Advertisement

Mutation Detection in Tumor Suppressor Genes Using Archival Tissue Specimens

  • Aristotelis Astrinidis
  • Elizabeth Petri Henske
Part of the Methods in Molecular Medicine book series (MIMM, volume 126)

Abstract

Tuberous sclerosis complex (TSC) is a neurocutaneous syndrome characterized by seizures, mental retardation, and benign tumors of many organs, including the brain, kidneys, skin, retina, and heart. TSC is caused by mutations in the TSC1 and TSC2 tumor suppressor genes. The genes follow the two-hit model for tumorigenesis, with germline mutations inactivating one allele and somatic mutations inactivating the remaining wild-type allele. Allelic loss (also called loss of heterozygosity [LOH]) in the 9q34 and 16p13 regions has been found in many tumor types from TSC patients. Cardiac rhabdomyomas are frequently found in infants with TSC. Because rhabdomyomas often spontaneously regress, access to fresh tissue is limited. In this chapter, we present methodology for detection of genetic inactivation of TSC1 and TSC2 in paraffin-embedded archival tissues. The template DNA is obtained either by direct scraping of tissue or after laser capture microdissection. LOH analysis is performed after polymerase chain reaction amplification of microsatellite markers in the 9q34 and 16p13 regions and denaturing polyacrylamide gel electrophoresis. Mutation detection is performed using single-strand conformation polymorphisms on mutation detection enhancement gels. Finally, variant bands are amplified and analyzed by direct sequencing.

Key Words

Tuberous sclerosis complex (TSC) TSC1 TSC2 tumor suppressor genes rhabdomyomas laser capture microdissection (LCM) loss of heterozygosity (LOH) singlestrand conformation polymorphisms (SSCP) direct sequencing variant bands 

