Rapid mutation scanning of genes associated with familial cancer syndromesusing denaturing high-performance liquid chromatography

Germline mutations in tumor suppressor genes, or less frequently oncogenes, have been identified in up to 19 familial cancer syndromes including Li-Fr aumeni syndrome, familial paraganglioma, familial adenomatous polyposis col i and breast and ovarian cancers. Multiple genes have been associated with some syndromes as approximately 26 genes have been linked to the developmen t of these familial cancers. With this increased knowledge of the molecular determinants of familial cancer comes an equal expectation for efficient g enetic screening programs. We have trialled denaturing highperformance liqu id chromatography (dHPLC) as a tool for rapid germline mutation Scanning of genes implicated in three familial cancer syndromes-Cowden syndrome (PTEN mutation), multiple endocrine neoplasia type 2 (RET mutation) and von Hippe l-Lindau disease (VHL mutation). Thirty-two mutations, including 21 in PTEN , 9 in RET plus a polymorphism, and 2 in VHL, were analyzed using the WAVE DNA fragment analysis system with 100% detection efficiency. In the case of the tumor suppressor gene PTEN, mutations were scattered along most of the gene. However, mutations in the RET proto-oncogene associated with multipl e endocrine neoplasia type 2 were limited to specific clusters or "hotspots ." The use of GC-clamped primers to scan for mutations scattered along PTEN exons was shown to greatly enhance the sensitivity of detection of mutant hetero- and homoduplex peaks at a single denaturation temperature compared to fragments generated using non-GC-clamped primers. Thus, when scanning tu mor suppressor genes for germline mutation using dHPLC, the incorporation o f appropriate GC-clamped primers will likely increase the efficiency of mut ation detection.