRCA1-mutation carriers. Breast Cancer Linkage Consortium. Lancet. 1994 Mar 19;343(8899):692–695. doi: 10.1016/s0140-6736(94)91578-4. [DOI] [PubMed] [Google Scholar]
  5. Ford D., Easton D. F., Stratton M., Narod S., Goldgar D., Devilee P., Bishop D. T., Weber B., Lenoir G., Chang-Claude J. Genetic heterogeneity and penetrance analysis of the BRCA1 and BRCA2 genes in breast cancer families. The Breast Cancer Linkage Consortium. Am J Hum Genet. 1998 Mar;62(3):676–689. doi: 10.1086/301749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Friedman L. S., Ostermeyer E. A., Szabo C. I., Dowd P., Lynch E. D., Rowell S. E., King M. C. Confirmation of BRCA1 by analysis of germline mutations linked to breast and ovarian cancer in ten families. Nat Genet. 1994 Dec;8(4):399–404. doi: 10.1038/ng1294-399. [DOI] [PubMed] [Google Scholar]
  7. Friend S. H. Breast cancer susceptibility testing: realities in the post-genomic era. Nat Genet. 1996 May;13(1):16–17. doi: 10.1038/ng0596-16. [DOI] [PubMed] [Google Scholar]
  8. Gayther S. A., Warren W., Mazoyer S., Russell P. A., Harrington P. A., Chiano M., Seal S., Hamoudi R., van Rensburg E. J., Dunning A. M. Germline mutations of the BRCA1 gene in breast and ovarian cancer families provide evidence for a genotype-phenotype correlation. Nat Genet. 1995 Dec;11(4):428–433. doi: 10.1038/ng1295-428. [DOI] [PubMed] [Google Scholar]
  9. Li D., Vijg J. Multiplex co-amplification of 24 retinoblastoma gene exons after pre-amplification by long-distance PCR. Nucleic Acids Res. 1996 Feb 1;24(3):538–539. doi: 10.1093/nar/24.3.538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Li F. P., Fraumeni J. F., Jr, Mulvihill J. J., Blattner W. A., Dreyfus M. G., Tucker M. A., Miller R. W. A cancer family syndrome in twenty-four kindreds. Cancer Res. 1988 Sep 15;48(18):5358–5362. [PubMed] [Google Scholar]
  11. Liaw D., Marsh D. J., Li J., Dahia P. L., Wang S. I., Zheng Z., Bose S., Call K. M., Tsou H. C., Peacocke M. Germline mutations of the PTEN gene in Cowden disease, an inherited breast and thyroid cancer syndrome. Nat Genet. 1997 May;16(1):64–67. doi: 10.1038/ng0597-64. [DOI] [PubMed] [Google Scholar]
  12. Miki Y., Swensen J., Shattuck-Eidens D., Futreal P. A., Harshman K., Tavtigian S., Liu Q., Cochran C., Bennett L. M., Ding W. A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science. 1994 Oct 7;266(5182):66–71. doi: 10.1126/science.7545954. [DOI] [PubMed] [Google Scholar]
  13. Narod S. A., Ford D., Devilee P., Barkardottir R. B., Lynch H. T., Smith S. A., Ponder B. A., Weber B. L., Garber J. E., Birch J. M. An evaluation of genetic heterogeneity in 145 breast-ovarian cancer families. Breast Cancer Linkage Consortium. Am J Hum Genet. 1995 Jan;56(1):254–264. [PMC free article] [PubMed] [Google Scholar]
  14. Narod S. A., Risch H., Moslehi R., Dørum A., Neuhausen S., Olsson H., Provencher D., Radice P., Evans G., Bishop S. Oral contraceptives and the risk of hereditary ovarian cancer. Hereditary Ovarian Cancer Clinical Study Group. N Engl J Med. 1998 Aug 13;339(7):424–428. doi: 10.1056/NEJM199808133390702. [DOI] [PubMed] [Google Scholar]
  15. Nelen M. R., Padberg G. W., Peeters E. A., Lin A. Y., van den Helm B., Frants R. R., Coulon V., Goldstein A. M., van Reen M. M., Easton D. F. Localization of the gene for Cowden disease to chromosome 10q22-23. Nat Genet. 1996 May;13(1):114–116. doi: 10.1038/ng0596-114. [DOI] [PubMed] [Google Scholar]
  16. Rines R. D., van Orsouw N. J., Sigalas I., Li F. P., Eng C., Vijg J. Comprehensive mutational scanning of the p53 coding region by two-dimensional gene scanning. Carcinogenesis. 1998 Jun;19(6):979–984. doi: 10.1093/carcin/19.6.979. [DOI] [PubMed] [Google Scholar]
  17. Sidransky D., Tokino T., Helzlsouer K., Zehnbauer B., Rausch G., Shelton B., Prestigiacomo L., Vogelstein B., Davidson N. Inherited p53 gene mutations in breast cancer. Cancer Res. 1992 May 15;52(10):2984–2986. [PubMed] [Google Scholar]
  18. Smith T. M., Lee M. K., Szabo C. I., Jerome N., McEuen M., Taylor M., Hood L., King M. C. Complete genomic sequence and analysis of 117 kb of human DNA containing the gene BRCA1. Genome Res. 1996 Nov;6(11):1029–1049. doi: 10.1101/gr.6.11.1029. [DOI] [PubMed] [Google Scholar]
  19. Smith W. M., Van Orsouw N. J., Fox E. A., Kolodner R. D., Vijg J., Eng C. Accurate, high-throughput "snapshot" detection of hMLH1 mutations by two-dimensional DNA electrophoresis. Genet Test. 1998;2(1):43–53. doi: 10.1089/gte.1998.2.43. [DOI] [PubMed] [Google Scholar]
  20. Van Orsouw N. J., Li D., van der Vlies P., Scheffer H., Eng C., Buys C. H., Li F. P., Vijg J. Mutational scanning of large genes by extensive PCR multiplexing and two-dimensional electrophoresis: application to the RB1 gene. Hum Mol Genet. 1996 Jun;5(6):755–761. doi: 10.1093/hmg/5.6.755. [DOI] [PubMed] [Google Scholar]
  21. Van Orsouw N. J., Vijg J. Design and application of 2-D DGGE-based gene mutational scanning tests. Genet Anal. 1999 Feb;14(5-6):205–213. doi: 10.1016/s1050-3862(98)00028-x. [DOI] [PubMed] [Google Scholar]
  22. Wooster R., Bignell G., Lancaster J., Swift S., Seal S., Mangion J., Collins N., Gregory S., Gumbs C., Micklem G. Identification of the breast cancer susceptibility gene BRCA2. Nature. 1995 Dec 21;378(6559):789–792. doi: 10.1038/378789a0. [DOI] [PubMed] [Google Scholar]
  23. van Orsouw N. J., Dhanda R. K., Rines R. D., Smith W. M., Sigalas I., Eng C., Vijg J. Rapid design of denaturing gradient-based two-dimensional electrophoretic gene mutational scanning tests. Nucleic Acids Res. 1998 May 15;26(10):2398–2406. doi: 10.1093/nar/26.10.2398. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Medical Genetics are provided here courtesy of BMJ Publishing Group

RESOURCES