title: Rapid mutation scanning of genes associated with familial cancer syndromes using denaturing high-performance liquid chromatography.
abstract: Germline mutations in tumor suppressor genes, or less frequently oncogenes, have been identified in up to 19 familial cancer syndromes including Li-Fraumeni syndrome, familial paraganglioma, familial adenomatous polyposis coli and breast and ovarian cancers. Multiple genes have been associated with some syndromes as approximately 26 genes have been linked to the development of these familial cancers. With this increased knowledge of the molecular determinants of familial cancer comes an equal expectation for efficient genetic screening programs. We have trialled denaturing high-performance liquid 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 Hippel-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 multiple endocrine neoplasia type 2 were limited to specific clusters or "hot spots." 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 tumor suppressor genes for germline mutation using dHPLC, the incorporation of appropriate GC-clamped primers will likely increase the efficiency of mutation detection.
authors.affiliation: Cancer Genetics, Kolling Institute of Medical Research, Royal North Shore Hospital, St Leonards, Sydney, NSW 2065, Australia. debbie_marsh@med.usyd.edu.au
authors.forename: D J
authors.initials: DJ
authors.lastname: Marsh
authors.forename: G
authors.initials: G
authors.lastname: Theodosopoulos
authors.forename: V
authors.initials: V
authors.lastname: Howell
authors.forename: A L
authors.initials: AL
authors.lastname: Richardson
authors.forename: D E
authors.initials: DE
authors.lastname: Benn
authors.forename: A L
authors.initials: AL
authors.lastname: Proos
authors.forename: C
authors.initials: C
authors.lastname: Eng
authors.forename: B G
authors.initials: BG
authors.lastname: Robinson
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D016147:Genes, Tumor Suppressor; D018095:Germ-Line Mutation / Q000235:genetics*; D006223:Hamartoma Syndrome, Multiple / Q000235:genetics / Q000473:pathology; D006801:Humans; D009377:Multiple Endocrine Neoplasia / Q000235:genetics / Q000473:pathology; D009386:Neoplastic Syndromes, Hereditary / Q000235:genetics*; D009691:Nucleic Acid Denaturation; D010641:Phenotype; D016133:Polymerase Chain Reaction; D000090063:Proto-Oncogene Mas; D013696:Temperature; D006623:von Hippel-Lindau Disease / Q000235:genetics / Q000473:pathology