Mutation detection in tumor suppressor genes using archival tissue specimens

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.