Rapid Screening of 4000 Individuals for Germ-line Variations in the BRAF Gene

Abstract

Background:The BRAF gene is frequently somatically altered in malignant melanoma. A majority of variations are at the valine 600 residue leading to a V600E substitution that constitutively activates the kinase. We screened 4000 patient and control DNAs for germ-line variations at the valine 600 residue. Methods: We developed a novel assay by adapting single-base variation assays and software for MALDI-TOF (matrix-assisted laser desorption/ionization timeof-flight) mass spectrometry to screen for all 5 reported variants at codon 600 of the BRAF gene. We screened a case-control collection comprising samples from 1082 melanoma patients and 154 of their unaffected relatives from 1278 families and from 2744 individuals from 659 unselected twin families with no history of melanoma. A panel of 66 melanoma cell lines was used for variation-positive controls. Results: All melanoma cell lines that we had found previously to carry a codon 600 variation were verified in this study. Three of the 4 possible variants (V600E n ‫؍‬ 47, V600K n ‫؍‬ 2, V600R n ‫؍‬ 1) were detected, but no case of V600D was available. No germ-line variants were found in the samples from the 3980 melanoma patients or from the control individuals. Conclusions: This new assay is a high-throughput, automated alternative to standard sequencing and can be used as a rapid initial screen for somatic variants associated with melanoma. Germ-line variants at valine 600 are unlikely to exist and do not contribute to the reported role of the BRAF gene in melanoma predisposition.

BRAFV600E Immunohistochemistry Facilitates Universal Screening of Colorectal Cancers for Lynch Syndrome

“…[35] This platform has been specifically validated for BRAFV600E, V600R, V600K and V600M and the laboratory holds full National Association of Testing Authorities Australia accreditation for this assay. The paraffin blocks of discordant cases were macro-dissected a second time and re-analyzed using a real-time PCR (rt-PCR) based assay (Roche COBAS 4800 BRAF V600 Mutation Test).…”

Genetic risk factors for melanoma

“…These mutations are also the most common somatic CDK4 mutations found in melanoma (Bennett 2008a;Dahl and Guldberg 2007;de Snoo and Hayward 2005;Forbes et al 2008) and have been shown to convert CDK4 into an autosomal dominant oncogene. Other oncogene mutations frequently observed in melanoma specimens, such as those in BRAF and NRAS, are not found in the germline of melanomaprone families (James et al 2006). Germline BRAF mutations have been identiWed in the cardio-facio-cutaneous (CFC) syndrome; however, this syndrome is not associated with a higher risk of cancer (Niihori et al 2006;RodriguezViciana et al 2006).…”

Molecular Markers of Tumor Progression in Melanoma

“…However, benign nevi show similar or higher rates of the V600E BRAF mutation [20, 21], and cell line and transgenic mouse models of melanoma do not clearly demonstrate the transforming power of this mutation. Germline BRAF mutations do not occur in melanoma [22]. …”