References

  1. 1.
    Webb, D., Thomas, R. D., and Osborne, J. (1993) Cardiac rhabdomyomas andtheir association with tuberous sclerosis. Arch. Dis. Child. 68, 367–370.CrossRefPubMedGoogle Scholar
  2. 2.
    Roach, E. S., Gomez, M. R., and Northrup, H. (1998) Tuberous sclerosis complex consensus conference: revised clinical diagnostic criteria. J. Child. Neurol. 13, 624–628.CrossRefPubMedGoogle Scholar
  3. 3.
    Gomez, M., Sampson, J. R., and Whittemore, V. H. (1999) Tuberous SclerosisComplex, 3rd ed, Oxford University Press, New York.Google Scholar
  4. 4.
    The European Chromosome 16 Tuberous Sclerosis Consortium. (1993) Identificationand characterization of the tuberous sclerosis gene on chromosome 16. Cell 75, 1305–1315.CrossRefGoogle Scholar
  5. 5.
    van Slegtenhorst, M., de Hoogt, R., Hermans, C., et al. (1997) Identification of the tuberous sclerosis gene TSC1 on chromosome 9q34. Science 277, 805–808.CrossRefPubMedGoogle Scholar
  6. 6.
    Carsillo, T., Astrinidis, A., and Henske, E. P. (2000) Mutations in the tuberous sclerosis complex gene TSC2 are a cause of sporadic pulmonary lymphangioleiomyomatosis. Proc. Natl. Acad. Sci. USA 97, 6085–6090.CrossRefPubMedGoogle Scholar
  7. 7.
    Sato, T., Seyama, K., Fujii, H., et al. (2002) Mutation analysis of the TSC1 and TSC2 genes in Japanese patients with pulmonary lymphangioleiomyomatosis. J.Hum. Genet. 47, 20–28.CrossRefPubMedGoogle Scholar
  8. 8.
    Knudson, A. (1971) Mutation and cancer: statistical study of retinoblastoma. Proc.Natl. Acad. Sci. USA 68, 820–823.CrossRefPubMedGoogle Scholar
  9. 9.
    Bosi, G., Lintermans, J. P., Pellegrino, P. A., Svaluto-Moreolo G., and Vliers A.(1996) The natural history of cardiac rhabdomyoma with and without tuberous sclerosis. Acta. Paediatr. 85, 928–931.CrossRefPubMedGoogle Scholar
  10. 10.
    Jones, A. C., Shyamsundar, M. M., Thomas, M. W., et al. (1999) Comprehensive mutation analysis of TSC1 and TSC2-and phenotypic correlations in 150 families with tuberous sclerosis. Am. J. Hum. Genet. 64, 1305–1315.CrossRefPubMedGoogle Scholar
  11. 11.
    Niida, Y., Lawrence-Smith, N., Banwell, A., et al. (1999) Analysis of both TSC1 and TSC2 for germline mutations in 126 unrelated patients with tuberous sclerosis. Hum. Mutat. 14, 412–422.CrossRefPubMedGoogle Scholar
  12. 12.
    van Slegtenhorst, M., Verhoef, S., Tempelaars, A., et al. (1999) Mutational spectrum of the TSC1 gene in a cohort of 225 tuberous sclerosis complex patients: no evidence for genotype-phenotype correlation. J. Med. Genet. 36, 285–289.PubMedGoogle Scholar
  13. 13.
    Dabora, S. L., Jozwiak, S., Franz, D. N., et al. (2001) Mutational analysis in a cohort of 224 tuberous sclerosis patients indicates increased severity of TSC2, compared with TSC1, disease in multiple organs. Am. J. Hum. Genet. 68, 64–80.CrossRefPubMedGoogle Scholar
  14. 14.
    Carbonara, C., Longa, L., Grosso, E., et al. (1994) 9q34 loss of heterozygosity in atuberous sclerosis astrocytoma suggests a growth suppressor-like activity also for the TSC1 gene. Hum. Mol. Genet. 3, 1829–1832.CrossRefPubMedGoogle Scholar
  15. 15.
    Henske, E. P., Neumann, H. P., Scheithauer, B. W., Herbst, E. W., Short, M. P.,and Kwiatkowski, D. J. (1995) Loss of heterozygosity in the tuberous sclerosis(TSC2) region of chromosome band 16p13 occurs in sporadic as well as TSCassociated renal angiomyolipomas. Genes Chromosomes Cancer 13, 295–298.CrossRefPubMedGoogle Scholar
  16. 16.
    Smolarek, T. A., Wessner, L. L., McCormack, F. X., Mylet, J. C., Menon, A. G., and Henske, E. P. (1998) Evidence that lymphangiomyomatosis is caused by TSC2 mutations:chromosome 16p13 loss of heterozygosity in angiomyolipomas and lymph nodes from women with lymphangiomyomatosis. Am. J. Hum. Genet. 62, 810–815.CrossRefPubMedGoogle Scholar
  17. 17.
    Yu, J., Astrinidis, A., and Henske, E. P. (2001) Chromosome 16 loss of heterozygosity in tuberous sclerosis and sporadic lymphangiomyomatosis. Am. J. Respir.Crit. Care. Med. 164, 1537–1540.PubMedGoogle Scholar
  18. 18.
    Carbonara, C., Longa, L., Grosso, E., et al. (1996) Apparent preferential loss of heterozygosity at TSC2 over TSC1 chromosomal region in tuberous sclerosis hamartomas. Genes Chromosomes Cancer 15, 18–25.CrossRefPubMedGoogle Scholar
  19. 19.
    Emmert-Buck, M. R., Bonner, R. F., Smith, P. D., et al. (1996) Laser capture microdissection. Science 274, 998–1001.CrossRefPubMedGoogle Scholar
  20. 20.
    Hornigold, N., Devlin, J., Davies, A., Aveyard, J., Habuchi, T., and Knowles, M.(1999) Mutation of the 9q34 gene TSC1 in sporadic bladder cancer. Oncogene 18,2657–2661.CrossRefPubMedGoogle Scholar
  21. 21.
    Au K.-S., Rodriguez J., Finch, J., et al. (1997) Germ-line mutational analysis of the TSC2 gene in 90 tuberous sclerosis patients. Am. J. Hum. Genet. 62, 286–294.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2006

Authors and Affiliations

  • Aristotelis Astrinidis
    • 1
  • Elizabeth Petri Henske
    • 1
  1. 1.Department of Medical OncologyFox Chase Cancer CenterPhiladelphia

Personalised recommendations